Rice-based Diets in Pigs–for protection against intestinal bacterial infections

A report for the Rural Industries Research and Development Corporation by Associate Professor John Pluske and Professor David Hampson September 2005 RIRDC Publication No 05/143 RIRDC Project No. UMU-30A

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© 2005 Rural Industries Research and Development Corporation All rights reserved. ISBN1 74151 206 9 ISSN 1440-6845 Rice-based Diets in Pigs – for protection against intestinal bacterial infections Publication No. 05/143 Project No. UMU-30A The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable industries. The information should not be relied upon for the purpose of a particular matter. Specialist and/or appropriate legal advice should be obtained before any action or decision is taken on the basis of any material in this document. The Commonwealth of Australia, Rural Industries Research and Development Corporation, the authors or contributors do not assume liability of any kind whatsoever resulting from any person's use or reliance upon the content of this document. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186. Researcher Contact Details Associate Professor J.R. Pluske School of Veterinary and Biomedical Sciences Murdoch University Murdoch WA 6150 Phone: (08) 9360 2012 Fax: (08) 9360 2487 Email: J.Pluske@murdoch.edu.au

In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6272 4819 Fax: 02 6272 5877 Email: rirdc@rirdc.gov.au. Website: http://www.rirdc.gov.au Published in September 2005 Printed on environmentally friendly paper by Canprint

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Foreword In Australia, post–weaning diarrhoea (PWD) represents a major constraint to efficient and profitable pig production. Antibiotic resistance caused by the routine inclusion of antibiotics in diets is becoming a major reason for the perseverance of this disease. The European Union has banned the use of growth promoting antibiotics in diets for pigs from 1st January 2006, although in some EU countries such as Sweden and Denmark, a voluntary (industry) ban on the use of dietary growth promoting antibiotics has been enforced for some years. Growth promoting antibiotics in diets for pigs are still permitted in Australia, however in countries where growth-promoting antibiotics are banned, the incidence of PWD has increased dramatically, concomitant with an increase in mortality, compromised welfare and deterioration in feed conversion efficiency. The increased use of antibiotics for therapeutic use together with the persistent occurrence of PWD has increased costs, contributes further to antibiotic resistance, and has highlighted the need for greater understanding of the aetiology of PWD. This research package was designed to identify the effects of cooked white rice in diets for weanling pigs as a nutritional strategy for controlling PWD without reliance on growth promoting antibiotics. The general hypothesis tested was that the incidence of PWD could be reduced by strategic nutritional interventions using cooked white rice in the post-weaning period. The overall aim of this research project was to investigate the scientific bases of interactions between diet composition and the occurrence of PWD, focusing specifically on rice processing and ingredient interactions to ultimately develop specialised rice-based diets that can be fed to young pigs to control PWD in Australia. This would have potential value-adding benefits for the Australian rice industry. This publication summarises a series of experiments conducted in vitro and in vivo to ascertain the potential beneficial properties of Australian-grown rice in ameliorating PWD in piglets and maintaining/enhancing production in the post-weaning period. The experiments investigated the physico-chemical properties of rice, again both in vitro and in vivo, to determine the most suitable type(s) of rice for potential use in the pig industry to overcome PWD without the use of growth promoting antibiotics. This project was funded from industry revenue that is matched by funds provided by the Australian Government. This report is an addition to RIRDC’s diverse range of over 1200 research publications. It forms part of the Rice R&D Sub-program which aims to improve the profitability and sustainability of the Australian rice industry. Most of our publications are available for viewing, downloading or purchasing online through our website: • downloads at www.rirdc.gov.au/fullreports/index.html • purchases at www.rirdc.gov.au/eshop Peter O’Brien Managing Director Rural Industries Research and Development Corporation

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Acknowledgments The technical assistance of Ms Fiona Cavaney and Dr Jae-Cheol Kim from Murdoch University is thanked throughout the course of this research project. Associate Professor David Pethick, School of Veterinary and Biomedical Sciences, Murdoch University, is also thanked for critical input and discussion. Dr Melissa Fitzgerald, NSW Agriculture, Yanco, NSW, is thanked for early helpful discussions and chemical analysis in the screening component of the project. Dr Bruce Mullan, WA Department of Agriculture, is thanked for assistance with diet formulation and diet preparation. The staff at the Medina Research Station, particularly Messrs Bob Davis and Richard Seaward, are thanked also for assistance with running of pig feeding trials. Dr Robert van Barneveld, Barneveld Nutrition, is thanked for arranging the extrusion of rice used in the feeding trials in this project. Dr Lucile Montagne, a post-doctoral fellow from INRA in Rennes, France, is also thanked for her involvement in the work during her visit to Murdoch University.

Abbreviations ANOVA: analysis of variance. AOAC: Association of Official Agricultural Chemists. CP: crude protein. CTTAD: coefficient of total tract apparent digestibility. DE: digestible energy. DF: dietary fibre. DM: dry matter. FCR: feed conversion ratio (grams of feed per gram of daily bodyweight gain). FDS: fast digestible starch. GE: gross energy. PWC: post-weaning colibacillosis. PWD: post-weaning diarrhoea. N: nitrogen. NE: net energy. NH3: ammonia. NSP: non-starch polysaccharides. P: phosphorus. RS: resistant starch. SED: standard error of difference. SEM: standard error of the mean. VFA: volatile fatty acids.

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Contents Foreword................................................................................................................................iii Acknowledgments.................................................................................................................iv Abbreviations ........................................................................................................................iv Executive Summary .............................................................................................................vii 1. Introduction .....................................................................................................................1

1.1 Background ........................................................................................................................... 1 1.2 Post-weaning diarrhoea (PWD)............................................................................................. 2 1.3 Associations between diet and PWD..................................................................................... 3 1.4 Digestibility of rice and effects of processing....................................................................... 4 1.5 Interactions with other feed ingredients ................................................................................ 4

2. Objectives ........................................................................................................................6 3. Methodology ....................................................................................................................7

3.1 Database of physical and chemical characteristics of Australian rice for pigs...................... 7 3.2 Selection of rice varieties and processing methods on physico-chemical effects in the

weaned pig............................................................................................................................. 7 3.3 Interactive effects of cooked white rice with vegetable and animal protein sources on

digesta and fermentation characteristics and the faecal shedding of haemolytic E. coli....... 8 3.4 Effects of extrusion of rice and dietary protein sources on production, digestibility and

PWD...................................................................................................................................... 8 3.5 Effects of added oat hulls to extruded rice-based diets on production, digestibility and the

incidence of PWD ................................................................................................................. 9 3.6 The nutritive value of extruded rice and cooked (autoclaved) rice for weaner and grower

pigs ........................................................................................................................................ 9 3.7 On-farm testing of processed rice-based diets..................................................................... 10

4. Screening and selection of rice varieties for in vitro and feeding trials ..................11 4.1 Summary ............................................................................................................................. 11 4.2 Introduction ......................................................................................................................... 11 4.3 Materials and Methods ........................................................................................................ 11 4.4 Results and Discussion........................................................................................................ 12

5. Selection of rice varieties and processing methods on physico-chemical effects in the weaned pig ..............................................................................................................14 5.1 Summary ............................................................................................................................. 14 5.2 Introduction ......................................................................................................................... 14 5.3 Materials and Methods ........................................................................................................ 15 5.4 Results ................................................................................................................................. 16 5.5 Discussion ........................................................................................................................... 20

6. Selection of rice varieties and processing methods on physico-chemical effects in the weaned pig ..............................................................................................................22 6.1 Summary ............................................................................................................................. 22 6.2 Introduction ......................................................................................................................... 22 6.3 Materials and Methods ........................................................................................................ 23 6.4 Results ................................................................................................................................. 27 6.5 Discussion ........................................................................................................................... 34

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7. Interactive effects of cooked white rice with vegetable and animal protein sources on digesta and fermentation characteristics and the faecal shedding of haemolytic E. coli..............................................................................................................................37 7.1 Summary ............................................................................................................................. 37 7.2 Introduction ......................................................................................................................... 37 7.3 Materials and Methods ........................................................................................................ 38 7.4 Results ................................................................................................................................. 40 7.5 Discussion ........................................................................................................................... 43

8. Effect of extrusion of rice and dietary protein sources on production, digestibility and faecal shedding of E. coli ......................................................................................46 8.1 Summary ............................................................................................................................. 46 8.2 Introduction ......................................................................................................................... 46 8.3 Materials and Methods ........................................................................................................ 47 8.4 Results ................................................................................................................................. 50 8.5 Discussion ........................................................................................................................... 54

9. Effect of added oat hulls to extruded rice- and wheat-based diets on production, digestibility and the incidence of PWD .......................................................................56 9.1 Summary ............................................................................................................................. 56 9.2 Introduction ......................................................................................................................... 56 9.3 Materials and Methods ........................................................................................................ 57 9.4 Results ................................................................................................................................. 60 9.5 Discussion ........................................................................................................................... 67

10. The nutritive value of extruded rice and cooked (autoclaved) rice for weaner and grower pigs ....................................................................................................................70 10.1 Summary ............................................................................................................................. 70 10.2 Introduction ......................................................................................................................... 70 10.3 Materials and Methods ........................................................................................................ 71 10.4 Results ................................................................................................................................. 72 10.5 Discussion ........................................................................................................................... 75

11. Implications and Recommendations...........................................................................79 12. References .....................................................................................................................81

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Executive Summary Seven experiments were conducted in this research programme to test the general hypothesis that the incidence of post-weaning diarrhoea (PWD) could be reduced by strategic nutritional interventions in the post-weaning period using Australian-grown rice. An associated purpose of the study was to examine the use of rice-based diets on production indices in pigs and the physico-chemical properties of rice in the gastrointestinal tract. The major objectives of this research project were the development of a database describing the physical and chemical characteristics of Australian rice and their suitability in diets for pigs, the commercial development and uptake by the Australian pig Industry of specialty processed rice-based diets for protection against PWD caused by enterotoxigenic Escherichia coli (E. coli) and increased production, an increased understanding of the mechanisms whereby such diets afford protection, and potentially, although outside the scope of this project, a biomedical avenue into the use of such diets for control of enteric conditions in man. The potential benefits of this research programme were an increased utilisation of Australian rice particularly in value-added markets, the development of high-value specialty or ‘boutique’ diets based on processed rice for feeding the young pig and (or) pigs in states of enteric disease, and a possible avenue for rice into the biomedical industries, i.e. “functional foods”. Rice is a staple food for much of the world’s human population but has received relatively little attention as a possible feedstuff for the animal industries, in this case the Australian pig industry. Rice, once gelatinised, is highly digestible within the gastrointestinal tract of the pig that, in turn, could enhance performance over pigs fed wheat, the most commonly used grain used in Australia. Furthermore, and based on previous studies conducted in Australia and overseas, rice has potential to ameliorate PWD, a disease which costs the Australian pig industry millions of dollars annually. The increasing pressure that governments are facing worldwide to ban the use of dietary growth promoting antibiotics and dietary heavy metals such as zinc and copper in pig diets has meant that alternative strategies for the control of PWD, and indeed other enteric diseases, need to be found. This has already occurred in the European Union, for example. Based on these previous studies, cooked white rice appears to offer promise as an alternative nutritional strategy to the current use of antimicrobial compounds for mitigating PWD. However, more research was needed in order to refine further dietary recommendations that could be made to the rice and pig industries regarding the feeding of rice to pigs to reduce PWD without recourse to antimicrobials in the diet. The seven experiments conducted in this programme are presented as chapters, as follows: 1. Screening and selection of rice varieties for feeding and in vitro trials. 2. In vitro assessments of starch-related properties in response to rice type, cooking methods and

cooling after cooking. 3. Effect of rice type fed to piglets after weaning on starch digestion, digesta and fermentation

characteristics and the faecal shedding of haemolytic E. coli. 4. Interactive effects of cooked rice products with vegetable and animal protein sources on digesta

and fermentation characteristics and the faecal shedding of haemolytic E. coli. 5. Effect of extrusion of rice and dietary protein sources on production, digestibility and faecal

shedding of E. coli. 6. Effect of added oat hulls to extruded rice- and wheat-based diets on performance and diarrhoea

after weaning. 7. The energy value of extruded rice and cooked (autoclaved) rice for weaner and grower pigs. Results obtained in this research project have demonstrated that cooked (processed) white rice, either in medium-grain or long-grain form, included in diets for weanling pigs can be used as a replacement for wheat without a loss of production in the immediate post—weaning period. The decision to replace a cereal such as wheat in diets for weanling pigs, therefore, is likely to be one of price differential. Cooking broken white rice, particularly in medium-grain and waxy rice that have lower amylose levels than long-grain rice, increases starch digestibility when measured at the end of the small intestine. This

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could be predicted with accuracy in vitro using a “fast digestible starch” assay modified for rice in our laboratory. Regardless of the type and variety of rice used, however, pigs fed cooked white rice partition more digested nutrients into carcass gain than pigs fed other cereals such as wheat and barley, although the type of proteins fed to pigs will also influence this. In this regard, the use of extruded rice plus sources of animal protein (eg, milk powders, fishmeal, meat and bone meal) appear the best dietary combination for production purposes. Feeding vegetable (plant) proteins typically increased the weight of the gastrointestinal tract as a consequence of increased fermentative activity in the large intestine, and reduced bodyweight gain and FCR. Determination of the energy (DE and NE) values of extruded medium-grain (Amaroo) and long-grain (Doongara) rice confirmed the superior energy value of these two rice types over existing cereals used in Australian feeding of pigs, such as wheat. The effects of feeding cooked white rice on reducing faecal shedding of the bacterium (E. coli) responsible for causing PWD were generally unchanged, or even exacerbated, when the rice plus animal protein diets were fed compared to commercially-based diets that were considered a contributing factor to the incidence of PWD. The extent and duration of faecal shedding of enterotoxigenic E. coli found in the studies conducted was generally low, and this might have influenced the capacity of the rice-based diets to exhibit protective effects. It was hypothesised also that an imbalance in the amounts of carbohydrate versus protein entering the large intestine might have predisposed the pig to PWD, due to a change in the types of microbiota and subsequent production of compounds implicated in non-infectious diarrhoea. The results of Chapter 9 advocate the inclusion of a quantity of slowly or moderately fermentable dietary fibre to extruded rice-based diets consisting of animal protein to ameliorate the diarrhoea that is sometimes observed when feeding this diet, although in this instance 20 g kg- oat hulls depressed digestibility and production after weaning. Nevertheless, this proposition is consistent with European experiences of feeding processed rice to piglets after weaning. In this respect, it is feasible that the addition of rice bran and (or) rice hulls, or possibly the use of brown rice, in diets for piglets after weaning could achieve similar results. An unfortunate consequence of the drought in the rice-growing regions of NSW for this particular project, however, was the inability to perform an on-farm trial implementing some of the findings and conclusions arising from this research project. The major recommendations arising from this project are as follows: 1. Medium-grain rice (variety Amaroo) or long-grain rice (variety Doongara) was identified as being

the most suitable rice cultivars for utilisation in piglet feeds in Australia. Waxy rice, but not parboiled rice, would also be suitable, but its lower production tonnage in Australia at present would increase its price relative to other rice types and other cereals and hence limit its usefulness.

2. Processed (extruded) medium-grain rice (variety Amaroo) or long-grain rice (variety Doongara) is a suitable replacement for cereals currently fed to weanling pigs in Australia such as wheat and barley. Adoption of processed rice by the pig industry will be predominately driven by the price differential between processed rice and these alternative cereals.

3. Starch digestion at the end of the small intestine, as well as the colon, can be predicted accurately with a “fast digestible starch” assay modified for use in our laboratory. This test could be used by the rice industry as part of a broader screening process for potentially new varieties of rice suitable for the pig industry, however the assay is capable of being tailored for use in other species, including man.

4. Sources of animal protein in diets containing processed (extruded) rice generally cause superior production after weaning compared to vegetable (plant) sources of protein, although vegetable proteins showed reduced faecal shedding of haemolytic E. coli compared to animal sources of protein.

5. Producers feeding extruded rice-based diets with animal protein sources are encouraged to include some slowly or moderately fermentable dietary fibre, such as oat hulls, wheat bran and (or) beet pulp, to ameliorate the diarrhoea that is sometimes observed when feeding this diet. Future studies should investigate the addition of rice bran and (or) rice hulls, or possibly the use of brown rice, in diets for piglets after weaning that could accomplish similar results.

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6. The average (mean) digestible energy (DE) content (MJ/kg as-fed) of extruded rice is 15.3 MJ/kg as-fed. Medium-grain (Amaroo) rice has a 0.4 MJ/kg higher DE content than the long-grain rice (Doongara).

7. Pig producers should use different DE values for pigs of different ages/weights. Weanling pigs (8 kg) extracted less energy from both extruded rices than grower (55 kg) pigs (up to 0.5 MJ/kg difference). Producers using a net energy (NE) system should use a common value of 11.5 MJ/kg as-fed.

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1. Introduction 1.1 Background Rice is the staple cereal consumed by much of the world’s population, and a plethora of studies exist investigating the physical and chemical properties of cooked rice for man. The vast majority of these studies relate to the starch properties of rice, presumably because starch constitutes in excess of 75% of rice’s composition (Marsono and Topping, 1993), and hence forms the major carbohydrate consumed. The high starch content of cooked rice coupled with a very low non-starch polysaccharide (NSP) level makes cooked rice a ready source of absorbable glucose, and hence energy, for the human population. More recently, there is interest in the use of rice-based oral rehydration formulas for controlling enteric diseases in children (eg, Iyngkaran and Yadav, 1998; Ramakrishna et al., 2000) and animals (eg, Wingertzahn et al., 1999; Hampson et al., 2001). In contrast, there is less information pertaining to the feeding of rice to animals, especially the pig, with respect to effects on production and intestinal “health”, which incorporates enteric disease. This is predominately because other cereal sources, such as wheat, barley, corn and sorghum, are used in pig production and can be fed to pigs cheaper than rice. Nevertheless, and given the information available from the human literature with respect to the cooking and milling properties of rice, potential exists for the use of processed (cooked) rice in certain diets for pigs, especially the young pig. This is particularly when the intestine is compromised by enteric pathogens such as Escherichia coli, the agent of post-weaning colibacillosis (PWC) or, as it is more commonly recognised, post-weaning diarrhoea (PWD). Incorporation of processed rice into such diets has potential to add value to the Australian rice industry and reduce the pig Industry’s reliance on the use of growth promoting antibiotics. Furthermore, spin-offs into the biomedical field in the control of human enteric pathogens may be possible. The Australian pig industry currently has approximately 300,000 sows producing around 6 million pigs per year. The gross value of pig meat production is estimated at $900 million. Yearly feed consumption for pigs in Australia is estimated at 1.5 million tonnes, with weaner pigs (i.e., those from weaning to around 20 kg) consuming around 12%, or 180,000 tonnes, of this amount. Of this, around 20% is fed in the first two weeks after weaning, or 36,000 tonnes, which at present-day diet prices is worth between $25 and $30 million annually. Currently young pigs are fed predominantly wheat-based diets, however wheat is associated with increased incidence of PWD (McDonald et al., 1999). Protective diets based on processed rice, therefore, are ideally positioned to capture a considerable proportion of this market, particularly if antimicrobials such as growth promoting antibiotics are banned. Enteric bacterial infections such as PWD cause extensive morbidity and loss of production in the pig industry, and losses are currently valued in excess of $60 million annually (Cutler, 1992). Post-weaning diarrhoea (PWD), which is the locus of this research proposal, costs the Australian pig Industry between $22 and $26 million annually (Cutler, 1992). More recently, Cutler (2001) estimated that a 1% increase in post-weaning mortality, which incidentally occurred in Denmark following their ban on antimicrobials in weaner diets (Larsen, 2004), would reduce profit by $18 per sow per year; this equates to a total Australian industry cost of approximately $6 million. Antimicrobial agents are presently the main tool used for control of PWD, and are provided to pigs to treat overt disease, to provide prophylaxis in situations where disease is liable to occur, and to improve growth rates in the absence of disease. However, problems are arising over the use of antimicrobials in the pig industry. Their long-term use eventually selects for the survival of resistant bacterial species or strains, and genes encoding this resistance also can be transferred to other formerly susceptible bacteria. Currently a variety of bacterial pathogens of pigs are showing resistance to a range of antimicrobial drugs. Not only is this reducing the number of antimicrobials available to control

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bacterial diseases in pigs, but this resistance also poses risks to human health. Risks include the transfer of multidrug resistant zoonotic pathogens (eg, Salmonella spp. and Campylobacter spp.) from pigs to humans, the direct or indirect transfer of resistance genes from the porcine intestinal microflora to human bacterial strains, and the presence of antimicrobial drug residues in pig meat (Hampson et al., 2001). Public concern about these issues is leading to reduced availability or the complete banning of certain antimicrobial agents for use in pig production, as has occurred in certain parts of Europe. Consequently it is import to develop alternative means, such as the use of nutrition, both of controlling bacterial infections and promoting growth in pigs without recourse to the use of antimicrobials. In countries where growth-promoting antibiotics are banned, such as Sweden and Denmark, the incidence of PWD has increased dramatically, concomitant with an increase in mortality, compromised welfare and deterioration in feed conversion efficiency in the period after weaning (Larsen, 2004). Presently, where growth-promoting antibiotics cannot be included in diets, producers therapeutically treat pigs displaying overt signs of diarrhoea. However, the increased use of antibiotics for therapeutic use together with the persistent occurrence of PWD has increased costs, contributes further to antibiotic resistance, and has highlighted the need for greater understanding of the aetiology of PWD. There are currently no bans on the use of growth promoting antibiotics in the Australian pig Industry. However, Australian Pork Limited (APL), which represents the interests of Australian pork producers and is responsible for formulating policies on their behalf, has outlined strategies for the use of antibiotics. In this document (available at www.australianpork.com.au), APL stated: "The elimination or prudent use of antibiotics……is vital for success in this sophisticated, exacting and globalised marketplace, and should be embraced". This concern has arisen, at least partly, in recognition of the growing problem of antibiotic resistance in Australia (Barton, 1999, 2000) of several economically important enteric pathogens, such as E. coli, the causative agent of PWD. Barton (1999), for example, found that approximately 90% of the porcine E. coli isolates tested in Australia were resistant to three or more classes of antibiotics, with around 20% of isolates resistant to six or more classes. Collectively, there is a need to investigate alternative ways to ameliorate enteric conditions such as PWD without antibiotics. The use of nutritional intervention is one important means by which this could be achieved. 1.2 Post-weaning diarrhoea (PWD) PWD is a diarrhoeal disease that typically starts 3-4 days after weaning and continues until 9-12 days post-weaning. Piglets usually develop watery diarrhoea and show a rapid loss of condition, with most members of a litter being affected. PWD is endemic on some farms, being present in many litters, and repeatedly occurring in successive litters over many years. Besides potential mortalities, and the cost of treatment, piglets fail to gain weight immediately after weaning, and this could extend the total time to reach slaughter weight (Williams, 2003). PWD is a complex and multifactorial disease, incorporating many aspects of management (Madec et al., 1998), but important aetiological agents include E. coli, and sometimes rotaviruses (Lecce, 1983). The main aetiological agent(s) associated with PWD is (are) specialised strains of E. coli, which differ from common non-pathogenic types that occur in the intestinal tracts of healthy pigs. Unlike non-pathogenic strains, these pathogenic strains can adhere to the luminal surface of small intestinal enterocytes or the mucus covering the villi, particularly in the anterior small intestine, preventing them being flushed away to the more distal parts of the tract by normal peristaltic movement of the luminal contents. Attachment is through bacterial rod-like surface structures called fimbriae or pili. In pathogenic E. coli strains, adhesins K88 (also known as F4), and F18 (formerly F107) are most commonly associated with PWD, and these both exhibit several antigenic variants (Francis, 2002). At this site adjacent to the enterocyte surface they deliver powerful toxins that disrupt the normal functionality of the enterocytes. As the anterior small intestine has a critical function in both digestion and absorption, disruption of function here is particularly harmful.

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The most common and significant pathogenic types associated with PWD are enterotoxigenic E. coli (ETEC). Different ETEC strains release different combinations of two toxin types, heat labile toxin (LT) and heat stable toxin (ST; variants STa and STb), both of which provoke hypersecretory diarrhoea as a result of loss of water and electrolyte into the intestinal lumen. These processes result in an excess volume of fluid and electrolyte in the gut lumen of infected pigs. This volume can only be fully reabsorbed if the colon is healthy, has a stable, well-balanced microflora, and is not physically overloaded (Argenzio, 1992). In addition, the E. coli strains that cause PWD are usually able to lyse red blood cells present in the blood agar plates that are used for their isolation, and consequently these bacteria are known as β-haemolytic E. coli. The haemolytic activity is a useful marker of strains that are liable to be involved in PWD. There is a small number of O-serotypes that are repeatedly observed in association with PWD, of which the most common are: O149, O138, O139, O141 and O8 (Hampson, 1994). Haemolytic E. coli are uncommon in the intestinal tract of healthy unweaned pigs, although occasionally these strains are present in unweaned diarrhoeic pigs. Following weaning, these organisms frequently proliferate in the gastrointestinal tract of both healthy pigs and pigs that go on to develop diarrhoea. The key difference is that the number and proportion of potentially pathogenic strains of E. coli in the gastrointestinal tract and faeces is higher in pigs with PWD, compared against those that remain healthy (Hampson, 1994). Pigs with PWD have up to 109 colony forming units of such haemolytic E. coli in the small intestine, whilst there is minimal change in other resident bacterial populations at this time (Smith and Jones, 1963). Richards and Fraser (1961) first reported the link between excessive multiplication of haemolytic E. coli in the small intestine and the development of diarrhoea in weaner pigs. The associated disease, which has been called post-weaning colibacillosis (PWC), is characterised by diarrhoea, dehydration, rapid loss of weight, metabolic acidosis, poor condition and shivering (Bertschinger, 1999). The terms PWC and PWD tend to be used interchangeably, but PWC is a more specific term where the disease is completely or predominantly attributable to the E. coli infection. On the other hand, the term PWD acknowledges that the diarrhoea that often occurs in piglets after weaning may have other aetiologies and (or) complex interactions superimposed on the E. coli infection (Hampson, 1994). Faecal-oral spread between animals is the primary means of transmission (Bertschinger, 1999). 1.3 Associations between diet and PWD Research conducted previously at Murdoch University has shown that feeding cooked (ie, 121° C in an autoclave) white rice to pigs experimentally-infected with a number of economically-significant enteric pathogens, including the causative agent of PWD, E. coli, offers protection against the proliferation of such bacteria in the intestines with a subsequent reduction in the clinical expression of diarrhoea (see papers by Siba et al., 1996; Pluske et al., 1996; Pluske et al., 1998; McDonald et al., 1999; Hampson et al., 2000; McDonald et al., 2001). This is believed to be related to the type of diet fed after weaning. The main source of growth substrate for the gastrointestinal microflora comes from the diet. Simple sugars tend to act as the main growth substrate in the upper part of the gastrointestinal tract, whilst in the large intestine, where the main bacterial biomass is located, dietary fibre (DF) serves as the major bacterial substrate. Our data at present suggests that it is the soluble non-starch polysaccharide (NSP) component of feedstuffs that most likely promotes proliferation of E. coli in the gut and causes diarrhoea, although the role of resistant starch (RS) cannot be dismissed without further studies. This may be mediated by enhanced viscosity of the intestinal contents. As such, cooked rice with its low soluble NSP content, high concentration of readily digestible starch and low viscosity-inducing properties, offers promise as a grain that may be used to control PWD.

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While it is recognised that different types of fibre in the diet can broadly influence the composition and metabolic activity of the large intestinal microflora in pigs (Varel et al., 1982; Varel and Pond, 1985; Bach Knudsen et al., 1991; Jensen and Jorgensen, 1994; Reid and Hillman, 1999), little is known about the way in which these bacteria interact with pathogenic species of bacteria. This lack of information makes it difficult to predict how a given dietary component could be used to indirectly influence a given enteric pathogen. Besides influencing the normal gastrointestinal microflora, diet could also influence colonization by pathogens through other routes. For example it could act by modulating the amount of specific substrate available for the pathogen at a given site, by influencing viscosity of the intestinal contents and hence altering accessibility of receptor sites and (or) affecting intestinal motility, and by direct or indirect effects on the intestinal mucosa. As an example of the latter effect, different cereal types and particle size have been shown to alter epithelial cell proliferation and lectin binding patterns of the epithelium in the large intestine of pigs (Brunsgaard, 1998). Similar changes may occur in specific colonization sites or bacterial receptors on the enterocytes. The diet also might influence intestinal function; for example, components in boiled rice inhibit secretion in the small intestine, and hence reduce the magnitude of secretory diarrhoea due to pathogens such as enterotoxigenic E. coli (Mathews et al., 1999). Many of these questions remain unanswered. 1.4 Digestibility of rice and effects of processing Despite the plethora of information pertaining to the physical and processing characteristics of rice, information concerning the pattern of digestibility in vivo along the gastrointestinal (GI) tract in the pig is scarce. It is important to assess the digestibility of rice along the GI tract because proliferation of E. coli generally occurs more anteriorly in the small intestine. Hence it is necessary to maximise digestion of carbohydrate as anteriorly (ie, towards the mouth) as possible so that little or no substrate is available for the bacteria. Marsono and Topping (1993) demonstrated that the choice of cultivar as well as milling and further processing influences the level and types of DF (particularly NSP) in Australian rice. However, and as highlighted by these authors, it remains to be seen whether these chemical changes modify the physiological effects in vivo of processed rice products, although Devi and Geervani (2000) reported that in vitro starch digestibility values in puffed, boiled and parboiled-boiled rice samples were higher than in raw and parboiled rice samples. In addition, Sagum and Arcot (2000) reported some redistribution from insoluble to soluble NSP after heat processing, while Tetens et al. (1997) reported that the rate of starch digestion was influenced by both variety and method of cooking when assessed using an in vitro starch digestibility assay. In this particular study it was parboiling, however other forms of processing such as extrusion are also likely to influence the rate of starch digestion. Again, and in the pig, this type of information is unknown but is crucial if rice-based diets are to be used in the control of PWD. This is especially the case given our previous data implicating soluble NSP in the aetiology of PWD (McDonald et al., 1999). It is imperative, therefore, that these possible effects are investigated in vivo and that there is no increase in the proliferation of E. coli and increased incidence of PWD in (processed) rice-fed pigs. A pivotal study to answer this question, therefore, is an investigation of the sites and rates of starch digestion along the GI tract study in several rice cultivars in association with the processing method. 1.5 Interactions with other feed ingredients An important consideration in this work will be the interaction between rice and other dietary ingredients. These ingredients will predominantly be protein-based feedstuffs of either animal or plant origin, and generally comprise 25-30% of a weaner diet. There is some evidence that PWD is influenced by the protein content and quality of the diet (Hampson et al., 2001), and we suspect that this is attributable to vegetable protein sources that can contain up to 300 g kg-1 NSP. It is crucial,

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therefore, that any likely interactions between rice type, rice processing and different protein feedstuffs are studies to minimise the risk of pigs succumbing to PWD with processed rice diets. The general hypothesis tested in this research programme was that the incidence of PWD could be reduced by strategic nutritional interventions based on the incorporation of Australian-grown, cooked white rice in diets in the post-weaning period. The overall aim of this research project was to investigate the scientific bases of interactions between diet composition, the use of cooked white rice and the occurrence of PWD, focusing specifically on rice processing and ingredient interactions to ultimately develop specialised rice-based diets that can be fed to young pigs to control PWD in Australia. This would have potential value-adding benefits for the Australian rice industry.

