NUTRITIONAL AND MANAGEMENT STRATEGIES TO ENHANCE THE REPRODUCTIVE PERFORMANCE OF THE COWHERD

By

REINALDO FERNANDES COOKE

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2008

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© 2008 Reinaldo Fernandes Cooke

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To my wife Flavia, and also to my entire family.

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ACKNOWLEDGMENTS

Firstly, I would like to thank my wife, Flavia Cooke, and my family for all their love,

support and patience. I also would like to thank Dr. John Arthington for the opportunity,

guidance, and excellent mentorship. I would like to thank the members of my committee (Dr.

Cliff Lamb, Dr. Matt Hersom, Dr. Joel Yelich, Dr. Ramon Littell, and Dr. Peter Hansen) for their

support with my research and classwork. Other faculty members not involved in my committee

(such as Dr. José Eduardo Portela Santos, Dr. William Thatcher, Dr. Charles Staples, Dr. João

Vendramini, and Dr. Richard Miles) were fundamental for the accomplishment of my goals. Last

but not least, I would like to thank Dr. Alan Ealy for all the help and guidance during the period

that I spent working in his lab.

I would like to thank all the staff who assisted me with my research projects, with special

thanks to Mr. Austin Bateman, Ms. Idania Alvarez, Mrs. Joyce Hayen, and Mrs. Andrea Dunlap.

My former advisor from Brazil, Dr. José Luiz Moraes Vasconcelos, is also very important to my

academic and research career, since he is the one that introduced me to the scientific world.

Special thanks are extended to my fellow graduate students and friends, particularly Brad Austin,

Dr. Jeremy Block, and Davi Araujo for all their friendship and collaboration during my research

program.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS ...............................................................................................................4 

LIST OF TABLES ...........................................................................................................................8 

LIST OF FIGURES .......................................................................................................................10 

ABSTRACT ...................................................................................................................................11 

CHAPTER

1 INTRODUCTION ..................................................................................................................13 

2 LITERATURE REVIEW .......................................................................................................14 

Summary of the Endocrine Control of Reproduction in Cattle ..............................................14 Onset of Puberty in Heifers .............................................................................................15 Reproductive Efficiency of Mature Cows .......................................................................17 

Supplementation Programs for Grazing Cow-Calf Operations ..............................................19 Frequency of Supplementation ........................................................................................21 Energy Metabolism and Reproduction ............................................................................24 Glucose ............................................................................................................................25 Insulin ..............................................................................................................................28 Insulin-like Growth Factor I ............................................................................................30 Progesterone ....................................................................................................................35 

Temperament, Acclimation, and Reproduction of Cattle .......................................................36 Assessment of Temperament in Beef Cattle ...................................................................36 Stress and Excitable Temperament .................................................................................39 The Hypothalamic-Pituitary-Adrenal axis ......................................................................39 Physiologic Responses to Excitable Temperament in Cattle ..........................................42 Temperament and Performance of Beef Cattle ...............................................................46 Temperament and Reproduction of Beef Cattle ..............................................................48 Cattle Acclimation ...........................................................................................................51 

Novel Strategies to Enhance the Reproductive Efficiency of Cow-Calf Operations .............52 

3 EFFECTS OF SUPPLEMENTATION FREQUENCY ON PERFORMANCE, REPRODUCTIVE, AND METABOLIC RESPONSES OF BRAHMAN-CROSSBRED FEMALES ..............................................................................................................................54 

Introduction .............................................................................................................................54 Materials and Methods ...........................................................................................................55 

Animals ............................................................................................................................55 Experiment 1 ............................................................................................................55 Experiment 2 ............................................................................................................56 

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Diets .................................................................................................................................56 Experiment 1 ............................................................................................................56 Experiment 2 ............................................................................................................57 

Sampling ..........................................................................................................................57 Experiment 1 ............................................................................................................57 Experiment 2 ............................................................................................................58 

Blood Analysis ................................................................................................................59 Tissue Analysis ................................................................................................................60 

Tissue Collection and RNA Extraction ....................................................................60 Real-time RT-PCR ...................................................................................................60 

Statistical Analysis ..........................................................................................................61 Experiment 1 ............................................................................................................61 Experiment 2 ............................................................................................................62 

Results and Discussion ...........................................................................................................63 Experiment 1 ...................................................................................................................63 Experiment 2 ...................................................................................................................70 

4 EFFECTS OF ACCLIMATION TO HANDLING ON PERFORMANCE, REPRODUCTIVE, AND PHYSIOLOGICAL RESPONSES OF BRAHMAN-CROSSBRED HEIFERS ........................................................................................................85 

Introduction .............................................................................................................................85 Materials and Methods ...........................................................................................................86 

Animals ............................................................................................................................86 Diets .................................................................................................................................87 Acclimation Procedure ....................................................................................................88 Sampling ..........................................................................................................................88 Blood Analysis ................................................................................................................90 Statistical Analysis ..........................................................................................................91 

Results and Discussion ...........................................................................................................92 

5 EFFECTS OF TEMPERAMENT AND ACCLIMATION ON PERFORMANCE, PHYSIOLOGICAL RESPONSES, AND PREGNANCY RATES OF BRAHMAN-CROSSBRED COWS ..........................................................................................................108 

Introduction ...........................................................................................................................108 Materials and Methods .........................................................................................................109 

Animals and Diets .........................................................................................................109 Acclimation Procedure ..................................................................................................110 Sampling ........................................................................................................................111 Breeding Season ............................................................................................................111 Blood Analysis ..............................................................................................................112 Statistical Analysis ........................................................................................................113 

Results and Discussion .........................................................................................................114 

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LIST OF REFERENCES .............................................................................................................130 

BIOGRAPHICAL SKETCH .......................................................................................................155 

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LIST OF TABLES

Table Page 3-1 Ingredient composition and nutrient profile of the supplement offered in Exp. 1 and 2 ...74 

3-2 Primer sets used for quantitative real-time RT-PCR .........................................................75 

3-3 Blood urea nitrogen and plasma glucose concentrations of heifers offered an energy supplement based on fibrous byproducts daily or 3 times weekly, at a weekly rate of 18.2 kg of DM per heifer ...................................................................................................76 

3-4 Plasma concentrations of insulin and IGF-I of heifers offered an energy supplement based on fibrous byproducts daily or 3 times weekly, at a weekly rate of 18.2 kg of DM per heifer .....................................................................................................................77 

