Testing hay treated with mould-inhibiting, biocontrol inoculum · Testing hay treated with...

Testing hay treated with mould-inhibiting, biocontrol inoculum

Microbial inoculant for hay

A report for the Rural Industries Research and Development Corporation by Sharon Brown and Peter Dart

July 2005 RIRDC Publication No. 05/103 RIRDC Project No UQ-82A

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© 2005 Rural Industries Research and Development Corporation. All rights reserved. ISBN 1 74151 166 6 ISSN 1440-6845 Testing hay treated with mould-inhibiting, biocontrol inoculum Publication No. 05/103 Project No. UQ-82A 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 Dr Sharon Brown School of Integrative Biology University of Queensland QLD 4072 Phone: 07 3365 1573 Fax: 07 3365 1177 Email: [email protected]

Dr Peter Dart School of Land and Food Sciences University of Queensland. QLD 4072 Phone: 07 3365 2867 Fax: 07 3365 1177 Email [email protected]

Becker Underwood Pty Ltd RMB 1084 Pacific Highway Somersby, NSW 2250 Phone: 02 4340 2246 Fax: 02 4340 2243 Email: [email protected]

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: [email protected]. Website: http://www.rirdc.gov.au

Published in July 2005 Printed on environmentally friendly paper by Canprint

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Foreword Hay production is a major rural industry in Australia with some 20,000 enterprises producing some 5 to 6 million tonnes per year worth about $900 million annually. Most of this hay is used to feed animals on-farm but some 25-30% is sold off–farm to feed dairy and beef cattle, and horses. About 500,000 tonnes is exported to countries such as Japan and the Middle East. Baling hay too moist, largely because of variable weather, can lead to spoilage through overheating, fungal contamination and rotting of the hay. An antibiotic-producing, microbial inoculant, developed by research at UQ and Bio-Care Technology P/L, now Becker Underwood P/L and supported by RIRDC funding, had shown promise in earlier field trials in controlling mould development on hay baled moist.

This report outlines the final stages of testing of the microbial formulation, HayRiteTM before release for commercial application. The project addressed quality control issues including those of animal safety and accumulation of antibiotics produced by the inoculum bacteria in animal tissues and undertook rigorous pre-release testing of the formulation in on-farm trials using farmers’ equipment.

The project involved collaboration between the University of Queensland who coordinated the research, Bio-Care Technology P/L who funded the field research and undertook research in-house to develop the formulations used in the field trials, and RIRDC as the funding agency supporting research by the fodder industry closely linked to the Australian Fodder Industry Association Inc.

This publication reports on the field inoculation trials and animal feeding trials undertaken by the project during 2001-2003.

This report is an addition to RIRDC’s diverse range of over 1200 research publications and forms part of our Fodder Crops R&D program, which aims to facilitate the development and maintenance of a viable fodder crops industry. Most of our publications are available for viewing, downloading or purchasing online through our website: . • downloads at www.rirdc.gov.au/fullreports/Index.htm • purchases at www.rirdc.gov.au/eshop Peter O’Brien Managing Director Rural Industries Research and Development Corporation

http://www.rirdc.gov.au/reports/Index.htm

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Acknowledgements This project was a collaboration between the Industry partner Bio-Care Technology P/L1 and the University of Queensland School of Land and Food Sciences, with farmer support in several states where hay is grown and without which the field work could not have been conducted. Mr Graham Thomson from south west Victoria in particular has enthusiastically conducted several experiments with HayRiteTM over several years. We thank the Cobram factory of the Murray Darling Cooperative for conducting the antibiotic tests on milk from cows fed HayRiteTM. RIRDC provided partial funding for this field testing phase of the research. The research was coordinated by Dr Sharon Brown and Dr Peter Dart. Mr Gary Bullard and Mr David Pulsford oversaw the research on the formulation conducted at Bio-Care Technology P/L. We also wish to thank Mr Jim Hales, Manager, Mt Cotton Research Facility and his staff for their assistance with this project, Mr Michael Neilsen for the analysis of the samples and Biocare Australia the provision of the HayRiteTM.

1 Note all work conducted during the project was with Bio-Care Technology P/L. After the project was completed Bio-Care Technology P/L became part of Becker Underwood P/L. Throughout this report Bio-Care Technology P/L was used.

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Contents Foreword................................................................................................................................ iii Acknowledgements............................................................................................................... iv Executive Summary .............................................................................................................vii 1. Background to the development of HayRiteTM .............................................................1

1.1 The hay industry ..................................................................................................................... 1 1.2 Improving hay quality.............................................................................................................. 2 1.3. Development of an inoculant for control of hay spoilage........................................................ 2

2. Field inoculation trials ....................................................................................................4 2.1. Lucerne hay trials..................................................................................................................... 4 2.2 Sorghum hay trial..................................................................................................................... 6 2.3. Rye grass and clover mixtures ................................................................................................. 6 2.4. Oats for export in Western Australia ..................................................................................... 10 2.5. Oats for export in South Australia ......................................................................................... 10 2.6 Oats for export in Victoria ..................................................................................................... 11 2.7 Clover hay in Victoria............................................................................................................ 11 2.8. Wheaten hay in New South Wales......................................................................................... 11 2.9. Implications............................................................................................................................ 12 2.10. Recommendations.................................................................................................................. 12

3. Inoculum formulation ...................................................................................................13 3.1. Introduction............................................................................................................................ 13 3.2 Objectives .............................................................................................................................. 13 3.3 Methodology and Results ...................................................................................................... 13 3.4 Discussion of Results ............................................................................................................. 14 3.5 Implications............................................................................................................................ 14 3.6 Recommendations.................................................................................................................. 14

4. Animal feeding trials.....................................................................................................15 4.1 Introduction............................................................................................................................ 15 4.2 Objectives .............................................................................................................................. 15 4.3 Methodology.......................................................................................................................... 15 4.4 Detailed Results and Discussion ............................................................................................ 16 4.5 Implications............................................................................................................................ 16 4.6 Recommendations.................................................................................................................. 16

5. Sheep feeding and metabolism trials..........................................................................17 5.1 Introduction............................................................................................................................ 17 5.2 Objectives .............................................................................................................................. 17 5.3 Methodology.......................................................................................................................... 17 5.4 Detailed Results ..................................................................................................................... 19 5.5 Discussion of Results ............................................................................................................. 21 5.6 Conclusions............................................................................................................................ 21 5.7 Implications............................................................................................................................ 22

6. References.....................................................................................................................23

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Executive Summary The project was initiated in 1989 following discussions between Bio-Care Technology P/L2 and the University of Queensland on the possibility of preserving hay through the biocontrol of spoilage fungi. The premise was that in good quality hay, growth of spoilage fungi may be inhibited by the presence of antagonistic bacteria present on the plant surfaces. If this were true, inoculation of plant material to be made into hay with such bacteria may prevent its spoilage. Initial funding of the project was provided by Bio-Care Technology and the University of Queensland, and a Post-graduate Industry Award allowed the appointment of a Post-Graduate scholar to work on the project. Subsequent funding was received from the RIRDC for further isolations of effective bacterial strains and a series of large scale field trials in WA, SA, NSW, Victoria and Queensland. The first stage of the project involved the identification and selection of bacterial strains that effectively inhibited the growth of spoilage fungi on hay baled too moist. A suite of bacteria were found on the leaf surfaces of lucerne which inhibited the in vitro growth of more than 30 types of fungi found on the leaves of spoilt hay. The most potent bacterial strains were selected for further laboratory investigations. Because of the need for an inoculum to survive in storage and to survive desiccation and UV irradiation after application to the cut crop, strains of bacillus, aerobic bacteria that produce spores which are resistant to these hazards, were selected for further testing. For field evaluation, a method was developed by Bio-Care Technology to grow the most potent strains in a fermenter and increase many fold the number of spores produced, and then formulate an inoculum for field use. Bacteria in the fermentation broth are separated from the broth by centrifuging and then freeze dried to a powder. The formulation is registered as HayRiteTM. This formulation resulted in very large increases in the survival rate of the two strains of potential inoculum bacteria H57 and H60, in storage and transport. The formulation is easy to use involving only mixing with clean water in the spray tank. The bacillus bacteria can even multiply in the spray tank, further increasing the inoculum potential. They can also grow on substances in the cuticle of the plant from which the hay is being made and in the juice expressed after conditioning the cut fodder. The bacteria produce a surfactant which facilitates their spread on the fodder tissue. Lastly, farmer-friendly field inoculation methods were developed in conjunction with cooperating farmers in Queensland, New South Wales, Victoria, South Australia and Western Australia who conducted the field tests of the HayRiteTM involving spraying 50 to 100L /ha of the inoculum mixture onto the crop just before cutting. Methods were also developed for inoculating the dried hay as it was picked up by the baler, with a maximum of 10L/ha of inoculum applied. Trials were conducted by the researchers in collaboration with the farmers and some were conducted by farmers themselves. The trials involved spraying inoculum onto the fodder crop at cutting and then the hay baled with more moisture than normal (25% cf 14 -18% moisture). Measuring the amount of moisture in hay is difficult. Commercial moisture probes can be bought but require experience in interpreting the variability usually found in the readings. Cores can be taken from the bale with a metal coring device about 2.6cm diameter and these can be used to measure the moisture content from the weight loss after drying or used to determine the fungal infection load in the hay. In some of the trials automatic, continuous recording temperature probes connected to a data logger were inserted into the stacked bales to measure the effect of inoculation on bale internal temperature changes after stacking. This provided a measure of the metabolic activity of the spoilage organism in the hay and residual respiration by the incompletely dried hay. Marked increases in temperature were found in bales that were becoming spoilt by the fungal growth on the hay and this is the process that sometimes leads to “caramelisation” of the sugars in the hay and fire can also develop in extreme