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2. Objectives The major objectives of this research project were as follows: • development of an Australian database describing the physical and chemical characteristics of

processed rice, and their suitability in diets for pigs • commercial development and uptake by the pig Industry of specialty processed rice-based diets for

protection against post-weaning diarrhoea caused by enterotoxigenic Escherichia coli and increased production

• understanding of the mechanisms whereby such diets afford protection • a biomedical avenue into the use of such diets for control of enteric conditions in man. The potential benefits of this research programme can be summarised as follows: • increased utilisation of rice in added-value markets • development of high-value specialty or ‘boutique’ diets based on processed rice for feeding the

young pig and (or) pigs in states of enteric disease • a possible avenue for rice into the biomedical industries, ie “functional foods”. An important, and

potentially lucrative, spin-off from this research proposal is the development of specialty products/diets for use in the treatment of enteric infections in the human population.

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3. Methodology This research programme comprised several distinct but interrelated approaches to assess the utilisation of cooked white rice in pig diets against intestinal bacterial infections, specifically enterotoxigenic E. coli, the agent of post-weaning colibacillosis (PWC). This research plan was developed following discussions with Dr Keith Hutton, General Manager of Coprice Feeds and By-Products Group, Dr Melissa Fitzgerald, NSW Agriculture Yanco, feed manufacturers and representatives from the Australian pig industry. Discussions were also held with Dr Jeff Davis, RIRDC Rice R&D Program Manager, throughout the project. This summary provides an overview of the major methodological approach to the experiments conducted. Methodologies and techniques relevant to specific experiments/procedures are detailed in the experimental chapters that follow this section. 3.1 Database of physical and chemical characteristics of

Australian rice for pigs The major aim of this part of the programme was to characterise the starch-related properties of a number of different rice types and rice varieties grown by NSW Agriculture. This process acted as a way of ‘screening’ varieties/types of rice for their suitability in diets for piglets, as well as acting as a valuable composition database for the Australian rice industry. A monitoring process such as this will also aid in selection of rice for subsequent trials in this project. Unfortunately the persistent drought that affected the rice-growing regions in NSW over the duration of this particular project restricted this study to samples harvested only in 2001, because it was not possible to continue with such work given the lack of rice available and the time-scale of the project. Nevertheless, the wide range of samples examined provided a valuable screening database and allowed progression of the project onto its next stage. It met its aim in allowing the selection of rice types deemed suitable for use in piglet feeding trials and associated in vitro studies. The results from this experiment are outlined in Chapter 4. 3.2 Selection of rice varieties and processing methods on

physico-chemical effects in the weaned pig On the basis of 3.1 (above), and considering the relative contribution of the different rice types produced in Australia to the total tonnage produced annually, a common medium-grain variety (cv. Amaroo) and a common long-grain variety (cv. Doongara) were selected as ‘candidate’ rice types for use in the pig industry. Both in vitro (Experiment 3.2a) and in vivo (Experiment 3.2a) studies were examined. 3.2a In vitro assessments of starch-related properties in response to rice

type, cooking methods and cooling after cooking The first part of this experiment examined in vitro the starch-related properties of two rice varieties both before and after cooking. This experiment permitted the establishment of a number of chemical techniques in our laboratory, such as the analysis of total starch, resistant starch, amylose:amylopectin ratio and fast digestible starch (FDS), and provided a foundation for 3.2b (below) with respect to determining the optimum rice:water ratio and method of cooling after cooking that would allow for maximum digestibility of starch in the GI tract of weanling pigs. Furthermore, a sample of extruded rice allowed a preliminary examination of its potential physico-chemical properties in vivo in the newly-weaned piglet.

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The results from this experiment are outlined in Chapter 5. 3.2b Effect of rice type fed to piglets after weaning on starch digestion,

digesta characteristics and the incidence of PWD The aim of the second part of this study was to ascertain in vivo the feeding of rice of different chemical composition to pigs on aspects of production and PWD. The rice types selected were (i) an Australian-grown medium-grain variety (cv. Amaroo), (ii) an Australian-grown long-grain variety (cv. Doongara), and (iii) waxy rice sourced from Thailand. The waxy rice was chosen because of its high amylopectin content, which is thought to have a higher starch digestibility in the GI tract than rices of lower amylopectin (higher amylose) content. It was necessary to source the imported waxy rice because sufficient quantity of an Australian-grown waxy rice was unavailable. In combination with this study was the development of a technique (based on that of Tetens et al., 1997) to measure starch digestion in vitro; this can then be verified against the in vivo data that will be gathered. The results from this experiment are outlined in Chapter 6. 3.3 Interactive effects of cooked white rice with vegetable and

animal protein sources on digesta and fermentation characteristics and the faecal shedding of haemolytic E. coli

Previous studies using cooked white rice in diets for young pigs have always included other dietary ingredients that are low in NSP and (or) RS. These ingredients are primarily animal-based, such as meat and bone meal, fishmeal, blood meal, and milk powders. Vegetable protein sources, such as lupins, and peas, or oilseed meals such as canola meal and soybean meal, were specifically excluded from diets since the hypothesis has been that diets low in soluble NSP prevent PWD. Furthermore, German data derived in the 1970’s shows that ‘resistant protein’ (protein escaping digestion in the small intestine into the large intestine) might be a contributor to PWD in its own right. However, the inclusion of vegetable proteins in diets for young pigs is commonplace due to their cost effectiveness, but these tend to be ingredients containing high quantities of soluble NSP such as lupins and soybean meal. In addition, future pressure on the use of animal protein products such as meat and bone meal will place greater reliance on the use of vegetable proteins in young pig diets It was necessary, therefore, to examine other sources of vegetable proteins such that the benefits of cooked rice are not diminished by other ‘non-beneficial’ dietary ingredients. This study examined the effects of the sources of protein, that is, plant (vegetable) protein sources versus animal protein sources. This comparison was made because the animal protein sources (eg, fishmeal, meat and bone meal) are believed to be less likely to cause PWD since they do not contain NSP. Part of this study necessitated the oral infection of pigs with a virulent strain of E. coli and subsequent monitoring of bacterial shedding and diarrhoea after weaning. The results from this experiment are outlined in Chapter 7. 3.4 Effects of extrusion of rice and dietary protein sources on

production, digestibility and PWD The intention in this experiment was to use information acquired from the previous experiments to assist further in the commercial development of specialty (processed) rice-based diets against PWD and to enhance production. A number of different processing options for rice are available to the Australian pig industry, such as micronisation, expansion and extrusion. Some form of heat and

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moisture processing of rice is required before the grain can be fed to pigs to gelatinise the starch and improve its feeding and nutritive value. Discussions were held with Australian feed manufacturers and the pig industry regarding the relative merits of these processing techniques, and it was considered that extrusion was the most viable processing option for rice and its subsequent incorporation into a diet. Discussions were held with Dr Robert van Barneveld, Barneveld Nutrition, Queensland, who has previously extruded rice at the Roseworthy Campus of the University of Adelaide. Two-tonne batches each of a medium-grain rice (predominately Amaroo) and a long-grain rice (Doongara) were bought from a trading company in Melbourne, because Coprice Feeds Pty Ltd was unable to supply us with the rice. The rice was then transported to Roseworthy, extruded, and then transported to the Medina Research Station in Western Australia, where it was stored in sealed bulka-bags ready for use in animal experiments. Based on results achieved in Chapters 4 and 5, it was decided to compare the two extruded rice types against wheat, the most common cereal included in diets for young pigs in Australia. Further, the experiment allowed an opportunity to test each rice type with either plant (vegetable) or animal sources of protein. Production indices, digestibility and diarrhoea were monitored in this three-week feeding experiment, as were the number of antibiotic treatments given to pigs for post-weaning diarrhoea. The results from this experiment are outlined in Chapter 8. 3.5 Effects of added oat hulls to extruded rice-based diets on

production, digestibility and the incidence of PWD Results described in Chapters 7 and 8 indicated that pigs fed a diet based on cooked white rice and animal protein sources generally resulted in better performance, particularly in the first week after weaning, but unfortunately the incidence of diarrhoea appeared to be exacerbated. Recent work originating from Europe, where many pig-producing countries are not permitted to use growth-promoting antibiotics, demonstrates that modulation of the diet is a key tool in the amelioration of PWD. Researchers have been investigating the influence of added insoluble/moderately fermentable sources of DF to reduce PWD. On the basis of this work, and after discussions with Dr Jeff Davis, it was decided to conduct an additional experiment using oat hulls, a rich source of insoluble DF, to establish its influence on post-weaning performance and PWD. The results from this experiment are outlined in Chapter 9. 3.6 The nutritive value of extruded rice and cooked (autoclaved)

rice for weaner and grower pigs The supply of extruded rice (Chapter 8) provided an ideal opportunity to examine the energy value of extruded medium-grain and extruded long-grain rice in pigs of two different ages – weaner and grower pigs. An accurate estimate of the energy value of rice was deemed important for the Australian feed manufacturing industry because accurate diet formulation for piglet diets depends, in part, on the accuracy of the estimates manufactures have for energy value. To our knowledge, there are no current estimates of the energy values of extruded rice for pigs; establishment of these estimates would be of value to the pig industry and potentially mean fewer barriers to the use of rice in pig diets. The results from this experiment are outlined in Chapter 10.

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3.7 On-farm testing of processed rice-based diets The final part of the research project was the conduct of some on-farm trials to test the efficacy of specialty rice-based diets against PWD and their effects on production in a practical setting. The formulation of the diets was to be based on the information derived from the aforementioned trials. The studies were planned for Australia’s largest producer of pigs, QAF Meat Industries (ex Bunge Meat Industries) in Corowa, NSW, and an experiment was also planned for Wandalup Farms in Western Australia. After discussions with Dr Jeff Davis, RIRDC Rice R&D Programme manager, these studies could unfortunately not be performed. The persistent drought in NSW over the past few years severely reduced the total quantity of rice harvested and therefore available for use in such trial work. The industry (in-kind) partner in the project, Coprice Feeds Pty Ltd in Leeton, advised that they could not supply sufficient rice for use in such trials.

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4. Screening and selection of rice varieties for in vitro and feeding trials

4.1 Summary A number of different rice varieties representing waxy, medium-grain and long-grain types were obtained from the 2001 harvest at NSW Agriculture, Yanco, for assessment of potential suitability for use in specialty diets for young pigs after weaning, based on in vitro chemical characteristics. The waxy and medium-grain rice varieties were judged to be the most suitable for use in piglet diets due to their lower amylose contents, which should result in higher starch digestibility in vivo. However, and based on quantities of the different rice varieties produced in Australia at the present time, the medium-grain rice Amaroo was considered the most promising to pursue for feeding to piglets. Waxy rice varieties, although most probably affording high starch digestibility coefficients, are produced in lesser quantities and the ‘waxy’ nature of the starch would most likely cause feed manufacturing problems on a commercial basis. 4.2 Introduction Rice is used mainly as a human foodstuff around the world, and only rice by-products (eg, broken rice) are generally used in the animal industries. A plethora of studies exist investigating the physical and chemical properties of cooked rice for man. The high starch content of cooked rice and its low DF level makes cooked rice a ready source of absorbable glucose, and hence energy. To the converse, less information exists regarding rice fed to pigs regarding influences on production and the prevention of enteric disease. However, and given information that exists in the human literature about the cooking and milling properties of rice, potential exists for the use of cooked rice in diets for young pigs. This is because increasing pressure is being placed on governments to restrict/ban the use of growth-promoting antibiotics in the intensive livestock industries for the control of enteric pathogens such as E. coli, the agent of PWD (Pluske et al., 2002). Use of processed rice in ‘specialty’ diets has potential to add value to the Australian rice industry and reduce the pig Industry’s reliance on the use of antibiotics. The major aim of this particular study was to characterise and ‘screen’ the starch-related properties of different rice varieties grown in NSW for potential use in diets for pigs. The three major types of rices considered most suitable for inclusion in Australian pig diets were waxy, medium-grain and long-grain. 4.3 Materials and Methods Dr Melissa Fitzgerald sourced a variety of different rice samples from the 2001 rice harvest at NSW Agriculture, Yanco. The waxy rice varieties were Shimuzi and Tarra 140, the medium-grain rice varieties were Amaroo, Millin and Langi, and the long-grain rice varieties were Doongara and L203. 4.3.1 Chemical analyses The amylose content was determined at NSW Agriculture, Yanco, according to a PCR-based method established in Dr Fitzgerald’s laboratory at Yanco, NSW. The amylopectin content of rice was determined by difference from total starch. Total Starch and Megazyme Resistant Starch kits (Megazyme International Ireland, Ltd., Wicklow, Ireland) were used for analysis of total starch and RS, respectively. For total starch analysis, 100-mg samples were pre-incubated with 2 mL DMSO in a boiling water bath for 5 min prior to incubation with the thermostable α-amylase and then with amyloglucosidase. The starch content was determined at 510 nm using a spectrophotometer (UVmini-

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1240, Shimadzu, Kyoto, Japan). For RS determination, 100-mg samples were incubated at 37 °C for 16 hrs with pancreatic α-amylase and amyloglucosidase. The hydrolysed sugars were washed several times with ethanol. The remainder was then hydrolysed with 2M KOH and then with amyloglucosidase. The RS content was determined at 510 nm. The fast digestible starch (FDS) content of the raw rices was determined using the method of Zarrinkalam et al. (2001), which is a modification of the AOAC official method 996.11 (AOAC 1997). 4.4 Results and Discussion The starch-related characteristics of the rice samples are presented in Table 4.1. The amylose content varied five-fold, ranging from 5.1% in the waxy Shimuzi variety to 25.7% in the long-grain variety L203. The amylose:amylopectin ratio in these two varieties similarly differed, ranging between 0.05 to 0.35. Gelatinisation temperature varied between 61.6 ºC for Shimuzi to 78.8 ºC for the long-grain variety Langi. The amylose content of rice provides an indication of the texture of cooked rice, and the higher the amylose content, then in general the firmer the rice but the less digestible the rice will be. During starch digestion in the pig, the α–amylase produced in the pancreas breaks down amylose to maltose and maltotriose and amylopectin is broken down to maltose, maltotriose and α–limit dextrins (Li et al., 2004). Amylopectin is regarded as being more digestible than amylose in starch because its branched structure allows more complete digestion by α–amylase, so in this regard, and for the young pig that has a lower capacity to digest amylose (Black, 2001), it is suggested that a waxy rice or medium-grain rice would be most suitable for incorporation into diets for young pigs, at least for production purposes. The digestibility of starch in the long-grain varieties, ie, Doongara and L203, might be reduced given the higher amylose content. This, in turn, could cause poorer performance and (or) result in increased susceptibility to enteric pathogens such as E. coli because of a higher bypass of RS entering the distal regions of the GI tract. Table 4.1 Starch-related characteristics of selected rice varietiesA. Type of rice

Variety Total starch, g/kg

Amylose, % Amylopectin, % Am:ApB Gelatinisation temperature, ºC

Waxy Shimuzi 776 5.1 94.9 0.05 61.6 Tarra 140 774 8.6 91.4 0.09 78.7 Medium grain

Amaroo 799 18.2 81.8 0.22 67.9

Millin 791 16.4 83.6 0.20 69.3 Langi 772 17.2 82.8 0.21 78.8 Long grain Doongara 775 23.8 76.2 0.31 75.7 L203 801 25.7 74.3 0.35 73.8 AAll analyses conducted by Dr Melissa Fitzgerald, NSW Agriculture, Yanco (except total starch). BAm:Ap: amylose to amylopectin ratio. Gelatinisation temperature is the temperature required to melt the amylopectin crystals within starch (Fitzgerald et al., 2003). This is important information to know for the pig feed industry given that rice used in diets will need to undergo some form of heat processing. In this regard, it would appear that in all varieties examined, the temperature at which all starch crystals ‘melt’ (are gelatinised) will present no difficulties to pig feed manufacturers. This is because pelleting of feeds generally occurs at temperatures ranging between 75 ºC and 90 ºC. Extrusion temperatures commonly exceed 120 ºC.

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In addition to these measurements, an in vitro ‘fast digestible starch’ (FDS) technique adapted in our laboratory was used as a rapid screening method for the suitability of rice in diets for young pigs. Analysis performed using this technique showed that the amount of glucose released in vitro correlated to the amylose content in the rice, suggesting that this method has possible merit as a means of screening rices for potential inclusion in diets. Further implementation and validation of this technique is described in Chapter 5. It was the intention of this study to track these particular varieties over three successive growing seasons to examine the variation that occurred in the selected measured parameters, and hence establish a nutrient database for the Australian rice and pig industries. Unfortunately the drought that affected the rice-growing regions in NSW over the duration of this particular project restricted this study to samples harvested only in 2001, because it was not possible to continue with such work given the lack of rice available and the time-scale of the project. In conclusion, the wide range of samples examined in the harvest from 2001 provided a valuable screening database and allowed progression of the project onto its next stage. This study met its aim in allowing the selection of rice types deemed suitable for use in piglet feeding trials and associated in vitro studies, considering the tonnage of rice produced in Australia, the quantities of ‘brokens’ available for use in the Australian pig industry, and the requirements for processing of rice by the feed manufacturing industry.

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5. Selection of rice varieties and processing methods on physico-chemical effects in the weaned pig

Experiment A: In vitro assessments of starch-related properties in response to rice type, cooking methods and cooling after cooking 5.1 Summary Three in vitro experiments were conducted to examine the effect of rice type, rice:water ratio and cooling on the resistant starch (RS) content of autoclaved rice. The three types of rice used were Amaroo (lower amylose, medium-grain variety), Doongara (higher amylose, long-grain variety) and parboiled rice. In Experiment 1, total starch, amylose, fast digestible starch (FDS) and RS contents in three uncooked rice samples were examined. The variety Amaroo contained less amylose (18.8 g 100g-1, P=0.001), a higher FDS content (21.7 g 100g-1, P<0.001) and less RS (0.1 g 100g-1, P<0.001) than Doongara (25.6, 15.9, 0.4, respectively). Parboiled rice contained the highest FDS (33.9 g 100g-1) and RS (0.72 g 100g-1) contents with an amylose content of 25.4 g 100g-1. In Experiment 2, the effects of rice type, rice:water ratio (1:1 or 1:2 w/w) and cooling (freshly dried or dried after refrigeration at 4 °C for 24 hrs) on the RS content of autoclaved rice were examined. The RS contents differed according to rice type (0.6, 1.4, 3.7 g 100g-1 for Amaroo, Doongara and parboiled, respectively, P<0.001). Decreasing the rice:water ratio (1:2) and cooling (24 hours at 4oC) after autoclaving increased the RS content (P<0.001). In Experiment 3, extrusion of rice using a single-screw extruder decreased the RS content only in Doongara, which had the highest RS content in its unextruded (raw) form (0.42 to 0.16 g 100g-1, P=0.02). The results indicate that more amylose, a lower rice:water ratio and cooling after autoclaving increase the RS content of rice, while extrusion decreases the RS content. 5.2 Introduction Resistant starch (RS) has been defined as starch that is resistant to enzymatic digestion in the small intestine but is fermented by the microbiota in the large intestine of non-ruminant animals (Englyst et al., 1992). Debate still exists as to whether RS should be included as part of dietary fibre (DF) because while it resists enzymatic digestion in the small intestine and behaves similarly to DF in the gut, it has the chemical structure of hydrolysable alpha-linked hexoses (Trowell et al., 1976; Englyst and Cummings, 1987). It is evident that some feed ingredients containing RS are fermentable in the small intestine (eg, Berggren et al., 1995; Heijnen and Beynen, 1997; Govers et al., 1999). Resistant starch is generally classified as RS1, physically inaccessible starch due to structural encapsulation which is mainly found in raw forms of grain; RS2, enzyme-resistant, B-type starch granules found mainly in unripe bananas and raw potato; and RS3, retrograde starch that is found mainly in processed grains and also occurs after cooking and cooling (Englyst et al., 1992). Rice is characterised by its high starch content, low non-starch polysaccharide (NSP) content and lower protein content in comparison to other cereals (Juliano, 1992). Rice has been investigated in diets for young pigs because of its positive effects on production relative to cereals such as maize, wheat and sorghum (Alcantara et al., 1989; Mateos et al., 2001, 2002; Martin et al., 2003; Vicente et al., 2004). Furthermore, the use of cooked rice has been associated with reductions in PWC and swine dysentery (see review by Pluske et al., 2002; Pluske et al., 2003; Hopwood et al., 2004). Moreover, Reid and Hillman (1999) reported lower coliform populations and higher Lactobacillus populations when retrograde RS was fed to pigs in the form of raw potato starch, suggesting that RS might play a role in altering the microbial ecology of the lower gut. Resistant starch is also known to influence ileal

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starch and faecal crude fat and energy digestibility, and the proliferation of colonic microbes (De Schrijver et al., 1999). It is most likely that rice requires some form of cooking and drying, such as extrusion, to enable gelatinisation of the starch and its incorporation into a commercial piglet diet. Generally, the RS content of rice increases with cooking and the amylose content is positively correlated with the increase in RS content in cooked rice (Juliano, 1992). Quantifying the RS contents of rice having different amylose contents and with various cooking and cooling combinations, therefore, should assist in the selection of rice types and processing conditions for the formulation of diets for piglets. A number of in vitro studies were conducted to examine whether: 1) rice with a higher amylose content contains a higher RS content, 2) autoclaving (cooking) will increase the RS content, 3) cooling after autoclaving will increase the RS content of rice, and 4) extrusion reduces the RS content of rice. 5.3 Materials and Methods 5.3.1 Rice varieties Three varieties of commercially available rice were obtained: (i) Amaroo (a lower amylose, medium-grain rice; Australian Ricegrowers’ Cooperative, Leeton, NSW, Australia), (ii) Doongara (a higher amylose, long-grain rice; Australian Ricegrowers’ Cooperative, Leeton, NSW, Australia), and (iii) parboiled rice (‘Tastic’ premium parboiled rice; Riviana Foods Pty Ltd, Australia). 5.3.2 Experimental design, cooking and cooling Three in vitro experiments were conducted. In Experiment 1, the total starch and RS contents were compared in the three uncooked rice samples. In Experiment 2, the effects of rice type, rice:water ratio and cooling combinations on the RS content of autoclaved rice were tested using a 3 x 2 x 2 factorial design. The respective factors in the study were the three rice types (Amaroo, Doongara, parboiled), two rice:water ratios (1:1 or 1:2 w/w) and two cooling treatments (non-refrigerated, or refrigerated at 4 °C for 24 hrs). All samples of rice were cooked in an autoclave (121 °C, 20 min steaming plus 20 min drying) with rice: water ratios of either 1:1 or 1:2 (w/w). After cooking, the rice was divided into two portions. The first portion was sampled immediately after cooking (‘Fresh’) whilst another sample was taken following refrigeration at 4 °C for 24 hours (‘Cooled’). These samples of rice were then dried at 70 °C for 48 hours to enable chemical analyses. After drying, the samples were milled to pass through a 1 mm screen twice. The milled samples were later analysed for total starch and RS contents. In Experiment 3, the effect of extrusion was examined by comparing the RS contents in raw and extruded forms of Amaroo and Doongara. The extruded rice samples were obtained from the Roseworthy Campus of the University of Adelaide. 5.3.3 Chemical analyses Dry matter (DM) was measured using the AOAC official method 930.15 (AOAC, 1997). Total starch and RS were determined as described previously (Chapter 4.2.1). The fast digestible starch (FDS) content of the raw rices was determined using the method of Zarrinkalam et al. (2001), which is a modification of the AOAC official method 996.11 (AOAC 1997). Ten sub-samples of each treatment combination were analysed in duplicate for the analysis of total starch, RS and the FDS contents. The amylose content of raw rice was determined by the calorimetric measurement of the iodine binding capacity of the amylose using a Megazyme Amylose/Amylopectin Assay Kit (Megazyme International Ireland, Ltd., Wicklow, Ireland). Amylopectin content was determined as the difference between the measured amylose and total starch contents.

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5.3.4 Statistical analysis For Experiment 1, the effect of rice type on the total starch, amylose, FDS and RS contents were assessed by one-way ANOVA. In Experiment 2, treatment effects were assessed by ANOVA for a factorial arrangement with the main effects being rice type, rice:water ratio and refrigeration. The effects were considered as fixed effects in the model. For Experiment 3, the effect of extrusion cooking on the RS content was assessed by ANOVA for a factorial arrangement with the main effects being rice type and extrusion cooking. All statistical analyses were conducted using the statistical package StatView 5.0 for Windows, SAS Inc. (AddSoft Pty. Ltd., Woodend, Vic., Australia). Statistical significance was accepted at P < 0.05. 5.4 Results 5.4.1 Experiment 1 Total starch, amylose, FDS and RS content in three uncooked rice samples are presented in Table 5.1. Total starch contents of uncooked rice were similar in Amaroo and Doongara, but were higher in parboiled rice (P<0.001). Amylose content was lower in Amaroo than in Doongara and parboiled rice (P<0.001). The FDS content decreased in the order of parboiled, Amaroo and Doongara (P<0.001). The RS content decreased in the order of parboiled, Doongara and Amaroo (P<0.001). The RS contents of raw rice ranged from 0.1 to 0.7 g 100-1. 5.4.2 Experiment 2 The main effects of rice type, rice:water ratio, refrigeration on RS content of autoclaved rice are presented in Table 5.2. The total starch contents in autoclaved rice were not influenced by any treatment. However, the RS content was significantly different in the three autoclaved rice types (P<0.001). The range in RS content was 0.6 g to 5.1 g 100g-1 DM. Decreasing the rice:water ratio and refrigeration (‘Cooled’, ie, 24 hours at 4oC) after autoclaving significantly increased the RS content (P<0.001). There were significant three-way interactions between rice, rice:water ratio and refrigeration (P<0.002) (Figure 5.1). 5.4.3 Experiment 3 The effect of extrusion on RS content is presented in Table 5.3. Extrusion decreased the RS content in Doongara to the level of the lower amylose, medium-grain variety Amaroo. However, the RS content was not influenced by extrusion in variety Amaroo as evidenced by a significant rice by extrusion interaction (P=0.005).

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Table 5.1 Starch-related properties (DM) in three types of uncooked (raw) rice (Experiment 1)A. 1

Rice Amaroo Doongara Parboiled Pooled mean SEMB Probability, P=

Total starch (g 100g-1) 86.1a 85.6a 89.4b 87.0 0.33 <0.001

Amylose (g 100g-1) 18.8a 25.6b 25.4b 22.8 1.15 <0.001

Amylopectin (g 100g-1) 67.3a 60.0c 64.0b 64.5 0.98 0.002

Amylose:amylopectin ratio (%) 0.28a 0.43b 0.40b 0.36 0.022 <0.001

FDS (g 100g-1)C 21.7b 15.9a 33.9c 20.5 1.37 <0.001

Resistant starch (g 100g –1)C 0.10a 0.42b 0.72c 0.41 0.058 <0.001

Prop. RS of total starch (%) 0.11a 0.49b 0.81c 0.47 0.066 <0.001 AValues are mean of 10 observations, except amylose (3) and FDS (8). 2 BSEM: standard error of the mean. 3 CFDS: fast digestible starch; RS: resistant starch. 4 abcValues within a row with different superscripts are significantly different (P<0.05). 5

18

Table 5.2 Main effects of rice type, rice:water ratio and refrigeration on starch-related properties (DM; Experiment 2)A. 1

Treatment Total starch (g 100g-1) Resistant starch (RS)

(g 100g–1)

Prop. RS of total starch (%)

Amaroo 90.2 0.60a 0.66a

Doongara 90.4 1.42b 1.57b Rice

Parboiled 90.1 3.74d 4.15d

1:1 90.3 1.52b 1.69b Rice:water ratio

1:2 90.1 2.31c 2.56c

Fresh 90.2 1.54b 1.71b RefrigerationB

Cooled 90.2 2.29c 2.54c

Pooled mean 90.2 1.92 2.13

SEMC 0.07 0.149 0.157

StatisticsD Probability, P=

Rice 0.327 <0.001 <0.001

Rice:water ratio 0.245 <0.001 <0.001

Refrigeration 0.669 <0.001 <0.001 AValues from mean of 10 observations for each treatment combination. 2 BFresh’: autoclaved and dried at 70 °C, 48 hrs; ‘Cooled’: autoclaved and stored at 4 °C, 24 hrs and dried at 70 °C, 48 hrs. 3 CSEM: Standard error of the mean. 4 DThree way interactions were significant for RS (P=0.002) and prop. RS of total starch (P=0.005); abcValues within a column with different superscripts are 5 significantly different (P<0.05). 6 .7

19

Table 5.3 Effect of extrusion on the RS content (g 100g-1 DM) of rice (Experiment 3)A.