3-5 Expression of hepatic genes associated with nutritional metabolism and growth of heifers offered an energy supplement based on fibrous byproducts daily or 3 times weekly, at a weekly rate of 18.2 kg of DM per heifer .......................................................78 

3-6 Blood urea nitrogen and plasma insulin concentrations of cows offered an energy supplement based on fibrous byproducts daily or 3 times weekly, at a weekly rate of 20.3 kg of DM per cow ......................................................................................................79 

3-7 Expression of hepatic genes associated with nutritional metabolism and status of cows offered an energy supplement based on fibrous byproducts daily or 3 times weekly, at a weekly rate of 20.3 kg of DM per cow ..........................................................80 

3-8 Plasma IGF-I concentrations of cows offered an energy supplement based on fibrous byproducts daily or 3 times weekly, at a weekly rate of 20.3 kg of DM per cow .............81 

4-1 Average daily gain, pregnancy rates, and concentrations of plasma ceruloplasmin, haptoglobin, and IGF-I of heifers exposed or not to handling acclimation procedures ....99 

4-2 Pearson correlation coefficients among ADG, plasma measurements, and temperament of heifers ....................................................................................................100 

4-3 Temperament measurements of heifers exposed or not to handling acclimation procedures ........................................................................................................................101 

4-4 Pearson correlation coefficients among measurements of temperament and plasma cortisol concentrations of heifers .....................................................................................102 

4-5 Effects of breed on performance, temperament, and physiologic parameters of replacement heifers ..........................................................................................................103 

5-1 Mean BW and BCS of Brahman-crossbred cows exposed or not to acclimation procedures ........................................................................................................................121 

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5-2 Body weight change, temperament measurements, and plasma concentrations of cortisol, haptoglobin, and IGF-I of Brahman-crossbred cows exposed or not to acclimation procedures ....................................................................................................122 

5-3 Body weight, BCS, and plasma ceruloplasmin concentrations of Brahman-crossbred cows exposed or not to acclimation procedures ..............................................................123 

5-4 Pearson correlation coefficients among measurements of temperament and concentrations of plasma cortisol, ceruloplasmin and haptoglobin of Brahman-crossbred cows during yr 1 of the study ..........................................................................124 

5-5 Pearson correlation coefficients among measurements of temperament and concentrations of plasma cortisol, ceruloplasmin and haptoglobin of Brahman-crossbred cows during yr 2 of the study ..........................................................................125 

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LIST OF FIGURES

Figure Page 3-1 Proportion of prepubertal heifers by week during Exp. 1 ..................................................82 

3-2 Proportion of non-pregnant heifers by week during the 60-d breeding season of Exp. 1..........................................................................................................................................83 

3-3 Expression of muscle myostatin, as relative fold change, of heifers in Exp. 1 .................84 

4-1 Body weight of heifers exposed or not to handling acclimation procedures ...................104 

4-2 Puberty attainment of heifers exposed or not to handling acclimation procedures .........105 

4-3 Plasma cortisol concentrations of heifers exposed or not to handling acclimation procedures ........................................................................................................................106 

4-4 Plasma progesterone concentrations of prepubertal heifers exposed or not to handling acclimation procedures ....................................................................................................107 

5-1 Pregnancy rates during the breeding season of Brahman x British and Braford cows exposed or not to acclimation procedures ........................................................................126 

5-2 Effects of BCS and plasma IGF-I concentrations on the probability of Brahman-crossbred cows to become pregnant ................................................................................127 

5-3 Effects of temperament score and plasma cortisol concentrations on the probability of Brahman × British and Braford cows to become pregnant. ........................................128 

5-4 Effects of plasma ceruloplasmin and haptoglobin concentrations on the probability of Brahman-crossbred cows to become pregnant. ...............................................................129 

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

NUTRITIONAL AND MANAGEMENT STRATEGIES TO ENHANCE THE

REPRODUCTIVE PERFORMANCE OF THE COWHERD

By

Reinaldo Fernandes Cooke

December 2008 Chair: John D. Arthington Major: Animal Sciences

Four experiments were conducted to evaluate nutritional and management strategies to

enhance the reproductive performance of forage-fed Brahman-crossbred females. Experiment 1

compared performance and physiological responses of replacement heifers consuming an

energy-based supplement daily or 3 times per week at similar weekly amounts. Daily

supplementation resulted in a normalized mRNA expression pattern of genes associated with

nutritional metabolism and growth, reduced daily variation in plasma concentrations of urea

nitrogen, glucose, and insulin, and increased hepatic mRNA expression and plasma

concentrations of IGF-I. These beneficial physiological effects of daily supplementation were

translated into the greater BW gain and hastened attainment of puberty and pregnancy detected

in daily-fed heifers compared to heifers supplemented thrice weekly.

Experiment 2 evaluated physiological responses of brood cows offered supplements

similar to those of Experiment 1. Daily supplementation resulted in a normalized pattern of

gluconeogenic enzyme mRNA expression, reduced variation in plasma concentrations of glucose

and insulin, and improved cow nutritional status as observed by increasing plasma IGF-I

concentrations.

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Experiment 3 evaluated the effects of acclimation to handling procedures on growth,

temperament, physiological responses, and reproductive performance of replacement heifers.

Acclimation reduced growth rates because of the additional exercise that heifers were exposed

to, but reduced plasma concentrations of cortisol and hastened onset of puberty.

Experiment 4 evaluated the effects of acclimation to human interaction on temperament,

physiological responses, and pregnancy rates of brood cows. Acclimation did not influence

temperament and concentrations of plasma cortisol and acute phase proteins. Nevertheless,

reproductive performance of cows appeared to be influenced by acclimation, although additional

evaluations are still required. Further, measurements associated with cow temperament, acute

phase response, and energy status influenced the probability of cows to become pregnant during

the breeding season.

In summary, daily supplementation of energy-based supplements enhances performance,

reproduction, and physiological responses of forage-fed Brahman-crossbred females.

Acclimation to human handling may be an additional alternative to enhance performance of

Brahman-crossbred females because strategies that improve cattle disposition and reduce the

physiological responses to handling stress appear to be beneficial to the reproductive function of

replacement heifers and brood cows.

CHAPTER 1 INTRODUCTION

The beef cattle industry in Florida is mainly constituted by grazing cow-calf operations.