2 Note all work conducted during the project was with Bio-Care Technology P/L. After the project was completed Bio-Care Technology P/L became part of Becker Underwood P/L. Throughout this report Bio-Care Technology P/L was used.

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cases of spoilage. The temperature probe measures provided a good indication of the baled hay quality and corresponded well with other measures of quality such as the smell and colour of the hay and the presence of fungal spores. Another bioassay method for assessing the control of fungal development in the hay was developed. This consisted of counting the number of fungal pieces and spores on the surface of the hay and growing in the hay tissue that could grow in vitro when extracted from the hay. The fungi on the surface of the hay are washed-off and plated on agar where they grow and numbers of colonies are counted. The washed hay tissue is then macerated in a blender and small pieces of the hay about 0.6mm long are placed on agar in petri plates to encourage growth of any fungi contained in the pieces. The pieces are then observed under a microscope to determine ones that are infected. The percentage of pieces infected gives a measure of the amount of spoilage and its control by inoculation. The bacterial inoculum is now being produced in commercial quantities by Bio-Care Technology as HayRiteTM and is being distributed for farmer managed tests in the 2003 production season with the aim to release commercially during 2003.

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1. Background to the development of HayRiteTM

1.1 The hay industry Hay and silage production is a major rural industry in Australia that is rapidly evolving, the product being worth more than $1bn annually. Between 5 and 6 million tonnes of hay are produced annually and about 2 million tonnes of silage. Most of the preserved fodder is retained on farm but between 25 and 30% is sold off-farm. The recent development of plastic wrapped, high dry matter silage is a new product for which quality control issues are just emerging. Hay is produced in every State for commercial sale as well as on-farm use. Hay is made from a variety of fodder crops with a range of feed values and end uses. The production of high quality legume hays in the tropical north of Australia is another new development. The recent change in the structure of the dairy industry with increase in herd size particularly in southern Australia, has led to an increased market for grass/clover hay. Lucerne is made into hay throughout the year in northern Australia and during late spring to late autumn in southern regions and the production of pellets is a new approach to preserving lucerne fodder. About 500,000 tonnes of mainly oaten hay is now exported as bales or pellets with principal markets in Japan and the Middle East. The advent of large bales weighing around 400 to 500 kg is another development affecting the way hay is made and the problems of maintaining quality. Hay quality is a major factor determining the price of hay and its value as an animal feed. Hay quality is determined by the feed value, colour, fungal contamination level and fodder species involved. The end use of the hay as a roughage and/or as a supplier of protein or nitrogen for the animal, and the distance to be hauled also determines the price of hay. In the recent 2002-2003 drought, hay was hauled long distances to new markets where it was used to keep animals alive. If the hay is very dry at baling there is a risk that the material will shatter and particularly if the hay includes legumes, leaf shatter becomes a quality issue. For small bales, moisture content around 18% is considered adequately dry to enable safe baling. For larger bales this decreases to around 14% moisture. However sometimes hay is baled moister to avoid impending rain or to preserve more leaf. This could result in improved quality if there is less shatter, fungi do not grow in the hay, and heating after baling is limited. If the cut fodder drying in the field is rained upon, as occurred repeatedly in south western Victoria in the 2000-2001 season, then quality of the baled hay is drastically affected as nutrients and the colour are leached out, and the fungal load on the hay increases. Although moisture content is a major determinant of hay quality, it is quite difficult to measure accurately. Stems dry slower that leaves and nodes slower than the rest of the stem. Moisture probes are produced commercially but readings from the one bale can be highly variable and interpretation of the results to predict the “true” moisture status of the hay requires much skill in interpretation of the readings obtained. To increase drying rate the cut fodder is now often conditioned by squeezing it through rollers that expresses the moisture from the hay tissue. This expressed juice is an ideal substrate for growth of microorganisms which could be beneficial, or harmful such as the hay spoilage fungi. Hay in the swathe is often raked but this also has implications for quality as the green, chlorophyll-based colour of the hay can be lost from the exposure to sunlight and leaf may also shatter. Every machine pass also adds to the cost of making hay.

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1.2 Improving hay quality Fodder always has a mixture of microorganisms on the plant surface, both bacteria and fungi. In most environments these populations are limited in size mainly by moisture and substrate availability. The balance within the populations is also likely to be controlled by the mix of organisms which reach an equilibrium stasis dependent on the environment and the plant species. There are more than 30 different types of fungi both pathogenic and saprophytic living on leaf surfaces of any fodder plant and these fungi multiply after cutting if the moisture conditions are conducive often resulting in spoilage from change in the appearance of the hay as it becomes “mouldy”. Mouldy hay contains harmful spores and may also produce aflatoxins which are harmful to animals and humans. Such moist conditions can occur in the field if the temperatures are cool and the swathe is large in height, or if the cut fodder is rained upon as it is drying. They can also occur if hay is baled too moist and the inherent moisture within the bale allows growth of the spoilage fungi from the natural inoculum always present on the leaf surface. As the fungi grow within this moist bale metabolic energy is produced which heats up the bale and this is often coupled with residual respiration of the plant tissues. Both processes produce water as a product and this further increases the moisture level within the bale, making it more conducive to growth of microorganisms. In some situations this results not only in fungal spoilage but also in the increased temperature affecting the chemical composition of the hay eg caramelising of sugars, but the temperature can become high enough for self ignition of the bale and this can lead to catastrophic fires. Besides the fungi present on the fodder leaf surfaces there are a range of bacterial strains. Some of these produce antibiotics, perhaps to give the strain a competitive advantage with respect to the other microbes living on the leaf. The production of antifungal antibiotics may inhibit the growth of the fungi that cause spoilage of the hay when they multiply under moist conditions. Increasing the number of these potentially beneficial bacteria on the leaf surface by inoculating them onto the leaf surface may limit the growth of the spoilage fungi under moist conditions that can occur during hay making. This hypothesis was the basis for the research that has led to the development of the HayRiteTM inoculum for controlling fungal spoilage in hay. Bio-Care Technology P/L is the largest producer of high quality microbial inoculants for use in agriculture in Australia. The Rhizobium inoculants they produce for legume nodulation have been acknowledged for over 50 years to be the highest quality such inoculants produced in the world. Australia has a Government and Industry sponsored Rhizobium Inoculant Research and Control Service that helps maintain this quality. Bio-Care Technology approached the University of Queensland in 1989 with a request to undertake research in collaboration with them to develop an inoculum that would control the growth of spoilage fungi in hay production systems. The RIRDC then also agreed to financially support the research. 1.3. Development of an inoculant for control of hay spoilage. Not all hay is spoilt when it becomes wet during production or storage. This may be because the bacteria present on the hay surfaces multiply with the change in moisture conditions and produce antibiotics which inhibit the growth of the fungi also present on the plant surfaces. Bacteria were isolated from a range of lucerne hays which had become moist and were, or were not, spoiled, and from fresh lucerne leaves growing in a hay production field at Gatton Campus farm of the University of Queensland. The bacteria were washed off the leaf by agitation and the solution plated onto a range of media solidified with agar. After several days growth, colonies with different appearance were isolated and purified by plating onto agar. A further selection criterion was to isolate only strains that formed spores because they are resistant to the UV radiation that can kill bacteria that are exposed to sunlight. Strains that produce spores are also more resistant to death during storage. Several hundred bacterial strains were obtained in this manner. At the same time fungi found on leaf surfaces were isolated by plating washings from leaves of field grown lucerne and spoiled and good quality lucerne hay. More than 30 different types of fungi were