Resistant starch (g 100g-1) DM

Raw Extruded

Amaroo 0.10 0.13

Doongara 0.42 0.16

Pooled mean 0.26 0.14

SEMB 0.051 0.028

Statistics Probability, P=

Rice (R) <0.001

Extrusion (E) 0.020

R x E interaction 0.005 AValues are mean of 10 observations for raw rice and mean of 5 observations for extruded rice. BSEM: standard error of the mean.

0

1

2

3

4

5

6

UC F R F R UC F R F R UC F R F R

Res

ista

nt s

tarc

h (g

100

g-1 D

M)

Rice:waterRice Amaroo Doongara Parboiled

Refrigeration

1:1 1:1 1:11:2 1:2 1:2

Figure 5.1 Interaction plot for the RS content (g 100g-1 DM) against rice type (Amaroo, Doongara and parboiled), rice:water ratio (1:1 versus 1:2) and cooking (‘Fresh’ versus ‘Cooled’) combinations in autoclaved rice (Experiment 2). The UC, F and R represents uncooked, fresh and refrigerated (‘Cooled’) rice, respectively.

20

5.5 Discussion Experiment 1 examined the content of RS1 in three types of rice, although parboiled rice presumably contains both RS1 and RS3. As hypothesised, the higher amylose rice variety Doongara had a higher RS content than the lower amylose variety Amaroo, which is in agreement with previous reports (Marsono and Topping, 1993; Sagum and Arcot, 2000). The RS contents in the three types of raw rice were within the range reported previously (Muir and O’Dea, 1993; Tetens et al., 1997), with the parboiled rice having the highest RS content. Parboiling leads to the formation of very heat resistant amylose-lipid complexes (Juliano, 1992), which have been shown to reduce the rate of starch digestion in rats (Holm et al., 1983). Tetens et al. (1997) reported that the amylose content of rice was negatively correlated to the degree in vitro of starch digestion, and that parboiling caused significantly lower rates of starch digestion compared to non-parboiled rice varieties. Data from our study only partly supports these findings because although Amaroo had lower amylose and RS contents than the parboiled rice, Doongara had a similar amylose content to the parboiled rice but had significantly less RS and a lower FDS content. Tetens et al. (1997) commented, however, that the extent to which rice is parboiled varies considerably and hence this could be an explanation for this discrepancy. Starch with a high amylose:amylopectin ratio has a dense and rigid structure due to the extensive hydrogen bonding between amylose molecules and possible amylose-lipid complexes, which results in hard starch gels in cooked rice (Tetens et al., 1997). The tight helical twists of amylose chains encapsulate some starch that makes it less accessible to pancreatic α-amylase in the small intestine (Zobel and Stephen, 1995; Black 2001). Therefore, the starch in waxy or lower amylose rice is generally hydrolysed more rapidly and more completely than that in a higher amylose rice variety (Hu et al., 2004). The highest RS content found in uncooked parboiled rice is due mainly to the formation of RS3 during the parboiling process (Marsono and Topping, 1994; Devi and Geervani, 2000). Experiment 2 demonstrated that the type of rice, rice:water ratio and refrigeration influenced the formation of RS. The higher amylose variety Doongara had a higher RS content after autoclaving, which is in agreement with previous reports by Juliano (1992), Marsono and Topping (1993), Mangala et al. (1999b) and Sagum and Arcot (2000). Autoclaving causes gelatinisation of the starch, but then retrogradation to RS3 occurs if the rice is cooled after the cooking process. Gelatinisation disorders the molecular structure of starch and the dispersed starch polymers reform semi-crystalline structures (retrogradation) when cooled (Garcia-Alonso et al., 1998). The presence of higher amounts of linear amylose increased RS formation upon cooking and cooling because the amylose molecules align themselves or associate with each other during retrogradation (Sagum and Arcot, 2000), and retrograded amylose is particularly resistant to hydrolysis by pancreatic α-amylase (Gee et al., 1991). Although long unbranched chains of amylopectin are possibly involved in the formation of RS when cooked starch was stored for up to 20 days (Mangala et al., 1999a), structural studies revealed that the majority of RS formed upon cooking and cooling was a linear (1 4)-a-D-glucan, originating from amylose (Mangala and Tharanathan, 1999). Also, amylose chain length was positively related to the RS formation in a study with potato starch (Eerlingen et al., 1993). Therefore, RS formation in autoclaved rice most likely increases with the increasing amount of amylose in rice, as espoused by Juliano (1992). Highest RS contents have been observed when parboiled rice was autoclaved since repeated cooking further increases the RS content in rice (Mangala et al., 1999a). In addition, a decreased rice:water ratio increased RS formation after cooking. With excess water, the amorphous phase of the starch granule swells and the crystalline regions become even more disrupted. This higher disruption contributes to the higher RS formation when the starch retrogrades (Sagum and Arcot, 2000). In the current study, refrigeration of cooked rice after autoclaving (‘Cooled’) increased RS formation by promoting retrogradation of starch polymers. Temperature plays an important role during the retrogradation process. Marsono and Topping (1993) found significantly increased RS contents in rice cooked in a rice-cooker and in microwave-cooked rice when they were stored at 0-4 °C compared to

21

freshly cooked rice. Mitsuda (1993) found that rice stored at –20 °C retrogrades more than one stored in a refrigerator at approximately 4 °C. In a more detailed study, Kavita et al. (1998) found that the RS content of cooked rice was 1.96 g 100g-1 DM. When samples were stored for 24 hrs or 48 hrs at 4 °C, the RS contents increased to 3.37 g and 4.38 g 100g-1 DM, respectively. In their next experiment, Kavita et al. (1988) reported that the RS content increased to 14%, 20% and 27%, when the cooked rice (RS content of 1.14 g 100g-1 DM) was stored for 12 hrs at 27 °C, for 12 hrs at 4 °C and for 24 hrs at 4 °C, respectively. In our study, the significant three-way interaction between rice, the rice:water ratio and refrigeration (Figure 5.1) was due to disparity in the RS values with different combinations of cooking and refrigeration. For example, the increase in RS content in Amaroo after cooking (autoclaving) and refrigeration attained the same level as that in the ‘Fresh’ high amylose rice. Extrusion decreased the RS content in Doongara but not in the lower amylose rice Amaroo, which caused a significant statistical interaction (P=0.005; Experiment 3). Parchure and Kulkarni (1997) reported that extruded rice had a lower RS content compared to that of raw and cooked rice, a finding found also by Pluske et al. (1996) but in a comparison of whole (ground) wheat versus extruded wheat. In conclusion, the range in RS formation as a consequence of the various cooking and cooling combinations under test was from 0.6 g to 5.1 g 100g-1 DM. The extent of RS formation was significantly influenced by the amylose content of rice, the volume of water used for cooking, and cooling after cooking. Further work is required using pigs to ascertain the importance, or otherwise, of these treatments on pig production and resistance/susceptibility to PWC. This was investigated in part in the next experiment.

22

6. Selection of rice varieties and processing methods on physico-chemical effects in the weaned pig

Experiment B: Effect of rice type fed to piglets after weaning on starch digestion, digesta and fermentation characteristics and the faecal shedding of haemolytic E. coli 6.1 Summary An experiment was conducted in male pigs weaned at approximately 21 days of age to examine the effects of different types of cooked white rice mixed with animal protein sources on starch digestion, fermentation characteristics, shedding of E. coli and performance in the 14 days following weaning. Pigs were allocated in a completely randomised block design having four dietary treatments, with 12 pigs allocated to each diet. The three rice types used were a medium-grain rice (Amaroo; AM), a long-grain rice (Doongara; DOON) and a waxy rice (WAXY), which were included in a diet at an inclusion level of 703 g kg-1. These three diets were compared against a diet comprising predominately wheat, barley and lupins (WBL). All diets contained the marker titanium dioxide (TiO2; 1 g/kg of diet) for subsequent determination of apparent starch digestibility. On days 1, 3, 7 and 8/9 after weaning, a faecal swab was taken for assessment of β–haemolytic E. coli and faecal consistency. Pigs were euthanased for sample collection after 14 days. Apparent digestibility of starch measured at the terminal ileum was highest (P=0.004) in AM (≈ 96%) and WAXY (≈ 99%) and lowest, but the same, in diets DOON and WBL (≈ 88%). Starch digestibility measured in the distal colon was highest (P<0.001) in all three rice-based diets. Pigs fed cooked rice generally consumed more dry matter and grew favourably relative to pigs fed diet WBL in the 14 days after weaning, and diverted more absorbed nutrients to carcass gain (P<0.001) than to growth of the visceral organs than pigs fed diet WBL. Pigs fed diet WBL produced more VFA in the gastrointestinal tract than pigs fed rice (P<0.05). Relatively low levels of infection with haemolytic E. coli were observed in this experiment making interpretation of the dietary effects on PWD difficult to interpret, however pigs fed diets AM and WBL appeared to shed less haemolytic E. coli than pigs fed diets DOON and WAXY. It is thought that the lack of a slowly/moderately fermentable source of DF in the rice-based diets could have shifted the balance of the bacteria to proteolytic rather than saccharolytic and predisposed the pigs to PWD. 6.2 Introduction Restrictions in some countries on the use of growth promoting antibiotics and heavy minerals such as zinc and copper in diets for young pigs has seen the development of alternative feed- and management-related strategies to maintain production and prevent enteric disorders, such as PWC, in the absence of antimicrobials (Pluske et al., 2002). A large number of products are marketed to the pig industry as alternatives/replacements to growth promoting antibiotics and heavy minerals, but these sometimes show inconsistent and variable effects. Another, or complementary, approach to feeding the weanling pig without dietary antimicrobials is to modify the ingredients used. Most pig starter diets are based on cereals, such as wheat, barley, oats and (or) maize, and a combination of animal and vegetable proteins. Some of these feedstuffs are high in certain NSP, which have been shown to reduce live weight gain (eg, McDonald et al., 1999), increase growth of the gastrointestinal organs at the expense of body gain (eg, McDonald et al., 1999; McDonald et al., 2001; Hopwood et al., 2003; Pluske et al., 2003) and predispose pigs to PWC (McDonald et al., 1999; McDonald et al., 2001; Hopwood et al., 2002). For example, Hopwood et al.

23

(2004) demonstrated that pearl (dehulled) barley fed to weanling pigs increased digesta viscosity and reduced starch digestion concomitant with greater proliferation of β–haemolytic Escherichia coli, in comparison to feeding cooked white rice. Furthermore, Pluske et al. (2003) reported beneficial effects of feeding a diet based on cooked white rice to weanling pigs in terms of reduced antibiotic treatments and empty bodyweight gain. Rice would appear to offer, therefore, a promising alternative to other cereals because of its higher starch and lower NSP contents, and its association with reduced shedding of enterotoxigenic E. coli. Unfortunately the higher price of rice relative to other cereals such as wheat may preclude its widespread use. Nevertheless, work from Spain (eg, Mateos et al., 2001, 2002) has shown that cooked white rice fed to weanling pigs, either alone or in combination with oats, enhances performance after weaning and reduces diarrhoea. Many types of rice are grown and, as would be expected, they differ in chemical characteristics such as amylose:amylopectin, starch and RS levels and gelatinisation temperature (Marsono and Topping, 1993; Fitzgerald et al., 2003). These differences, in turn, are likely to alter their physico-chemical characteristics in the gastrointestinal tract of pigs. If rice is to be considered for use in young pig diets, then it is important to examine different rice types that could be available to the feed manufacturing industry. This experiment investigated a number of cooked rice-based diets on apparent starch digestibility, fermentation and digesta characteristics, shedding of β–haemolytic E. coli and performance after weaning. The general hypothesis tested was that rice containing a lower amylose:amylopectin ratio would cause a higher rate of starch digestion concomitant with decreased shedding of β–haemolytic E. coli from the gastrointestinal tract. 6.3 Materials and Methods 6.3.1 Animals and housing Forty-eight entire male pigs (Large White x Landrace) aged 19-24 days of age and weighing 6.7 ± 0.24 kg (mean ± SE) were used in the experiment. Pigs were obtained from a commercial farm on the day of weaning and transported in an insulated horse float to the experimental facility at Murdoch University. Upon arrival, the pigs were ear-tagged, weighed, and stratified into pens of four pigs each according to treatment and liveweight. Pens were of wire-mesh construction with slatted metal floors, and measured 2.5 m2 in floor area. Each pen had an enclosed wooden box containing a heat lamp, and was equipped with a nipple water drinker and a feed trough. The ambient temperature varied between 19 and 26°C throughout the study. The Murdoch University Animal Ethics Committee and the Animal Ethics and Experimentation Committee of the WA Department of Agriculture approved this experiment. 6.3.2 Experimental design, diets and feeding Pigs were allocated in a completely randomised block design having four experimental (dietary) treatments, with 12 pigs allocated to each treatment. The experiment was conducted in three replicates, with 16 pigs (ie, one pen of four pigs per treatment combination) constituting a single replicate. The three rice-based diets in the experiment comprised: (i) medium-grain rice (cultivar Amaroo) plus an animal protein supplement (AM), (ii) long-grain rice (cultivar Doongara) plus an animal protein supplement (DOON), (iii) waxy rice plus an animal protein supplement (WAXY), and (iv) commercially based weaner diet (WBL) (Table 6.1). The rice types were chosen on the basis of results in Chapter 4 because they each differed in their chemical characteristics (eg, amylose:amylopectin) and would therefore be expected to cause different physico-chemical effects in the gastrointestinal tract. The three diets based on rice were fed daily by mixing the cooked rice with the remainder of the diet (on an as-fed basis) immediately before feeding. A small amount (150 to 200 mL) of warm water was

24

used to assist with mixing. Each rice type was cooked in an autoclave at 121°C for 20 minutes using a ratio of 2:1 water: dry rice, and was left to cool overnight in a cool room (4° C) prior to feeding the following day. Diet WBL was fed as a meal. Pigs were fed between 0900 and 1030 daily, and any residue was recorded the following day. Pigs were fed the experimental diets on an ad libitum basis for 14 days, after which they were euthanased for sample collection (see below). Antibiotics, zinc oxide or copper sulphate were not included in the diets. 6.3.3 Faecal shedding of β-haemolytic E. coli All pigs were swabbed upon arrival at Murdoch University by inserting a soft cotton bud into the anus of the pig. Swabs were then cultured for the presence of β-hemolytic E. coli on sheep blood agar plates (after McDonald et al., 2001). Swabs from all pigs were also collected and subsequently cultured on day 1, day 3, day 6, day 7 and day 8 of the experiment. Plates were scored and the faecal consistency determined according to the methods of Montagne et al. (2004). 6.3.4 Post-mortem procedures Pigs in each pen were fed their morning meal on the day of sampling in a staggered fashion across each dietary treatment, such that the period of time between feeding and euthanasia was 3-6 hours. Pigs were transported a distance of approximately 200 metres from the experimental facility to a necropsy room. Pigs were euthanased with a lethal dose of sodium pentobarbitone solution administered intravenously (Lethobarb; May and Baker Pty. Ltd., Australia). Cervical dislocation and exsanguinations followed, reducing the amount of blood present as a potential contaminant during sample collection and weighing. A faecal sample was taken at this time. The abdomen was then opened from the sternum to the pubis, and the gastrointestinal tract was removed. It was then divided into four sections (stomach, small intestine, caecum and colon) that were tied off with light twine before being separated. The small intestine was stripped free of its mesentery and laid on a table into sections of equal length. The full and empty weights of the small intestine, caecum and colon were determined by first weighing the organ containing its contents and then reweighing the organ after the contents were removed and the organ was blotted dry. Samples of digesta from the ileum, caecum, and proximal and distal colon were collected into plastic jars, placed immediately on ice, and later frozen at –20° C for subsequent chemical analyses (see below). The pH of digesta was measured by inserting the electrode of a calibrated portable pH meter (Schindengen pH Boy-2; Schindengen Electric MFG, Tokyo, Japan) into the collected sample. A sub-sample of digesta from the ileum and caecum was collected for determination of viscosity. Finally, a segment of adipose tissue was collected adjacent to the longissumus dorsi, placed in a sterile container, and immediately placed in liquid nitrogen for subsequent determination of ATP-citrate lyase (see below). 6.3.5 Analytical methods The DM content of the digesta and faecal samples and the starch, FDS and amylose contents were measured as described previously (Chapter 4.2.1). Titanium dioxide (TiO2) was measured according to the methods described by Short et al. (1996).

25

Table 6.1 Composition of experimental diets (air-dry basis, g kg-1). DietA,B

Ingredient AM DOON WAXY WBL Rice 702.7 702.7 702.7 - Wheat - - - 508.5 Barley - - - 100 Australian sweet lupins

- - - 100

Skim milk powder - - - 50 Soybean meal - - - 42.8 Whey powder 100 100 100 50 Blood meal 30 30 30 - Fish meal 100.5 100.5 100.5 70 Meat and bone meal

51.5 51.5 51.5 14.4

Canola oil 5 5 5 40 L-lysine 2.8 2.8 2.8 5.1 DL-methionine 0.4 0.4 0.4 0.8 L-threonine 1.4 1.4 1.4 1.9 L-tryptophan 0.3 0.3 0.3 10.9 Choline chloride (60%)

4 4 4 0.4

Dicalcium phosphate

- - - 2.1

Limestone - - - 1.6 Salt 1 1 1 - Vitamin and mineral premixC

0.7 0.7 0.7 1.5

Calculated analysis:

DE (MJ/kg) 15.3 15.3 15.3 15.2 Crude protein, g kg-1

200 200 200 220

Total lysine, g kg-1 13.0 13.0 13.0 12.9 ADiets were formulated to contain 8 g Ca/kg and 4.5 g available P/kg. BRefer to the text for details of diets. CPremix provided (mg/kg air-dry diet): retinyl acetate 3.44, cholecalciferol 0.065, α-tocopheryl acetate 20, menadione 4.4, riboflavin 4, pyridoxine 1.6, cyanocobalamin 0.02, pantothenic acid 14, nicotinic acid 20, Co 0.2, I 0.6, Fe 120, Mn 60, Zn 100, Cu 10, Se 0.13.

26

To determine volatile fatty acid (VFA) concentration (C2: C6), thawed digesta samples from the ileum, caecum, proximal colon and distal colon were diluted either 1:1 (w:v) (ileal digesta) or 1:2 (w:v) (caecal and colonic digesta) with distilled water, mixed, centrifuged and the supernatant fraction analysed chromatographically. The supernatant fraction (0.1 mL) was added to 1 mL internal standard solution containing valeric acid before processing on a capillary GC. A working standard and a control (distilled water) were included in each run of the analysis, with the working standard containing acetic acid (60 mM), propionic acid (20 mM), isobutyric acid (6.67 mM), butyric acid (20 mM), isovaleric acid (10 mM), valeric acid (10 mM) and caproic acid (4 mM). The Hewlett Packard 5890A capillary GC (Agilent Technologies, Forrest Hill, Victoria, Australia) was maintained at injector and detector FID settings of 260 °C and 265 °C respectively, and an initial and final oven temperature of 120 °C and 240 °C, respectively. The carrier gas flow rate was 5 mL/min and the split-flow rate was 70 mL/min. The Hewlett Packard Chemstation integration system was used to calculate the VFA concentrations from the area of the peaks. For viscosity, an aliquot of the digesta from the ileum was placed in an Eppindorf tube, mixed on a vortex, and centrifuged at 12000 g for 8 min (Quantum Scientific Pty. Ltd., Milton, QLD, Australia). The supernatant fraction (0.5 mL) was placed in a Brookfield LVDV-II+ cone-plate rotational viscometer (CP40; Brookfield Engineering Laboratories Inc., Stoughton, MA, USA), and the viscosity of all samples was measured. The viscosity was measured in mPas. The specific activity of ATP-citrate lyase in adipose tissue was determined according to the methodology established in the Biochemistry Laboratory at Murdoch University. ATP-citrate lyase was used in this study as a biomarker of starch digestion and hence glucose availability, since this enzyme is the rate-limiting step in the conversion of glucose to adipose tissue. Briefly, stock premix buffer was prepared just prior to the assay. Assay ingredients were added to one blank and two duplicate, 1-ml cuvettes (path length = 1 cm). The assay protocol is summarised in Table 5.2. The assay was started after 10 minutes pre-incubation at 37 ºC by adding the assay mix, which contained 3.025 mg ATP and 380 μl NaOH (1M) made up to 2 ml with deionised water. The rate of NADH disappearance was measured over 5 minutes. Table 6.2 Conditions used in the ATP-citrate lyase assay. Reagent Blank cuvette (μl) Duplicate sample cuvettes (μl) Premix bufferA 750 750 Cytosol extractB 200 200 Water 50 - Assay mix - 50 A0.1 ml of 0.15 M Tris buffer (pH 7.4), 0.1 ml of MgCl2 (0.1 M), 0.1 ml of K3citrate (0.2 M), 0.4 mg of CoA, 0.21 mg of NADH, 0.7 μl of mercaptoethanol, 2 μl of malic dehydrogenase, made up to 0.95 ml using deionised water. BPrepared by pulverising frozen sample of adipose tissue and then sonicating to release cytosolic contents. 6.3.6 Calculations and statistical analyses Apparent starch digestibility at the terminal ileum and in the colon was calculated using ratios of the TiO2 concentration in the feed and digesta according to the following calculation: Apparent starch digestibility = 1-[(ID x AF) / (IF x AD), where ID is the concentration of marker (TiO2) in the diet, AF is the starch concentration in the digesta, IF is the marker concentration (TiO2) in the digesta, and AD is the starch concentration in the diet. All digestibilities are presented as percentages.

27

Empty body weight (EBW) was determined as the liveweight of the pig minus the contents (digesta) contained in the stomach, small intestine, caecum, colon and bladder (if applicable). The percentage of the pig that was carcass was calculated as: [(Liveweight of pig minus weight of gastrointestinal tract)/liveweight] x 100. Statistical analyses were performed using Statview for Macintosh (version 5.0; SAS Institute Inc., Cary, NC, USA). Replicate was included as an independent variable for all dependent variables analysed, but was removed whenever found to be non-significant (P>0.05). Following removal of this term, one-way ANOVA determined significant differences between treatment groups, and the mean values were compared using Fisher’s-protected least significant difference test. Statistical significance was accepted at P<0.05. The unit of replication was the individual pig for all measurements, except in the case of dry matter intake where the pen was used as the experimental unit. 6.4 Results 6.4.1 Chemical characteristics of rice Amylose:amylopectin ratios for rice types AM, DOON and WAXY were 0.22, 0.31 and 0.03, respectively. The low ratio for WAXY indicates that this rice type is almost exclusively amylopectin, with AM also having a higher level of amylopectin than DOON. The FDS content, expressed as a percentage of total starch, mirrors the amylose content of the rice types in both raw and cooked forms. Gelatinisation temperature was highest in DOON (75.8 °C) and lowest in WAXY (69.6 °C). The level of RS varied from 0.6 g kg-1 in Amaroo to 1.4 g kg-1 in Doongara (Table 6.3). Table 6.3 Selected chemical characteristics of the three rice types used. Type of rice Characteristic Medium-grain

(Amaroo; AM) Long-grain (Doongara;

DOON) Waxy (WAXY)

Total starch, g kg-1 DMA

Raw 878 860 883 Cooked 913 903 914

Resistant starch, g kg-1 DM

0.60 1.42 0.75

FDSB, % total starch Raw 23.2 18.3 26.5 Cooked 86.7 84.0 90.0

Amylose content, % 18.2 23.8 6.1

Gelatinisation temperature, °C

69.8 75.8 69.6

ADM: dry matter. BFDS: fast digestible starch.

28

6.4.2 Apparent digestibility of starch and viscosity Apparent digestibility of starch measured at the terminal ileum was lowest in pigs fed DOON and diet WBL and highest in pigs fed AM and WAXY (P=0.004). Starch had virtually been fully digested in the distal region of the colon but was still about 2% digestibility units lower in pigs fed diet WBL compared to pigs fed any of the rice types (P<0.001). Residual starch content reflected apparent starch digestibility, although pigs fed diet WBL had between 6 and 12 times the level of starch in their distal colons compared to pigs fed the three rice-based diets (P<0.001) (Figure 6.1). Digesta viscosity was highest in pigs fed diet WBL in both the ileum (P<0.001) and the caecum (P=0.027). The specific activity of ATP-citrate lyase varied between 37 μmol/mg protein for pigs fed diet WBL to 148 μmol/mg protein for pigs fed WAXY. Pigs fed WAXY had activities higher than pigs fed AM and WBL (P=0.005) but were similar (P>0.05) to pigs fed DOON. Pigs fed AM had similar ATP-citrate lyase activities to pigs fed DOON and WBL (P>0.05) (Table 6.4).

0

2

4

6

8

10

12

Calrose Doongara Double �Elephant

Commercial

Diet Type

Starch content (g/100 g digesta)

Ileum

Colon

Figure 6.1 The starch content in the terminal ileum (end of small intestine) and colon (large intestine) of pigs fed different rice types (medium-grain - AM; Long-grain – DOON; WAXY – Double Elephant) and WBL for 14 days after weaning. 6.4.3 Performance data There were no statistically significant differences in the liveweight of pigs or rate of daily gain between diets after weaning, although there was a trend (P=0.085) for pigs in the first week eating rice types AM and WAXY to grow faster than pigs eating DOON and WBL. There was a significant replicate effect on daily gain, with pigs in replicate 1 performing better (P=0.032) than pigs in replicates 2 and 3 (204 g/day versus 139 and 180 g/day, respectively). Similarly, replicate was significant (P<0.001) for the liveweight of pigs after 14 days, with pigs in replicate 1 weighing more (10.4 kg) than pigs in replicate 3 (9.4 kg), with pigs in replicate 2 being lightest (7.5 kg). These weights reflected differences in starting weight (7.6, 6.9 and 5.5 kg for replicates 1, 3 and 2, respectively). The EBW of pigs was greatest (94.6-95.4%) in pigs fed the three rice-based diets and lowest (90.1%; P<0.001) for pigs fed diet WBL (Table 6.5).

29

Table 6.4 Apparent digestibility of starch, residual starch content, viscosity and the ATP-citrate lyase activity of the digesta in pigs fed different diets after weaning. DietA Item AM DOON WAXY WBL SEDB P= Starch digestibility, %

Ileum 96.2 a 88.6 b 99.1 a 88.5 b 5.78 0.004 Distal colon 99.8 a 99.8 a 99.9 a 97.6 b 0.55 <0.001

Residual starch, mg/g DMC

Ileum 44 a 110 b 20 a 98 b 26.4 <0.001 Distal colon 6 a 7 a 3 a 36 b 7.9 <0.001

Viscosity, mPas Ileum 2.9 a 2.9 a 3.8 a 8.2 b 1.77 <0.001 Caecum 2.8 a 2.9 a 2.6 a 4.7 b 1.19 0.027

ATP-citrate lyase, μmol/mg protein

79 ac 105 ab 148 b 37 c 24.5 0.005

ARefer to the text for details of diets. BSED: standard error of difference between treatment means. CDM: dry matter. abcMeans in the same row lacking a common superscript are significantly different. Table 6.5 The performance of pigs fed different diets after weaning. DietA Item AM DOON WAXY WBL SEDB P= Body weight, kg

Weaning 6.8 6.6 6.7 6.7 0.75 0.979 Day 7 7.6 7.0 7.6 7.3 0.79 0.785 Day 14 9.4 8.6 9.5 9.2 1.24 0.621 EBWC at

slaughter, kg 8.9 8.2 9.1 8.4 1.12 0.465

Pig weight expressed as % of EBWC

95.4 a 94.6 a 95.3 a 90.1 b 1.08 <0.001

Average daily gain, g

Day 1-7 117 54 123 84 49.8 0.085 Day 8-14 253 228 270 280 90.0 0.770 Day 1-14 185 141 196 182 57.2 0.865

Dry matter disappearance, g/pen (n=3)

Day 1-7 977 709 979 481 291.1 0.434 Day 8-14 1812 1608 1816 1347 440.7 0.770 Day 1-14 1395 1159 1397 913 358.3 0.621

ARefer to the text for details of diets. BSED: standard error of difference between treatment means. CEBW: empty body weight; calculated as bodyweight of pig minus weight of gastrointestinal tract contents. abcMeans in the same row lacking a common superscript are significantly different.

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6.4.4 PWD and faecal shedding of E. coli Only sporadic diarrhoea was observed in this particular study, with only a few pigs developing serious PWD. There were no significant differences between diets on days 1, 3, and 7 regarding the faecal E. coli score (P>0.05), although on days 8/9, pigs fed diets AM, WAXY and WBL shed less haemolytic E. coli in the faeces than pigs fed diet DOON (P=0.011). The consistency of faeces varied considerably in pigs fed different diets after weaning, which prevented statistical differences between diets. Nevertheless, it was evident that pigs fed WBL had more moist faeces on days 7 and 8/9 after weaning than pigs fed any of the rice-based diets (Table 6.6). Table 6.6 The haemolytic E. coli score in faeces and the faecal consistency in pigs assessed at various time points after weaning. DietA Item AM DOON WAXY WBL SEDB P= E. coli score in faecesC

Day 1 0.58 0.25 0.45 0.33 0.121 0.767 Day 3 0.42 0.50 0.27 - 0.107 0.309 Day 7 0.50 1.67 2.00 0.25 0.204 0.069 Day 8/9 0.33 a 1.75 b 0.91 ab 0.50 a 0.169 0.011

Faecal consistency, %D

Day 3 42 38 29 33 6.0 > 0.05 Day 7 25 36 30 39 4.6 > 0.05 Day 8/9 33 44 31 50 5.3 > 0.05

ARefer to the text for details of diets. BSED: standard error of difference between treatment means. CFaecal score: agar plates were scored from 0-5 acording to the number of streaked sections that had viable growth of haemolytic E. coli, where 0 = no growth, 1 = E. coli in first section, and so on (5 = heaviest growth of haemolytic E. coli). DFaecal consistency: expressed as % cumulative score per day of pigs having more liquid faeces; higher values are associated with more liquid faeces. abcMeans in the same row lacking a common superscript are significantly different. 6.4.5 Fermentation characteristics The acidity of digesta in the ileum (P=0.055), and the caecum, proximal colon and distal colon (all P<0.001), was highest for pigs fed diet WBL and not statistically significant between any of the three rice diets. Faecal pH was similar (P>0.05) in pigs fed all diets. The total VFA concentration was similar for pigs fed all diets in the ileum and distal colon. In the caecum, pigs fed the three rice diets had VFA concentrations ranging from 140-175 mM, but only pigs fed AM and WAXY had less VFA than pigs fed diet WBL (196 mM; P=0.026). In the proximal colon, pigs fed diet WBL had a significantly higher concentration of VFA than pigs fed the three rice-based diets (P<0.001). The concentration of acetate was similar (P>0.05) at all sites of the gastrointestinal tract in the four diets, but the concentration of propionate was highest in the caecum of pigs fed diets DOON and WBL (P=0.044) and in the proximal colon of pigs only fed WBL (76 mM; P=0.010). Pigs fed WBL had a 2-3-fold higher concentration of butyrate at all gastrointestinal sites than pigs fed the three rice-based diets (0.017>P<0.001). Conversely, pigs fed any of the three rice-based diets consistently had higher concentrations (0.030>P<0.001) of isovalerate and isobutyrate in their digesta than pigs fed WBL (Table 6.7).