According to the USDA census (USDA, National Agricultural Statistics Service, 2002), Florida

contains approximately 983,000 beef cows, ranking 11th nationally, and leads the country in

number of operations with more than 2,500 cows. The two most important factors that affect the

profitability of cow-calf operations are reproduction and nutrition (Hess et al, 2005).

Reproduction efficiency of the herd is optimal when replacement heifers attain puberty as

yearlings and calve at 2 yr of age (Bagley, 1993), and brood cows are able to become pregnant

early during the annual breeding season (Rae, 2006). Nutrition is the environmental factor that

most influences reproductive efficiency of cattle (Bagley, 1993; Diskin et al., 2003); therefore

the cowherd should be always maintained at adequate planes of nourishment. In southern

Florida, where the majority of the state’s beef herd resides, cattle have a high Brahman influence,

and beef females containing Brahman-breeding typically reach puberty at older ages (Martin et

al., 1992) and exhibit excitable temperament (Hearnshaw and Morris, 1984; Fordyce et al., 1988;

Voisinet et al., 1997), which may further compromise their performance and reproductive

function (Plasse et al., 1970). Additionally, the forages grown in the state usually do not have

adequate nutrient density to meet the requirements of developing heifers and brood cows (Moore

et. al., 1991). Consequently, management strategies to offset the genetic and nutritional

disadvantages encountered in typical Florida cow-calf operations are required to optimize the

reproductive efficiency of the cowherd.

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CHAPTER 2 LITERATURE REVIEW

Summary of the Endocrine Control of Reproduction in Cattle

The mechanisms associated with acquisition, resumption, and maintenance of reproductive

ability in cattle are mediated by the hypothalamic-pituitary-ovarian axis (Hess et al., 2005). The

hypothalamus synthesizes and releases GnRH via neurosecretory neurons to the pituitary portal

blood system, which transports GnRH to the anterior pituitary gland, resulting in synthesis and

secretion of gonadotropins (LH and FSH) to the circulation (Anderson et al., 1981; Hess et al.,

2005). In the ovaries, recruitment and growth of follicles are stimulated by FSH until follicle

deviation is reached (Ginther et al., 1996). Following deviation, the maturation and consequent

ovulation of the dominant follicle are mediated by LH (Ginther et al., 2001; Yavas et al., 2000).

Estradiol is mainly produced by the ovarian follicles, and regulates gonadotropin synthesis and

release both positively and negatively (Kesner et al., 1981). Estradiol has been shown to

feedback negatively onto the hypothalamus and suppress GnRH synthesis prior to puberty and

during late gestation in cattle (Day et al., 1984; Yavas and Walton, 2000). Conversely, synthesis

of estradiol increases significantly as follicles grow, influencing follicle deviation (Ginther et al.,

2000) and eventually reaching a threshold that stimulates GnRH synthesis, increases the number

of GnRH receptors in the pituitary gland (Gregg et al., 1990; Hess et al., 2005), and induces the

LH surge required for ovulation (Kesner et al., 1981). Progesterone (P4), produced by the corpus

luteum formed following ovulation, suppresses GnRH release and consequent gonadotropin

synthesis in the brain; therefore the corpus luteum needs to be regressed for the ovulation process

to be repeated, or preserved for maintenance of pregnancy (Hess et al., 2005). Additionally, P4

appears to be required for the onset of normal estrous cycles in cattle because transient increases

in blood P4 concentrations were detected prior to puberty in heifers (Gonzalez-Padilla et al.,

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1975) and prior to the first ovulation following parturition in mature cows (Werth et al., 1996;

Looper et al., 2003).

The same endocrine mechanisms that control reproduction are present in Bos taurus and B.

indicus females. However, Brahman-influenced cows have a decreased responsiveness in

gonadotropin release to GnRH and estradiol stimuli (Griffin and Randel, 1978; Rhodes and

Randel, 1978), indicating that B. indicus females may have a decreased sensitivity to GnRH and

estradiol in the brain compared to B. taurus females, which may lead to differences in

reproductive ability between species.

Onset of Puberty in Heifers

The inclusion of replacement heifers into the cowherd is one of the most important factors

for the overall efficiency of cow-calf operations (Bagley, 1993), and strategies to maximize the

number of heifers conceiving early during their first breeding season has been shown to improve

the profitability of these operations (Leismeister at al., 1983). Ideally, replacement heifers should

be able to conceive at 14 to 16 mo of age, and consequently calve at approximately at 2 yr of age

(Schillo et al., 1992). Conception rates have been shown to be greater during the third estrus

compared to the pubertal estrus (Byerley et al., 1987), thus replacement heifers should attain

puberty at 12 mo of age to experience optimal reproductive performance during the beginning of

their first breeding season (Schillo et al., 1992; Bagley, 1993).

Puberty is attained when a heifer expresses her first estrous behavior and ovulates a fertile

oocyte followed by a luteal phase (Larson, 2007). The physiological processes associated with

follicle growth to preovulatory stages are primarily regulated by gradual increases in LH

secretion (Kinder et al., 1995). Although heifers are capable of synthesizing significant amounts

of LH prior to puberty (Schilo et al., 1982), basal quantities of estradiol secreted by ovarian

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follicles during the prepubertal phase inhibit the LH surge required for ovulation (Day et al.,

1984). As puberty approaches, the number of estradiol receptors available in the hypothalamus

and anterior pituitary decreases (Kinder et al., 1987), resulting in reduced negative feedback of

estradiol on LH secretion. Synthesis and pulse frequency of LH are thus enhanced, and reach the

threshold necessary to stimulate ovulation (Schillo et al., 1992). As commented previously, P4

also seems to be required for the establishment of puberty in heifers. Although the major sources

of this hormone are the corpus luteum and the pregnant placenta (Hoffmann and Schuler, 2002),

significant increases in blood concentrations of P4 are detected in heifers 2 wk prior to the onset

of puberty (Gonzalez-Padilla et al., 1975). The adrenal gland and luteal structures found within

the ovary were credited as the prepubertal sources for this hormone (Gonzalez-Padilla et al.,

1975; Berardinelli et al., 1979). Progesterone seems to stimulate the onset of puberty by priming

the hypothalamic-pituitary-ovarian axis (Looper et al., 2003) and suppressing estradiol receptors

in the hypothalamus, resulting in enhanced LH secretion (Anderson et al., 1996).