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obtained in pure culture. The potential bacterial biocontrol strains were then tested for antifungal antibiotic production by testing their ability to inhibit the growth of leaf surface fungi on agar plates in vitro. The potential bacterial biocontrol strains were tested against 30 different fungal strains. Those with the largest production of diffusible antibiotic and which produced the largest fungal growth inhibition zone around the bacterial colony on the plate were selected for further characterisation. This included the ability to survive in culture at room temperature, to produce spores which would aid in survival in harsh environments and to produce surfactants which would encourage the spread of the inoculum bacteria on the leaf surface. The next phase involved preliminary field testing with a broth inoculant. Bacterial cultures were grown in flasks on a shaker and then taken to the field, diluted in water and applied with a boom spray to lucerne just before cutting. The hay was then baled at higher moisture than usual (at c.25% moisture) and compared with uninoculated hay baled at the same moisture. The temperature within the bales was monitored. The uninoculated bales became hotter than the inoculated bales and after some weeks storage the uninoculated bales had a very large development of spoilage fungi which were visibly absent from the inoculated bales. Further experiments with lucerne at the Gatton Campus farm of the University of Queensland confirmed that inoculation could reduce the growth of fungal spoilage organisms on hay when it was rained on during drying or baled moist when compared with uninoculated hay. Field inoculation experiments were then conducted on farmers’ fields interstate, supported in part by the RIRDC. These trials are described in Chapter 2. Research on the formulation of the product then took place at Bio-Care Technology and the outcome was a product that has a long shelf life, is easy to use and is active in reducing fungal spoilage. The development of this formulation is addressed in Chapter 3. Because the inoculant strains were selected on the basis of their antifungal, antibiotic production it was important to check whether there was any residual carry over into animal products such as milk and in blood plasma which could lead to uptake by muscle tissues. Further the inoculant could affect palatability and the quality of the hay as a feed. These two issues were addressed and no deleterious effect occurred from the inoculant use (Chapter 4).

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2. Field inoculation trials 2.1. Lucerne hay trials The initial field trials were conducted at the Gatton Campus of The University of Queensland on commercial lucerne crops using standard, commercial, New Holland cutting and baling equipment for small rectangular bales approx 25kg each in weight. Several trials were conducted at first using broth inoculant produced at University of Queensland and thereafter broths produced by Bio-Care Technology P/L and finally freeze dried inoculant. The trials tested different methods of applying the inoculant; time of application, whether at cutting or at baling; strain of bacteria in the inoculant; and moisture of the hay at baling. These trials were completed before the start of this project but were the progenitors of the project under report. In these small scale trials, suitable conditions were developed for the spraying e.g. spray nozzle type, pressure applied. The inoculated bales were stacked in a mini stack of 20 to 30 bales to contain any heat generated in the bale whose temperature was being measured, in an attempt to simulate commercial stack conditions. Bale temperatures were monitored for up to a month after baling. The inoculum was applied at cutting to the crop before the swathe was cut or after the cutting but in the chamber where the lucerne is channelled into the swathe before laying on the ground. The volume of inoculant applied was also varied from approx 200 L per ha to approx 50 L per ha. A portable spray kit was developed to enable ready testing of the inoculants on privately owned farms. These trials with lucerne have shown that inoculation can reduce the rise in temperature associated with baling at high moisture levels of approx 30% (Figure 2.1, below). The inoculated bales also had a much lower fungal population (Table 2.2, below) and appeared to be of higher quality visually and by smell. Inoculated hay is also softer to the touch. The following experiment illustrates the kind of response obtained. Strain H60 was grown in tryptone soya broth. In the field, 3 L of broth culture containing c 108 cells per mL) was added to 100L of water mounted at the front of a tractor and inoculated onto the lucerne crop at a rate of 300L Ha. A mower mounted on the same tractor was used to cut a total of 0.5 ha of lucerne. The hay was not raked before being wet by 17.2mm of rain two days after cutting and 1.8mm three days after cutting. The hay was subsequently raked and baled four days after cutting.

At baling moisture levels were determined using the oven - dry method (table 2.1). Six samples were collected to represent a complete cross section of the swath for each treatment. In turn, three sub samples were taken from the original sample and oven dried. The moisture contents were calculated to be 32.2%, SE 0.03 moisture in inoculated hay and 32%, SE 0.01 in uninoculated hay. Ten inoculated and ten uninoculated control bales were transferred to a shed and the bales from each treatment were stacked together. After nine days storage, thermocouples connected through an AM416 Multiplexer connected to a Campbell CR10 data logger were installed to record the temperature of the bales. After 17 days storage in the shed, hay moisture was taken using a Delmhorst DBL- moisture probe (25cm) to allow non - destructive sampling. All readings (6 per bale) were taken from the end of the bales, across the biscuit slices. Hay samples were collected and assessed for fungal contamination (table 2.2). Table 2.1. Hay moisture levels.

Treatment % Moisture at Baling % Moisture after 17 days

Inoculated hay 32.0 (SE 0.03) 22.96 (SE 0.7)

Uninoculated hay 32.2 (SE 0.01) 37.04 (SE 5.1)

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Table 2.2. Fungal infection of lucerne hay following inoculation at cutting.

Inoculated plants Stem Leaf

No. infected No. tissue pieces

% infected No. infected No. tissue pieces

% infected

0 22 0 1 12 8 0 18 0 0 14 0 0 26 0 1 22 5 0 19 0 0 33 0 0 10 0 0 48 0 0 8 0 0 21 0

Total sampled 103 150 Mean % 0 2 SE 0 1.44

Uninoculated plants Stem Leaf

No. infected No. tissue pieces

% infected No. infected No. tissue pieces

% infected

6 22 27 3 11 27 5 17 29 9 16 56 3 51 6 33 41 80 7 17 41 4 9 44 7 9 78 21 26 81 6 40 15 18 16 59

26 29 90 Total sampled 156 158 Mean % 33 70 SE 10.29 6.96 It is proposed that the high temperature and high moisture levels of the uninoculated hay, relative to the inoculated hay, was due to metabolic activity of mould fungi. This pattern of activity has been observed in several other trials.

Hay Temperature Change

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

0 100 200 300 400 500 600 700 800 900 1000

Ti me ( h)

Ambient

Inoculat ed

Uninoculat ed

Figure 2.1. Temperature changes measured by platinum resistance probes in Lucerne hay inoculated with HayRiteTM or untreated at cutting and baled at high moisture content. The small bales were left to dry in a stack. The ambient temperature was also automatically recorded by the data logger.

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2.2 Sorghum hay trial

The effectiveness of HayRiteTM on sorghum hay was tested on the farm of Mr Graham Pennel, Fasifern Valley, Qld. Sorghum is a difficult crop to bale and often suffers from mould damage due to the difficulty of drying thick stems and leaves and the high sugar levels in the plant. In this trial, HayRiteTM was applied as 60g freeze dried inoculum in 50L of water using the portable spray unit developed at the University of Queensland. Treatments were: uninoculated, inoculated at mowing only, inoculated at baling only, inoculated at mowing and at baling. The temperature response was similar to the lucerne experiment described above. Hay with the best visual appearance had the smallest temperature rise and the lowest rate of moisture loss. Spraying at both mowing and baling was the best treatment and was similar to the dry uninoculated hay. Spraying at mowing was almost as good but spraying only at baling was not successful. Later trials on other crops in Southern Australia where inoculum was added at baling were successful. However, it is suggested for best results in the warmer climates of Queensland., hay should be inoculated at mowing, not at baling.

The visual appearance of each bale was examined and recorded (table 2.3). The experiment showed that under the environmental conditions and farming practice used in SE Queensland, HayRiteTM was effective in controlling mould in sorghum hay when applied at mowing but not at baling.

Table 2.3. Condition of sorghum hay after 3 weeks storage.