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6.4.6 Organ weights The percentage of pig that was carcass was 4-6 points higher in pigs fed the three rice-based diets than in pigs fed WBL (P<0.001). This difference reflected lower weights of the stomach (P=0.002), caecum (P=0.024) and colon (P=0.032) commensurate with significantly less digesta (0.025>P<0.001) being present in these organs. There was also significantly less digesta found in the small intestine (P=0.010) of pigs fed the rice diets compared to pigs fed diet WBL. When expressed on a relative basis (% EBW), pigs fed AM had a significantly lighter caecum and colon than pigs fed either DOON or WBL, whereas the relative weights of the caecum and colon of pigs fed WAXY was comparable to those in pigs fed DOON. The relative weight of the colon was heaviest (1.63%; P<0.001) in pigs fed WBL. Pigs fed diet WBL had the heaviest pancreas (P=0.002; Table 6.8). 6.4.7 Prediction of residual starch content using fast digestible starch (FDS) The data in Figure 6.2 show that more starch remained at the end of the small intestine (ileum) in the digesta of pigs fed DOON and diet WBL compared to pigs fed AM and WAXY. These data concur with the apparent starch digestibility figures expressed in Table 6.4. In the colon, where significant microbial fermentation of starch can occur, the amount of starch remaining in pigs fed all rice types was not different, but remained elevated in pigs fed WBL. The digestibility of starch in the small intestine could be predicted from the ‘fast digestible starch’ in vitro test (Figure 6.2).

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Table 6.7 Fermentation characteristics of digesta in pigs fed different diets after weaning. DietA Item AM DOON WAXY WBL SEDB P= pH

Ileum 6.7 a 6.7 a 6.7 a 6.2 b 0.36 0.055 Caecum 6.3 a 6.1 a 6.3 a 5.4 b 0.30 <0.001 Proximal

colon 6.5 a 6.4 a 6.5 a 5.6 b 0.30 <0.001

Distal colon 6.8 a 6.7 a 6.9 a 6.3 b 0.30 0.006 Faeces 6.9 7.0 7.0 6.9 0.29 0.730

Total VFAC, mM

Ileum 19 27 25 30 10.9 0.372 Caecum 140 a 175 ab 145 a 196 b 34.2 0.026 Proximal

colon 121 a 137 a 136 a 213 b 39.4 <0.001

Distal colon 103 110 96 150 35.7 0.071 Acetate, mM

Ileum 12 16 16 10 7.8 0.480 Caecum 75 79 69 58 13.3 0.061 Proximal

colon 61 61 62 64 15.3 0.984

Distal colon 42 43 34 52 12.4 0.126 Propionate, mM

Ileum 0 2 0.4 2 2.88 0.582 Caecum 42 a 64 bc 48 ab 75 c 20.8 0.044 Proximal

colon 37 a 47 a 46 a 76 b 20.5 0.010

Distal colon 29 34 30 49 15.9 0.142 Butyrate, mM

Ileum 7 a 7 a 8 a 18 b 6.4 0.017 Caecum 16 a 22 a 17 a 52 b 10.3 <0.001 Proximal

colon 16 a 19 a 18 a 57 b 12.1 <0.001

Distal colon 16 a 17 a 17 a 37 b 10.9 0.008 Isobutyrate, mM

Caecum 1 a 1 a 2 a 0.1 b 1.2 0.014 Proximal

colon 1 a 1 a 2 a 0.1 b 1.04 0.008

Distal colon 5 a 4 a 5 a 0.9 b 2.04 0.010 Isovalerate, mM

Caecum 3 a 3 a 4 a 0.6 b 1.28 <0.001 Proximal

colon 4 ab 3 a 5 b 1 c 1.2 <0.001

Distal colon 7 a 6 a 7 a 3 b 2.4 0.030 ARefer to the text for details of diets. BSED: standard error of difference between treatment means. CVFA: volatile fatty acids (expressed as mM, or mmol/kg wet digesta). abcMeans in the same row lacking a common superscript are significantly different.

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Table 6.8 Weight of the carcass at euthanasia, organ weight (empty, and as a % of empty bodyweight) and the weight of organ contents in pigs fed different diets after weaning. DietA Item AM DOON WAXY WBL SEDB P= Carcass, %C 88.0 a 86.8 a 87.7 a 82.5 b 1.41 <0.001 Empty organ weight, g

Stomach 71 a 64 a 71 a 92 b 12.2 0.002 Small intestine 487 460 499 490 69.3 0.779 Caecum 22 a 24 a 23 a 28 b 3.8 0.024 Colon 104 a 108 a 108 a 137 b 21.0 0.032

Pancreas, g 17 a 17 a 16 a 24 b 3.0 0.002 % EBWD

Stomach 0.78 a 0.80a 0.78 a 1.10 b 0.096 < 0.001 Small intestine 5.44 5.68 5.57 5.90 0.559 0.542 Caecum 0.24 a 0.30 bc 0.26 ab 0.34 c 0.044 0.003 Colon 1.15 a 1.35 b 1.20 ab 1.63 c 0.177 <0.001

Contents, g Stomach 216 a 189 a 205 a 341 b 91.7 0.025 Small intestine 122 a 134 a 127 a 218 b 53.7 0.010 Caecum 26 a 40 ab 41 b 77 c 14.3 <0.001 Colon 83 a 96 a 82 a 217 b 27.9 <0.001

ARefer to the text for details of diets. BSED: standard error of difference between treatment means. CCarcass, %: Calculated as [(Liveweight of pig minus weight of gastrointestinal tract)/liveweight] x 100. DEBW: empty body weight (calculated as liveweight of pig minus total weight of gastrointestinal tract contents. abcMeans in the same row lacking a common superscript are significantly different.

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0

2.5

5

7.5

10

Starch content g/100 g digesta

74 76 78 80 82 84

Fast digestible starch %

Colon

Ileum

DoongaraDoongara

Doongara

DoongaraDoongaraDoongara

Elephant

Calrose

Figure 6.2 The relationship between fast digestible starch content (%) and the amount of starch remaining in the terminal ileum and colon of pigs killed 14 d after weaning. 6.5 Discussion These data clearly demonstrate that there are differences between different types of cooked rice in the digestibility of starch. The major findings of this study were that (i) pigs fed Amaroo (AM) and Double Elephant (WAXY, ie, ≈ 97% amylopectin) had a higher apparent starch digestibility at the end of the small intestine (ileum) and performed best (growth rate) in the study; (ii) pigs fed Doongara (DOON) and the commercial wheat-and barley-based diet (WBL) had inferior starch digestion in the small intestine, presumably because of the higher amylose content in DOON and the anti-nutritional effects of NSP in the wheat and barley in diet WBL; (iii) pigs fed all rice types ate more energy and were more efficient at converting energy from the feed into carcass tissue than pigs fed the commercial diet, indicating a strong, positive effect for feeding a cooked white-rice-based diet after weaning; and (iv) the faecal score of E. coli (incidence of diarrhoea) was reduced in pigs fed AM and the commercial diet WBL, but was higher in pigs fed DOON and WAXY. Collectively, these data suggest that piglet diets in Australia should preferably be based on the use of medium-grain rather than long-grain rice, although a mixture of the two cannot be precluded from possible use. In addition, increased digestion of starch in the medium- (AM) and waxy-rice (WAXY) types suggests that any diets based on these rices will cause better performance than those currently used. Few reports have investigated the effect of cooked white rice fed to piglets on apparent starch digestion. Hopwood et al. (2004) reported an apparent starch digestibility of 96% and 100%, respectively, in the ileum and faeces of piglets killed 10 days post-weaning after being fed a diet based on cooked white medium-grain rice and animal protein sources. Incorporation of increasing amounts of pearl barley at the expense of cooked white rice by Hopwood et al. (2004) significantly reduced starch digestibility in the ileum, but faecal digestibility of starch was unaffected (P=0.096) by the

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incorporation of pearl barley. Feeding pigs diet WBL in the current study significantly depressed small intestinal starch digestion to approximately 88% but only to the level of pigs fed the diet using DOON (Table 6.4). Presumably the higher amylose content of DOON, which is regarded as being less digestible by pancreatic amylase in the small intestine (Black, 2001), accounted for the lower disappearance of starch than in pigs fed AM and WAXY, which have more amylopectin. The resultant excess starch present within the lumen of pigs fed diets DOON and WBL would pass directly into the caecum and colon and potentially cause considerable microbial fermentation, resulting in a lowering of pH and higher VFA production, and organ hypertrophy, although this was only evident in pigs fed diet WBL (Tables 6.7 and 6.8). The greater NSP content of diet WBL together with greater viscosity of the digesta and the undigested starch undoubtedly caused the significantly higher level of VFA production, greater gut weights and lower carcass percentage compared to pigs fed all three rice-based diets, which concurs with previous work (eg, McDonald et al., 1999, 2001; Pluske et al., 2003). The lack of any differences in growth performance between diets, except for a trend (P=0.085; Table 6.5) for improved daily gain in pigs fed diets AM and WAXY in the first seven days of the study, suggests that cooked rice-based diets can be used successfully to maintain weight gain in the immediate post-weaning period. Piglets generally suffer a period of sub-optimal growth caused by low feed intake in the period after weaning (Pluske et al., 1997), and feeding diets with AM and WAXY assisted in alleviating the period of temporary anorexia that generally accompanies weaning under commercial conditions. The compensation seen in pigs fed diet WBL and the failure of pigs eating diets AM and WAXY to continue their weight gain advantage could be due to a lower energy value ascribed to rice during feed formulation, which would reduce growth rate. The final study in this project (Chapter 10) showed a DE value of ≈ 15.3 MJ/kg, whereas the DE value used to calculate the DE content of these particular diets was ≈ 14.6 MJ/kg (Dr B. Mullan, personal communication). The lower DE value ascribed to rice limited energy supply to the pig and restricted growth to below genetic potential. Commercial use of the determined DE value of rice described in Chapter 10 will improve the accuracy of diet formulation and provide feed manufacturers with more confidence to use rice in piglet diets to attain maximum growth. The in vitro test developed herein using fast digestible starch (FDS) as a predictor of starch digestion at the ileum (Figure 6.2) offers promise as a screening tool also to the feed manufacturing industry. Nevertheless, pigs consuming all three rice-based diets ate more dry matter (albeit not significantly) and converted absorbed nutrients more efficiently to body gain (ie, in the carcass) than pigs fed diet WBL, as evidenced by the higher percentage of carcass weight. This reflects, in part, lower visceral weights recorded for pigs eating rice (Table 6.8). These data agree with previously published work from this University (eg, McDonald et al., 1999, 2001; Pluske et al., 2003). These data are further supported, again in part, by the measured ATP-citrate lyase activities (Table 6.4) that show a lower level in adipose tissue in pigs fed the commercial WBL diet. This most probably indicates a slower/reduced incorporation of the absorbed glucose into adipose tissue that, in turn, mirrors the reduced digestion of starch seen in the small intestine. The higher ATP-citrate lyase activity for DOON, which had a similar ileal starch digestibility value to pigs fed diet WBL, might be explained by the higher level of dry matter intake observed in these pigs, and hence an overall greater absorption of glucose. Only sporadic instances of spontaneous diarrhoea were observed in this study, and in general the health of all pigs remained high. It is difficult to draw conclusions from a study such as this where a natural infection is relied upon to examine dietary effects on PWD, and hence the results obtained are ambiguous compared to a study where pigs are experimentally infected. However, pigs fed all three rice-based diets, particularly diets DOON and WAXY, shed haemolytic E. coli in their faeces, and in general at higher levels than pigs fed diet WBL. For example, on day 8/9 of the study, pigs fed DOON and WAXY had E. coli scores of 1.75 and 0.91 respectively compared to 0.33 and 0.50 for pigs fed AM and WBL, respectively. These data compare favourably to those presented by Hopwood et al. (2004), who also reported no differences in faecal E. coli swab score in experimentally-infected pigs fed cooked white rice or pearl barely; however, these authors used an infection model that causes a greater infection pressure in the gastrointestinal tract, and subsequently detected a significant

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amelioration of haemolytic E. coli in the small intestine in pigs fed cooked white rice. It was not possible to do this in the current study because pigs were not experimentally infected. However, the consistency of faeces was firmer (lower value) in pigs fed rice than in pigs fed WBL, albeit non-statistically (Table 6.6). The production of iso (branched chain) VFA and other nitrogenous compounds such as NH3 and biogenic amines have been implicated in the aetiology of PWD (eg, Bolduan et al., 1988; Aumaitre et al., 1995; Awati, unpublished PhD Thesis, Wageningen University, The Netherlands). Bolduan et al. (1988) hypothesised that DF added to piglet diets, particularly insoluble DF, was beneficial in reducing PWD because the production of such nitrogenous compounds was reduced. There is some recent support for this proposition, and it was noticeable in the current study that pigs fed all three rice-based diets produced significantly greater quantities of isobutyrate and isovalerate than pigs fed diet WBL (Table 6.7). It is difficult to implicate cause and effect with respect to the greater shedding of haemolytic E. coli observed, however the lack of a slowly/moderately fermentable source of DF in the rice-based diets could have shifted the balance of the bacteria to proteolytic rather than saccharolytic and predisposed the pigs to PWD. In conclusion, data in this experiment showed that pigs fed either the medium-grain rice (AM) or the waxy rice (WAXY) had a superior starch digestibility in the small intestine than pigs fed the long-grain rice (DOON) or the commercial WBL diet. Pigs fed rice consumed more dry matter and grew favourably relative to pigs fed diet WBL in the 14 days after weaning, and diverted more absorbed nutrients to carcass gain than to growth of the visceral organs than pigs fed diet WBL. Relatively low levels of infection with haemolytic E. coli were observed in this experiment and hence dietary effects on PWD were difficult to interpret, however pigs fed diets AM and WBL appeared to shed less haemolytic E. coli than pigs fed diets DOON and WAXY. It is postulated that the lack of a a slowly/moderately fermentable source of DF in the rice-based diets could have shifted the balance of the bacteria to proteolytic rather than saccharolytic and predisposed the pigs to PWD.

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7. Interactive effects of cooked white rice with vegetable and animal protein sources on digesta and fermentation characteristics and the faecal shedding of haemolytic E. coli

7.1 Summary Sixty-four piglets weaned at approximately 21 days of age and fed different diets for a period of 10 days after weaning. Diets were based on either cooked white rice or wheat as the two cereal types, and these were supplemented with protein derived from either animal sources or plant sources. Pigs were inoculated with an enterotoxigenic serotype of E. coli (O149;K91;K88) at 48, 72, 96 and 120 h after weaning. At 10 days after weaning, all pigs were euthanased, and samples were taken for bacteriological assessment and measures of fermentation and digestive physiology. Pigs fed cooked white rice consumed more feed than pigs fed wheat (P<0.001). At euthanasia, pigs fed wheat were found to have a higher viscosity in their ileal digesta than pigs fed rice (P<0.001). A higher viscosity did not correlate to a greater colonisation by E. coli of the wheat-fed pigs and, overall, levels of colonisation in pigs fed all diets were low. A number of possible reasons for the results obtained are discussed. 7.2 Introduction Different diets appear to influence the colonisation of enterotoxigenic E. coli in the gastrointestinal tract of weaner pigs (Pluske et al., 2002). Colonisation and proliferation of, in particular haemolytic strains, of enterotoxigenic E. coli in the small intestine characterises PWD, which generally results in diarrhoea, dehydration, weight loss, and sometimes death in the first two weeks following weaning. Some authors have viewed diarrhoea, an excretion of cellular solutes, as a protective defence mechanism in response to this infective agent in the gut (Stewart et al., 2001). Regardless, PWC/PWD is a significant disease causing major economical loss to the pig industry. Diet influences pH, nutrient composition, and flow of digesta, which in turn influences distribution, composition and metabolic activity of gut flora (Stewart et al., 2001; Pluske et al., 2002). It is important to find effective methods of control of specific enteric bacterial pathogens that are cost effective and plausible, and diet provides an excellent medium. This is especially relevant given the ban placed on a number of antibiotics in Europe recently, and reported cases in Australia of antibiotic resistance to strains of E. coli causing PWC (Barton, 1999). One of the components of dry matter digested mainly in the large intestine is known as dietary fibre (DF). Key components of DF are soluble NSP that are found in plant cell walls, and are broken down into metabolites, gases, and microbial biomass in the large intestine and terminal ileum (Pluske et al., 1999). Pigs are unable to release the energy portion of the complex carbohydrate, as it is resistant to the pigs’ own endogenous enzymes. The volatile fatty acids (VFA) that are produced from the breakdown of NSP, mainly acetate, propionate and butyrate (Mosenthin et al., 2001), decrease the pH of digesta. In addition, a diet that has high levels of soluble NSP takes up more water in the intestine, making it more viscous. It is likely that diets containing plant protein are more viscous than those with animal protein sources, due to the soluble NSP in plant proteins. Previous studies conducted at Murdoch University (eg, McDonald et al., 1999) have used cooked white rice in diets for young pigs with other dietary components that are low in NSP and (or) resistant

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starch (RS). These supplements are predominantly animal-based, for example fishmeal, meatmeal, bloodmeal, bonemeal and skimmed milk powder. Vegetable protein sources had deliberately been left out as these contain soluble NSP, and the hypothesis tested was that diets low in soluble NSP prevent PWC. However, because of their availability and cost effectiveness, vegetable proteins, derived from lupins and peas for example, are often used in diets. Also, the continued use of animal proteins worldwide in diets is under recurrent scrutiny and their use is actually banned in the European Union. There is likely to be a greater reliance on vegetable proteins in young pig diets in the future, so potential benefits of cooked rice combined with this needs to be investigated. Wheat contains about 10 g kg-1 viscous soluble NSP (Kim et al., 2003). In comparison, rice contains very little soluble NSP (Marsono and Topping, 1993). Increased intestinal viscosity is believed to be associated with increased microbial fermentation and microbial numbers, and greater microbial numbers may cause more microbial fermentation (McDonald et al., 1999). In addition, increased viscosity slows transit time of digesta through the upper small intestine, which allows more time for microbial pathogens to proliferate. McDonald et al. (2001) have demonstrated increased numbers of enterotoxigenic E. coli in the small intestine by using carboxymethylcellulose to increase viscosity in diets. Increasing intestinal viscosity also decreases absorption of glucose (Rainbird et al., 1984) and other nutrients (Ehrlein and Stockmann, 1998). In other studies performed by Pluske et al. (1996), it was found that a diet higher in NSP content (based on wheat) caused swine dysentery in 75% of pigs after infecting with S. hyodysenteriae, whereas there were no cases of swine dysentery in pigs fed cooked white rice. Collectively, these data suggests that diets based on wheat might predispose pigs to a number of enteric bacterial diseases, with viscosity being a causative or associative factor in the aetiology of the diseases. In regard to PWC, it is possible that newly-weaned pigs fed a wheat diet will have a higher colonisation of E. coli in the small intestine than pigs fed a rice based diet, as a result of greater viscosity in the small intestine. In addition, weaner pigs fed a diet supplemented by plant protein could have a higher number of E. coli colonised in the small intestine than a diet supplemented with animal protein, due to higher levels of soluble NSP. Therefore, the hypotheses tested in this experiment were as follows: a) diets based on wheat, irrespective of the mixture of supplementary protein sources, will cause a higher colonisation of E. coli in the small intestine than pigs fed rice-based diets, and b) a rice diet supplemented with a plant protein mixture will cause a higher colonisation of E. coli in the small intestine, when compared to a rice-animal protein diet. 7.3 Materials and Methods 7.3.1 Animals and procedures A total of 64 mixed-sex pigs (Large White x Landrace) were used in the trial. The trial was run in two replicates of 32 pigs. Pigs were weaned at approximately 21 days of age and transported to Murdoch University from Wandalup Farms, Western Australia. Pigs were then randomly divided into four groups on the basis of gender and liveweight, so that average weights in each pen were similar. Each of these groups (n=4 per treatment) was fed a different diet. Daily records were kept of voluntary food intake in each pen. Pigs were housed in wire-mesh pens with slatted raised pens, four per pen, in an isolation animal house at Murdoch University. Heating was provided by the use of electric heaters. The Animal Ethics Committee of Murdoch University approved this experiment. On arrival at Murdoch University, pigs were weighed and faecal swabs were taken to record initial E. coli presence. Faecal swabs were cultured for the presence of haemolytic E. coli on day 2 of the experiment, which was the first day of infection, and then on days 6 to 9 after weaning. Pigs were infected orally with 107 CFU/mL of E. coli serotype O149;K91;K88 at 48, 72, 96 and 120 hours after arrival.

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7.3.2 Experimental design, diets and feeding The experiment was arranged as a 2 x 2 factorial arrangement of treatments with the respective factors tested being cereal type (cooked white rice vs. wheat) and type of protein source (plant, or vegetable, protein sources vs. animal protein sources). The wheat-plant protein (WPP) diet consisted of wheat and was made up to a complete diet only with plant protein sources. The wheat-animal protein (WAP) diet contained wheat mixed with animal protein sources. The rice-based diets used cooked, medium-grain, white rice (Sunwhite Calrose®; Australian Rice Growers Co-operative, Australia; variety Amaroo), mixed with a plant protein RPP) or animal protein (RAP) supplement (Table 7.1). The rice was cooked with water (1:2 rice:water ratio) in an autoclave for 20 minutes at 121 ºC, and was then allowed to cool overnight at 4 ºC before incorporation into the diet with either the plant or animal protein supplement. A small amount of warm water was added to facilitate mixing the rice and either the plant or animal protein mixture. Pigs were fed one of the four diets (Table 7.1) on an ad libitum basis for 10 days after weaning, by placing the mixed feed in open troughs once daily. Water was available at all times through a nipple drinker located in each pen. Table 7.1 Composition of the experimental diets (g kg-1 on as-fed basis). Wheat-based diets Rice-based diets Ingredient

Plant Protein Animal Protein

Plant Protein Animal Protein

Wheat 750 780 - - Rice - - 660 750 Canola meal 50 - 100 - Lupins 50 - 100 - Full fat soybean 74.9 - 87.2 - Meatmeal (50% CP) - 50 - 50 Whey powder - 50 - 95.4 Blood meal (85% CP) - 25 - 26.4 Fishmeal (65% CP) - 50 - 50 Canola oil 30 28.3 5 5 Salt 1 1 1 1 Vitamin/mineral premixA 1.5 1.5 1.5 1.5 L-Lysine 5.2 2.9 3.3 1.1 DL-Methionine 2.2 1.2 2 1.5 L-Threonine 4.6 2.7 4.4 2.9 Tryptosine 6.6 4.2 7.5 6.3 Dicalcium phosphate 18.5 0.7 20.3 8.4 Choline chloride 0.4 0.4 0.4 0.4 Calculated analysis: DE (MJ/kg) 15.00 15.00 15.15 15.20 Available Lysine (g/MJ) 0.85 0.85 0.85 0.85 Crude protein (%) 16.7 19.7 16.0 17.1 APremix provided (mg/kg air-dry diet): retinyl acetate 3.44, cholecalciferol 0.065, α-tocopheryl acetate 20, menadione 4.4, riboflavin 4, pyridoxine 1.6, cyanocobalamin 0.02, pantothenic acid 14, nicotinic acid 20, Co 0.2, I 0.6, Fe 120, Mn 60, Zn 100, Cu 10, Se 0.13.

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7.3.3 Post-mortem procedures Refer to the previous experiment (Chapter 6.3.4) for details of procedures used. In addition, and after the small intestine was stripped of its mesentery, it was laid out in four sections and keyhole incisions were made. Individual sterile swabs were wiped along the mucosal surface at distances approximately 25% and 75% along the small intestine. Swabs were also applied to the digesta in the caecum, proximal colon and the faeces. Swabs were then rolled onto Sheep Blood Agar (SBA) plates. Plates remained at room temperature until they were streaked using standard procedures, after which they were incubated overnight at 37 °C and scored the following day for the presence of haemolytic E. coli colonies (see 7.3.4 below). Furthermore, a 10-cm section was cut out halfway along the small intestine (mid jejunum), and 1g of epithelium were stripped from its mucosal surface. This was added to 9mL of sterile PBS. Further 1/10 serial dilutions were performed, and 100 µL was dropped onto SBA plates. These were also incubated overnight in air at 37 °C before being counted for haemolytic E. coli colonies the following day (see 7.3.4 below). Samples of digesta were taken from the stomach, ileum, caecum, proximal colon and faeces, and pH was measured immediately (Boy-2 pH meter). The viscosity of the digesta was determined according to methods described in Chapter 6.3.5. 7.3.4 Microbial assessment Plates were assessed for β-haemolytic colonies displaying morphology characteristic of E. coli, after overnight incubation. Some representative colonies of E. coli were selected and streaked onto sterile nutrient agar slopes to be serotyped by the National E. coli Reference Laboratory at the Department of Natural Resources and Environment Agriculture, Bendigo, Victoria, Australia, by slide co-agglutination. 7.3.5 Statistical analyses Statistical analyses were calculated using SuperANOVA (version 1.11; Abacus Concepts, 1989-1991) according to a two-way ANOVA. The unit of replication for all measurements of body weight, internal measurements, and microbiological data was the individual pig. The unit of replication for food intake was each pen of four pigs. Statistical significance was accepted at P<0.05. Differences between treatments for significant main effect means were examined using Fisher’s-protected least significant difference test. 7.4 Results 7.4.1 Microbial assessments There was no significant difference between faecal swabs taken from pigs on any day, except day 8 after weaning. It was expected that there would be low counts from faecal swabs for days 1 and 3, as these were taken before infection of pigs with E. coli. All counts were low, except day 6, which is also the 4th and final day of infection. Generally, pigs fed rice had a higher colonisation of E. coli than those fed wheat. Also, the organs of pigs fed rice had higher averages of swab counts than organs of pigs fed wheat. Most averages between the two different protein types were quite similar. Significantly more bacteria were found in pigs fed rice than those fed wheat, in the digesta 25% along the length of the small intestine. There was no significant difference between numbers of bacteria detected at any of the other sites measured along the digestive tract (Table 7.2).

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Table 7.2 β–haemolytic E. coli swab score assessed from faecal swabs taken at different times after weaning, the swab score assessed from different sites along the gastrointestinal tract at euthanasia on day 10, and the number of colony forming units (CFU) found in the small intestine at euthanasia. Cereal Source Protein Source Probability, P=A,B

Rice Wheat Animal Plant C P C*P (i) Swab scoreC

Day 1 0.31 0.30 0.45 0.16 NS NS NS Day 3 0.28 0.20 0.32 0.16 NS NS NS Day 6 2.40 1.70 2.00 2.00 NS NS NS Day 7 1.90 1.60 1.80 1.80 NS NS NS Day 8 1.90 1.20 1.50 1.60 * NS ** Day 9 0.78 0.93 1.00 0.67 NS NS NS

(ii) Swab score at day 10 collected from:

Caecum 1.00 0.60 0.81 0.82 NS NS NS Colon 1.00 0.70 0.84 0.86 NS NS NS Small intestine -

25% along tract 0.34 0.00 0.12 0.22 ** NS NS

Small intestine - 75% along tract

0.47 0.20 0.29 0.38 NS NS NS

Faeces 0.69 0.40 0.51 0.57 NS NS NS (iii) CFU (x 106) 1.9 0.16 1.0 1.9 NS NS NS

AC- Cereal; P- Protein; C*P- Cereal by protein interaction. BNS- Not Significant; *- P≤0.05 to P<0.01; **- P≤0.01 to P<0.001; and ***- P≤0.001 CMethod of scoring swabs: 0= no colonies grown on the SBA plate exhibit morphology characteristic of haemolytic E. coli; 1= the first swab of primary inoculum displays colonies that are characteristic of haemolytic E. coli; 2= the second streak with a flamed wire loop, diluting the primary inoculum, displays colonies' characteristic of haemolytic E. coli; 3= the third streak with a flamed wire loop, diluting the primary inoculum, displays colonies' characteristic of haemolytic E. coli; 4= the fourth streak with a flamed wire loop, diluting the primary inoculum, displays colonies' characteristic of haemolytic E. coli; 5= the fifth streak with a flamed wire loop, diluting the primary inoculum, displays colonies' characteristic of haemolytic E. coli. 7.4.2 Performance data Pigs fed the rice diets ate, on average, more than double the amount of DM in the 10 days after weaning than their counterparts offered the wheat-based diets (310 vs. 150 g DM per pig per day, P<0.01). However this increase in DM intake failed to translate into an improvement in liveweight and average daily liveweight gain after weaning, as there were no significant differences in bodyweight between experimental treatment diets before and after the experiment. Growth rates were generally very poor, with newly-weaned pigs only growing between 26 and 44 g per day (Table 7.3).