Breed type highly influences the age at which beef heifers attain puberty. In the case of B.

indicus vs. B. taurus, Martin et al. (1992) compiled data from several experiments and reported

that Brahman-influenced heifers reach puberty at older ages and greater body size compared to

B. taurus heifers. However, the same breed effect was not observed for pregnancy rates after

heifers were exposed to their first breeding season (Gregory et al., 1979), indicating that after

reaching puberty, heifers with Brahman influence have similar capability of conceiving as B.

taurus heifers. The differences observed for age at puberty between B. indicus and B. taurus

heifers may be attributed to the nutritional and environmental differences among the regions of

the world where these breed types were developed and were adapted to (Frisch and Vercoe,

1984; Rodrigues et al., 2002). However, even when heifers of similar age from both breed types

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are reared in the same locality and have similar growth rates, B. taurus heifers still reach puberty

at younger ages and lighter BW (Rodrigues et al., 2002). The same authors reported that the

physiological processes related to onset of puberty were similar between breed types but

occurred later for B. indicus, leading to the differences in age at puberty.

Reproductive Efficiency of Mature Cows

The major objective of cow-calf operations is to produce one calf per cow annually. The

average gestation length of Brahman-crossbred cattle is 286 d (Reynolds et al., 1980); therefore

brood cows should be able to become pregnant within 80 d after calving in typical Florida

operations. However, nursing cows experience a period of ovarian inactivity following

parturition that may last 60 d or more (Yavas and Walton, 2000), when they are unable to

ovulate and consequently conceive. The length of this period, denominated postpartum interval,

significantly determines the likelihood of cows becoming pregnant during the breeding season

(Wiltbank, 1970). Consequently, one of the main objectives to be accomplished in order to

maximize the reproductive efficiency of cow-calf operations is to minimize the postpartum

interval of the cowherd.

During late pregnancy, circulating concentrations of gonadotropins are reduced in cows

because the constant production of estradiol and P4 by maternal tissues impairs the synthesis of

GnRH by the hypothalamus (Short et al., 1990; Lucy, 2003), and thus depletes pituitary reserves

of LH and FSH (Wettemann et al., 2003). However, LH reserves and follicular growth are re-

established weeks prior to the resumption of cyclicity in most beef cows (Yavas and Walton,

2000; Day, 2004), but postpartum anestrous is sustained because estradiol still exerts a negative

feedback on gonadotropin synthesis (Short et al., 1990) and is not produced by the dominant

follicle in sufficient amounts to trigger the LH surge required for ovulation (Day, 1994).

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Consequently, the first postpartum ovulation highly depends on sufficient production of estradiol

by the dominant follicle to signal the preovulatory gonadotropin surge (Day et al., 2004),

whereas alternatives to alleviate the estradiol negative feedback, stimulate GnRH delivery to the

pituitary, and anticipate the ovulatory LH surge are options to decrease the postpartum interval

of beef cows.

Similar to the processes associated with onset of puberty, transient increases in P4 prior to

the first postpartum ovulation seems to influence the endocrine changes required by the

reproductive system of mature cows to attain normal and fertile estrous cycles (Kinder et al.,

1987). During postpartum anestrous, P4 concentrations are depressed because the major sources

of P4 production - functional corpus luteum and pregnant placenta (Hoffmann and Schuler,

2002) - are absent. Transient increases in P4 concentrations are typically observed in mature

cows within 10 d prior to the first observed postpartum estrus (Humphrey et al., 1983; Werth et

al., 1996; Looper et al., 2003), and this P4 may have originated from luteinized follicles (Corah

et al., 1974; Rawlings et al., 1980), or short-lived corpus luteum tissue originating from

undetected ovulations (Perry et al., 1991). Looper et al. (2003) reported normal estrous cycles for

81% of cows detected with transient increases in P4 concentrations during the anestrous period

compared with 36% for cows without P4 increase. Werth et al. (1996) detected transient

increases in P4 concentrations prior to first postpartum estrus in 70% of cows from their study,

and reported greater conception rates to AI at the first estrus for those cows (76%) compared

with cows that did not experience transient P4 increases (41%).

Circulating P4 is critical for the establishment and maintenance of pregnancy (Spencer and

Bazer, 2002). Progesterone produced by the corpus luteum originated from the ovulated

dominant follicle suppresses gonadotropin synthesis, release, and consequent ovulation during

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gestation (Hess et al., 2005). More importantly, P4 prepares the uterine environment for

conceptus growth and development, and modulates the release of hormones that may regress the

corpus luteum and thus disrupt pregnancy (Bazer et al., 1998). Consequently, the corpus luteum

has to be maintained so circulating P4 levels are adequate for maintenance of gestation, while

circulating P4 are required in certain levels to prevent corpus luteum regression. Several

researchers have reported that blood concentrations of P4 in cattle prior to or after breeding have

been positively associated with conception rates. Folman et al. (1990) reported that plasma

progesterone concentrations prior to AI were highly correlated (r = 0.87) with conception rates in

dairy cows. Fonseca et al. (1983) indicated that conception rates to AI increased by

approximately 13 % for each ng/mL increase in average blood P4 concentration during the 12-d

period preceding AI. Robinson et al. (1989) reported that dairy cows treated with exogenous P4

from d 5 to 12 or d 10 to 17 following AI had increased pregnancy rates compared to untreated

cows (60 vs. 30%, respectively). Research by Lopez-Gatius et al. (2004) indicated that

supplementation of exogenous P4 to pregnant dairy cows from d 40 to 68 after AI decreased the

risk of pregnancy loss by approximately 60%. These findings are also supported by other studies

(Wiltbank et al., 1956; Xu et al., 1997; Starbuck et al., 2001), indicating the importance of P4 for

the establishment and maintenance of pregnancy in cattle.

Supplementation Programs for Grazing Cow-Calf Operations

The cow-calf industry in Florida relies on tropical forages as the main source of feed for

beef cattle. Bahiagrass (Paspalum notatum) is the primary pasture forage available in the state,

covering an area of approximately 1 million ha (Chambliss and Sollenberger, 1991). Other

grasses such as limpograss (Hermathria altissima), bermudagrass (Cynodon dactylon), and

stargrass (Cynodon spp.) are also commonly used as a forage source for cattle (Arthington and

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Brown, 2005). In Florida, temperature and drought may limit tropical forage production during

the fall and winter (Sollenberger and Chambliss, 1991), but methods to conserve summer forage

surplus and offer it during periods of shortage are typical alternatives to provide adequate

amounts of forage to cattle throughout the year. Nevertheless, tropical forages do not always

have the adequate nutrient composition and quality to meet the requirements of developing

heifers and lactating brood cows, even when these animals consume sufficient amounts of forage

DM (Moore et al., 1991; Pate, 1991). Therefore, deficiencies in forage quality or quantity must

be corrected by nutrient supplementation to maintain cattle at adequate levels of nourishment.