Treatment Colour/smell Spore level Moisture loss after 3 weeks

storage

Uninoculated wet hay (23% moisture)

Brown, musty Very high 11.6 (SE 0.8)

Inoc at mowing only

23% moisture)

Light green/brown, fresh smell

Low 11.4 (SE 0.7)

Inoc at baling only

(23% moisture)

Brown, musty Moderate to high 5.8 (SE 0.9)

Inoc at mowing and baling (23% moisture)


Very low 3.3 (SE 0.2)

Uninoculated dry hay (15%)


Low 3.2 (SE 0.9)

Colour and smell assessed by Sharon Brown, Peter Dart and Graham Pennel

2.3. Rye grass and clover mixtures

These trials were a collaborative effort jointly planned and executed by University of Queensland staff and Mr Graham Thomson, Condah, Western Victoria. The experiments were designed to test the effectiveness of HayRite™ in preventing spoilage of rye grass and clover hay when baled in 550kg large square bales at high moisture levels. Rye grass clover was selected for testing because Western Victoria is the largest producer of hay in Australia and rye/clover is the most common hay baled in that region. Hay is made from one cut per year and difficulties are often encountered in making hay during this spring period because the weather can still be cold and rain often occurs reducing hay quality considerably. The trials followed the usual pattern of making hay for the district.

One hectare of rye grass / clover mixture yielding 7.7 tonnes hay/ ha. (14 x 550kg bales ha.) was inoculated with HayRite™ and another 1 ha in the same field remained uninoculated. HayRite™ was applied at mowing, baling, and mowing + baling. Control treatments were dry hay inoculated at

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baling, uninoculated hay baled when dry and uninoculated hay baled when wet. The plant sap was allowed to dry and inoculated hay was baled at high moisture levels caused by morning dew fall. A 12m boom spray with the spray nozzles positioned just above the crop was used to spray 80L/ha of the inoculum mixture (100g HayRite™ powder in 100L water). The HayRiteTM contained 1.35x109

live bacterial cells/g and these numbers were not affected by storage and transport. Weather conditions were cool with a temperature of 18ºC and 50% cloud cover. After mowing the hay, two cuts were raked into one large windrow and allowed to lay undisturbed in the field before raking a second time directly before baling. For all treatments, a New Holland D1210 baler was used to produce 550kg large square bales. All treatments were baled at the same density (3.5) and load (75%) and within 1 hr of baling were stored in a shed for the duration of the experiment. Three or four replicate bales of each treatment were stacked. Each treatment stack of 3 bales was about one metre apart. Moisture levels were estimated in the field before storage (within 1 hr of baling) using a Farmscan hay moisture tester. 10 readings were taken from each side of the bale and the results averaged (Table 2.4). Changes in the temperature of the inoculated or uninoculated hay were monitored at hourly intervals by inserting a temperature probe 30 cm into the centre of the top surface of each test bale. After 90 days storage, seven 3cm diameter x 30cm long core samples were collected from each bale in a uniform manner from the ends and sides of the bales. Both high moisture and dry hay was inoculated just before baling by spraying onto the windrow. After spraying, the hay was immediately raked and baled (finished within 30 min of spraying). The inoculum consisted of 150g powder dissolved in 120L water with 100L applied to 4 bales worth of hay (c. 2.5 tonnes hay). A portion of the crop that had been inoculated at mowing was inoculated again at baling. The hay field was flat and hay quality appeared to be very uniform (table 2.5), however moisture levels within the bale varied between bales. Table 2.4. Hay moisture levels (mean of 20 Farmscan moisture meter readings).

Treatments Rep A Rep B Rep C Rep D 1. Wet – uninoculated 30 31 32 2. Wet - inoculated at mowing 23.4 25 32.7 31.2 3. Wet - inoculated at mowing and baling 30.2 26.2 29.1 4. Wet - inoculated at baling 29.8 29.8 31.6 5. Dry - inoculated at mowing 23.6 14.4 6. Dry - uninoculated 16.4

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Table 2.5. Condition of rye/clover hay stored for 3 months, 2001.

Treatment Colour/smell* Spore level

1. Wet - uninoculated Brown, musty Very high

2. Wet - inoculated at mowing


Low

3. Wet - Inoculated at mowing and baling

Brown, musty Moderate to high

4. Wet - inoculated at baling


Very low

5. Dry - Inoculated at mowing


Low

6. Dry uninoculated

* colour and smell assessed by Sharon Brown, Graham Thomson and Bill Goff

Table 2.6. Fungal colonization of pieces of rye grass/clover hay, 2001.

Treatment % pieces infected

Wet – uninoculated 74

Wet – inoculated at Mow + Bale 25

Wet - inoculated at Mowing 5

Wet - inoculated at Baling 0

Dry - inoculated at Mowing 8

Dry uninoculated 0

The change in moisture levels during storage followed the pattern for lucerne. The better quality inoculated or uninoculated hay had a smaller moisture loss than spoiled hay. Inoculated hay had a better appearance, smell and lower level of fungal colonization (table 2.6). An observation made at the time by Mr Graham Thomson was that the sprayed hay dried faster in the field than the unsprayed hay. It was also softer to the touch. This was confirmed by the operator of the hay baler who recorded a difference in reading on the tractor “there was reduced resistance at baling (Charge Meter reading for inoculated of 50Bar & uninoculated 50-100Bar)”.

A similar large scale trial on rye grass and clover conducted at the same time on Mr Graham Thomson’s farm in early January 2002 gave similar results. Three different rates of HayRiteTM application were used. 50g of HayRiteTM was dissolved in 100L of water and this was applied at 100L ha, 75L ha and 50L ha. On this occasion HayRiteTM was severely challenged as rain continued to fall on the mown crop for 27 days after spraying and mowing. The hay was baled at the conventional moisture levels (c. 14% moisture) and stored in a shed. Twenty bales of each treatment were examined thoroughly for colour, smell (table 2.7) and fungal contamination (table 2.8).

9

Table 2.7. Condition of rye/clover hay stored for 3 months, 2001 trial.

Treatment Colour/smell* Spore level

Uninoculated Brown, musty Very high

inoculated @ 100L /ha


Low

inoculated @ 75L/ ha


Low

inoculated @ 50L/ ha


Low

Table 2.8. % Fungal colonization of leaf pieces – rye grass/clover, 2001 trial.

Treatment %

inoculated @ 100L /ha 10

inoculated @ 75L/ ha 14

inoculated @ 50L/ ha 50

Uninoculated 68

In all cases, the treated hay was of higher quality than the untreated hay. One bale each of inoculated and uninoculated hay was fed to about 100 dairy heifers. They appeared to show a preference for the treated hay. The importance of this trial is that it showed that HayRiteTM applied at mowing offered protection to hay when subsequently rained on in the field during drying. 2.3.1. Trials conducted at Condah, Victoria November 2002. Comprehensive, large-scale trials were conducted with grass/clever hay. The weather was relatively favourable for hay making with about 70% cloud cover during the period with little rain. Temperatures were in the low 20’s during the day. Two trials were conducted. In the first HayRiteTM plus surfactant was sprayed on the standing crop of Concord rye grass and clover in the morning when the dew was still on the plants and then cut while the vegetation was still damp. 50g of HayRiteTM containing only strain H57 dissolved in 80L water was applied to a crop estimated to yield 4 tonnes baled hay per ha. The treated hay was baled at 6 days after cutting with eight bales resulting from the area sprayed (1ha). The untreated hay was not ready for baling until 11 days after cutting. The sprayed hay had very good green colour and its appearance was superior to the untreated hay. In another trial there was 25mm rain 3 days after mowing. The HayRiteTM (containing H57 and H 60 strains) treatment was baled at 11 days after mowing. The uninoculated control was baled at 13 days with similar moisture probe readings. There was a marked difference in colour of the bales with the treated hay having a brighter appearance that lasted until March. The bales were then sampled with a corer. From smelling the hay it was obvious that there were more fungal spores present in the untreated bales. On this farm the only raking occurs to consolidate two cuts into one windrow just after cutting, and sometimes just before baling but nothing in between. Samples of hay were examined in the laboratory for fungal infection level.