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Table 7.3 The performance of pigs fed different diets for the first 10 days after weaning. Cereal Source Protein Source Probability, P=A,B Item Rice Wheat Animal Plant C P C*P DMIC, kg/pig/day

0.31 0.15 0.24 0.22 *** NS NS

Bodyweight, kg

Start 6.1 6.3 6.2 6.2 NS NS NS Finish 6.4 6.6 6.6 6.4 NS NS NS

Daily liveweight gain, grams

36 34 44 26 NS NS NS

AC- Cereal; P- Protein; C*P- Cereal by protein interaction. BNS- Not Significant; *- P≤0.1 to P<0.05; **- P≤0.05 to P<0.01; ***- P≤0.01 to P<0.001. CDMI: dry matter intake. 7.4.3 Fermentation characteristics Pigs fed cooked whole rice had a higher pH than pigs fed wheat-based diets in the ileum, caecum and proximal colon (P<0.01). A significant difference in the pH of the caecum contents and faeces was also found in pigs fed different protein diets. Pigs fed animal protein had a higher pH (P<0.05) in the caecum, but a lower pH in the faeces (P<0.05). A significant interaction occurred for digesta pH in the faeces, with pigs fed diet RPP having the highest pH and pigs fed diet WAP had the most acidic pH (P<0.1) (Table 7.4). Table 7.4 The pH of digesta in the gastrointestinal tract of pigs fed different diets. Cereal Source Protein Source Probability, P=A,B

Rice Wheat Animal Plant C P C*P pH

Stomach 2.2 2.4 2.4 2.3 NS NS NS Ileum 7.1 6.4 6.7 6.9 *** NS NS Caecum 6.1 5.8 6.0 5.8 ** ** NS Proximal colon 6.2 5.9 6.1 5.9 ** NS NS Faeces 7.1 6.8 6.7 7.2 NS ** *

AC- Cereal; P- Protein; C*P: Cereal by protein interaction. BNS- Not Significant; *- P≤0.1 to P<0.05; **- P≤0.05 to P<0.01; ***- P≤0.01 to P<0.001. Differences between full and empty organ weights were significant when expressed as a percentage of (live) bodyweight. Most of the statistical differences in organ weights were between pigs fed cooked white rice or wheat. The weights of the full stomach, caecum and colon were heavier (P<0.05) in pigs fed wheat when compared to those that ate rice, however only the empty stomach weight was heavier in pigs fed wheat (P<0.01). Weights of the full caecum and colon were heavier (P<0.01) in pigs fed plant protein sources compared to animal protein sources, however on an empty bodyweight basis, only the colon was heavier in pigs fed plant protein. Pigs fed a wheat-based diet had more than a twofold higher digesta viscosity in the ileum (4.5 vs. 2.1 cP, P<0.01) than pigs fed rice (Table 7.5).

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Table 7.5 Organ weights of pigs fed different diets and digesta viscosity in the ileum of pigs fed different diets. Cereal Source Protein Source Probability, P=A,B

Rice Wheat Animal Plant C P C*P FOW3, g

Stomach 2.1 4.1 3.1 3.1 *** NS NS Caecum 0.83 1.0 0.73 1.1 * *** NS Colon 2.4 2.9 2.3 3.0 ** *** NS Small

intestine 6.0 6.6 6.5 6.1 NS NS NS

EBWC, g Stomach 0.75 0.87 0.77 0.85 *** ** NS Caecum 0.31 0.31 0.30 0.32 NS NS NS Colon 1.3 1.4 1.2 1.4 NS * NS Small

intestine 5.1 5.2 5.2 5.1 NS NS NS

Viscosity, cPD 2.1 4.5 3.0 3.6 *** NS NS AC- Cereal; P- Protein; and C*P- Cereal * Protein BNS- Not Significant; *- P≤0.1 to P<0.05; **- P≤0.05 to P<0.01; ***- P≤0.01 to P<0.001. CFOW- Full organ weight expressed as a percentage of pig liveweight; EBW: Empty organ weight expressed as a percentage of liveweight. DCP: centipoise (mPas). 7.5 Discussion The low faecal swab scores and low jejunal counts of haemolytic E. coli contributed to a general lack of any significant differences between diets in this study, illustrating the low degree of colonisation that occurred in these pigs despite experimental infection. More bacteria were found in the digesta 25% of the way along the small intestine in pigs fed rice when compared to wheat (Table 7.2), and the general trend was that pigs fed a rice-based diet had higher counts of haemolytic E. coli than wheat-fed pigs. This occurred despite pigs fed wheat having a significantly higher viscosity than pigs fed cooked rice, and does not support the first hypothesis proposed in this study that increased intestinal viscosity will increase susceptibility of pigs to E. coli colonisation, and hence a higher incidence of PWD. In addition, there were no significant differences in colonisation or scores of E. coli found between pigs fed different protein sources, which does not support the second hypothesis that pigs fed plant protein sources containing more NSP will also show greater colonisation of the gastrointestinal tract with haemolytic E. coli. Data from this study showed clearly that wheat is a more viscous cereal than rice when assessed at the terminal ileum (2.1 vs. 4.5 cP; Table 7.5). Dietary composition, the time of measurement after feeding and the extent of hydration of the dietary NSP all influence the viscosity value (McDonald et al., 2001). However, and based upon previous work done at Murdoch University by McDonald et al. (2001) and Hopwood et al. (2003), the expectation in this experiment was that weaner pigs fed wheat would be more susceptible to PWC since their digesta was more viscous. There was little difference between feacal swabs from day to day, apart from day 8, in the colonisation of the gastrointestinal tract by the inoculated E. coli, with diet RPP having the highest faecal swab score and diet WAP the lowest faecal swab score. Nevertheless pigs fed the plant protein sources had numerically higher numbers of haemolytic E. coli than pigs offered the animal protein sources, but the large variation between pigs meant that this difference was not significant either.

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The results of this experiment, therefore, are contrary to a previous study performed by McDonald et al. (2001), where effects of viscosity were given as a possible reason for a marked increase in the numbers of colonisation of E. coli and incidence of PWC. A possible reason for this is that these piglets may have developed some prior immunity to the serotype that the pigs were inoculated with. Upon entry to Murdoch University from the donor piggery all pigs were swabbed, and it was found that just over 15% of the weaner piglets were positively identified as having colonies displaying characteristics of haemolytic E. coli. One of these pigs was positively identified as carrying serotype 0149;K91;K88. Pigs may also be immune through a genetic trait, that is, a lack of enterocyte receptors for enterotoxigenic E. coli, to match the K88 fimbrial attachment. It is highly unlikely, however, that the pigs sourced from Wandalup Farms for this study are genetically immune to this particular E. coli because piglets have successfully been inoculated and succumbed to PWD in previous experiments at Murdoch University. Another reason for the poor colonisation in this study might have been low levels of voluntary feed intake, such that the amount of diet consumed by the pigs may have been insufficient to predispose pigs to the enterotoxigenic effects of the bacterium. Kelly et al. (1984) noted that pigs eat insufficient food to provide energy for maintenance shortly after weaning, which may contribute to changes in intestinal morphology. Hampson (1987), Rantzer et al. (1995) and numerous other workers have reported previously that pigs eating less feed after weaning were less likely to develop PWC than pigs that consumed more feed, although Madec et al. (1998) reported the converse in a French study. Furthermore, diets used in this study were isoenergetic and contained the same lysine:DE ratio, however they contained less protein (≈ 170 g/kg) than would typically be found in a starter diet in Australia (210-230 g/kg). There is a growing body of evidence (eg, see review by Pluske et al, 2002) that pigs fed diets lower in crude protein (170-200 g/kg) shed less enterotoxigenic E. coli and are less likely to develop PWC than pigs fed commercial levels of protein (> 230 g/kg). Some authors have associated the production of products from bacterial protein digestion, such as biogenic amines and phenolics, with a higher incidence of PWC (Bolduan et al., 1988; Gaskins, 2001). A greater CP content of the diet might cause more of these products to be formed, even at low levels of feed intake. The low dietary CP levels coupled with low levels of feed consumption seen in this study might have contributed to the lack of E. coli colonisation observed. An increase in the DF, or NSP, level of the diet usually increases the amount of VFA produced in the large intestine. Acetate, propionate and butyrate are the main VFA resulting from the breakdown of NSP by bacteria (Mosenthin et al., 2001), hence an increase in VFA in the caecum and colon increases acidity of the gastrointestinal contents (McDonald et al. 2001). It was evident that pigs fed the wheat-based diets had greater levels of microbial fermentation in their caecum and proximal colon, and most likely higher VFA levels, than rice-fed piglets, which in turn caused lower pH values. The higher soluble NSP content in wheat would have allowed more substrate for fermentation by the microbes (Pluske et al., 1999). A lower pH in the ileum may also be due to a change in the relative populations of microbes, as is noted in chickens fed the viscous compound sodium carboxymethylcellulose (van der Klis et al., 1993; Smits et al., 1998). Concomitant with changes in acid production were changes in the weights of organs associated with feeding different cereal diets. Both full and empty organs from pigs fed wheat diets all weighed equal to, or more than, organs from rice-fed pigs (see Table 7.5). However, pigs fed the rice diets ate significantly more DM than those on the wheat diets, which might be attributable to the increased viscosity of the wheat-based diets and negative feedback effects on transit time through the gastrointestinal tract. Wheat is the more viscous of the two cereals, and requires more frequent and forceful peristaltic contractions to move digesta posteriorly (Cherbut et al., 1990). This leads to more muscle accretion and hence heavier organ weights. The plant (vegetable) proteins used also had high levels of soluble NSP, however the sources used also contained appreciable levels of oligosaccharides (eg, verbascose, stachyose and raffinose in lupins) that also contribute to fermentation. Microbial fermentation was the most likely reason for the increase in empty stomach and colon weights seen. Excepting the small intestine, organs, both full and empty, from pigs fed plant protein sources were heavier than those obtained from pigs fed animal protein organs. This is attributable to both the higher

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viscosity of plant protein, which most likely causes the development of smooth muscle in these organs, and the greater apparent digestibility of protein from the animal protein sources. A higher digestibility leaves fewer residues available for the proteolytic bacteria in the distal part of the small intestine and the large intestine.

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8. Effect of extrusion of rice and dietary protein sources on production, digestibility and faecal shedding of E. coli

8.1 Summary A 3 x 2 factorially arranged group of dietary treatments using 84 male weaned piglets aged approximately 21 days of age and weighing 6.7 ± 0.13 kg (mean ± SEM) was used to investigate the effects of diet on performance and PWD. The experimental factors were (a) three cereal types, ie, the medium-grain, lower-amylose rice Amaroo, the long-grain, higher-amylose rice Doongara, and wheat and (b) two protein sources, namely plant (vegetable) and animal protein types. Diets are subsequently referred to as: MGAP: medium-grain rice plus animal protein, MGPP: medium-grain rice plus plant protein, LGAP: long-grain rice plus animal protein, LGPP: long-grain rice plus plant protein, WAP: wheat plus animal protein, and WPP: wheat plus plant protein. The experiment lasted for 21 days, during which time performance indices were monitored, piglets were swabbed for the presence or absence of haemolytic E. coli, piglets were injected with antibiotics if deemed to require treatment by the stockperson, and the coefficient of total tract apparent digestibility (CTTAD) of selected dietary components was assessed. Pigs fed extruded medium-grain or long-grain rice performed equally (P>0.05) to pigs fed wheat as the sole cereal in diets in the 21 days after weaning. The inclusion of plant (vegetable) protein sources in the diet decreased growth rate (P<0.001) and feed intake (P=0.007) and increased FCR (P=0.028) in weeks two and three of the trial compared to the use of animal protein sources, indicating that potential users of extruded rice for specialty piglet diets need to select dietary protein sources judiciously in order to maximise the efficiency of rice. Significant interactions occurred for the CTTAD of dietary components when assessed in week three of the study. In general, pigs fed diets MGAP and LGAP had higher (P<0.001) faecal digestibilities than pigs fed all other diets, and plant proteins depressed (P<0.001) digestibility compared to animal proteins. Faecal shedding of haemolytic E. coli was low and similar (P>0.05) across all dietary treatments The number of antibiotic injections given by the stockperson for PWD was highest for pigs fed animal protein sources than vegetable protein sources (main effect of protein: P=0.057), although there was a tendency for an interaction (P=0.069) because pigs fed diet MGPP had a higher swab score than their counterparts fed MGAP (0.7 vs. 0.5). In summary, pigs tolerated the extruded-rice based diets well and performed equivalently to pigs fed wheat as the sole cereal. 8.2 Introduction Results obtained in Chapters 6 and 7 showed that pigs fed a diet after weaning based on cooked white rice and animal protein generally showed better performance, particularly in the first week after weaning, but effects on ameliorating the shedding of E. coli and incidence of PWD were equivocal. Furthermore, the results seen in Chapter 6 showed that pigs fed plant protein sources seemingly succumbed to more diarrhoea after weaning and grew slower than pigs fed animal protein sources. Whilst the latter finding is not unexpected, our findings that the cooked rice-based diet did not ameliorate the incidence of scouring after weaning is in contrast to earlier work reported from this laboratory (eg, McDonald et al., 1999, 2001). The reason(s) for these ambiguous findings are not known but warranted further investigation. Recent work from Spain (eg, Mateos et al., 2002; Solà-Oriol et al., 2004) has demonstrated that rice processed in a similar way to extrusion outperforms other cereals such as maize (corn), wheat and sorghum in terms of piglet performance and reduced mortality when growth promoting antibiotics and zinc oxide are not added to the diet. A recent Chinese study (Li et al., 2004), however, demonstrated

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that extrusion of Chinese stored brown rice did not influence performance after weaning compared to non-extruded Chinese stored brown rice, and in fact caused inferior feed conversion efficiency. Another Chinese study using whole brown rice fed to growing pigs (44 kg) reported improved nutrient digestibility (Piao et al., 2002). For Australian-grown rice to be used commercially in piglet diets in Australia, it was imperative that rice types identified in Chapter 4 as potentially being of benefit to young pigs be processed in such a way to simulate commercial reality. Furthermore, it was important to assess any possible interactions between extruded rice and different protein sources. The hypotheses tested in this experiment, therefore, were that (a) diets based on extruded rice will cause faster growth after weaning and less faecal excretion of haemolytic E. coli than a traditional, pelleted weaner diet based on wheat, (b) diets based on extruded Amaroo (medium-grain rice) will cause better performance, irrespective of the protein source, than diets based on Doongara (long-grain) rice, and (c) rice-based diets using a combination of animal protein sources will cause less excretion of haemolytic E. coli than diets based on vegetable proteins. 8.3 Materials and Methods 8.3.1 Animals, procedures and housing A total of 84 entire male piglets (Large White x Landrace) aged approximately 21 days of age and weighing 6.7 ± 0.13 kg (mean ± SEM) was used in this experiment. The piglets were obtained from Wandalup Farms, Mandurah, WA. On arrival at the Medina Research Station, the pigs were ear-tagged, weighed, and stratified into pens of three or four pigs each according to treatment and liveweight. Pigs were offered their respective diets (see below) in groups of four for the first seven days after weaning, to accustom them to their new surroundings. For the final two weeks of the study, pigs were housed individually. Pens were of wire-mesh construction with slatted metal floors, and measured 1.68 m2 in floor area (0.42 m2 per pig). Each pen was equipped with a nipple water drinker and a stainless steel feed trough. The ambient temperature was maintained between 26 and 28° C throughout the study using two reverse-cycle air conditioning units. The room containing the pens was cleaned daily. The Murdoch University Animal Ethics Committee and the Animal Ethics and Experimentation Committee of the WA Department of Agriculture approved this experiment. The experiment was conducted in two replicates. 8.3.2 Experimental design, diets, feeding and sample collection The experiment was designed as a 3 x 2 factorial arrangement of treatments with the respective factors being (a) three cereal types, ie, the medium-grain, lower-amylose rice Amaroo, the long-grain, higher-amylose rice Doongara, and wheat and (b) two protein sources, namely plant (vegetable) and animal protein types. Diets are subsequently referred to as: MGAP: medium-grain rice plus animal protein, MGPP: medium-grain rice plus plant protein, LGAP: long-grain rice plus animal protein, LGPP: long-grain rice plus plant protein, WAP: wheat plus animal protein, and WPP: wheat plus plant protein. Diets were formulated to contain adequate levels of energy and nutrients for pigs of this genotype and age. The extruded rice sourced from the University of Adelaide had to be passed through a hammer mill to reduce particle size before incorporation into the meal-based diets. The diet was offered to piglets in mash form, and the diet composition is presented in Table 8.1. Titanium dioxide (TiO2) was added as an inert marker for CTTAD estimation. Faecal samples were collected from the wire-mesh floor for weaner pigs at 0800, 1000, 1200, 1400, 1600 h on days 18-21 of the experiment. Samples collected over the three-day period were pooled, kept at –20 ºC and later thawed, mixed, freeze-dried and ground through a laboratory hammer mill (1 mm screen) prior to chemical analysis.

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8.3.3 Microbial assessment Faecal swabs were taken to record initial E. coli presence upon arrival, and then on days 2, 5, 6 and 8 after weaning. Faecal swabs were cultured for the presence of haemolytic E. coli, and plates were assessed for β-haemolytic colonies displaying morphology characteristic of E. coli, after overnight incubation. The presence of haemolytic E. Coli was then scored from no growth (0) to heavy colonisation (5), as has been described previously (Chapter 6.3.3). Piglets were monitored daily by the stockperson for clinical signs of diarrhoea. Affected pigs (as assessed by the stockperson, who was unaware of the treatment allocation of pigs) were treated for diarrhoea by intramuscular injection with Trisoprim-480 [(trimethropin 80 mg/mL, sulfadiazine, 400 mg/mL), 1.5 mL/30 kg body weight; Troy Laboratories, Smithfield, NSW, Australia]; treatment continued until the diarrhoea ceased. Records were kept of the duration of treatment required for each treated piglet. 8.3.4 Chemical analyses The dry matter (DM), nitrogen (N), gross energy (GE), total starch, RS, amylose and amylopectin contents of extruded rice were determined as described previously (Chapter 4.2.1). Crude protein (CP) content was calculated as N x 6.25. The DM, GE and TiO2 content of diets and faecal samples were determined for estimation of apparent digestibilities of GE and the DE content of the experimental diets. The GE content of the rice, diet, and faecal samples was determined using a Ballistic Bomb Calorimeter (SANYO Gallenkamp, Loughborough, UK). The TiO2 contents of diet and feacal samples were determined using the method described by Short et al. (1996). 8.3.5 Statistical analyses Treatment effects were assessed by two-way ANOVA for a factorial design with the main effects being cereal (medium-grain rice, long-grain rice and wheat) and protein type (plant and animal). Average daily gain in the first week after weaning was assessed using the pen as the unit of replication. Daily gain, voluntary feed intake and FCR in weeks two and three of the study used the individual pig as the experimental unit, because pigs were housed individually. For the CTTAD of DM, starch, GE and CP, the individual pig was considered the unit of replication. All effects were considered as fixed effects in the model. Fisher’s-protected least significant difference test were used (at 5% significance level) for comparison between mean values of different variables. All statistical analyses were conducted using the statistical package StatView 5.0 for Windows (AddSoft Pty. Ltd., Woodend, Vic., Australia).

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Table 8.1 Composition of the experimental diets used in the experiment (g kg-1 as-fed basis). Medium-grain rice Long-grain rice Wheat Ingredient Animal

protein Plant

protein Animal protein

Plant protein

Animal protein

Plant protein

Rice 705.6 528.4 705.6 528.4 - - Wheat - - - - 780 532.8 Meat and bone meal

51.6 - 51.6 - 50 -

Whey powder

100 - 100 - 50 -

Bloodmeal (85% CP)

30 - 30 - 25 -

Fishmeal (65% CP)

100.4 - 100.4 - 50 -

Lupins - 100 - 100 - 100 Canola meal - 150 - 150 - 150 Full-fat soybean meal

- 185.2 - 185.2 - 151.6

Canola oil 5 - 5 - 28.3 30 L-lysine 2.78 6.4 2.78 6.4 6.04 6.84 DL-methionine

0.36 1.12 0.36 1.12 1.2 1.47

L-threonine 1.43 2.68 1.43 2.68 2.7 2.54 L-tryptophan 0.28 0.34 0.28 0.34 0.42 0.23 Choline chloride

0.4 0.4 0.4 0.4 0.4 0.4

Dicalcium phosphate

- 18.7 - 18.7 0.7 17

Limestone - 4.4 - 4.4 - 5.2 Salt 1 1 1 1 1 1 Vitamin and mineral premixA

0.7 0.7 0.7 0.7 0.7 0.7

Titanium dioxideB

1 1 1 1 1 1

Calculated analysis:

DE (MJ/kg) 15.3 15.4 15.3 15.4 15.0 15.3 CP, g/kg 200 200 200 200 197 215 Available lysine, %

1.30 1.31 1.30 1.31 1.28 1.30

Calcium % 1.2 0.8 1.2 0.8 0.91 0.8 Available P, %

0.6 0.45 0.6 0.45 0.49 0.45

AProvided the following nutrients (per kg of air-dry diet): Vitamins: A 1500 IU, D3 300 IU, E 37.5 mg, K 2.5 mg, B1 1.5 mg, B2 6.25 mg, B6 3 mg, B12 37.5 μg, Calcium pantothenate 25 mg, Folic acid 0.5 mg, Niacin 30 mg, Biotin 75 μg; Minerals: Co 0.5 mg (as cobalt sulphate), Cu 25 mg (as copper sulphate), Iodine 1.25 mg (as potassium iodine), Iron 150 mg (as Ferrous sulphate), Mn 100 mg (as Manganous oxide), Se 0.5 mg (as Sodium Selenite), Zn 0.25 mg (as zinc oxide). (Hogro Bronze Weaner and Grower, Rhone-Poulenc Animal Nutrition Pty Ltd., Queensland, Australia). BTitanium dioxide (TiO2; Sigma Chemical Company, St. Louis, MO, USA).

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8.4 Results 8.4.1 Faecal shedding of haemolytic E. coli and antibiotic treatments The mean number of antibiotic treatments and the mean faecal swab score are shown in Table 8.2. The number of antibiotic treatments given by the stockperson for clinical PWD was similar (P>0.05) across all dietary treatments. However, shedding of haemolytic E. coli ascertained via faecal swabs showed a significant main effect of protein source on the faecal swab score, with pigs fed animal protein demonstrating a higher score than pigs fed vegetable protein (0.6 vs. 0.4, P=0.057). Furthermore there was a tendency for an interaction between cereal type and protein source (P=0.069), with pigs fed diet MGPP having a higher swab score than pigs fed diet MGAP (Table 8.2). Table 8.2 Interaction means for average number of antibiotic treatments and the average faecal swab score for piglets after weaning.

Dietary treatment Cereal type Protein source

Number of antibiotic treatments

Faecal swab scoreB

Animal 1.4 0.5 Medium-grain rice Plant 0.7 0.7

Animal 1.0 0.6 Long-grain rice Plant 0.6 0.3 Animal 1 0.8 Wheat Plant 1 0.3

Pooled mean 0.9 0.5 SEMA 0.0.72 0.30 Probability, P= Cereal type 0.792 0.461 Protein source 0.230 0.057 Cereal * Protein 0.638 0.069 ASEM: standard error of the mean. BFaecal swab score is the mean score per pig determined from swabs taken on days 2, 5, 6 and 8 after weaning. 8.4.2 Performance data Pigs generally adapted well to their new environment. There were no statistically significant differences between treatment groups for average daily gain in the first week after weaning, although there was a tendency for pigs offered animal protein to outperform pigs offered plant protein (78 vs. 43 g/day, P=0.152). Pigs fed rice numerically performed better than pigs fed wheat (an average of 69 vs. 44 g/day), however large variation between pens of pigs precluded statistical differences (Table 8.3). In weeks two and three, there was no significant main effect of cereal type on any of the indices measured, although there was suggestion of an improved FCR with wheat compared to rice (P=0.191). There was a significant main effect, however, of protein source. Pigs fed animal protein rather than plant (vegetable) protein were heavier at the end of the experiment (11.80 vs. 10.36) because they grew faster (317 vs. 242 g/day). This was a consequence of a higher voluntary feed intake (580 vs. 500 g/day) and improved FCR (1.87 vs. 2.31). No interactions occurred for any of the production indices (Table 8.4).

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Table 8.3 Interaction means for average daily gain of pigs kept in groups in the first week after weaning.

Dietary treatment Cereal type Protein source

Start Liveweight, kg

Liveweight after 7 days, kg

Daily gain, g

Animal 6.71 7.32 87 Medium-grain rice Plant 6.66 7.04 54

Animal 6.68 7.31 91 Long-grain rice Plant 6.69 6.98 42 Animal 6.97 7.46 55 Wheat Plant 6.81 7.03 32

Pooled mean 6.73 7.15 60 SEMA 0.131 0.177 110.2 Probability, P= Cereal type 0.900 0.991 0.625 Protein source 0.952 0.465 0.152 Cereal * Protein 0.996 0.984 0.903 ASEM: standard error of the mean. 8.4.3 Coefficient of total tract apparent digestibility The CTTAD of DM, starch, energy and CP are depicted in Table 8.5. Significant interactions between the cereal type and protein source occurred for DM, starch, energy and CP. The CTTAD for DM was higher in diets MGAP and LGAP than in the corresponding wheat-based diet (WAP) (0.92 and 0.92 vs. 0.85, P<0.001), however DM digestibility was similar between all three diets when plant proteins were fed to pigs instead of animal proteins (0.83, 0.82 and 0.80 for diets MGPP, LGPP and WPP, respectively). A similar interaction occurred between cereal type and protein source for the CTTAD of energy, with diets MGAP and LGAP having the highest coefficients of energy digestibility compared to WAP (0.92 and 0.91 vs. 0.83, P<0.001). Total-tract starch digestibility was very high in all diets (range 0.989 to 0.999). The CTTAD for starch was higher in diet WPP than in diet WAP (0.993 vs. 0.989), which resulted in a significant interaction (P<0.001). Surprisingly, the CTTAD for CP was higher in pigs fed diet WPP compared to those fed diets MGPP and LGPP (0.76 vs. 0.67 and 0.66 respectively, P=0.016) (Table 8.5).

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Table 8.4 Interaction means for performance of pigs fed different diets in weeks two and three of the experiment.

Dietary treatment Cereal type Protein source

Liveweight at start of Week 2, kg

Liveweight at end of Week 3, kg Daily gain, g Daily feed intake, g

day-1 FCR (g feed:g gain)

Animal 7.31 11.66 311 586 1.92 Medium-grain rice Plant 6.98 10.39 243 527 2.69 Animal 7.32 11.69 312 586 1.98 Long-grain rice Plant 7.04 9.97 219 488 2.32 Animal 7.46 12.06 329 569 1.71 Wheat Plant 7.03 10.71 264 485 1.92

Pooled mean 7.15 11.07 278 540 2.13 SEMA 0.170 0.284 10.4 15.1 0.122 Probability, P= Cereal type 0.981 0.723 0.330 0.712 0.191 Protein source 0.322 0.010 <0.001 0.007 0.028 Cereal * Protein 0.987 0.942 0.803 0.874 0.545 ASEM: standard error of the mean.

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Table 8.5 The coefficient of total tract apparent digestibility (CTTAD) of dry matter (DM), starch, energy and crude protein (CP) in pigs fed different diets after weaning.

Dietary treatment CTTAD of: Cereal type Protein source DMA Starch Energy CPA

Animal protein 0.92 0.999 0.92 0.79 Medium-grain rice Plant protein 0.83 0.998 0.82 0.67 Animal protein 0.92 0.999 0.91 0.78 Long-grain rice Plant protein 0.82 0.997 0.81 0.66 Animal protein 0.85 0.993 0.83 0.78 Wheat Plant protein 0.80 0.989 0.80 0.76

Pooled mean 0.86 0.996 0.85 0.74 SEMB 0.006 0.0012 0.006 0.009 Probability, P= Cereal type <0.001 <0.001 <0.001 0.022 Protein source <0.001 0.837 <0.001 <0.001 Cereal * Protein <0.001 0.016 <0.001 0.016 ADM: dry matter; CP: crude protein. BSEM: standard error of the mean.