Supplementation programs need to be designed according to the nutritional requirements

of the animal to be supplemented, inadequacies of the consumed forage, and economic viability

(DelCurto et al., 2000; Kunkle et al., 2000). Moore et al. (1991) compiled the nutritional analysis

of 637 samples of forages commonly grown in Florida (bahiagrass, bermudagrass, digitgrass,

stargrass, and limpograss) and reported that most of these grasses contained between 5 to 7% CP

(DM basis), and 48 to 51% TDN (DM basis). Developing heifers require at least 55% TDN and

8.5% CP of diet DM to sustain adequate growth rates (≥ 0.5 kg/d), whereas lactating brood cows

require approximately 60% TDN and 11% CP of diet DM (NRC, 1996). Therefore, nutritional

programs focusing on energy and protein supplementation are often required in typical Florida

cow-calf operations to maintain the herd in adequate levels of nutrition.

Intake of low-quality forages can be increased by protein supplementation (Köster el al.,

1996; Kunkle et al., 2000); however it does not result in adequate energy intake by the animal

(Bowman and Sanson, 1996). Supplemental energy is required in areas where energy availability

from grazed forages is limited (Caton and Dhuyvetter, 1997), and energy-based supplements

containing adequate amounts of protein has been shown to improve the performance of cattle

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grazing low-quality forages (Kunkle et al., 2000; Bodine and Purvis, 2003). Research has also

shown that energy intake positively influences the performance and reproductive efficiency of

brood cows and developing heifers (Mass, 1987; Schillo et al., 1992; Roberts et al., 1997).

Consequently, nutritional programs emphasizing energy-based supplements are required by cow-

calf operations based on low-quality forages to maintain the performance of the cowherd at

satisfactory levels.

Up to 63% of annual production costs in beef cow/calf operations are associated with cattle

feeding, including forage production and feed purchase (Miller et al., 2001). Additional expenses

associated with feeding, such as fuel and labor, also contribute significantly to these production

costs. Therefore, choosing feedstuffs that are economically viable and adoption of strategies that

minimize costs associated with the supplementation may contribute positively to the profitability

of cow-calf operations. Several feedstuffs are widely available in Florida to constitute energy-

based supplements, such as soybean hulls, citrus pulp, sugarcane molasses, and more recently

distillers grains. Determining which feedstuff to use depends on economics, management of

operation, and also on the effects of the feed on animal performance. This later factor is

important and has been thoroughly investigated previously, but will not be further addressed in

this document. Nevertheless, energy feedstuffs that favor propionate synthesis in the rumen

should be supplemented because they have been shown to improve the performance of beef

cattle (Pate, 1983; Ciccioli et al., 2005; Cooke et al., 2007a).

Frequency of Supplementation

Supplementing cattle infrequently, such as once or 3x weekly instead of daily, is a typical

strategy to decrease costs of production because the expenses associated with labor, fuel, and

equipment are reduced. Kunkle et al. (2000) compiled results from several experiments

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comparing different supplementation frequencies of protein-based feeds for cattle grazing low-

quality forages, and reported that supplementing protein as infrequently as once a wk instead of

daily did not alter cattle performance. Wettemann and Lusby (1994) reported similar

performance measures among range cows supplemented 6x/wk or 3x/wk with a protein-based

supplement. Similarly, Huston et al. (1999) reported no differences in BW and BCS changes

among beef cows offered cottonseed meal daily, 3x/wk, or once a wk, at a weekly rate of 5.6 kg

of DM. Bohnert et al. (2002a; 2002b) reported that steers consuming low-quality forages and

offered protein-based supplements as infrequently as once every 6 d had similar performance,

DMI, nutrient utilization, and ruminal microbial efficiency compared to steers supplemented

daily. Farmer et al. (2001) reported that mature cows and steers consuming dormant forages and

offered protein-based supplements at a weekly rate of 11.2 and 5.6 kg of DM, respectively, had a

linear improvement in performance (cows), forage intake and diet digestibility (steers) as

supplementation frequency increased. However, the authors stated that changes in performance

were small enough to support that reduced supplementation frequency of protein-based feeds is

an acceptable management alternative to decrease costs of production.

In contrast to protein-based supplements, decreasing the supplementation frequency of

energy-based feeds to cattle consuming low-quality forages has been shown to be detrimental to

animal performance. Research by Chase and Hibberd (1989) reported that cattle consuming low-

quality forages and offered corn-based supplements daily instead of every other day, at weekly

rates of 9.8 or 14.0 kg (as-fed), experienced slight improvements in DM digestibility and

consequent OM intake. The authors concluded that cattle offered corn-based supplements daily

utilized their diet more efficiently than cattle supplemented infrequently. Beaty et al. (1994)

reported that steers consuming wheat straw and offered grain-based supplements (sorghum in

22

addition to soybean meal) daily instead of 3x/wk, at a weekly rate of 14.0 kg of DM, had

increased digestible DMI but decreased DM digestibility. The same authors also reported that

mature cows grazing dormant tallgrass prairie and supplemented similarly as the steers

experienced alleviated BW and BCS losses during the calving period but had similar pregnancy

rates if supplemented daily instead of 3x/wk. In their review, Kunkle et al. (2000) reported

several research results indicating improved performance of cattle offered energy-based feeds

daily instead of infrequently, and attributed this effect to improved ruminal function and

consequent forage intake of daily-fed cattle. It is important to note that all these studies

comparing supplementation frequency of energy-based feeds utilized supplements containing

high-starch ingredients. These supplements are known to have negative effects on rumen health

and forage intake and digestibility in cattle even if offered daily (Sanson et al., 1990; Olson et

al., 1999). This negative impact is associated with decreased ruminal pH and activity of

cellulotic enzymes (Martin et. al., 2001), impaired bacterial attachment to fibrous material