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2.4. Oats for export in Western Australia These trials were made possible through the cooperation of Mr Barry McDonald, Mr Troy McDonald and Mr Mark Heaslip. In 2002, the McDonalds grew 2850ha of oats, mostly for the hay export market, and expected to average 5.5ton (at15%moisture)/ ha. Two trials were established on different farms in the Shire of Victoria Plains, approximately 150km north-west of Perth. A major problem for producers in WA is rainfall damage caused to the crop while drying in the paddock after mowing and before baling. Rainfall reduces hay quality and causes losses from mould growth. Hay produced for export must have moisture reduced to 12% before baling while moisture of 16% would be acceptable for the local market. The aim of these trials was to test the effectiveness of HayRiteTM on oaten hay produced for export and the local market. The large “square” bales weighed about 750kg. For both trials, at Yerecoin and New Norcia, inoculum was applied from a boom spray in front of the mower at the rate of 50L of water mixed with 50g HayRiteTM per hectare. The small amount of rain that fell during the hay drying period was insufficient to cause significant mould development, confirmed by analysing hay pieces for fungal contamination in the laboratory. Because it proved difficult to organize baling equipment to bale at higher than usual moisture levels, the hay was baled at the normal moisture levels. This meant that the hay had not been sufficiently challenged by either rain or high moisture at baling and there was no significant difference in hay quality between inoculated and uninoculated hay, either from appearance, smell or fungal colonisation of hay pieces tested in the laboratory. 2.5. Oats for export in South Australia These two trials were made possible by the collaboration of Mr Andrew Parkinson, Balco Pty Ltd., Balaclava, SA. The Balaclava Plant processes about 80,000 ton of hay for export each year. Most of this hay is sent to Japan where the high quality hay is fed to dairy cows and the lower grade hay is used for beef cattle. Farmers, mostly within a 30 km radius of the Plant, supply Balco under contract with large square (8X4X4 ft or 750kg) bales. The average yield for oaten hay is 6tonnes/ ha varying from 4 – 10 ton ha depending on the rainfall. The Plant accepts hay at 14% moisture and processing reduces this to 12% In 2001, 60% of the crop was damaged by rainfall after cutting. The most significant change in hay quality was the development of black spot mould, mostly on the surface of the windrow, yellowing of the hay, and a reduction in soluble carbohydrate levels by about 4%. The black spot mould is the most serious cause of loss of hay quality in this production system. Isolates of the fungus causing this mould were inhibited in growth in vitro by the HayRiteTM inoculant biocontrol bacteria. This will give HayRiteTM an important role in maintaining the hay quality in this production system. In this series of experiments HayRiteTM was inoculated onto windrows that had received a small amount of rainfall. More rainfall was expected the same day, very soon after spraying. This trial contrasted with the one at McDonald’s farm in WA where HayRiteTM was applied to a standing crop of oats immediately before cutting and where a small amount of rainfall was received after the crop had been drying for about 3 days. On Farm 1 (Cameron and Lachlan Wood, Owen), a heavy crop of oats, expected to yield 8ton ha had been cut 2 days earlier and had received 3mm of rain on the second night but had dried before the inoculum was sprayed onto the windrows on the next day. Two hundred g of HayRiteTM was mixed with 220L of water and applied onto a 2ha area with a boom spray. This meant that the spray was applied only to the surface of 7 windrows and some of the spray was wasted (between windrows). However, it was suggested that this method is the most likely to be adopted by farmers in this circumstance. Because it reduces unnecessary cost incurred if applied to all of crop at cutting and

11

rain does not occur, it is fast to apply, and farmers have the boom spray equipment available as it is used for other crop spraying purposes on the farm. On the second farm (Andrew Wilson, Balaclava) the outside windrows of oats cut 7 days previously were inoculated with a hand held boom after receiving about 3mm of rainfall overnight. 100g of HayRiteTM was dissolved in 200 L of water and 15L of the mixture inoculated onto 0.9 km of windrow (a windrow represented a 3m cut). The surface of the hay was moist at the time of application and more rainfall was received after spraying on the same day. The outcome of this trial series was similar to the one in WA. The drying hay was not subjected to enough environmental pressure and the hay was baled at the normal moisture content. 2.6 Oats for export in Victoria This trial was made possible through the collaboration of Mr Julian Kaye, Elmhurst, Southern Pyrenees District, Central Victoria. HayRiteTM was applied in a long strip by air at a rate of 25g ha, one week before cutting to two paddocks of oaten hay. The conditions in this region were thought by Mr Kaye to suit this strategy because the cut crop and soil remain wet throughout the day due to the cool temperatures, mist and heavy dew experienced at the time of the year when oaten hay is made. The idea was that the bacteria would multiply in the crop to cover the whole plant before cutting. It was hoped that HayRiteTM might also protect lodged plants from mould, a major problem in the area, After cutting, this crop received 2 months continual rainfall and all of the hay was ruined. HayRiteTM was not able to provide protection to oats in such harsh conditions. The failure of this trial contrasted with the success in other trials where HayRiteTM was applied just prior to cutting. 2.7 Clover hay in Victoria Trials were conducted with clover in collaboration with Mr Graham and Mr Jarad Lukies, and Mr Ray Donnen at Katamatite, northern. Victoria. These trials were designed to test HayRiteTM on clover baled at 13% and 19% moisture. The usual farming practice in this area for clover is to cut, after 1 week rake to turn the top to the bottom, after 2 more days rake 2 windrows into one, and then bale the same day. The Lukies grow flood irrigated sub clover for the dairy industry. The 450kg big squares are baled at 13 to18% moisture for dairy farm use. The hay was sprayed just prior to cutting (50g HayRiteTM/ha), baled and stored. The hay was not subjected to significant environmental stress during drying and was baled at 13 and 19% moisture levels. Although both treatments of hay were of good quality, some differences were observed at sampling after 3 months storage. Notably, the leaf shatter was less in the inoculated hay. 2.8. Wheaten hay in New South Wales This trial involved collaboration between the University of Queensland and Mr Bob McCormack, Lenton Park, Winchendonvale, NSW. The trial was designed to test if HayRiteTM could protect wheat against fungal infection when baled at high sap moisture. If successful, this would benefit farmers by allowing them to bale earlier than usual and should give better colour and higher nutritional value. The crop was sprayed with HayRiteTM (50g dissolved in 100L water per ha) just prior to cutting. The inoculated hay and the uninoculated control hay was baled at high moisture (over 30% determined by moisture meter readings) and stored in a shed. Mr McCormack reported that the uninoculated hay rose in temperature quickly, became very hot (a steel bar plunged into the bale could not be held) and the temperature reduced slowly. By contrast, the inoculated hay rose in temperature slowly and cooled more quickly. This hay did not reach the high levels attained by the uninoculated hay. When sampled the inoculated hay appeared to have lower spore levels and was “greener” than the uninoculated control. HayRiteTM appeared to have a significant beneficial effect on wheat hay baled at high sap levels. Cattle fed both treated and non-treated hay together seemed to prefer the treated hay. All feeding trials have shown that HayRiteTM inoculation does not affect palatability.

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2.9. Implications Hay spoilage from weather damage during drying is a major cause of loss of quality in hay production and can reduce the quality to the point where it is not worth baling the hay. This not only affects the incomes of the producer farmers but also the buyers who rely on the hay for their animal production systems, particularly the dairy industry. The export industry cannot tolerate low quality hay so that a system which helps ensure quality should be a valuable input. The farmers in these trials tried different systems of application of the HayRiteTM but the most reliable method was application just before cutting, and while dew is still on the plant. This considerably improved the quality of grass/clover hay that received much rain during the drying phase. Since the increased value from better quality hay as a result of inoculation at cutting is likely to exceed the cost of application, application of HayRiteTM would appear to be a cost effective input that will act as an insurance against bad weather. A range of hays - lucerne, grass/clover, wheat and sorghum – benefited from HayRiteTM application at cutting when they were baled moister than normal. This provides an advantage to hay producers because it gives them the flexibility to bale earlier than normal practice at a higher moisture content when rain is pending. Baling at higher moisture content also helps to reduce shattering of leaf. The implication is that HayRiteTM can not only protect hay from mould during the drying process but also after baling. Methods to apply the HayRiteTM at baling are still under test but it seems that both applying to the windrow as well as applying into the channelling shute of the baler just after pickup are feasible. This gives producers flexibility. 2.10. Recommendations It is recommended that HayRiteTM be made available to farmers to test in a wide range of situations to expand the experience from using HayRiteTM. Farmers have been encouraged to contact Bio-Care Technology to receive advice on how to use HayRiteTM and how to conduct farmer designed and managed trials. This activity will take place during the 2003 hay making season in all hay making states. For the first time HayRiteTM is scheduled to be tested in tropical hay production systems in the Northern Territory and in Western Australia as well as in Tasmania. Farmers attending the meeting of the Australian Fodder Industry Association in Canberra in August 2003 were informed of the current status of the trials which tested HayRiteTM and encouraged to conduct their own trials. More than 15 farmers wished to conduct their own tests. A newsletter produced by Bio-Care Technology and the University of Queensland will keep the farmers conducting the trials and the AFFIA members informed of the trial outcomes. The trials to date and the interest of AFFIA members indicate that it is now timely for the commercial release of HayRiteTM.