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8.5 Discussion Weaned piglets fed extruded medium-grain rice or long-grain rice performed equally to pigs fed wheat in the first three weeks after weaning, indicating that extruded rice can replace wheat as the sole cereal in piglet diets after weaning. There was a suggestion, however, that pigs fed wheat as the cereal converted feed to daily gain more efficiently than pigs fed the two extruded rice-based diets (P=0.191; Table 8.3). As alluded to in Section 7.5 and highlighted further in Chapter 10, however, it seems that the energy value of extruded rice might have been underestimated, which would account for the inferior feed conversion of the rice-fed piglets. This is further supported by the findings that pigs fed the extruded medium-grain rice or long-grain rice diets had higher CTTAD for starch, energy and CP that would have yielded more nutrients in the digestive tract for absorption and body growth, although the response of the cereal depended on the type of protein sources fed to the pigs. The inclusion of vegetable (plant) protein sources in the diet depressed growth rate (P<0.001), feed intake (P=0.007) and FCR (P=0.028) in weeks two and three of the trial compared to the use of animal protein sources. This is an important finding because it provides clear direction to potential users of extruded rice regarding the selection of dietary protein sources to maximise the efficiency of rice. Vegetable proteins such as the lupins, soybean meal and canola meal used in this study contain appreciable levels of NSP and oligosaccharides that are anti-nutritive in the gastrointestinal tract of the pig, reducing the extent of digestion and absorption of nutrients available for body growth (Pluske et al., 1999). Animal protein sources are more digestible than plant protein sources and hence more nutrients became available for growth and development. However, the presence of significant interactions between cereal and protein sources for CTTAD indicates that the dietary component responded differently to both dietary variables. For example, CTTAD for energy was significantly higher in both extruded rice-based diets irrespective of whether animal or plant protein was added compared to diets WAP and WPP, whereas for CP digestion, the significant interaction (P=0.016) was caused by an apparently higher digestibility in pigs fed diet WPP compared to pigs fed diets MGPP and LGPP. It is difficult to explain the higher CP digestibility in pigs fed diet WPP compared to pigs fed diets MGPP and LGPP given the higher DM and energy digestibilities observed in the extruded rice-based diets when plant protein sources were added, but might be attributable to a higher formation of microbial protein causing an overall depressed total tract digestibility (Pluske et al., 2003). Interpretation of total tract digestibility coefficients for CP is fraught, therefore, because the formation of protein by the microbiota provides no real indication of ileal digestibility of CP and absorption of amino acids, which cannot be absorbed by the pig in the large intestine. Seemingly in contrast to these findings, Montagne et al. (2004) reported no differences in average daily gain or FCR when pigs were fed diets based on cooked (autoclaved) white rice with either an animal or plant protein supplement. The reason(s) for this difference is (are) hard to explain, but could be attributable to the fact that Montagne et al. (2004) infected pigs experimentally with enterotoxigenic E. coli that could have disturbed the intestinal milieu associated with digestion and absorption, and (or) there was an effect of cooking form on digestibility and subsequent growth rate. Extruded rice contains very low levels of RS whereas cooked (autoclaved) white rice that is then cooled prior to feeding contains approximately 20 times the RS content of extruded rice (Chapter 5). The RS level of the extruded products were not considered in the derivation of the energy value of extruded rice used in the formulation of the diets, and hence there could have been a misbalance in energy contributions between the protein sources and the extruded rice that contributed to the inferior performance of the pigs fed plant protein. The number of antibiotic treatments given by the stockperson for clinical PWD in the 21 days after weaning was similar (P>0.05) in pigs across all treatment groups. In contrast, the faecal shedding of E. coli in the first eight days after weaning showed that vegetable proteins (as a main effect in the statistical analysis) reduced (P=0.059) the mean faecal swab score recorded in the first eight

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days after weaning compared to pigs fed animal protein sources. This difference was caused predominately by the greater swab score recorded in pigs fed diets LGAP and WAP, suggesting that in these two cereal sources the presence of vegetable proteins reduced colonisation and subsequent shedding of the haemolytic E. coli. Vegetable proteins contain considerable levels of DF, which are possibly thought to influence PWD and shedding of E. coli (Bolduan et al., 1988). These data contrast to the work of Pluske et al. (2003) where the addition of various sources of DF, which are present also in vegetable proteins, increased the number of antibiotic injections required in pigs that displayed PWD. However, the quantity and chemical composition of the DF sources used Pluske et al. (2003) contrasted greatly with the predominately uronic acid-based DF present in vegetable proteins (Pluske et al., 1999), which could help explain the difference. Moreover, the use of extruded rice instead of cooked (autoclaved) rice could also have contributed to the differences observed because of changes to the physico-chemical properties (Marsono and Topping, 1993). Montagne et al. (2004) noted a lack of difference in pigs fed diet WPP compared to pigs fed diets MGAP and MGPP for the faecal shedding of haemolytic E. coli, which again could reflect the difference in the form (ie, extrusion vs. autoclaving) of rice used in the experiments. Nevertheless, and as depicted in Table 8.4, pigs fed diets MGAP or LGAP performed equivalently to their counterparts fed wheat-based diets, and mortality on the rice-based diets was zero. Professor Gonzales Mateos (personal communication), who has conducted similar studies with processed rice in Spain, has sometimes observed a higher incidence of PWD in piglets fed a cooked rice-based diet, although this did not cause detrimental performance. Professor Mateos believes that the (transient) diarrhoea after weaning caused by feeding rice is a consequence of higher feed intake in the first week. It was not possible to measure individual feed intake in the first week post-weaning in our experiment because pigs were housed in groups of four, however there was a clear trend for pigs fed the extruded rice and animal protein-based diets (MGAP and LGAP) to numerically perform better than pigs fed wheat (an average of 69 vs. 44 g/day), a difference that undoubtedly reflects a higher voluntary feed intake. In conclusion, pigs fed the extruded rice-based diets with either animal protein sources or plant protein sources performed equivalently to pigs fed either of the wheat-based diets in the first three weeks following weaning. The digestibility of DM and the dietary components starch and energy was generally improved by the use of extruded rice compared to wheat, and was increased by the use of animal rather than vegetable proteins. The lack of difference in antibiotic treatments and faecal shedding of haemolytic E. coli might have been a general reflection of the low levels of PWD seen in this study, although the reduction in shedding seen in pigs fed vegetable proteins implicated a role for DF in the expression of PWD. This feature will be pursued in the next chapter.

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9. Effect of added oat hulls to extruded rice- and wheat-based diets on production, digestibility and the incidence of PWD

9.1 Summary This experiment was conducted in male weaner pigs (96 pigs with initial weight of 5.16 ± 0.08 kg) to examine the effects of added oat hulls (20 g kg-1) in either an extruded rice- or wheat-based diet on performance, the incidence and treatment of PWD, coefficients of total tract apparent digestibility (CTTAD) of dietary components and the levels of blood and faecal metabolic indicators of hindgut digestion in the post-weaning period. The diets used were: (i) extruded medium-grain rice (R; variety Amaroo) plus animal protein (RAP); (ii) diet (i) with added oat hulls (20 g kg-1; RAPOH); (iii) wheat (W) plus animal protein (WAP); and (iv) diet (iii) with added oat hulls (20 g kg-1; WAPOH). All diets were formulated to contain 14.4 MJ DE kg-1 and 0.80 g lysine MJ DE-1. Pigs (24 pigs per treatment combination; 4 pigs per pen and 6 pens per treatment) were randomly allocated based on initial live weight and fed their respective diets ad libitum. Blood and faecal samples were collected on days 7 and 14 after weaning. Pigs fed wheat diets retained more moisture in their faeces than pigs fed R diets (P<0.01). Pigs fed diet RAP had more diarrhoea and hence received more antibiotic treatment than pigs fed diet WAP. Addition of oat hulls to diet RAP decreased the incidence of PWD and the number of antibiotic treatments. The number of pigs shedding haemolytic E. coli measured five days after weaning was lower (P<0.05) in pigs fed oat hull-supplemented diets. Mean E. coli scores were lower in pigs fed wheat-based diets than in pigs fed extruded rice-based diets regardless of oat hull supplementation. The CTTAD of all dietary components (DM, starch and energy) were higher in extruded rice-based diets than in wheat-based diets (P<0.001). The addition of oat hulls decreased the CTTAD (P<0.001). Pigs fed extruded rice-based diets had higher plasma urea concentrations and lower faecal biogenic amine concentrations compared to pigs fed wheat-based diets. Plasma creatinine concentration was positively correlated to haemolytic E. coli scores after weaning (P<0.015). Supplementation of oat hulls tended to decrease biogenic amine concentrations (P=0.103). In conclusion, insoluble NSP added in the form of oat hulls in extruded rice and animal protein-based diets for weaner pigs reduced the incidence of PWD in the first three weeks after weaning. 9.2 Introduction Numerous recent studies in pigs have demonstrated that cooked white rice shows excellent potential for inclusion in diets as a replacement for more traditional cereals such as maize and sorghum (Alcantara et al., 1989; Mateos et al., 2001; 2002; Solà-Oriol et al., 2004; Vicente et al., 2004). Furthermore, the use of cooked white rice has been associated with reductions in PWC and swine dysentery (Pluske et al., 2002; 2003; Hopwood et al., 2004). However, results found previously in Chapters 7 and 8 showed an increased incidence of PWD, or certainly no reduction in PWD, when pigs were fed either extruded rice-based diets or a cooked rice-based diet with animal protein compared to pigs fed a WAP or WPP diet. It has been postulated that an imbalance between the reduced amount of (fermentable) carbohydrates entering the hindgut from rice, as it is all starch, and an excess of undigested nitrogen (N) relative to carbohydrate entering the large intestine in pigs fed diet RAP, may have contributed to PWD because the microbiota decarboxylate the N into diamines which are thought to be implicated in the aetiology of PWD (Bolduan et al., 1988; Gaskins, 2001; Pluske et al., 2002).

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Substrates such as carbohydrate and N entering the caecum most likely change the ratio of saccharolytic and proteolytic microbiota in the large intestine. For example, a diet containing a limited amount of fermentable carbohydrates (NSP and RS) and (or) is high in N will cause the proteolytic microbiota to predominate over saccharolytic bacteria (Piva et al., 1995; Reid and Hillman, 1999). The proliferation of proteolytic bacteria in the large intestine of pigs can produce many potentially toxic metabolic by-products including branched-chain VFA, NH3, volatile phenols, indoles and biogenic amines (Williams et al., 2001). When undigested N enters the caecum, deamination or decarboxylation processes produce NH3 and amines, respectively. Putrescine, cadaverine, histamine and β-phenylethylamine are produced in the pig’s large intestine from arginine, lysine, histidine and phenylalanine, respectively, by a number of bacterial groups including Bacteroides, Clostridium, Enterobacterium, Lactobacillus and Streptococcus (Gaskins, 2001). These microbially generated amines (biogenic amines) could contribute to the occurrence of diarrhoea after weaning. However, the addition of insoluble and (or) slowly fermentable NSP sources in weaner pig diets showed a linear reduction in biogenic amines and less PWD (Bolduan et al., 1988; Aumaitre et al., 1995). Given the unexpected results found in Chapters 7 and 8, the purpose of the current study was to (1) determine whether the type of cereal (low fermentable carbohydrate diet RAP vs. high fermentable carbohydrate diet WAP) influences the incidence of diarrhoea and excretion of haemolytic E. coli, (2) see whether the addition of insoluble NSP (20 g oat hulls kg-1) reduced the deleterious effects feeding an extruded rice plus animal protein diet appeared to have on PWD, and (3) examine further the digestibility of selected dietary components and performance of weaner pigs in response to cereal type and added oat hulls. 9.3 Materials and Methods 9.3.1 Experimental design A 2 x 2 factorially designed experiment was conducted, with the respective factors being cereal type (extruded medium-grain rice vs. wheat) and oat hull addition (with or without 20 g oat hulls kg-1), to test the effect of cereal source and insoluble NSP supplementation on faecal moisture content, the incidence of PWD, the number of antibiotic treatments, faecal scores of haemolytic E. coli, blood urea and creatinine, the formation of biogenic amines, CTTAD of dietary components and performance after weaning. The Murdoch University Animal Ethics Committee and the Animal Ethics and Experimentation Committee of the WA Department of Agriculture approved the experimental protocol. 9.3.2 Animals, diets, feeding, recording and sample collection Ninety-six male pigs (Large White x Landrace) weaned at approximately 18-22 days of age were obtained from a commercial supplier (Wandalup Farms, Mandurah, WA). The pigs were transported to an isolated animal house at Murdoch University where they housed in metal wire-mashed pens with a floor space of 2.5 m2. The experiment was conducted in two replicates with 48 pigs each. The average live weights (mean ± SEM) of pigs at arrival were 5.5 ± 0.05 kg for replicate 1 and 4.9 ± 0.08 kg for replicate 2. Pigs were randomly accommodated to a pen (4 pigs/pen, 6 pens/treatment = 24 pigs per treatment) based on their live weight at arrival. The ambient temperature was maintained at 29 ± 1°C. Water and feed were freely accessible during the whole experiment through a nipple drinker and feed trough, respectively, set in each pen. The pigs were offered their respective experimental diet ad libitum for three weeks. The diets used were: (i) extruded medium-grain rice (R; variety Amaroo) plus animal protein (RAP); (ii) diet (i) with added oat hulls (20 g kg-1; RAPOH); (iii) wheat (W) plus animal protein (WAP); and (iv) diet (iii) with added oat hulls (20 g kg-1; WAPOH). All diets were formulated to contain 14.4 MJ DE kg-1 and 0.80 g lysine MJ DE-1. Oat hulls (Glen Forrest Stockfeeds, Glen Forest, WA) were passed

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twice through a hammermill fitted with a 4.5 mm screen prior to incorporation into diets. The extruded rice was that used previously (Chapter 8.3.2), and was passed through a hammermill without the screen to achieve a more uniform particle size. The wheat was passed through the same hammermill but with a 4.5 mm screen, to reduce particle size. Titanium dioxide (TiO2) was added as an inert marker for estimation of the CTTAD. The composition and analysed chemical composition of experimental diets are presented in Table 9.1. Pigs were weighed every week and feed intake was recorded on a weekly basis as feed disappearance from the feeder. Pigs were monitored for the presence of diarrhoea at least twice daily. Pigs with diarrhoea were treated immediately with an intramuscular injection of Trisprim-480 (trimethropin 80 mg/mL, sulfadiazine, 400 mg/mL; Troy Laboratories, Smithfield, NSW, Australia) until the diarrhoea ceased. Faecal swabs were taken for culture on days 0, 2, 5 and 6 after weaning. Blood samples (10 ml) from two randomly selected pigs per pen were taken from the anterior vena cava into heparinised EDTA-vacutainer tubes on days 7 and 14. The samples were immediately transported and analysed for plasma urea and creatinine contents. Faecal ‘grab’ samples were collected from each pen at 0800, 1000, 1200, 1400, 1600 h on day 7 and 14 of the trial. The samples were then kept at –20 ºC and later thawed, mixed, freeze-dried and ground through a laboratory hammer mill (1 mm screen) prior to chemical analysis. 9.3.3 Chemical and microbial analyses All analyses were made in duplicate. Dry matter (DM) was measured using AOAC official method 930.15 (AOAC, 1997). Total starch was determined as described previously (Chapter 4.2.1). The N content was determined with a LECO FP-428 Nitrogen Analyser using a combustion method (AOAC official method 990.03; AOAC, 1997). The gross energy (GE) content was determined using a Ballistic Bomb Calorimeter (SANYO Gallenkamp, Loughborough, UL). NSP and their constituent sugar contents were determined as alditol acetates by gas-liquid chromatography at The University of New England, using the method of Theander and Westerlund (1993). Water holding capacity (WHC) of diets was determined using a method by Kyriazakis and Emmans (1995). Titanium dioxide (TiO2) was determined as described by Short et al. (1996). The E. coli score was determined by scraping faecal swabs on a sheep blood agar plate and grown overnight at 37 °C in air. The presence of haemolytic E. coli was then scored from no growth (0) to heavy colonisation (5.) Faecal consistency was measured and scored as normal, moist, wet and diarrhoea. Plasma urea content was determined using an enzymatic (urease) kinetic method (Randox). Plasma creatinine assay was performed on an automated analyser (Daytone RX, Randox, Northern Ireland) using alkaline picrate without deproteinisation. Both metabolites were determined in the Clinical Pathology laboratory at Murdoch University. The concentration of biogenic amines in the feed and faeces of pigs was determined by the State Chemistry Laboratories of the Department of Primary Industries, Victoria, Werribee. 9.3.4 Statistics The pen was considered as an experimental unit for all statistical analyses. For pig performance, the treatment effects were assessed by ANOVA for a factorial arrangement with the main effects being cereal type and oat hull supplementation. For CTTAD, blood metabolites and biogenic amine levels, treatment effects were assessed by repeated measures ANOVA. Pearson’s correlation study was performed to examine relationships between the plasma creatinine concentration and haemolytic E.coli scores. The effects were considered as fixed effects in the model. Fisher’s-Protected least significant difference test were used (at 5% significance level) for comparison of treatment differences. The digestibility at week one was compared with the results repeated after two weeks using one-way ANOVA. All statistical analyses were conducted using the statistical package StatView 5.0 for Windows, SAS Inc. (AddSoft Pty. Ltd., Woodend, Vic., Australia).

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Table 9.1 DietA composition (g kg-1, as-fed basis) and analysed nutrient content (g kg-1 DM). Ingredient RAP RAPOH WAP WAPOH Extruded rice 702 682 - - Wheat - - 782 762 Meat and bone meal 52 52 50 50 Whey powder 100 100 50 50 Bloodmeal 30 30 25 25 Fishmeal 100 100 50 50 Oat hulls - 20 - 20 Canola oil 5 5 28 28 L lysine-HCl 2.8 2.8 6 6 DL Methionine 0.4 0.4 1.2 1.2 L Threonine 1.5 1.5 2.7 2.7 Tryptophan 2.8 2.8 0.4 0.4 Choline chloride 0.4 0.4 0.4 0.4 Dicalcium phosphate - - 0.7 0.7 Salt 0.1 0.1 0.1 0.1 Vit/Min premixB 0.7 0.7 0.7 0.7 Titanium dioxide 0.1 0.1 0.1 0.1 Calculated analysis (g kg-1 DM): Crude protein 195 188 204 190 Starch 569 568 543 563 GE (MJ/kg DM) 19.03 19.03 19.32 19.35 Total NSP 11.94 23.02 76.99 94.07 Insoluble NSP 8.95 20.19 65.74 83.36 Soluble NSP 3.00 2.82 11.24 10.71 Free sugars 31.66 31.20 32.37 28.34 Biogenic amines (mg kg-1 DM) β-Phenylethylamine 15 19 22 22 Putrescine 24 24 17 17 Cadaverine 41 43 26 24 Histamine 4 4 3 3 Water holding capacity (g g-1 dried diet) 3.67 3.71 1.69 1.65 ARAP: Rice animal protein; RAPOH: Rice animal protein and oat hulls; WAP: wheat animal protein; WAPOH: wheat animal protein and oat hulls BProvided the following nutrients (per kg of air-dry diet): Vitamins: A 1500 IU, D3 300 IU, E 37.5 mg, K 2.5 mg, B1 1.5 mg, B2 6.25 mg, B6 3 mg, B12 37.5 �g, Calcium pantothenate 25 mg, Folic acid 0.5 mg, Niacin 30 mg, Biotin 75 �g; Minerals: Co 0.5 mg (as cobalt sulphate), Cu 25 mg (as copper sulphate), Iodine 1.25 mg (as potassium iodine), Iron 150 mg (as Ferrous sulphate), Mn 100 mg (as Manganous oxide), Se 0.5 mg (as Sodium Selenite), Zn 0.25 mg (as zinc oxide). (Hogro Bronze Weaner and Grower, Rhone-Poulenc Animal Nutrition Pty Ltd., Queensland, Australia).

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9.4 Results Faecal consistency scores on 2, 5 and 6 days after weaning are presented in Table 9.2. Pigs fed the extruded rice-based diet had firmer faeces than pigs fed the wheat-based diet on day 2 (P=0.011), but the difference was not significant on day 5 and 6 after weaning. Follow-up determination of faecal moisture content one and two weeks after weaning showed significantly higher moisture content in pigs fed wheat diets compared to pigs fed extruded rice diets (see Table 9.5, below). Table 9.2 Faecal consistency scoreA of pigs fed different diets after weaning.

DietsB

RAP RAPOH WAP WAPOH Pooled means SEM Probability, P=C

Day 2 1.2 1.6 2.0 1.7 1.6 0.087 0.011

Day 5 1.6 1.6 2.0 1.7 1.7 0.090 0.340

Day 6 1.7 1.9 2.0 1.7 1.8 0.094 0.429 AFaecal consistency scored defined as; 1: normal, 2: moist, 3: wet and 4: diarrhoea. BRefer text for details of diets. CFrom one-way ANOVA. The incidence of scouring and the number of pigs injected with antibiotics are presented in Table 9.3. Pigs in replicate 1 had no diarrhoea, so the data presented in Table 9.3 and Figure 9.1 refer only to observations in replicate 2. Pigs fed diet RAP diet showed a higher incidence of scouring and received more antibiotic injections than pigs fed diet WAP. Addition of oat hulls in the RAP diet significantly decreased the incidence of scouring and the number of antibiotic treatments. The presence of haemolytic E. coli during the first week after weaning is illustrated in Figure 9.1. The number of pigs shedding haemolytic E. coli on day 5 was significantly lower in oat hull-supplemented groups. Mean E. coli scores were lower in pigs fed wheat diets than in pigs fed extruded rice diets regardless oat hull supplementation. Table 9.3 Incidence of scouring and number of antibiotic treatments of pigs fed different diets after weaning.

DietA

RAP RAPOH WAP WAPOH

No. of pigs injected 4 2 0 1

Incidence of scouring 9 2 0 1

No. of antibiotic treatmentsB 21 6 0 3 ARefer text for details of diets. BIntramuscular injection of Trisoprim-480 (refer text for details).

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0

1

2

3

4

5

6

7

8

Day 0 Day 2 Day 5 Day 6

Day after weaning

No.

of p

igs

shed

ding

hae

mol

ytic

E. c

oli

RAP RAPOH WAP WAPOH

0.5

1

1.5

2

2.5

Day 0 Day 2 Day 5 Day 6

Day after weaning

Mea

n he

amol

ytic

E. c

oli s

core

RAP RAPOH WAP WAPOH

Figure 9.1 The number of pigs shedding haemolytic E. coli (left) and mean E. coli score of pigs fed different diets after weaning (right). Mean E. coli score calculated only from the pigs shedding E. coli on the measurement day

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Performance indices are summarised in Table 9.4. Daily gain (P<0.05) and FCR (P<0.01) were affected by the cereal source, while feed intake remained unchanged. Pigs fed wheat diets grew faster than pigs fed extruded rice-based diets. Supplementation of 20 g kg-1 oat hulls did not affect the performance of pigs. There were significant replicate effects for daily gain and intake (P<0.001) but not FCR because of the unavoidable difference in average starting weights between the two replicates (mean live weight of 5.5 ± 0.05 kg for replicate 1 and 4.9 ± 0.08 kg for replicate 2). The significant effect of replicate is not shown in the table because interactions between replicates and independent variables were not significant. Effects of cereal source and oat hull supplementation on the CTTAD are presented in Table 9.5. Cereal source significantly influenced the CTTAD of DM, starch, GE and the DE content of the total diet (P<0.001). Generally, the extruded rice-based diets were more digestible than the wheat-based diets. Supplementation with 20 g oat hulls kg-1 reduced the CTTAD of DM and GE (P<0.001). However, the CTTAD of starch was not affected by oat hull supplementation. The interaction between cereal source and oat hull supplementation for all measurements was not significant. Faecal moisture contents were higher in the wheat diets than in the extruded rice diets (P<0.01), and were not influenced by oat hull supplementation. Table 9.6 shows the difference in CTTAD when measured one or two week(s) after weaning. The CTTAD of DM (P=0.108) and energy (P=0.084) but not starch tended to increase as the pigs aged. The DE content of diet (P<0.05) was significantly improved as the pigs aged. The effects of cereal source and oat hull supplementation on blood metabolites and formation of biogenic amines are presented in Table 9.7. Plasma urea content (mmol L-1) tended to be higher in the extruded rice-based diets than in the wheat-based diets (P=0.064). There was no significant main effect (P=0.160) of oat hulls in lowering plasma urea levels, however a significant cereal source by oat hull interaction was observed, such that supplementation of oat hulls significantly reduced plasma urea concentration in the extruded rice-based diet (P<0.01) while no significant difference was observed in the wheat-based diet. Plasma creatinine content (mmol L-1) was significantly higher in the extruded rice-based diets than in the wheat-based diets (P<0.01). However, oat hull supplementation did not alter the plasma creatinine concentration. Plasma creatinine concentration was positively associated with mean haemolytic E. coli scores (Figure 9.2, P<0.015). There were significant increases in faecal contents of putrescine, cadaverine and histamine (P<0.001) in the wheat-based diets compared to the extruded rice-based diets. Also, the content of β-phenylethylamine tended to be higher in the wheat-based diets (P=0.066). Adding oat hulls tended to decrease the contents of β-phenylethylamine (P=0.066) and cadaverine (P=0.104) but not the contents of putrescine and histamine. There was a tendency for an interaction between cereal source and oat hull supplementation for the content of β-phenylethylamine (P=0.077), such that oat hull supplementation significantly decreased β-phenylethylamine content in the wheat-based diet (P<0.05) but did not alter β-phenylethylamine content in the extruded rice diet. Overall, total amine content was significantly higher in wheat-based diets (P<0.001), and oat hull supplementation tended to decrease total amine production in the large intestine (P=0.103).

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Table 9.4 Effect of cereal source and oat hull supplementation on performance indicesA of piglets for three weeks after weaning.

Cereal Extruded rice Wheat Probability, P=B

Oat hulls No +20 g oat hulls kg-1 No +20 g oat hulls kg-1 Pooled mean SEM Cereal source Oat hull Interaction

No. of pigs 24 24 24 24

Start wt. (kg) 5.20 5.21 5.06 5.17 5.16 0.08 NS NS NS

Finish wt. (kg) 9.88 9.81 10.77 10.99 10.36 0.30 * NS NS

Gain (g day-1) 223 219 271 277 248 11.6 * NS NS

Intake (g day-1) 443 437 423 440 436 13.0 NS NS NS

FCR (g g-1) 2.05 2.01 1.58 1.60 1.81 0.06 ** NS NS AValues are LS means from 24 observations. BRepeated measures ANOVA; NS: non-significant, *P>0.05; **P<0.01.

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Table 9.5 Effects of cereal source and oat hull supplementation on the CTTAD measured at one and two weeks after weaningA

Cereal Extruded rice Wheat Probability, P=B

Oat hull No +20 g oat hulls kg-1 No +20 g oat hulls kg-1 Pooled mean SEM Cereal source Oat hull Interaction

CTTAD of

DM 0.90a 0.87b 0.86c 0.84d 0.89 0.004 *** *** NS

Starch 0.999a 0.999a 0.994b 0.995b 0.997 0.004 *** NS NS

Gross energy 0.89a 0.87b 0.84c 0.83d 0.86 0.004 *** *** NS

DE (MJ/kg DM) 17.0a 16.5b 16.3b 16.0c 16.4 0.07 *** *** NS

Faecal moisture content (g per 100g)

64a 66ab 68ab 71b 67 0.8 ** NS NS

AValues are least-squares means from 24 observations (CTTAD was measured at week one and two from 12 pens). abcValues in row without common superscripts are significantly different at 5% significance level. BRepeated measures ANOVA; NS: non-significant, **P<0.01; ***P<0.001.

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Table 9.6 Effect of collection time after weaning on the CTTADA of selected dietary components.

Week after weaning Treatment

1 2 Pooled mean SEM

Probability, P=B

CTTAD of

DM 0.86 0.87 0.87 0.004 0.108

Starch 0.996 0.997 0.997 0.004 NS

Gross energy 0.85 0.86 0.86 0.004 0.084

DE (MJ/kg DM) 16.3 16.6 16.4 0.07 *

Faecal moisture content (g g-1) 67 68 67 0.8 NS

AValues are LS means from 12 observations. BANOVA; NS: non-significant, *P>0.05; ***P<0.001.

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70

80

90

0 1 2 3 4Total haemolytic E. coli score

Mea

n pl

asm

a cr

eatin

ine

( μmol

/L)

Figure 9.2 Relationship between plasma creatinine concentration and rectal haemolytic E. coli score of pigs during 14 days post-weaning (P<0.015).

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Table 9.7 Main effects of cereal source and oat hull supplementation on blood metabolitesA and formation of biogenic aminesB in weanling pigs.

Cereal Extruded rice Wheat Probability, P=C

Oat hull No +20 g oat hulls kg-1 No +20 g oat hulls kg-1 Pooled mean SEM Cereal source Oat hull Interaction

Blood metabolites

Urea (mmol L-1) 3.5a 2.6b 2.5b 2.7b 2.8 0.10 0.064 0.160 *

Creatinine (�mol L-1) 74.1ab 76.4a 69.2b 68.4b 72.1 1.00 ** NS NS

Biogenic amines (mg kg-1)

β-Phenylethylamine 28a 28a 41b 28a 31 1.93 0.066 0.066 0.077

Putrescine 43a 49a 177b 153b 106 11.19 *** NS NS

Cadaverine 353a 211a 868b 730b 504 59.54 *** 0.104 NS

Histamine 7a 6a 64b 63b 35 4.70 *** NS NS

Total amine 431a 292a 1150b 974b 712 72.42 *** 0.103 NS AValues are least-squares means means from 12 observations. abcValues in row without common superscripts are significantly different at 5% significance level. BValues are least-squares means from 24 observations (biogenic amines were measured at week one and two from 6 pens). CRepeated measures ANOVA; NS: non-significant, P>0.05; **P<0.01; ***P<0.001.

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9.5 Discussion 9.5.1 Incidence of diarrhoea and antibiotic treatment after weaning As was observed in the previous trial (Chapter 8), pigs fed an extruded rice diet with animal protein supplements (diet RAP) showed a higher incidence of diarrhoea after weaning. These two experiments are seemingly in contrast to previous reports from this laboratory showing that cooked white rice is associated with reductions in PWC. Previous experiments at this laboratory have always used autoclaving as the method for cooking rice. For example, Pluske et al. (2003) and Hopwood et al. (2004) autoclaved the rice and then cooled it at 4 ºC overnight before feeding it to pigs. It is well documented that cooking and cooling of rice can significantly increase the RS content by promoting retrogradation of starch polymers (Marsono and Topping, 1993). Studies conducted as part of this research project (Chapter 5) have shown that autoclave cooking and then cooling in a refrigerator increases the RS content by up to 12 times in the medium-grain (Amaroo) rice. Extrusion of the medium- and long-grain rice, in contrast, showed decreases in the RS content, due most likely to the solubilisation of starch by degradation of its macromolecular structure (Parchure and Kulkarni, 1997). Therefore, it is possible that the (readily fermentable) RS formed during the (autoclave) cooking and cooling of rice somehow prevented adhesion of haemolytic E. coli and (or) manipulated the hindgut microbiota to reduce coliform shedding in the faeces, as was observed by, for example, Pluske et al. (2003) and Hopwood et al. (2004). Reid and Hillman (1999) reported that feeding retrograded waxy maize to weaner pigs increased the Lactobacilli:coliform ratio in the large intestine, which the authors’ claim is beneficial to gut “health” and reduces infection by intestinal pathogens. These studies used diets containing very low amounts of RS and NSP, which may have limited the proliferation of health-promoting saccharolytic microbes and showed that fermentable RS could depress expression of pathogens (Pluske et al., 2003, Hopwood et al., 2004) and potentially harmful proteolytic microbes such as Bacteroides spp (Reid and Hilman, 1999). In this current study, it was evident that dietary supplementation with 20 g kg-1 insoluble/slowly digestible NSP as oat hulls to a low-fibre diet containing highly digestible ingredients, such as diet RAP, positively influenced piglet health after weaning because there was less diarrhoea and less antibiotic treatments. Martin et al. (2003) similarly reported that addition of 20 g oat hulls kg-1 in a rice-based diet reduced the incidence of PWD between 21 and 41 days of age. Surprisingly, the wheat-based diets used in this study were protective against PWD and pigs remained healthy throughout the study. We have no obvious explanation for this finding because it contrasts to previous work in our laboratory. However, it is well recognised that PWD is a very complex disease and its aetiology still remains relatively misunderstood. It is possible that the overall level of infection in this particular batch of pigs was generally low (as evidenced by the lack of disease in replicate 1) and (or) the level of immunity was high, and feeding diet RAP was sufficient to cause mild diarrhoea. The NSP in the wheat, under these infection conditions, might have been of just the correct quantity/fermentability to limit shedding of haemolytic E. coli. Nevertheless, when feeding extruded rice-based, low-fibre diets to weaner pigs, the addition of an appropriate insoluble / slowly digestible fibre source is recommended. Further work is required in this area. 9.5.2 Digestibility and performance The RAP diet showed significantly higher total-tract digestibility of all measured dietary components than the WAP diet. However, pigs fed diet WAP grew faster and had a better feed efficiency (FCR) than their counterparts fed diet RAP. Several reports showed higher digestibility of dietary components and better performance indices in pigs fed a cooked rice-based diet compared to corn-, wheat- and sorghum-base diets (Bonet et al., 2003; Lopez et al., 2003; Martin et al., 2003; Solà-Oriol et al., 2004; Vicente et al., 2004). In the current study, we suspect that the inferior performance shown in extruded rice-fed piglets was attributable to the use of a lower DE value and a higher CP value in the formulation of the experimental diets despite the higher CTTAD of dietary components observed.