(Hiltner and Dehority, 1983), and an increase in lag time for digestion (Mertens and Loften,

1980). Conversely, energy supplements based on low-starch ingredients, such as fibrous by-

products, has been shown to not depress forage intake (Bowman and Sanson, 2000) and maintain

animal performance at the same levels as when high-starch supplements are fed (Sunvold et al.,

1991; Horn et al., 1995). However, few studies have evaluated supplementation frequency of

low-starch feeds to cattle consuming low-quality forages. Loy et al. (2007) reported that forage-

fed heifers offered supplements based on distillers grains daily or in alternate days, at a weekly

rate of 10.5 kg of DM, had similar mean forage intake (1.69 vs. 1.66% of BW, respectively),

rumen pH (6.12 vs. 6.17, respectively), and in situ rate of NDF disappearance (4.09 vs. 4.01%

per h, respectively). Cooke et al. (2007b) reported that steers offered supplements based on

23

citrus-pulp daily had similar mean forage DMI (1.36 vs. 1.29% of BW, respectively), but

improved ADG (0.30 vs. 0.18 kg/d, respectively) compared to steers offered similar supplements

3x/wk, at a weekly rate of 16.1 kg of DM per steer for both treatments. Both studies also

reported a treatment × day interaction on forage DMI because forage intake of infrequently

supplemented cattle was reduced on days that supplements were offered, but greater on the

remaining days compared to that of cattle supplemented daily. Nevertheless, this outcome is

likely attributed to a substitution effect (Caton and Dhuyvetter, 1997; Bodine and Purvis, 2003)

rather than to compromised ruminal function. Cooke et al. (2007b) attributed the ADG

differences detected between cattle supplemented daily or 3x/wk to beneficial effects of frequent

supplementation on concentrations of blood hormones and metabolites associated with energy

intake and metabolism, such as glucose, insulin, and IGF-I. These same substances have been

positively associated with reproductive function of cows and heifers (Schilo et al., 1992; Hess et

al., 2005); therefore frequent supplementation of low-starch energy feeds is also expected to

benefit cattle reproduction.

Energy Metabolism and Reproduction

Energy is the primary nutrient consideration for optimal reproductive performance of cattle

(Mass, 1987). Attainment of puberty can be hastened by increasing energy intake and consequent

BW gain (Schillo et al., 1992). Cows with adequate energy status have decreased postpartum

interval (Richards et al., 1986; Looper et al., 1997) and greater pregnancy rates (Wiltbank et al.,

1962; Dunn et al., 1969; Bellows and Short, 1978) compared to cows with inadequate energy

status. These beneficial effects of energy on reproduction can be attributed, at least in part, to

plasma hormones and metabolites that are influenced positively by energy intake and by levels of

body energy reserves, such as glucose, insulin, and IGF-I (Wettemann et al., 2003).

24

Glucose

Glucose is a small, polar, and water-soluble monosaccharide (Nussey and Whitehead,

2001), essential for maintenance and productive functions of ruminants (Huntington, 1997;

Reynolds, 2005). Previous research indicated that blood glucose concentrations in beef cattle are

positively associated with feed intake and rates of BW gain (Vizcarra et al., 1998; Hersom et al.,

2004). Cooke et al. (2007a) reported greater mean glucose concentrations (83.3 vs. 74.7 mg/dL,

respectively) and ADG (0.40 vs. 0.30 kg/d, respectively) for grazing heifers consuming citrus

pulp-based supplements instead of iso-caloric and iso-nitrogenous supplements based on

sugarcane molasses. On the other hand, Cooke et al. (2007b) reported that forage-fed steers

supplemented daily with a citrus pulp-based supplement had greater ADG (0.30 vs. 0.18 kg/d,

respectively) but lesser mean glucose concentrations (66.0 vs. 76.2 mg/dL, respectively)

compared to steers offered supplements 3x/wk, but at similar weekly amounts. Metabolic fates of

glucose include generation of ATP via glycolysis and TCA cycle, and generation of NADPH

through the hexose monophosphate shunt (Huntington, 1997). Cattle consuming forage-based

diets ferment the majority of the dietary carbohydrates to VFA in the rumen, and often obtain

less than 10% of their glucose requirements from the diet (Young, 1977). Consequently, forage-

fed cattle depend significantly on liver gluconeogenesis to meet their metabolic glucose

requirements (Young, 1977; Huntington, 1997).

The major substrate for liver gluconeogenesis in cattle is propionate originated from rumen

fermentation (Reynolds et al., 1994; Reynolds, 2005), followed by other substrates originated

from digestion processes such as lactate, carbon skeletons from glucogenic aminoacids

deamination, and glycerol originated from TAG breakdown (Huntington, 1997). Following

digestion processes, these substrates are removed by the liver from the portal blood flow that

25

drains the gut (Huntington, 1997). In the liver, propionate is oxidized into succinyl-CoA, a TCA

cycle intermediate, and then converted to oxaloacetate (Nelson and Cox, 2005). Aminoacids can

be either degraded into TCA cycle intermediates, pyruvate or oxaloacetate, whereas lactate is

converted into pyruvate as part of the Cori cycle (Nelson and Cox, 2005). The initial steps of the

gluconeogenesis pathway are the conversion of pyruvate into oxaloacetate, catalyzed by the

enzyme pyruvate carboxylase (PC), and oxaloacetate into phosphoenolpyruvate, catalyzed by

phosphoenolpyruvate carboxykinase (PEPCK; Nelson and Cox, 2005). These two enzymes have

been shown to highly regulate the rate of gluconeogenesis in mammals, including cattle

(Drackley et al., 2006). Furthermore, PEPCK has two isozymes of similar activities and kinetic

properties, compartmentalized to the cell mitochondria (PEPCK-M) and cytosol (PEPCK-C;

Agca et al., 2002)

Research with cattle has shown that activity and mRNA expression of PC and PEPCK

influence positively glucose synthesis via gluconeogenesis. The availability of gluconeogenic

precursors was positively associated with mRNA expression of PC and PEPCK-C in the liver of

dairy cattle (Greenfield et al. 2000; Hammon et al., 2003; Karcher et al., 2007), whereas mRNA

expression of these enzymes in hepatic tissue was correlated with enzyme activity (Greenfield et

al. 2000; Agca et al. 2002) and consequently glucose synthesis (Bradford and Allen, 2005).