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3. Inoculum formulation

3.1. Introduction

Formulation of the HayRiteTM will be a key to its success. Early field trials using broths as inoculants showed that the difficulty in maintaining aeration of the cultures during transport would rule out this method as the inoculant strains died rapidly in broths that were not well aerated. Two alternatives were to use a paste of bacteria centrifuged out of the broth, held cold during transport and resuspended in the spray tank just before application to the crop. The cold chain aspect of this approach made it less desirable than one where a relatively “dry” inoculum was produced. Early trials with peat based inocula where broths of the bacterial cultures were mixed with sterile peat and the peat was then suspended in water in the spray tank just before application showed that the bacteria survived very well in the peat and even multiplied but clogging of spray nozzles by the peat particles was a major impediment and this approach was abandoned. Another issue in formulation was the increase in the population of the spores produced by the Bacillus strains that are contained in HayRiteTM. These spores are an important part of the concept of HayRiteTM as they ensure a long shelf life of the formulated inoculant and survive UV irradiation after application, multiplying when the moisture conditions are conducive especially after rain. Thus an important objective was to increase the population of spores in the broths used to prepare the inoculant. 3.2 Objectives i) to improve the fermentation techniques for the bacterial cultures used in HayRiteTM to increase the populations levels of viable cells in the broths ii) to increase the numbers of spores in the broths iii) to develop a technique for concentrating the bacteria out of the broths to enable drying of the cultures iv) to develop a technique for drying the bacterial cultures in order to formulate a dry powder inoculum v) to develop a package that is robust enough for transport of the inoculum that prevents spoilage during storage vi) to develop a formulation that dissolves readily on mixing with water and which does not aggregate to clog spray nozzles. 3.3 Methodology and Results The key to obtaining large populations of viable cells in the broth in a media that was cost effective involved testing different ingredients that were readily available and inexpensive to purchase as well as to find fermentation conditions (aeration rate, temperature, broth pH etc) that were conducive to multiplication of the cells rapidly. A suitable media was developed which improved populations several fold especially of the spores, and a suitably large fermentor and conditions were identified from experimental fermentation runs. Scaling up production from a small to a large fermentor requires much technical expertise and Bio-Care Technology has achieved this in a very short time period. Many experiments were needed to develop a suitable system for the concentration of the cells from the broth. Cross flow filtration was not suitable for all strains and centrifugation proved satisfactory to produce a paste of cells from the broth. A technique for freeze drying was then developed which minimised death of cells on drying. This formulation approach resulted in a product that has an excellent shelf life with no loss in numbers of viable cells in the formulated product over several

14

months. A product with more than 5 x 109 viable cells/g inoculum powder is the result. At the recommended rate of application of 20g/tonne hay this provides more than100,000 bacteria per gram of hay. A suitable container for shipment of the inoculum, brochures for farmer information, and instructions for use have been developed by Bio-Care Technology and the University of Queensland. . 3.4 Discussion of Results The formulation achieved is suitable for commercial use and the release of the product is scheduled for October 2003. Inoculum is already being made available to cooperator farmers. They will be able to test the product in a range of potential applications varying from mould development in the bale at Kununara on the Ord in the Kimberleys, because the tropical humidity induces spoilage after baling (drying is rapid within 3 days and not a problem) to Tasmania where the problem is rain during drying. The challenge of new materials such as vetch hay in Victoria and centrosema hay in the Northern Territory is to be addressed by these trials. Bio-Care Technology P/L has achieved its objectives. 3.5 Implications The fodder industry is growing in Australia and becoming more sophisticated in its use of machinery. Large bales have brought a new set of requirements in making hay particularly with the moisture levels which it is safe to use to prevent over heating and shed fires. HayRiteTM should provide some insurance against such occurrences if used properly in conjunction with good management practice. Use of HayRiteTM should likewise reduce spoilage during drying due to rain. The product will need to become known about by farmers through word of mouth as it has to overcome negative perceptions towards additives from products on the market that have not worked or have corroded machinery. 3.6 Recommendations A series of farm trials are now needed to enable farmers to develop methods for using HayRiteTM successfully. These farmer managed trials are now underway. These experiences will need to be documented in order to develop a series of farmer training activities to increase awareness on how HayRiteTM can be used in a variety of situations. This will require extension of this knowledge to Government and private agencies concerned with supporting farmers by providing technical information. Farmers are increasingly aware that new technology can support their enterprise and are attending field days and employing technical support personnel from companies. Knowledge about HayRiteTM needs to be disseminated into this component of the knowledge transfer system. Production of a training video on how to use HayRiteTM would help this process. There is a willingness to accept well tested products such as HayRiteTM at all levels of the production chain but some effort is now needed to “spread the word” about the way it can be used and the management practices that will support its successful use. .

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4. Animal feeding trials

4.1 Introduction

There was a need to demonstrate that the antibiotics produced by HayRiteTM are not detectable in milk or other animal products and that animals like to eat the hay that has been treated and do not suffer any deleterious effects from having done so. A series of animal feeding trials was set up to show that HayRiteTM did not adversely affect animals that ate treated hay. The trials were conducted at the University of Queensland and with farmer cooperators and the Murray Goulburn Cooperative factory at Cobram for testing for any residual antibiotic in the milk. In the first animal feeding trial at the University of Queensland, the fungal biocontrol bacteria were prepared as a peat inoculum by injecting pure cultures of the broth into sterile peat powder and allowing the bacteria to further multiply in the peat. The peat was then used to spray on chaffed lucerne hay and an animal feeding preference trial established with 6 young steers. The cattle were offered treated and untreated hay together and the amounts eaten in a given period of time recorded. This trial showed that the cattle did not like peat on their chaff as both peat alone and peat plus the bacteria were equally unfavoured by the cattle over a 2 week feeding trial, when compared with the untreated hay. This trial prompted a new look at the formulation that was to be used for the hay, and one without peat was obviously required. In the second trial at the University of Queensland, steers were fed with chaffed lucerne hay with and without treatment with HayRiteTM at cutting and baled at the normal c.18% moisture and their preference for these hays established. In the third trial at the University of Queensland, pregnant ewes were fed chaffed grass/clover hay that had been prepared on a farm at Condah, South Western Victoria, and had been treated with HayRiteTM and untreated hay over a 6 week period and their dry weight gain and metabolism of nitrogen and cellulose determined. Samples of blood plasma and urine and were tested for presence of antibiotics. This chapter 4 deals with the initial trials. Chapter 5 deals with the longer term sheep feeding trials which dealt with the effects of HayRiteTM on animal metabolism of hay. 4.2 Objectives i) to show that there are no detectable antibiotics in milk from cows fed HayRiteTM treated hay. ii) to establish the effect of HayRiteTM treatment of hay on its palatability for cattle. 4.3 Methodology i) Dairy cow feeding trial. It is possible that cows fed with hay inoculated with HayRiteTM may ingest enough antibiotics to be detectable in the milk. This trial addressed quality assurance issues this raises for the dairy industry. The trial was designed to identify any antibiotic that is passed into the milk thereby reducing milk quality. The trial was a collaboration between the University of Queensland, Mr Ray Donnan, Hay Contractor, the Cameron family, dairy farmers, and the Murray Goulburn Cooperative laboratory in Cobram.