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The DE value determined with weaner pigs using the same extruded rice samples (Chapter 10) showed a higher value than the tabulated DE value that was used in the formulation, and hence the dietary DE content of diet RAP was higher than the WAP diet (see Table 9.4). Also, chemical analysis showed that the extruded rice had only 64 g CP kg-1 (DM), which is a much lower value compared to raw rice (73 g CP kg-1). Therefore, an unbalanced energy: protein ratio was the most likely cause of the disparity between CTTAD and performance figures. Supplementation of 20 g oat hulls kg-1 did not influence performance of piglets but significantly decreased CTTAD of DM, GE and dietary DE content, which is in agreement with Lopez et al. (2003) and was most likely due to the dilution effects of the insoluble fibre. Increasing age after weaning tends to increase the CTTAD of dietary components, as other studies have reported age-related developments in the small intestinal enzyme systems and enhanced colonisation of the microbiota in the large intestine (eg, Martin et al., 2003; Kim et al., 2005a). 9.5.3 Blood metabolites and biogenic amines Urea is synthesised in the liver through the catabolism of amino acids when either (1) excess dietary amino acids enter into the portal blood system, (2) the excess amino acids are not used for gluconeogenesis, or (3) dietary amino acids are not ideally balanced (Eggum, 1970). Plasma urea concentration can also be influenced when microbial fermentation of nitrogenous compounds are increased in the large intestine, either by increased undigested or endogenous nitrogen entering the large intestine and (or) by overwhelming proliferation of proteolytic bacteria over saccharolytic microbes (Younes et al., 1996, 1998). Catabolism of amino acids by microbes produces NH3 that crosses the colonic epithelium and diffuses into the portal blood system, where it is converted to urea in the liver. The urea synthesised in the liver is either excreted as urine or diffuses back into the caecum and is incorporated into microbial N (Younes et al., 1996, 1998). Therefore, the higher plasma urea content in pigs fed diet RAP compared to pigs fed diet WAP could be a reflection of either inefficient utilisation of dietary protein or increased protein fermentation due to the proliferation of proteolytic microbes in the pigs fed diet RAP. However, the latter is the most likely explanation because supplementation of oat hulls decreased the plasma urea level in the low-fibre RAP diet, possibly through the modification of the large intestinal microbiota. These data concur with the findings of Bolduan et al. (1998), who reported a linear decline in plasma urea content with increasing crude fibre levels in diets of weaner pigs. Furthermore, pigs fed diet WAP with oat hulls did not show a modified plasma urea concentration, presumably because there was already sufficient NSP from wheat to maintain saccharolytic bacterial activity in the large intestine. Creatine is synthesised from arginine, glycine and methionine, and is used as a high-energy phosphate reserve in muscle. Degradation from creatine and creatine phosphate in muscle is the main route of the creatinine fluxed into the portal blood system, although some originates from diets. Therefore a determinant of plasma creatinine levels is muscle mass (about 0.3 to 0.5% of muscle weight; Braun et al., 2003). However, when animals are dehydrated such as can occur with diarrhoea after weaning, plasma creatinine levels can be increased due either to simple dehydration of plasma contents or to increased mobilisation of protein reservoirs from muscle (eg, the viscera) to compensate for decreased nutrient intake and (or) absorption (Wannemacher, 1977; Segales et al., 1998). The association between plasma creatinine levels and shedding of haemolytic E. coli in the post-weaning period found in our study (Figure 9.2) adds testament to this theory, albeit that no assessment of total body water content was made in the work. Biogenic amines produced by the decarboxylation of amino acids in the large intestine have been considered by some authors to increase the incidence of PWD, particularly in pigs fed diets containing low DF and high CP levels (Aumaitre et al., 1995; Bolduan et al., 1988; Pluske et al., 2002). The biogenic amines are thought to produce proteolytic and osmotic diarrhoea due to their ability to irritate the colonic mucosa and to its high osmotic pressure (Nollet et al., 1999). However, our finding that the levels of biogenic amines were markedly lower in the RAP diet suggests that (a) the amount/composition of the protein entering the hindgut was different to that in the wheat-based diet

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and caused lower production of amines, and (or) (b) that the diarrhoea seen in diet RAP was unrelated to amine production. Hence it appears that the type and concentration of amines produced in the large intestine depends on the type of cereal that pigs received and the amino acid flow into the hindgut. Further, the significant interaction between oat hull supplementation and different responses of the individual amines (eg, decreased β-phenylethylamine and cadaverine but not putrescine and histamine) suggests that oat hulls depressed the microbiota metabolising phenylalanine and lysine but not the microbiota metabolising arginine and histidine. Higher biogenic amines in wheat-based diets could be the result of (a) increased endogenous secretions in the WAP diets due to its higher NSP content (Low, 1989), (b) increased entry of dietary sources of N (amino acids) into the large intestine because the higher NSP content in the wheat decreases N digestibility at the terminal ileum (Bedford et al., 1992; Baidoo et al., 1998) and (or) reduces digesta retention time in the small intestine (Freire et al., 2000), and (or) (c) increased decarboxylation of N rather than catabolism of amino acids in WAP diet due to the favourable environment for microbiota such as E. coli, Proteus and Clostridia (Nollet et al., 1999). Although it was not statistically significant, supplementation of oat hulls tended to decrease amine production, which is in general agreement with reports by Aumaitre et al. (1995) and Bolduan et al. (1988). This finding implies that supplemental oat hulls may have modified the activity of proteolytic microbes in the large intestine. In conclusion, and as indicated in the digestibility indices, extruded rice is an excellent replacement for the traditional cereals such as wheat fed to pigs. The next study was conducted with the aim of refining the nutritive value of extruded rice for the Australian pig industry, Furthermore, it appears that under certain (and not yet properly defined) conditions, feeding a highly-digestible, extruded rice-based diet with animal protein in the absence of a source (or sources) of insoluble/slightly fermentable DF could predispose weanling pigs to an increased risk of infection by intestinal pathogens such as E. coli.

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10. The nutritive value of extruded rice and cooked (autoclaved) rice for weaner and grower pigs

10.1 Summary An experiment was conducted to examine the digestible energy (DE) and calculated net energy (NE) contents of two varieties of extruded rice (medium-grain variety Amaroo and long-grain variety Doongara) in pigs of two body weight groups (8 and 55 kg). Diets contained 857 g rice kg-1, 50 g meat and bone meal kg-1, 82 g fish meal kg-1 and other trace ingredients. Diets were fed at 5% of body weight for 8 kg pigs and at 3.75% of body weight for 58 kg pigs. Digestibility of GE was determined using an indigestible marker (TiO2) and the DE content of rice was calculated by subtracting the DE content of ingredients other than rice. The mean (± SEM) CTTAD of gross energy and DE content (MJ/kg as-fed) of rice were 0.917 (0.003) and 15.26 (0.08), respectively. Variety significantly influenced the DE content of rice such that the medium-grain rice had a higher DE content than the long-grain rice (0.3 MJ difference, P<0.001). Body weight of the pig also significantly influenced the DE content of rice such that weaner pigs extracted less energy from rice than grower pigs (up to 0.5 MJ, P<0.001). In a separate trial, the CTTAD of GE and DE content (MJ/kg) of cooked medium-grain rice Amaroo (autoclaved for 20 min at 120 °C; rice:water ratio of 1:2 w/w) were examined with 10-kg pigs. The mean values (± SEM) were 0.918 (0.001) and 15.1 (0.031), respectively. Estimation of the net energy (NE) content of rice using CVB (Dutch) and INRA (French) prediction equations showed a mean NE content for both rice types of 11.5 MJ/kg (air-dry basis). The DE and NE values found for rice are higher than for other cereals used in weaner pig diets such as wheat. 10.2 Introduction Approximately 600 million tonnes of rice are produced annually in the world (FAO, 2003), with the overwhelming majority of this entering the human food market. Rice is characterised by its high starch content, low NSP content and lower CP content in comparison to other cereals (Juliano, 1992). Several recent studies in pigs, however, have demonstrated that cooked white rice shows excellent potential for inclusion in diets as a replacement for more traditional cereals such as maize and sorghum. For example, Alcantara et al. (1989), Mateos et al. (2001, 2002), Solà-Oriol et al. (2004) and Vicente et al. (2004) all reported positive effects on growth when rice was included in diets for pigs. Furthermore, the use of cooked rice has been associated with reductions in post-weaning colibacillosis and swine dysentery (Pluske et al., 2002, 2003; Hopwood et al., 2004). Aside from the NRC (1998) estimate of energy value for rice of 14.9 MJ DE/kg, further published data pertaining to the energy content of cooked rice for pigs is scarce. This information is necessary for nutritionists to more accurately formulate diets. The purpose of this study was to examine the DE content of two types of extruded rice (lower or higher amylose:amylopectin ratio) in weaner pigs and grower pigs. It is recognised though that body weight (Bell and Keith, 1989; Kim et al., 2005a) and starch structure influence starch digestibility in pigs (Black, 2001; Lindberg et al., 2003; Kim et al., 2005b), so in this experiment the hypotheses tested were that (i) rice with a lower amylose:amylopectin ratio will have a higher DE content compared to a variety with a higher amylose:amylopectin ratio, and (ii) heavier pigs will extract more energy than lighter pigs from rice.

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10.3 Materials and Methods 10.3.1 Animals and experimental design Thirty-two male pigs (Large White x Landrace, 16 pigs per each body weight groups) were used in this experiment. The experiment was designed as a 2 x 2 factorial arrangement of treatments with the respective factors being (a) two extruded rice varieties (medium-grain, lower amylose:amylopectin Amaroo vs. long-grain, higher amylose:amylopectin Doongara) and (b) two body weight groups (weaner and grower). The rice was extruded at described previously (Chapter 8.3.2). The average body weights for each group were mean (± SEM) of 7.9 (± 0.16) kg and 55.4 (±3.10) kg, respectively. Pigs in each body weight group were weighed and sub-divided randomly into two groups of eight pigs according to their body weight. The Murdoch University Animal Ethics Committee and the Animal Ethics and Experimentation Committee of the WA Department of Agriculture approved this experiment. 10.3.2 Housing, diet preparation, feeding and sample collection The weaner pigs were kept in individual wire-mesh floored metabolism crates in a room where the temperature was maintained as constant as possible (27 ± 1°C). The grower and finisher pigs were kept in individual concrete floor pens in a conventional room without heating. Water was freely accessible during the whole experiment through a nipple drinker set in each crate and pen. From the time of arrival, the pigs were offered their respective experimental diet at a rate of 5% of body weight for weaner pigs and 3.75% of body weight for grower pigs, for a period of 10 days. The diet was offered in mash form, and the diet composition is presented in Table 10.1. Titanium dioxide (TiO2) was added as an inert marker for CTTAD estimation. Pigs were adapted to experimental diets for 7 days and the faecal samples were collected from the wire-mesh floor for weaner pigs and from the concrete pan for grower pigs at 0800, 1000, 1200, 1400, 1600 h for the final 3 days. Samples collected over the three-day period were pooled, kept at –20 ºC and later thawed, mixed, freeze-dried and ground through a laboratory hammer mill (1 mm screen) prior to chemical analysis. An additional experiment was conducted to determine the DE content of cooked rice (medium-grain variety Amaroo) with 8 pigs weighing 10.3 kg (± 0.16). Rice was autoclaved for 20 min at 120 ºC with a rice:water ratio of 1:2 (w/w) and kept at 4 ºC overnight. The cooked rice was then mixed with a mixture of other ingredients (see Table 10.1) before feeding. The experimental conditions were the same with the weaner pigs in the first trial. 10.3.3 Chemical analyses The dry matter (DM), nitrogen (N), gross energy (GE), total starch, amylose and amylopectin contents of extruded rice were determined as described previously (Chapter 4.2.1). The RS contents of extruded rice were determined using a Megazyme Resistant Starch kit (Megazyme International Ireland, Ltd., Wicklow, Ireland). Crude protein (CP) content was calculated as N x 6.25. The DM, GE and TiO2 content of diets and faecal samples were determined for estimation of apparent digestibilities of GE and the DE content of the experimental diets. The GE content of the rice, diet, and faecal samples was determined using a Ballistic Bomb Calorimeter (SANYO Gallenkamp, Loughborough, UK). The TiO2 contents of diet and feacal samples were determined using the method described by Short et al. (1996). The DE content of rice was calculated by subtracting the DE content of ingredients other than rice from the DE content of the complete diet and expressed on a DM basis.

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Table 10.1 The composition of the experimental diets (g kg-1). Ingredient Amount (g kg-1) Test riceA 857 Meat and bone meal 50.5 Fish meal 82.3 Canola oil 1 Lysine 3.7 Threonine 1.8 Tryptophan 0.5 Choline 0.4 Salt 1 Vitamin/Mineral mixB 0.7 Titanium dioxideC 1 Calculated analysis: DE (MJ/kg) 15.29 Available Lysine (g/MJ DE) 0.60 Crude protein (g/kg) 144 Crude fat (g/kg) 20.5 Neutral Detergent Fibre (NDF, g/kg) 10.9 Available phosphorus (g/kg) 4.5 Calcium (g/kg) 8.8 AEither extruded Amaroo or Doongara. BProvided the following nutrients (per kg of air-dry diet):Vitamins: A 1500 IU, D3 300 IU, E 37.5 mg, K 2.5 mg, B1 1.5 mg, B2 6.25 mg, B6 3 mg, B12 37.5 μg, Calcium pantothenate 25 mg, Folic acid 0.5 mg, Niacin 30 mg, Biotin 75 μg; Minerals: Co 0.5 mg (as cobalt sulphate), Cu 25 mg (as copper sulphate), Iodine 1.25 mg (as potassium iodine), Iron 150 mg (as Ferrous sulphate), Mn 100 mg (as Manganous oxide), Se 0.5 mg (as Sodium Selenite), Zn 0.25 mg (as zinc oxide). (Hogro Bronze Weaner and Grower, Rhone-Poulenc Animal Nutrition Pty Ltd., Queensland, Australia). CTitanium dioxide (TiO2; Sigma Chemical Company, St. Louis, MO, USA). 10.3.4 Statistical analyses The pig was considered as the experimental unit. For CTTAD of GE and DE content of rice, the treatment effects were assessed by two-way ANOVA for a factorial design with the main effects being rice variety and body weight. The effects were considered as fixed effects in the model. Fisher’s-protected least significant difference test were used (at 5% significance level) for comparison of the CTTAD of GE and DE content between mean values of different variables. All statistical analyses were conducted using the statistical package StatView 5.0 for Windows (AddSoft Pty. Ltd., Woodend, Vic., Australia). 10.4 Results Pigs generally adapted well to the experimental diets by the end of the adaptation period. One pig from the weaner group was removed from the trial due to persistent low feed intake. The chemical composition of the two extruded rice varieties is presented in Table 10.2. Although total starch contents of the two rice varieties were the same, Doongara had a higher amylose, CP and lower crude fat and crude fibre content than the variety Amaroo. The RS contents of extruded Amaroo and Dongara were similar and lower than the RS contents in raw rice (0.1 and 0.4 g/100g, respectively).

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Table 10.2 Chemical composition of the extruded rice (g kg-1). Composition, g kg-1 (DM) Medium grain riceA Long grain riceA DM 859 863 Total starch 879 880 Amylose 193 253 Amylopectin 686

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Amylose:amylopectin 0.28 0.40 Resistant starch 1.3 1.6 Crude protein 64.0 76.0 Crude fatB 32.0 28.5 Crude fibre 5.0 3.8 Gross energy (MJ/kg DM) 18.7 18.9 AVariety: Amaroo and Doongara. BCanola oil was sprayed in the process of extrusion; the crude fat contents of corresponding raw rice were 10.9 g and 10.8 g/kg DM, respectively. The energy value of added fat was adjusted when estimating rice DE. The effects of variety and body weight of pigs on the DE content of extruded rice are presented in Table 10.3. The CTTAD of GE and the DE content of rice were 0.917 (range from 0.899 to 0.930) and 15.3 MJ/kg (range from 14.8 to 15.6), respectively. Variety influenced the DE content of rice (P<0.001) with it being higher in the lower amylose:amylopectin variety Amaroo than the higher amylose:amylopectin variety Doongara. Also, body weight of the pig significantly influenced the CTTAD of GE and the DE content of rice (P<0.001). Weaner pigs extracted less energy from a given rice than grower pigs. The interactions between variety and body weight of pigs were not significant for the CTTAD and DE. The DE content of (autoclaved) cooked Amaroo rice was within the range of extruded rice with a mean of 15.1 MJ/kg (Table 10.4). The net energy (NE) contents of the two extruded rice types, from the determined DE values and chemical compositions of the rices, was made using CVB (Dutch) and INRA (French) prediction equations. The results are presented in Table 10.5. The mean NE value of extruded Amaroo and Dongara was 11.5 MJ/kg as-fed.

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Table 10.3 Main effects of variety and body weight of pigs on the digestible energy (DE) content of extruded rice.

Treatment n1 Initial wt. (kg) Final wt. (kg) Growth (g/day) CTTAD of GE DE (MJ/kg as is) DE (MJ/kg DM)

Medium rice 16 0.92ab 15.4b 17.9b Rice variety2

Long rice 15 0.92ab 15.1a 17.5a

Weaner 15 7.9 8.7 80 0.90a 15.0a 17.4a Body weight

Grower 16 55.4 61.4 301 0.93b 15.5b 18.0b

Pooled mean 33.2 35.1 190 0.92 15.3 17.7

SEM 4.6 4.8 34 0.003 0.07 0.08

Statistics Probability, P =

Rice variety (V) 0.336 0.001 0.001

Body weight (BW) 0.001 0.001 0.001

V x BW 0.350 0.273 0.281 1One pig from weaner group was removed from the trial. 2Medium grain rice (lower amylose:amylopectin; variety Amaroo) and long grain rice (higher amylose:amylopectin; variety Doongara) Values within a column without common superscript are significantly different at P<0.05.

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Table 10.4 The DE content (MJ/kg) of cooked Amaroo rice in piglets.

Rice Cooked rice (Amaroo) SEM

n= 8

Initial wt. (kg) 10.3 0.16

Final wt. (kg) 12.6 0.17

Growth (g/day) 236 7.34

CTTAD of GE 0.92 0.001

DE (MJ/kg DM) 17.3 0.03

DE (MJ/kg as is) 15.1 0.03

10.5 Discussion The DE content of extruded rice reported in this paper (15.3 MJ/kg; range from 14.8 to 15.6) showed much higher values than the NRC value (14.9 MJ/kg). Eggum (1977) reported that the mean DE content of raw and cooked rice in the rat was 15.6 MJ/kg DM (range from 15.4 to 15.8 MJ/kg), which is also higher than the current NRC value for pigs. Extrusion most likely improves the energy digestibility and the DE content of rice, since the RS content significantly decreased with extrusion compared to polished raw rice (Parchure and Kulkarni, 1997; Faraj et al., 2004). However, the extrusion effect alone does not explain the higher DE value reported in this paper, because the energy contribution from the reduced RS by extrusion (0.3 g 100g-1) was less than 0.05 MJ/kg DM (MacDonald et al., 1995). Therefore, the current DE value of rice used for formulation of pig diet may be underestimated by as much as 0.4 MJ/kg. The lower amylose, medium-grain variety Amaroo had a 0.3 MJ/kg higher DE content than the higher amylose, long-grain variety Doongara. The amylose:amylopectin ratio is known to determine many physical and chemical properties of processed rice. Generally, increased amylose content is associated with the increased water holding capacity of the starch granule and increased capacity of retrogradation through increasing capacity of hydrogen bonding (Juliano, 1992). A higher amylose content in rice is known to inversely correlate to the in vitro starch digestion index (SDI; Tetens et al., 1997; Rashmi and Urooj, 2003) and to increase the RS content (Sagum and Arcot, 2000). Retrograded RS has been shown to decrease ileal starch digestibility and faecal fat and energy digestibility in pigs (De Schrijver et al, 1999). In the current study, the varietal difference in the DE content was higher in weaner pigs (0.4 MJ/kg) than in grower pigs (0.2 MJ/kg), suggesting that weaner pigs were less able to extract energy in response to differences in the chemical structure of starch, namely the higher amylose content and (or) the higher RS content. The lower DE content of the higher amylose rice used in the present study was most likely due to 1) the higher amount of undigested starch from RS reaching to the hindgut, and (or) 2) less fermentation capacity in the hindgut of weanling pigs. A similar observation was reported in a rat study where digestibility values were compared in five rice cultivars (raw and cooked) differing in amylose content (22–284 g/kg DM) (Eggum et al., 1993). In this particular study, rice with more amylose showed a lower energy digestibility coefficient compared to rice with less amylose when hindgut fermentation was suppressed by addition of the antibiotic Nabacitin. However, rats fed a non-antibiotic diet showed no differences in energy content. These data implicate the extent of hindgut

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fermentation by the microbiota in different aged pigs as a possible reason for differences between varieties in different aged pigs. Nevertheless, it is unlikely that the type of starch alone influenced the DE content of rice, because even retrograded RS is almost completely fermented by microbes in the large intestine of adult rats (CTTAD of rice starch >0.99 in rat, Eggum, 1977; Eggum et al., 1993). Instead the difference in starch, lipid and protein digestibility and in NSP content may be attributed to the overall slightly lower DE content in the higher amylose variety Doongara. The NSP content of rice was reported to be higher in a higher amylose rice variety. For example, variety Doongara (311 g amylose kg-1 DM) had 18 g NSP kg-1 DM, while the medium- and lower-amylose rice varieties Inga (202 g amylose kg-1 DM) and Japonica (115 g amylose kg-1 DM) had 16 g and 10 g NSP kg-1 DM, respectively (Sagum and Arcot, 2000). The DE content of the cooked (autoclaved) rice was similar to the value determined with extruded rice. Generally, autoclaving of rice is known to increase the RS content during cooling (Mangala et al., 1999), and cooked rice had a lower digestibility of energy and protein compared to raw rice in the rat (Eggum et al., 1993). However in the current study, the DE content of the cooked rice was within the range of the extruded rices. Body weight of pigs significantly influenced the DE content of extruded rice, which is in agreement with earlier studies investigating other grains such as wheat (Bell and Keith, 1989; Kim et al., 2004). The DE content determined with weaner pigs was significantly lower than that determined with grower pigs, which was most likely due to a less developed hindgut and hence a lower level of fermentation. However, the between-animal variation for the DE content (DM) was high in weaner pigs (standard deviation, SD, of 0.250) compared to grower pigs (SD 0.156). Since the weaner pigs were used only one week after weaning (aged approximately 35 ± 2 days), it is possible that variation in recovery rate from the damaged gut integrity may cause the variation in DE content (Pluske et al., 1997). The cooked rice experiment further supports this because pigs fed cooked rice (aged 49 ± 2 days) had a lower variation in the DE content (SD 0.09). An early report on the DE and NE values of rice by Robles and Ewan (1982) indicated figures of 13.9 MJ and 9.5 MJ/kg, respectively. Furthermore, the NRC (1998) has shown the NE value of rice to be 9.61 MJ/kg. However, the DE and NE values determined in our study showed higher values than in the abovementioned reports. The mean DE and NE values reported herein are in agreement with the values reported in the Dutch tables of composition and nutritional value of feed materials (Sauvant et al., 2004; 14.8 MJ DE and 11.8 MJ NE/kg) and in Valdivié (2004; 14.9 MJ ME/kg). The NE estimation using CVB (Dutch) and INRA (French) prediction equations showed that the INRA formulas gave a higher NE value than CVB formula (see Table 10.5). Similar results showing slightly higher NE values by using INRA formula compared to CVB formula for corn, wheat and barley were reported (Gowans, 2004). Nevertheless, both the CVB and INRA estimations of NE are widely accepted as valid equation as they correlated well, and values were 96% equivalent (Gowans, 2004).

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Table 10.5 Estimated net energy (NE) values of rice (MJ/kg as-fed).

Medium grain Long grain Remarks

NRC 9.61 - NRC 1998

CVBA 11.10 11.17 Sauvant et al., 2004

INRA meanB 11.72 11.83 Noblet et al., 2004

INRA-1C 11.74 11.83

INRA-2D 11.79 11.91 Using calculated DE

INRA-3E 11.63 11.76

INRA Weaner meanF 11.88 11.70 Using determined DE value

INRA-2 WeanerG 12.03 11.71

INRA-3 WeanerH 11.86 11.55

INRA Grower meanI 12.06 11.98 Using determined DE value

INRA-2 GrowerJ 12.31 12.14

INRA-3 GrowerK 12.14 11.99

Overall INRA meanL 11.89 11.84

Overall meanM 11.50 11.51 Formulas used to calculate NE are: ACVB= 0.108 x DCP + 0.361 x DEE + 0.137 x (Starch – fermentable starch) x Dstarch + 0.124 x Dsugars + 0.96 x (NSP + fermentable starch) x DNSP; 0.96 is the correction factor for disaccharides for rice. Digestible nutrient contents are expressed as g/100g as-fed and NE is expressed as MJ/kg as-fed. BMean of 3, 4, and 5. CINRA-1= 0.121 x DCP + 0.350 x DEE + 0.143 x starch + 0.119 x sugars + 0.086 x Dresidue; digestible residue= DOM – (DCP + DEE + starch + sugars). Digestible nutrient contents are expressed as g/100g DM and NE is expressed as MJ/kg DM. DINRA-2= 0.703 x DE + 0.066 x EE + 0.020 x starch – 0.041 x CP – 0.041 x CF. EINRA-3= 0.73 x ME + 0.005 x EE + 0.015 x starch – 0.028 x CP – 0.041 x CF; Crude nutrients are expressed as kg/kg DM and NE is expressed as MJ/kg DM. Abbreviations: D: digestible, CP: crude protein, EE: ether extract, NSP: non-starch polysaccharides, OM: organic matter, CF: crude fibre. Digestibility coefficients applied for CVB formula: OM: 0.94, CP: 0.67, EE: 0.34, CF: 0.96, NSP: 0.60, Starch: 1.0, sugars: 1.0. Digestibility coefficients applied for INRA formula: OM: 0.98, CP: 0.89, EE: 0.24, NSP: 1.0, Starch: 1.0, sugars: 1.0. FMean of 3, 7 and 8. GCalculated using INRA-2 formula and determined DE content with weaner pigs. HCalculated using INRA-3 formula and the ME value used in the formula was calculated from the determined DE content with weaner pigs. IMean of 3, 10 and 11. JCalculated using INRA-2 formula and determined DE content with grower pigs. KCalculated using INRA-3 formula and the ME value used in the formula was calculated from the determined DE content with grower pigs. LMean of 2, 6 and 9. MMean of 1 and 12. In conclusion, this study suggests that the DE and NE content of rice might be higher than originally thought, and this should be considered for practical diet formulation if rice is being used. The DE and NE values for rice are also higher than for other cereals used in weaner pig diets such as wheat. In addition, weaner pigs could extract approximately 0.5 MJ/kg less DE (as-fed) from a given rice

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compared to grower pigs. These data will be invaluable to nutritionists and feed formulators in the Australian pig industry who wish to incorporate rice into diets for piglets.

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11. Implications and Recommendations Results obtained in this research project have demonstrated that cooked (processed) white rice, either in medium-grain or long-grain form, included in diets for weanling pigs can be used as a replacement for wheat without a loss of production in the immediate post—weaning period. The decision to replace a cereal such as wheat in diets for weanling pigs, therefore, is likely to be one of price differential. Cooking broken white rice, particularly in medium-grain and waxy rice that have lower amylose levels than long-grain rice, increases starch digestibility when measured at the end of the small intestine and reduces shedding of haemolytic E. coli. This could be predicted with accuracy in vitro using a “fast digestible starch” assay modified for rice in our laboratory. Regardless of the type and variety of rice used, however, pigs fed cooked white rice partition more digested nutrients into carcass gain than pigs fed other cereals such as wheat and barley, although the type of proteins fed to pigs will also influence this. In this regard, the use of commercially manufactured extruded rice plus sources of animal protein (eg, milk powders, fishmeal, meat and bone meal) appear the best dietary combination for production purposes. Feeding vegetable (plant) proteins typically increased the weight of the gastrointestinal tract as a consequence of increased fermentative activity in the large intestine, and reduced bodyweight gain and FCR. Determination of the energy (DE and NE) values of extruded medium-grain (Amaroo) and long-grain (Doongara) rice confirmed the superior energy value of these two rice types over existing cereals used in Australian feeding of pigs, such as wheat. The effects of feeding cooked white rice on reducing faecal shedding of the bacterium (E. coli) responsible for causing PWD were generally unchanged, or even exacerbated, when the rice plus animal protein diets were fed compared to commercially-based diets that were considered a contributing factor to the incidence of PWD. The extent and duration of faecal shedding of enterotoxigenic E. coli found in the studies conducted was generally low, and this might have influenced the capacity of the rice-based diets to exhibit protective effects. It was hypothesised also that an imbalance in the amounts of carbohydrate versus protein entering the large intestine might have predisposed the pig to diarrhoea after weaning, due to a change in the types of microbiota and subsequent production of compounds implicated in non-infectious diarrhoea. The results of Chapter 9 advocate the inclusion of a quantity of slowly or moderately fermentable dietary fibre to extruded rice-based diets consisting of animal protein to ameliorate the diarrhoea that is sometimes observed when feeding this diet, although in this instance 20 g kg- oat hulls impacted upon digestibility and production after weaning. Nevertheless, this proposition is consistent with European experiences of feeding processed rice to piglets after weaning. In this respect, it is feasible that the addition of rice bran and (or) rice hulls, or possibly the use of brown rice, in diets for piglets after weaning could achieve similar results. An unfortunate casualty of the drought for this particular project, however, was the inability to perform an on-farm trial implementing some of the findings and conclusions arising from this research project. The major recommendations arising from this project are as follows:

1. Medium-grain rice (variety Amaroo) or long-grain rice (variety Doongara) was identified as being the most suitable rice cultivars for utilisation in piglet feeds in Australia. Waxy rice, but not parboiled rice, would also be suitable, but its lower production in Australia at present would increase its price relative to other rice types and other cereals and hence limit its usefulness.