Karcher et al. (2007) reported that transition dairy cows supplemented with monensin had

increased hepatic expression of PEPCK-C because of enhanced propionate synthesis in the

rumen, although no differences in blood glucose concentrations were detected compared to non-

supplemented cows. Expression of PEPCK-C and PC were observed to be inhibited directly by

elevated plasma concentrations of glucose and insulin (Agca et al. 2002; Hammon et al., 2003).

Conversely, PEPCK-M has been shown not to be regulated by mRNA levels and also not

26

responsive to hormones and substrates (Greenfield et al., 2000; Agca et al., 2002), although the

activity of this enzyme may be responsible for up to 61% of glucose synthesis in ruminant

hepatocytes (Aiello and Armentano, 1987), and Hammon et al. (2003) reported that neonatal

calves fed colostrum had greater PEPCK-M mRNA expression compared to cohorts fed milk-

based formulas.

Glucose is the main source of energy for the central nervous system; consequently

inadequate availability of glucose may reduce the synthesis and release of GnRH and

gonadotropins by the brain and impair reproduction. Previous studies have shown that ruminants

administered glucose antagonists experienced induced anestrus, anovulation (McClure et al.,

1978), and impaired LH secretion (Funston et al., 1995; Bucholtz et al., 1996; Rutter and Manns,

1987). In cattle, the hypothalamus may recognize low-blood glucose concentrations by a

threshold-dependent fashion, given that GnRH secretion is impaired when glucose availability is

inadequate, but resumed when glucose levels are adequate (Hess et al., 2005). On the other hand,

Wettemann and Bossis (2000) indicated that glucose concentrations in blood are probably not a

major factor controlling follicular growth and ovulation because glucose concentrations in

anestrous heifers were similar to those observed in cycling heifers at least two follicular waves

before resumption of cyclicity. Blood glucose concentrations in ruminants are fairly stable, and

this fact may partially explain the lack of a direct relationship between circulating glucose

concentrations and reproductive function reported by Wettemann and Bossis (2000). Further, the

positive effects of increased circulating glucose through enhanced gluconeogenesis on

reproduction of cattle may be associated with improvements in energy status and concentrations

of other blood metabolites and hormones instead of the increase in glucose availability itself

(Randel, 1990; Hess et al., 2005).

27

Insulin

Bovine insulin is a small peptide hormone containing 51 aminoacid residues and a

molecular weight of 5.7 kDa (Smith, 1966). Insulin is synthesized within the pancreas and

initially stored as a pro-hormone in secretory granules in the pancreatic beta-cells (Nelson and

Cox, 2005). Several stimuli trigger the conversion of proinsulin into active insulin, and

consequent insulin release into the bloodstream (Nelson and Cox, 2005). The main effects of

insulin are on liver, muscle and adipose tissues, where it increases anabolic processes and

consequently decreases tissue catabolism (Nussey and Whitehead, 2001). Insulin directly induces

the uptake of glucose by muscle and fat tissue by recruiting intracellular GLUT4 transporters and

increasing their cell-surface expression, consequently enhancing cell ATP production, glycogen

synthesis in muscle, and lipogenesis in adipose tissues (Nussey and Whitehead, 2001; Nelson

and Cox, 2005). In the liver, insulin promotes glycogenesis but does not modulate glucose

uptake by the hepatocytes via GLUT2 transporter. Insulin also modulates cellular uptake of

amino acids and some electrolytes (Austgen et al., 2003). Although insulin is secreted in pulses

approximately every 10 min, insulin secretion is mainly stimulated by high blood glucose

concentrations (Nussey and Whitehead, 2001), therefore maintaining metabolic homeostasis

because blood glucose is maintained within constant concentrations. Additionally, insulin

secretion is also stimulated by gastrointestinal hormones and neural/paracrine mechanisms

associated with feed intake (Nussey and Whitehead, 2001).

Because of the interaction between insulin and glucose metabolism, the effects of insulin

on performance and reproduction of cattle have been extensively investigated. Similar to

glucose, blood insulin concentrations have been positively associated with feed intake and rates

of BW gain in beet cattle (Vizcarra et al., 1998; Bossis et al., 2000; Lapierre et al., 2000). Cooke

28

et al. (2007a) reported greater mean insulin concentrations (0.89 vs. 0.75 ng/mL, respectively)

and ADG (0.40 vs. 0.30 kg/d, respectively) for grazing heifers consuming citrus pulp-based

supplements instead of iso-caloric and iso-nitrogenous supplements based on sugarcane

molasses. Conversely, Cooke et al. (2007b) reported that forage-fed steers supplemented daily

with a citrus pulp-based supplement had greater ADG (0.30 vs. 0.18 kg/d, respectively) but

inferior mean insulin concentrations (0.46 vs. 0.60 ng/mL, respectively) compared to steers

offered supplements 3x/wk. In terms of reproduction, Arias et al. (1992) reported that GnRH

release from perifused hypothalamic fragments of female rats was dramatically increased when

glucose and insulin were added simultaneously to the perifusion medium compared to those

containing no supplemental glucose, insulin, or both. On the other hand, Hileman et al (1993)

reported that short-term intracerebroventricular administration of insulin failed to increased LH

secretion in lambs, suggesting that insulin alone may not act as a nutritional signal regulating LH

secretion. Glucose uptake by brain cells are dependent on glucose transporters GLUT1, GLUT3,

and GLUT8 (Maher et al., 1994; Olson and Pessin, 1996; Sankar et al., 2002), and GLUT3 is

often considered the main neuronal glucose transporter because of its relative abundance in the

brain (Maher et al., 1994). These transporters, especially GLUT3, have been shown to be rather

insensitive to insulin; therefore brain cells are considered insulin unresponsive, and glucose

uptake in neurons rely on other mechanisms (Heidenreich et al., 1989). Conversely, Livingstone

et al. (1995) indicated that the hypothalamic neurons may also express GLUT4 and therefore

absorb glucose via insulin stimuli. Nevertheless, lack of insulin sensitivity by the majority of

brain cells might explain why LH secretion in lambs was not increased by insulin injections

(Hileman et al., 1993), and GnRH release from perifused hypothalamic fragments were not

increased by addition of only insulin to perifusion medium (Arias et al., 1992).