Hay produced in a trial with Mr Graham Thomson was transported to the Cameron farm and used in this trial (details of the Thomson trial is found under chapter 2, rye grass/clover). Three healthy cows were placed in a small paddock apart from the main herd and fed the inoculated hay at the normal rate used on farm of 5kg/cow/day, for a total of two weeks. These cows were milked as usual with the main herd and the milk diverted into 20L buckets and fed to calves. Milk samples were collected

16

before feeding of inoculated hay commenced, and 4 days and 2 weeks after commencement. The milk samples were then sent to the Cobram factory laboratory for standard laboratory testing using the Delvotest SP testing procedure.

ii) Cattle palatability feeding trial. The aim of this trial was to determine whether or not the inoculation of hay with freeze dried inoculum will adversely affect the palatability of hay as compared with uninoculated hay. Six Charbray weaner cattle weighing on average 235kg were be held at the University of Queensland Mt Cotton Farm feedlot in individual pens and fed pangola hay for two weeks as a settling period. For the next two weeks the cattle received both HayRiteTM inoculated lucerne hay and the control of uninoculated lucerne hay baled at c. 18% moisture in small bales at the University of Queensland Gatton Campus farm. The hay was presented in a single container divided in the middle to allow the animals equal access to either the inoculated or uninoculated hay. The steers were fed the hay ad libitum for a 2hr period twice daily at 8am and 12 am and refusals measured each day so that a total feed consumption of control and treatment was determined. Enough feed was supplied so that there was always feed left over at the end of the feed period. The feed positions of the treated and untreated hay in the trough were changed randomly at each feed to remove positional bias. The trial continued for a 12 day period after the animals had been kept for a 2 week settling period in which time the steers became comfortable with being kept in pens and eating from divided troughs. Samples of the control and treated lucerne hay refusals were analysed for dry matter content with no difference found between samples. 4.4 Detailed Results and Discussion i) Dairy cow feeding trial. The tests showed no detectable antibiotic in the milk. The farmer reported an increase in milk produced by the test cows. It is acknowledged however, that this may have been entirely due to the separation of the cows from the main herd thus reducing the stress of competition. ii) Cattle palatability feeding trial. The results of this trial showed that the animals did not have a preference for the uninoculated hay. This indicated that the HayRiteTM treatment of hay had no effect on its palatability. Thus treating hay will have no deleterious effect on the feed intake or as the next chapter will show, also have no harmful effect on digestion of the hay and subsequent metabolism by ruminants. Besides cattle and sheep, horses and cattle have also been fed in the field treated and untreated hay baled at the same normal moisture content and these ad hoc trials showed no effect of the HayRiteTM on preference 4.5 Implications HayRiteTM can be used safely on hay fed to dairy cows as it will not affect palatability or production. 4.6 Recommendations HayRiteTM effects on animal metabolism needed a more detailed investigation and this was pursued as detailed in Chapter 5.

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5. Sheep feeding and metabolism trials 5.1 Introduction The nutritive value of hay depends largely on the quality of the forage used and the conditions under which drying takes place. In ideal conditions, drying takes place quickly, inhibiting cellular metabolism and decreasing the opportunity for microbial spoilage. Where these conditions exist, high levels of soluble carbohydrate are maintained in the dried forage, and high digestibility hays are produced. Quality is lost when soluble carbohydrates are metabolized by either continuing plant respiration or when used by invading bacterial and fungal populations. These circumstances are most often caused by slow rates of drying or by wetting from rain during the drying period. The project had isolated a naturally occurring bacterium from hay which produces an inhibitor of fungal growth, and stabilized cultures of these organisms are now commercially available under the trade name of HayRiteTM. The application of this product in solution at baling has been shown to decrease the rate of heating and to improve the visual quality and smell of the hay. These effects are most obvious where the rate of drying has been compromised by rain or high humidity at the time of cutting and baling. However, the effects of this treatment on hay composition, palatability and nutritive value for animals is not known, and the following experiment was designed to provide information on effects of HayRiteTM on these parameters in pregnant sheep. This trial was established to see if HayRiteTM affected animal metabolism and weight gain. It was conducted by Dr Barry Norton in collaboration with Dr Dart and Dr Brown. The trial involved feeding sheep with grass clover hay from a farm at Condah inoculated with HayRiteTM at cutting and baled at normal dryness of around 14% moisture, and compared with the same hay treated with HayRiteTM just before feeding so that the populations of bacteria on the hay were maximised and hence able to exert their most effect on any animal metabolic function. These treatments were compared with untreated hay baled at the same moisture content. The animal intake of hay was monitored so that the effect of the HayRiteTM on palatability was also indirectly assessed. 5.2 Objectives i) to determine if there is any deleterious effect of the HayRiteTM treated hay on animal metabolism and any detectable presence of antibiotic in the tissues of animals fed such treated hay 5.3 Methodology The effects of microbial amendment of rye-grass clover hay at baling on intake and nutritive value for pregnant sheep were tested at the University of Queensland Mt Cotton farm. Experimental design and statistical analysis. Three treatments were applied to groups of 6 pregnant Border-Leicester x Suffolk ewes (4-6 years old) held in individual metabolism cages. Two large bales of ryegrass-clover hay from western Victoria were used, one bale was from hay which had been treated at baling with a microbial culture which inhibits fungal growth (HayRiteTM ) during the drying process (Treated with HayRiteTM), and the other bale was from the same paddock but was normally cured (untreated). Further details of the effects of this treatment on hay quality have been reported by Brown and Dart (2001). The first group of sheep were offered the treated hay, the second group offered the untreated hay and the third treatment group were given untreated hay to which had been added a daily supplement of HayRiteTM (1x109 bacteria), dissolved in 100 ml tap water and sprinkled over the feed (HayRiteTM at Feeding). This treatment was designed to test the direct effects of HayRiteTM on animal metabolism and performance. The experiment was statistically analysed as completely random design of three treatments with six sheep per treatment. The significance of differences between treatments was tested by analysis of variance and detection of least significant differences between treatments (Steel and Torrie 1960).

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Animal management and feeding. The pregnant ewes (73-97 days pregnant) were drawn at random from the Mt Cotton breeding flock, treated for intestinal parasites (SCANDA TM), weighed and offered a diet of Pangola grass hay ad libitum while being held in individual metabolism cages one week prior to the beginning of the trial. All sheep were offered the experimental feeds ad libitum (110% previous days intake), water was freely provided and each sheep had continual access to a mineral block in its feed bin. The feeding period lasted for 35 days, and digestibility and balance measurements were taken during the last 5 days of the experiment. At this time, all sheep were weighed again, and returned to the farm flock for lambing at pasture. Measurements and analytical methods. Feed was offered on continuous basis, and all refusals collected and weighed at the end of the digestibility period. During the last five days of the trial, urine and faeces were collected from each animal, weighed, sub-sampled (10%) and stored at –20oC until analysis. Urine was collected into buckets containing 100 ml 25% V/V sulphuric acid which effectively maintained urine pH below 3. At the end of the trial, collected feed, refusal and faecal samples were dried to constant weight in a forced-draught oven (63-48 hours at 65oC), ground through a 1 mm sieve, and stored for later analysis. The organic matter content of feed, refusals and faeces was determined by loss on ignition in a muffle furnace at 600 OC for 3 hours. Neutral detergent and acid detergent fibre in these samples were determined by the methods of Goering and Van Soest (1970) and Van Soest and Wine (1967) respectively. Total N in these samples and in urine was determined by the Kjeldahl method (AOAC 1960). Purines (allantoic acid, creatinine, xanthine, hypoxanthine and uric acid) were determined in urine and estimates of microbial N absorbed calculated by the methods described by Chen et al., (1995). On the last day, rumen fluid samples were collected by stomach tube from each animal, and strained aliquots taken for analysis. Samples for ammonia determination were preserved in 0.1M HCl prior to distillation and titration (Buchi System). Volatile fatty acids (VFA) were stored in a solution containing 4 ml rumen fluid and 1 ml protein precipitant (25% metaphosphoric acid containing 2mg/ml iso-caproic acid) and analysed for VFA concentrations and proportions by gas-liquid chromatography (Hewlett-Packard 5830A)(Cottyn and Boucque 1968). Blood samples were collected by veni-puncture and plasma samples stored at –20oC until analysed. Testing for antibiotics in tissues. A bioassay was used to determine if the antibiotics produced by the bacteria in HayRiteTM persisted after the treated hay was fed to animals. Dried faeces, urine, muscle, rumen fluid and plasma samples were tested for the presence of fungal anti-biotics by applying samples to agar plates of Potato Dextrose Agar containing established colonies of the fungus Fusarium oxysporum whose growth is inhibited by the bacterial strains H57 and H60 which are the component strains in the HayRiteTM used for treating the hay used in these trials. The radial growth of the fungus from the central fungal inoculum plug was measured with the test materials being placed as a spot at the edge of the plates and compared with the growth when no materials were placed on the edge of the plate and with the inhibition achieved when colonies of H57 and H60 were established at the same position on the edge of the plates to serve as a positive control. If fungal growth inhibition occurs, it can be assumed that it was due to the antibiotics contained in the animal samples.