2. Processed (extruded) medium-grain rice (variety Amaroo) or long-grain rice (variety Doongara) are a suitable replacement for cereals currently fed to weanling pigs in Australia such as wheat and barley. Adoption of processed rice by the pig industry will be predominately driven by the price differential between processed rice and these alternative cereals.

3. Starch digestion at the end of the small intestine, as well as the colon, can be predicted accurately with a “fast digestible starch” assay modified for use in our laboratory. This test could be used by the rice industry as part of a broader screening process for potentially new

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varieties of rice suitable for the pig industry, however the assay is capable of being tailored for use in other species, including man.

4. Sources of animal protein in diets containing processed (extruded) rice generally cause superior production after weaning compared to vegetable (plant) sources of protein, although vegetable proteins showed reduced faecal shedding of haemolytic E. coli compared to animal sources of protein.

5. Producers feeding extruded rice-based diets with animal protein sources are encouraged to include some slowly or moderately fermentable dietary fibre, such as oat hulls, wheat bran and (or) beet pulp, to ameliorate the diarrhoea that is sometimes observed when feeding this diet. Future studies should investigate the addition of rice bran and (or) rice hulls, or possibly the use of brown rice, in diets for piglets after weaning that could accomplish similar results.

6. The mean digestible energy (DE) content (MJ/kg as-fed) of extruded rice is 15.26 MJ/kg as-fed. Medium-grain (Amaroo) rice has a 0.4 MJ/kg higher DE content than the long-grain rice (Doongara).

7. Pig producers should use different DE values for pigs of different ages/weights. Weanling pigs (8 kg) pigs extracted less energy from both extruded rices than grower (55 kg) pigs (up to 0.5 MJ/kg difference). Producers using a net energy (NE) system should use a common value of 11.5 MJ/kg as-fed.

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12. References Alcantara, P.E., Cordova, E.D., Villeta, M.O. and Naldo, M.E. (1989). Substitution value of rice bran

(D1) and rough rice (Palay) for corn in growing finishing swine rations. Philippine Journal of Veterinary Animal Science 15, 1-22.

AOAC (1997). Official methods of Analysis, 16th ed. Association of Official Analytical Chemists, Washington, DC.

Argenzio, R.A. (1992). Pathophysiology of diarrhea. In Veterinary Gastroenterology, 2nd ed. (ed N. Anderson), pp. 163-172. Lea and Febiger, Philadelphia.

Aumaitre, A., Peiniau, J. and Madec, F. (1995). Digestive adaptation after weaning and nutritional consequences in the piglet. Pig News and Information 16, 73N-79N.

Bach Knudsen, K.E., Jensen, B.B., Andersen, J.O. and Hansen, I. (1991). Gastrointestinal implications in pigs of wheat and oat fractions. 2. Microbial activity in the gastrointestinal tract. British Journal of Nutrition 65, 233-248.

Baidoo, S.K., Liu, Y.G. and Yungblut, D. (1998). Effect of microbial enzyme supplementation on energy, amino acid digestibility and performance of pigs fed hulless barley based diets. Canadian Journal of Animal Science 78, 625-631.

Barton, M.D. (1999). The down-side of antibiotic use in pig production: The effect on antibiotic resistance of enteric bacteria. In Manipulating Pig Production VII (ed. P.D. Cranwell), pp. 194-199. Australasian Pig Science Association, Victoria.

Barton, M.D. (2000). Antibiotic use in animal feed and its impact on human health. Nutrition Research Reviews 13, 279-299.

Bedford, M.R., Patience, J.F., Classen, H.L. and Inborr, J. (1992). The effect of dietary enzyme supplementation of rye and barley-based diets on digestion and subsequent performance in weanling pigs. Canadian Journal of Animal Science 72, 97-105.

Bell, J.M. and Keith, M.O. (1989). Factors affecting the digestibility by pigs of energy and protein in wheat, barley, sorghum diets supplemented with canola meal. Animal Feed Science and Technology 24, 253-265.

Berggren, A.M., Björck, I.M.E., Margareta, E. and Nyman, G.L. (1995). Short-chain fatty acid content and pH in caecum of rats fed various sources of starch. Journal of the Science of Food and Agriculture 68, 241-248.

Bertschinger, H.U. (1999). Postweaning Escherichia coli diarrhoea and oedema disease. In Diseases of Swine, 8th ed. (eds. B.E. Straw, S. D'Allaire, W. L. Mengeling and D.J. Taylor), pp. 441-454. Iowa State University Press, Ames, Iowa.

Black, J.L. (2001). Variation in the nutritional value of cereal grains across livestock species. Proceedings of the Australasian Poultry Science Symposium 13, 22-29.

Bolduan, G. Jung, H., Schnable, E. and Schneider, R. (1988). Recent advances in the nutrition of weaner piglets. Pig News and Information 9, 381-385.

Bonet, J., Coma, M., Cortés, M., Medal, P. and Mateos, G.G. (2003). Rice vs wheat feeding and protein level of the diet on performance of piglets from 10 to 16 kg BW. Journal of Animal Science 81 (Suppl. 1), 47.

Braun, J.P., Lefebvre, H.P. and Watson, A.D.J. (2003). Creatinine in the dog: a review. Veterinary Clinical Pathology 32, 162-179.

Brunsgaard, G. (1998). Effects of cereal type and feed particle size on morphological characteristics, epithelial cell proliferation, and lectin binding patterns in the large intestine of pigs. Journal of Animal Science 76, 2787-2798.

Cherbut C., Albina E., Champ M., Coublier J.L. and Lecannu G. (1990). Action of guar gums on the viscosity of digestive contents and on the gastrointestinal motor function in pigs. Digestion 46, 205-213.

Cutler, R. (1992). The cost of disease on Australian farms. In Pig Production (186), University of Sydney Post-Graduate Committee in Veterinary Science, Proceedings 1992 (186): 47-54.

82

Cutler, R.S. (2001). A review – The prevalence and eradication of pig diseases in Australia. In Manipulating Pig Production VIII (ed. P.D. Cranwell), pp. 125-140. Australasian Pig Science Association, Werribee, Victoria.

CVB, (2004). Veevoedertabel 2004. Centraal Veeveoder Bureau, Lelystad, The Netherlands. De Schrijver, R., Vanhoof, K. and Vande Ginste J. (1999). Nutrient utilization in rats and pigs fed

enzyme resistant starch. Nutrition Research 19, 1349-1361. Devi, K. and Geervani, P. (2000). Rice processing- effect on dietary fibre components and in vitro

starch digestibility. Journal of Food Science and Technology 37, 315-318. Eerlingen, R.C., Deceuninck, M. and Delcour, J.A. (1993). Enzyme-resistant starch. II. Influence of

amylose chain length on resistant starch formation. Cereal Chemistry 70, 345-350. Eggum, B.O. (1970). Blood urea measurement as a technique for assessing protein quality. British

Journal of Nutrition 24, 983-988. Eggum, B.O. (1977). Effect of cooking on nutritional value of milled rice in rats. Nutrition Reports

International 16, 649-655. Eggum, B.O., Juliano, B.O., Perez, C.M. and Acedo, E.F.A. (1993). The resistant starch, undigestible

energy and undigestible protein contents of raw and cooked milled rice. Journal of Cereal Science 18, 159-170.

Ehrlein, H., and Stockmann, A. (1998). Intestinal absorption of nutrients is not influenced by soy fiber and does not differ between oligomeric and polymeric enteral diets. Digestive Diseases and Sciences 43, 2099-2110.

Englyst, H.N. and Cummings, J.H. (1987). Digestion of polysaccharides of potato in the small intestine of man. American Journal of Clinical Nutrition 45, 423-431.

Englyst, H.N., Kingman, S.M. and Cummings, J.H. (1992). Classification and measurement of nutritionally important starch fractions. European Journal of Clinical Nutrition 46 (Suppl 2), S33-S50.

FAO (2003). Summary of food and agricultural statistics 2003. Food and Agriculture Organization of the United Nations, Rome.

Faraj, A., Vasanthan, T. and Hoover, R. (2004). The effect of extrusion cooking on resistant starch formation in waxy and regular barley flours. Food Research International 37, 517-525.

Fitzgerald. M.A., Martin, M., Ward, R.M., Park, W.D. and Shead, H.J. (2003). Viscosity of rice flour: A rheological and biological study. Journal of Agricultural and Food Chemistry 51, 2295-2299.

Francis, D.H. (2002). Enterotoxigenic Escherichia coli infections in pigs and its diagnosis. Journal of Swine Health and Production 10, 171-175.

Freire, J.P.B., Guerreiro, A.J.G., Cunha, L.F. and Aumaitre, A. (2000). Effect of dietary fibre source on total tract digestibility, caecum volatile fatty acids and digestve transit time in the weaned piglet. Animal Feed Science and Technology 96, 456-478.

Garcia-Alonso, A., Saura-Calixto, F. and Delcour, J.A. (1998). Influence of botanical source and processing on formation of resistant starch type III. Cereal Chemistry 75, 802-804.

Gaskins, H.R. (2001). Intestinal bacteria and their influence on swine growth. In Swine Nutrition, 2nd edition (eds. A.J. Lewis, L.L. Southern), pp. 585-608. Florida, CRC Press LLC.

Gee, J.M., Faulks, R.M. and Johnsson, I.T. (1991). Physiological effects of retrograded alpha-amylase resistant corn starch in rats. Journal of Nutrition 121, 44-49.

Govers, M.J.A.P., Gannon, N.J., Dunshea, F.R., Gibson, P.R. and Muir, J.G. (1999). Wheat bran affects the site of fermentation of resistant starch and luminal indexes related to colon cancer risk: a study in pigs. Gut 45, 840-847.

Gowans, J. (2004). Adoption of the net energy system - the Canadian pea perspective. In Proceedings of the 25th Western Nutrition Conference, pp. 11-18. Saskatoon, SW, Canada.

Hampson, D.J. (1987). Dietary influences on porcine postweaning diarrhoea. In Manipulating Pig Production (eds. J.L. Barnett, E.S. Batterham, G.M. Cronin, C. Hansen, P.H. Hemsworth, P.E. Hughes, N.E. Johnston and R.H. King), pp. 202-214. Australasian Pig Science Association, Victoria.

Hampson, D.J. (1994). Postweaning Escherichia coli diarrhoea in pigs. In Escherichia coli in Domestic Animals and Humans (ed. C.L. Gyles), pp. 171-191. CAB International, Wallingford, UK.

83

Hampson, D.J., Pluske, J.R., and Pethick, D.W. (2001). Dietary manipulation of enteric disease. Pig News and Information 22, 21N-28N.

Hampson, D.J., Robertson I. D., La, T., Oxberry, S. L. and Pethick, D. W. (2000). Influences of diet and vaccination on colonisation of pigs with the intestinal spirochaete Brachyspira (Serpulina) pilosicoli. Veterinary Microbiology 73, 75-84.

Heijnen, M.L.-A. and Beynen, A.C. (1997). Consumption of retrograded (RS) but not uncooked (RS) resistant starch shifts nitrogen excretion from urine to feces in cannulated piglets. Journal of Nutrition 127, 1828-1832.

Holm, J., Björck, I., Ostrowska, S., Eliasson, A.-C., Asp, N.-G., Larsson, K. and Lindquist, I. (1983). Digestibility of amylose-lipid complexes in-vitro and in-vivo. Starch 35, 294-297.

Hopwood, D.E. and Hampson, D.J. (2003). Interactions between the intestinal microflora, diet and diarrhoea, and their influences on piglet health in the immediate post-weaning period. In Weaning the Pig: Concepts and Consequences (eds. J.R. Pluske, J. Le Dividich and M.W.A. Verstegen), pp. 199-218. Wageningen Academic Publishers, Wageningen.

Hopwood, D.E., Pethick, D.W. and Hampson, D.J. (2002). Increasing the viscosity of the intestinal contents stimulates proliferation of enterotoxigenic Escherichia coli and Brachyspira pilosicoli in weaner pigs. British Journal of Nutrition 88, 523-532.

Hopwood, D.E., Pethick, D.W., Pluske, J.R. and Hampson, D.J. (2004). Addition of pearl barley to a rice-based diet for newly weaned piglets increases the viscosity of the intestinal contents, reduces starch digestibility and exacerbates post-weaning colibacillosis. British Journal of Nutrition 92, 419-427.

Hu, P., Zhao, H., Duan, Z., Linlin, Z. and Wu, D. (2004). Starch digestibility and estimated glycemic score of different types of rice differing in amylose contents. Journal of Cereal Science 40, 231-237.

Iyngkaran, N. and Yadav, M. (1998). Rice-starch oral rehydration therapy in neonates and young infants. Journal of Tropical Pediatrics 44, 199-203.

Jensen, B.B., and Jorgensen, H. (1994). Effect of dietary fibre on microbial activity and microbial gas production in various regions of the gastrointestinal tract of pigs. Applied Environmental Microbiology 60, 1897-1904.

Juliano, B.O. (1992). Structure, chemistry and function of the rive grain and its fractions. Cereal Foods World 37, 772-779.

Kavita, V., Verghese, S., Chitra, G.R. and Prakash, J. (1998). Effects of processing, storage time and temperature on the resistant starch of foods. Journal of Food Science and Technology 35, 299-304.

Kelly, D., Greene, J., O’Brien, J.J., and McCracken, K.J. (1984). Gavage of feeding of early-weaned pigs to study the effect of diet on digestive development and changes in intestinal microflora. In Proceedings of the VIIIth International Pig Veterinary Society Congress, p. 317. Ghent, Belgium (abs.).

Kim, J. C., Mullan, B. P., Simmins, P. H. and Pluske, J. R. (2003). Variation in the chemical composition of wheats grown in Western Australia as influenced by variety, growing region, season and post-harvest storage. Australian Journal of Agricultural Research 54, 541-550.

Kim, J. C., Simmins, P. H., Mullan, B. P., Pluske, J. R. (2005a). The digestible energy value of wheat for pigs; with special reference to the post-weaned animal [review]. Animal Feed Science and Technology (In press).

Kim, J.C., Mullan, B.P. and Pluske, J.R. (2005b). A comparison of waxy vs non-waxy wheats in diets for weaner pigs: Effects of particle size, enzyme supplementation, and collection day on total tract apparent digestibility and pig performance. Animal Feed Science and Technology (In press).

Kyriazakis, I. and Emmans, G.C. (1995). The voluntary feed intake of pigs given feeds based on wheat bran, dried citrus pulp and grass meal, in relation to measurements of feed bulk. British Journal of Nutrition 73, 191-207.

Larsen, P.B. (2004). Consequences of termination of AGP use for pig health and usage of antimicrobials for therapy and prophylaxis. In Working Papers from the International

84

Symposium Beyond Antimicrobial Growth Promoters in Food Animal Production, pp. 67-71. Danish Institute of Agricultural Sciences, Foulum, Denmark.

Lecce, J.G. (1983). Dietary regimen, rotavirus, and hemolytic enteropathogenic Escherichia coli in weanling diarrhea of pigs. Annales Recherches Veterinaires 14, 463-8.

Li, D., Zhang, D.F., Piao, X.S., Han, I.K., Yang, C.J., Li, J.B. and Lee, J.H. (2002). Effects of replacing corn with Chinese brown rice on growth performance and apparent faecal digestibility of nutrients in weanling pigs. Asian-Australasian Journal of Animal Science 15, 1191-1197.

Li, J., Li, D., Yin, Y.-L., Piao, X.S., He, J.H., Chen, G.P. and Shu, J.C. (2004). Performance, nutrient digestibility and intestinal disaccharidase activity of weaner/grower pigs given diets containing extruded Chinese stored brown rice with exogenous enzyme supplements. Animal Science 79, 429-438.

Lindberg, J.E., Arvidsson, A. and Wang, J. (2003). Influence of naked barley cultivar with normal, amylose-rich or amylopectin-rich starch and enzyme supplementation on digestibility and piglet performance. Animal Feed Science and Technology 104, 121-131.

Lopez, E., Latorre, M.A., Valencia, D.G., Lazaro, R. and Mateos, G.G. (2003). Inclusion of oat hulls in diets for piglets based on native or cooked cereals. Journal of Animal Science 81 (Suppl. 1), 47.

Low, A.G. (1989). Secretory response of the pig gut to non-starch polysaccharides. Animal Feed Science and Technology 23, 55-65.

Madec, F., Bridoux, N., Bounaix, S. and Jestin, A. (1998). Measurement of digestive disorders in the piglet at weaning and related risk factors. Preventative Veterinary Medicine 35, 53-72.

Mangala, S.L. and Tharanathan, R.N. (1999). Structural studies of resistant starch derived from processed (autoclaved) rice. European Journal of Food Research and Technology 209, 38-42.

Mangala, S.L., Malleshi, N.G., Mahadevamma and Tharanathan, R.N. (1999b). Resistant starch from differently processed rice and ragi (finger millet). European Journal of Food Research and Technology 209, 32-37.

Mangala, S.L., Udayasankar, K. and Tharanathan, R.N. (1999a). Resistant starch from processed cereals: the influence of amylopectin and non-carbohydrate constituents in its formation. Food Chemistry 64, 391-396.

Marsono, Y. and Topping, D.L. (1993). Complex carbohydrates in Australian rice products- Influence of microwave cooking and food processing. Lebensm.-Wiss. u.-Techno. 26, 364-370.

Martin, F., Lotorre, M.A., Gonzalez-Alvarado, J.M., Lazaro, R. and Mateos, G.G. (2003). Oat hulls in diets for young pigs based on cooked rice or corn without antibiotics. Journal of Animal Science 81 (Suppl. 1), 47.

Mateos, G.G., Alcantarilla, A., Latorre, M.A., Lázaro, R., Gómez, E. and Laso, N. (2001). Influence of type of cereal and level of fiber on performance of early-weaned pigs. Journal of Animal Science 79 (Suppl. 1), 106-107.

Mateos, G.G., Latorre, M.A., Martín, F., Gracia, M.I. and Lázaro, R. (2002). Influence of type of processed cereal and oat hulls inclusion in the diet on nutrient digestibility and productive performance of young pigs. Journal of Animal Science 80 (Suppl. 1), 199.

Mathews, C.J., MacLeod, R.J., Zheng, S.X., Hanrahan, J.W., Bennett, H.P. and Hamilton, J.R. (1999). Characterization of the inhibitory effect of boiled rice on intestinal chloride secretion in guinea pig crypt cells. Gastroenterology 116, 1342-1347.

McDonald D.E., Pethick D.W., Mullan B.P., and Hampson D.J. (2001). Increasing viscosity of the intestinal contents alters small intestinal structure and intestinal growth, and stimulates proliferation of enterotoxigenic Escherichia coli in newly-weaned pigs. British Journal of Nutrition 86, 487-498.

McDonald, D.E., Pethick, D.W., Pluske, J.R. and Hampson, D.J. (1999). Adverse effects of soluble nonstarch polysaccharide (guar gum) on piglet growth and colibacillosis immediately after weaning. Research in Veterinary Science 67, 245-250.

Mitsuda H. (1993). Retrogradation of cooked rice. Journal of Food Quality 16, 321-325. Montagne, L., Cavaney, F.S., Hampson, D.J., Lallès, J.-P. and Pluske, J.R. (2004). Effect of diet

composition on postweaning colibacillosis in piglets. Journal of Animal Science 82, 2364-2374.

85

Mosenthin R., Hambrecht E., and Sauer W.C. (2001). Utilisation of different fibres in piglet feeds. In Recent Developments in Pig Nutrition 3 (eds. J. Wiseman and M.A Varley), pp. 293-322. Nottingham University Press, Nottingham, UK.

Muir, J.G. and O’Dea, K. (1993). Validation of an in vitro assay for predicting the amount of starch that escapes digestion in the small intestine of humans. American Journal of Clinical Nutrition 57, 540-546.

Nollet, H., Deprez, P., Van Driessche, E. and Muylle, E. (1999). Protection of just weaned pigs against infection with F18+ Escherichia coli by non-immune plasma powder. Veterinary Microbiology 65, 37-45.

NRC (1998). Nutrient requirements of swine (10th ed). National Academy Press, Washington, DC. Parchure, A.A. and Kulkarni, P.R. (1997). Effect of food processing treatments on generation of

resistant starch. International Journal of Food Science and Nutrition 48, 257-260. Piao, X.S., Li, D., Han, I.K., Chen, Y., Lee, J.H., Wang, D.Y., Li, J.B. and Zhang, D.F. (2002).

Evaluation of Chinese brown rice as an alternative energy source in pig diets. Asian-Australasian Journal of Animal Science 15, 89-93.

Piva, A., Panciroli, A., Meola., E. and Formigoni, A. (1995). Lactitol enhances short-chain fatty acid and gas production by swine cecal microflora to a great extent when fermenting low rather than high fibre diets. Journal of Nutrition 126, 280-289.

Pluske, J.R., Black, B., Pethick, D.W., Mullan, B.P. and Hampson, D.J. (2003). Effects of different sources and levels of dietary fibre in diets on performance, digesta characteristics and antibiotic treatment of pigs after weaning. Animal Feed Science and Technology 107, 129-142.

Pluske, J.R., Durmic, Z., Pethick, D.W., Mullan, B.P. and Hampson, D.J. (1998). Confirmation of the role of non-starch polysaccharides and resistant starch in the expression of swine dysentery in pigs following experimental infection with Serpulina hyodysenteriae. Journal of Nutrition 128, 1737-1744.

Pluske, J.R., Hampson, D.J. and Williams, I.H. (1997). Factors influencing the structure and function of the small intestine in the weaned pig: a review. Livestock Production Science 51, 215-236.

Pluske, J.R., Pethick, D.W., Durmic, Z., Hampson, D.J. and Mullan, B.P. (1999). Non-starch polysaccharides in pig diets and their influence on intestinal microflora, digestive physiology, and enteric disease. In Recent Advances in Animal Nutrition (eds P.C. Garnsworthy and J. Wiseman), pp. 189-225. Nottingham University Press, Loughborough, UK.

Pluske, J.R., Pethick, D.W., Hopwood, D.E. and Hampson, D.J. (2002). Nutritional influences on some major enteric bacterial disease of pigs. Nutrition Research Reviews 15, 333-371.

Pluske, J.R., Siba, P.M., Pethick, D.W., Durmic, Z., Mullan, B.P. and Hampson, D.J. (1996). The incidence of swine dysentery in pigs can be reduced by feeding diets that limit the amount of fermentable substrate entering the large intestine. Journal of Nutrition 126, 2920-2933.

Rainbird, A.L., Low, A.G., and Zebrowska, T. (1984). Effect of guar gum on glucose and water absorption from isolated loops of jejunum in conscious growing pigs. British Journal of Nutrition 52, 489-498.

Ramakrishna, B.S., Venkataraman, S., Srinivasan, P., Dash, P., Young, G.P. and Binder, H.J. (2000). Amylase-resistant starch plus oral rehydration solution for cholera. New England Journal of Medicine 342, 308-313.

Rantzer, D., Svendsen, J. and Westrom, B. (1995). Effect of strategies restriction on pig performance and health during the post-weaning period. Acta Agriculturae Scandinavica 46A, 219-225.

Rashmi, S. and Urooj, A. (2003). Effect of processing on nutritionally important starch fractions in rice varieties. International Journal of Food Science and Nutrition 54, 27-36.

Reid, C.-A. and Hillman, K. (1999). The effects of retrogradation and amylose/amylopectin ratio of starches on carbohydrate fermentation and microbial populations in the porcine colon. Animal Science 68, 503-510.

Richards, W.P.C. and Fraser, C.M. (1961.) Coliform enteritis of weaned pigs. A description of the disease and its association with haemolytic Escherichia coli. Cornell Veterinarian 51, 245-257.

Robles, A. and Ewan, R.C. (1982). Utilization of energy of rice and rice bran by young pigs. Journal of Animal Science 55, 572-577.

86

Sagum, R. and Arcot, J. (2000). Effect of domestic processing methods on the starch, non-starch polysaccharides and in vitro starch and protein digestibility of three varieties of rice with varying levels of amylose. Food Chemistry 70, 107-111.

Sauvant, D., Perez, J.-M. and Tran, G. (2004). Tables of Composition and Nutritional Value of Feed Materials. Wageningen Academic Publishers, The Netherlands.

Segales, J., Piella, J., Marco, E., Matue-de-Antonio, E.M., Espuna, E. and Domingo, M. (1998). Porcine dermatitis and nephropathy syndrome in Spain. Veterinary Record 142, 481-486.

Short, F.J., Gorton, P., Wiseman, J. and Boorman, K.N. (1996). Determination of titanium dioxide added as an inert marker in chicken digestibility studies. Animal Feed Science and Technology 59, 215-221.

Siba, P.M., Pethick, D.W. and Hampson, D.J. (1996). Pigs experimentally infected with Serpulina hyodysenteriae can be protected from developing swine dysentery by feeding them a highly digestible diet. Epidemiology and Infection 116, 207-216.

Smith, H.W. and Jones, J.E.T. (1963). Observations on the alimentary tract and its bacterial flora in healthy and diseased pigs. Journal of Pathology and Bacteriology 86, 387-412.

Smits C.H., Veldman A., Verkade H.J. and Beynen A.C. (1998). The inhibitory effect of carboxymethylcellulose with high viscosity on lipid absorption in broiler chickens coincides with reduced bile salt concentration and raised microbial numbers in the small intestine. Poultry Science 77, 1534-1539.

Solà-Oriol, D., Roura, E. and Torrallardona, D. (2004). Piglets at weaning or three weeks post-weaning prefer rice to sorghum. Journal of Animal Science 87 (Suppl.1), 22.

Stewart C.S., Hillman K., Maxwell F., Kelly D. and King T.P. (2001). Recent advances in probiosis in pigs: observations on the microbiology of the pig gut. In Recent Developments in Pig Nutrition 3 (eds. J. Wiseman and M.A. Varley), pp. 51-78 Nottingham University Press, Nottingham UK.

Tetens, I., Biswas, S.K., Glitsø, L.V., Kabir, K.A., Thilsted, S.H. and Choudhury, N.H. (1997). Physico-chemical characteristics as indicators of starch availability from milled rice. Journal of Cereal Science 26, 355-361.

Theander, O. and Westerlund, E. (1993). Determination of individual components of dietary fiber. In Dietary fiber in human nutrition, 2nd ed. (ed. A.G. Spiller), pp. 77-98. CRC Press, Inc., Boca Raton, Florida.

Trowell, H., Southgate, D., Wolever, T., Leeds, A., Gasull, M. and Jenkins, D. (1976). Dietary fiber redefined. Lancet 1, 967.

Van Beers-Schreurs, H.M.G., Vellenga, L., Wensing, T. and Breukink, H.J. (1992). The pathogenesis of the post-weaning syndrome in weaned piglets; a review. Veterinary Quarterly 14, 29-34.

Van der Klis J.D., van Voorst A. and van Cruyningen C. (1993). Effect of a soluble polysaccharide (carboxymethylcellulose) on the physico-chemical conditions in the gastrointestinal tract of broilers. British Poultry Science 34, 971-983.

Varel, V.H. and Pond, W.G. (1985). Enumeration and activity of cellulytic bacteria from gestating swine fed various levels of dietary fibre. Applied Environmental Microbiology 49, 858-862.

Varel, V.H., Pond, W.G., Pekas, J.C. and Yen, J.T. (1982). Influence of high-fibre on bacterial populations in gastrointestinal tracts of obese and lean genotype pigs Applied Environmental Microbiology 44, 107-112.

Vicente, B., Valencia, D.G., Lázaro, R., Latorre, M.A. and Mateos, G.G. (2004). Use of rice in substitution of corn in diets for young pigs. Journal of Animal Science 87 (Suppl. 1), 465.

Wannemacher, R.W. (1977). Key role of various individual amino acids in host response to infection. Journal of Clinical Nutrition 30, 1269-1271.

Williams, B.A., Verstegen, M.W.A. and Tamminga, S. (2001). Fermentation in the large intestine of single-stomached animals and its relationship to animal health. Nutrition Research Reviews 14, 207-227.

Williams, I.H. (2003). Growth of the weaned pig. In Weaning the Pig: Concepts and Consequences (eds. J.R. Pluske, J. Le Dividich and M.W.A. Verstegen), pp. 17-35. Wageningen Academic Publishers, Wageningen.

Wingertzahn, M.A., Teichberg, S. and Wapnir, R.A. (1999). Modified starch enhances absorption and accelerates recovery in experimental diarrhoea in rats. Pediatric Research 45, 397-402.

87

Younes, H., Demigné, C., Behr, S.R., Garleb, K.A. and Rémésy, C. (1996). A blend of dietary fibers increases urea disposal in the large intestine and lowers urinary nitrogen excretion in rats fed a low protein diet. Journal of Nutritional Biochemistry 7, 474-460.

Younes, H., Garleb, K.A., Behr, S.R., Demigné, C. and Rémésy, C. (1998). Dietary fiber stimulates the extra-renal route of nitrogen excretion in partially nephrectomised rats. Journal of Nutritional Biochemistry 9, 613-620.

Zarrinkalam, M.R., Tivey, D.R., Choct, M. and van Barneveld, R.J. (2001). Fast digestible starch content as a measure of available energy in barley fed to pigs and poultry. In Manipulating Pig Production VIII (ed. P.D. Cranwell), p. 207. Australasian Pig Science Association, Werribee.

Zobel, H.F. and Stephen, A.M. (1995). Starch: structure, analysis and application. In Food polysaccharides and their applications (ed A.M. Stephen), pp. 19-66. Marcel Dekker Inc., New York.