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5.4 Detailed Results Table 5.1. Chemical composition of ryegrass-clover hay treated and untreated with HayRiteTM during baling

Chemical composition (g/kg DM)

Treatment

Dry Matter (% as fed)

Organic Matter

NDF

ADF

Crude Protein (Nx6.25)

Treated with “HayRiteTM”

Untreated

878

902

938

925

656

676

418

430

76

102

Effects of HayRiteTM on composition of hay. Table 5.1 shows mean values for some measurements of the botanical and chemical composition of the treated and untreated hays. While the differences in chemical composition were small, do suggest that treated hays had higher concentrations of soluble carbohydrates (206 g/kg) than did the untreated hays (147 g/kg) and do not indicate any major differences in nutritive value. However, the protein content of both hays was relatively small, and for this reasons, both hays are likely to be of relatively low quality. During feeding, there was a distinct physical difference between the two bales of hay, the treated hay was easily separated into friable wafers for chaffing, whereas the untreated hay formed stiff and difficult to separate wafers, and tended to produce more fine dust on chaffing that the treated hay. Effects of treatments on feed intake and digestibility Sheep given the untreated hay generally consumed less feed, and this hay was also significantly lower in dry matter, organic matter and N digestibility when compared with the treated hay. However there were no significant differences in the digestibility of the fibrous fractions (NDF and ADF) suggesting that the small differences digestibility may be accounted for by the differences in soluble carbohydrate contents of the two hays (Table 5.2). It is also possible that the lower digestibility is related to the lower feed intakes of sheep consuming the untreated hay. Table 5. 2. Mean values for the intake and digestibility of dietary components in sheep offered ryegrass-clover hay treated with HayRiteTM at baling, untreated hay plus daily addition of HayRiteTM and untreated hay.

Voluntary Feed Intake

Digestibility (g /kg)

Hay Treatment

gDM

/d

gDM

/kg0.75/d

Dry

Matter

Organic matter

Neutral

Detergent Fibre

Acid

Detergent Fibre

Crude Protein

(N x6.25)

HayRiteTM at Baling HayRiteTM at feeding Untreated SE Mean

1049a

1135b

936a

+ 43.6

60.7ab

65.3a

56.7b

+ 2.19

633a

653a

567b

+11.4

646a

641a

596b

+ 12.3

653a

696b

658ab

+ 13.2

697a

731b

693a

+ 12.7

559a

565a

510b

+ 17.2

Values in a column with different letters differ significantly (P<0.05)

20

Table 5.3 shows some indices of N metabolism in the sheep on the different diets. As expected, ruminal ammonia concentrations were low, but sufficient to maintain effective fermentations in the rumen (ie > 70 mgN/L). The excretion patterns of purines (allantoin, uric acid, hypoxanthine and xanthine) in urine were used to estimate microbial N digested in the small intestines. There were no significant differences found for these measurements between treatments, suggesting that microbial metabolism was not markedly affected by the treatments. However, sheep given either hay treated with HayRiteTM at baling or direct supplements of HayRiteTM showed increased N retention and efficiencies of N utilization. This aspect requires further study because it does suggest a metabolic effect of HayRiteTM on N metabolism in these animals. Table 5.3. Mean values for some measures of N metabolism in sheep offered ryegrass-clover hay treated with HayRiteTM at baling, untreated hay plus daily addition of HayRiteTM and untreated hay.

Treatment Rumen Ammonia (mgN/l)

Urinary Purine

Excretion (mM/day

Urinary Creatine Excreted (mM/day)

Microbial N

absorbed (g/d)

N retained

(g/d)

Percentage of Absorbed

N Retained


83a

136b

126b

+ 13.4

6.68

6.90

6.42

+ 0.816

6.74

5.31

6.53

+ 0.776

5.35

5.60

5.17

+ 0.766

3.88a

6.10b

2.89a

+ 0.538

54.0a

56.7a

36.5b

+ 4.21

Values in a column with different letters differ significantly (P<0.05)

Table 5.4 shows mean values for the concentrations and proportions of volatile fatty acids in the rumen of sheep given the three treatments. Total VFA concentrations are low because animals were sampled before feeding, and there appear to be few significant differences in VFA proportions. However, hay treatment and direct consumption of HayRiteTM shifted the fermentation pattern slightly towards acetic acid production and away from propionic acid, suggested that there may have been some effect of HayRiteTM on the activity of micro-organisms in the rumen. Table 5.4. Mean values for the concentrations and proportions of volatile fatty acids (VFA) in the ruminal fluid of sheep offered ryegrass-clover hay treated with HayRiteTM at baling, untreated hay plus daily addition of HayRiteTM and untreated hay.

Molar Proportions of VFA in rumen fluid (m mole/mole)

Treatment

Total VFA

(mM/L) Acetic acid Propionic acid

Butyric acid Branched chain acids


29.8 27.0 26.6

+ 2.58

716 728 707

+ 10.7

168 168 179

+ 4.0

83 73 90

+ 7.1

33 32 24

+ 3.4

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The effects of HayRiteTM treatment on animal production. Although the period of study was too short to accurately measure effects on live-weight gain, the results in Table 5.5 show that sheep given treated hay and the untreated hay supplemented with HayRiteTM gained weight over the whole experimental period, whereas those given untreated hay only maintained weight. These trends are supported by earlier observations where sheep given untreated hay had lower voluntary intakes of less digestible hay, and they also retained less and lower proportions of feed N than did sheep in the other two groups. There did not appear to be a significant presence of residual microbial antibiotics from the HayRiteTM, since no inhibition of fungal growth on plates was observed for blood, urine or faeces. There was no opportunity to select only pregnant ewes for this experiment, and it was expected that if there was a negative effect of HayRiteTM on sheep metabolism, then this effect would be shown by abnormalities in lambs and lambing. However, there appeared to be no effect of these treatments on lambing. Table 5.5. Mean values for liveweights, and liveweight changes of sheep offered ryegrass-clover hay treated with HayRiteTM at baling, untreated hay plus daily addition of HayRiteTM and untreated hay.

Treatment

Mean

Live-weight (kg)

Live-weight

Change (g/d)


44.8 44.8 42.4

+ 1.73

81 71 5

+ 27.3

Presence of Antibiotic in Animal Tissues. The plates inoculated with the animal tissues did not inhibit the Fusarium growth showing that they

did not contain any of the antibiotics that the pure cultures of bacteria were able to produce. If any residual antibiotics had been produced by the bacterial biocontrol inoculum on the hay tissue then they did not survive in the rumen after ingestion or pass into the blood or muscle tissue of the sheep. Likewise if any of the inoculum bacteria had survived on the hay then they did not produce any significant amount of antibiotic in the rumen fluid after the hay was ingested. 5.5 Discussion of Results This trial showed that HayRiteTM had no deleterious effect on the growth and metabolism of pregnant ewes. The indications were that HayRiteTM may improve metabolism. As in the trial feeding dairy cows and testing for antibiotic presence in milk, there was no carry over of any antibiotic produced by the HayRiteTM bacteria into sheep muscle, plasma, urine or faeces. 5.6 Conclusions. The treatment of hay with HayRiteTM at either baling or by direct consumption had no deleterious effects on the health of either the ewes treated or their lambs born 3-6 weeks after feeding. There were some minor effects of these treatments on nutritive value and live-weight gains of ewes given HayRiteTM in all cases, beneficial. It may be concluded that the substantial beneficial effects of treating hay with HayRiteTM during drying may be further enhanced by minor improvements in hay quality and animal performance on these diets.

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5.7 Implications These trials demonstrate that HayRiteTM is safe to use on hay because it will have no harmful effect on the animals which are fed on treated hay and there will be no carry over of any antibiotic the bacteria in HayRiteTM may produce either on the hay or in the rumen, into milk or into animal blood or tissues. The possibility exists that HayRiteTM may even improve intake and metabolism of ruminants. Thus the whole hay production system can use HayRiteTM safely and this is a very large market indeed. There are some types of hay and haylage production where more field tests are required and these will be undertaken in the next 2003 hay making season.

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6. References AOAC (1960). Official Methods of Analysis, 9th Edition. Association of Official Agricultural Chemist. Washington, DC. Chen, XB., Meijia, AT., Kyle, DJ. And Orskov, ER. (1995) J Agr Sci, 125, 137-143 Cottyn, B.G. and Boucque, C.V. (1968). J. Agr. Food Chem., 16, 105. Goering, H.K. and Van Soest, P.J. (1970). Agricultural Handbook 379 ARS USDA, Washington, DC. Steel, R.G.D. and Torrie, J.H. (1960). Principles and Procedures of Statistics. McGraw-Hill Book Company, Inc. New York. Van Soest, P.J. (1963). J. Assoc. Official Agric. Chem. 46, 829. Van Soest, P.J. and Wine, RH. (1967) J. Assoc. Official. Anal. Chem. 50, 50-55

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