29

Other studies, however, reported a positive effect of insulin on reproductive efficiency of

cattle. Sinclair et al. (2002) reported that postpartum anestrous beef cows with low plasma

insulin concentrations (< 5 mIU/L) failed to ovulate the first dominant follicle in response to

restricted calf access compared to cows with moderate plasma insulin (5 to 8 mIU/L), and

attributed this result to an inadequate responsiveness of the dominant follicle to the LH surge that

frequently accompanies calf removal. Gong et al. (2002) fed lactating dairy cows with diets to

stimulate (propiogenic ingredients) or maintain insulin concentrations at normal levels

(acetogenic ingredients) from d 0 to d 50 post-calving, and reported that gonadotropin

concentrations, milk yield, and changes in BW and BCS were similar between treatments, but

cows fed insulin-stimulating diets had greater plasma insulin concentrations (0.40 vs. 0.27

ng/mL) and reduced postpartum interval (34 vs. 48 d) compared to cows fed the acetogenic diet.

Therefore, it can be speculated that insulin influences reproductive efficiency of cattle at the

ovarian level by modulating the activity of gonadotropins rather than influencing gonadotropin

synthesis by the brain. Insulin has been shown to be a potent stimulator of estradiol synthesis in

the ovary of cattle (Spicer and Echternkamp, 1995), and estradiol is required for the ovulatory

LH surge (Kesner et al., 1981) and also synthesis of LH receptors in the membranes of granulosa

cells within the dominant follicle (Bao and Garverick, 1998). Consequently, cows with low

insulin concentrations may have impaired LH surge, and/or reduced numbers of LH receptors in

the dominant follicle, failing to ovulate even if the LH surge is present (Diskin et al., 2003).

Insulin-like Growth Factor I

Bovine IGF-I is a single chain polypeptide hormone that structurally resembles proinsulin

(Gluckman et al., 1987). The IGF-I molecule contains 70 aminoacid residues with a molecular

weight of approximately 7.6 kDa (McGuire et al., 1992; Etherton, 2004), and is synthesized by

30

most, if not all, body tissues (Le Roith et al., 2001). Liver is the main source of circulating IGF-I

(D’Ercole et al., 1984), and IGF-I synthesized by other tissues mainly exert autocrine and

paracrine effects (McGuire et al., 1992; Johnson et al., 1998). Synthesis of IGF-I is mainly

regulated by GH (McGuire et al., 1992), and its secretion into the circulation occurs in a constant

pattern (Thissen et al., 1994). The majority of IGF-I found in blood or other body fluids is bound

with high affinity to one of six different IGFBPs, which vary in length from 201 to 289

aminoacid residues, and molecular weight from 24 to 44 kDa (Thissen et al., 1994; Beattie et al.,

2006). These IGFBPs serve mainly as circulatory transport vehicles, retard IGF-I degradation,

and modulate the actions of IGF-I in target cells by enhancing or blocking IGF-I activity (Le

Roith et al., 2001). In the circulation, more than 90% of IGF-I is bound to IGFPB-3 (Martin and

Baxter, 1992), whereas the remainder of circulating IGF-I is either free (< 5 %) or bound to

IGFBP-1, IGFBP-2, or IGFBP-4 (Clemmons, 1991). The complex formed by IGF-I and IGFBP-

3 also contains an acid-labile unit, and has a molecular weight of approximately 150 kDa,

whereas IGFBP-1 and IGFBP-2 associate directly with IGF-I into smaller complexes (30 to 40

kDA; Thissen et al., 1994). Due to its large molecular size, the IGFBP-3/IGF-I complex cannot

leave the circulatory system and reach target tissues. Therefore, IGF-I is released from this

complex and associates with IGFBP-1 and IGFBP-2 in the circulation, forming smaller

complexes that can cross the capillary endothelium and deliver IGF-I to target tissues (Thissen et

al., 1994; Le Roith et al., 2001). The presence of IGFBP-5 and IGFBP-6 is not abundant in the

blood; however, IGFBP-5 has been associated with bone and muscle development because of its

role in cell survival, differentiation, and apoptosis (Beattie et al., 2006; Dayton and White, 2008).

The major anabolic effects of IGF-I are inherent to protein and carbohydrate metabolism,

and also cell replication and differentiation (Jones and Clemmons, 1995; Le Roith et al., 2001).

31

At target tissues, IGF-I binds to its receptor, which is 60% alike the insulin receptor at the

aminoacid level (Ulrich et al., 1986), and initiates a cascade of cellular events beginning with

IRS-1 phosphorylation (Jones and Clemmons, 1995). As a result, anabolic effects are stimulated

via pathways such as PI3 kinase and MAPK kinase, whereas catabolism is inhibited via the BAD

phosphorylation (Le Roith et al., 2001). Other processes such as cell function, cell secretory

activity, and also immune response are influenced positively by IGF-I (Jones and Clemmons,

1995). To fuel all of these activities, IGF-I has insulin-like stimulatory actions on most cells with

IGF-I receptors, stimulating glucose and aminoacid uptake, and also glycogen synthesis (Jacob et

al., 1989; Dimitriadis et al., 1992).

In beef cattle, feed intake and BW gain have been associated positively with IGF-I

(Bossis et al., 2000; Armstrong et al., 2001; Rausch et al., 2002), but negatively with GH

concentrations (Ellenberger et al. 1989; Granger et al., 1989; Lapierre et al., 2000). Therefore,

IGF-I synthesis in cattle at adequate levels of nutrition relies on additional mechanisms besides

direct GH stimuli. Insulin facilitates IGF-I synthesis in the liver by enhancing binding of GH to

hepatic GH receptors (Houston and O’Neill, 1991; McGuire et al., 1995; Molento et al., 2002),

whereas concentrations of insulin typically correlate positively with IGF-I concentrations in

cattle (Keisler and Lucy, 1996; Webb et al., 2004; Cooke et al., 2007a). Other studies have

shown that IGF-I synthesis is also regulated by thyroid hormones. Hypothyroid animals have

impaired IGF-I synthesis (Burnstein et al., 1979), whereas hepatic GH binding is enhanced by

thyroid hormones, implicating their ability to potentiate IGF-I synthesis via GH stimuli (Barash

and Posner, 1989; Hochberg et al., 1990). Additionally, aminoacid restriction, especially

tryptophan, decreased IGF-I synthesis in rats with adequate glucose, insulin, and GH levels

(Maiter et al., 1989; Phillips et al., 1991). These nutritional and physiological modulations of

32

IGF-I synthesis mainly occur at a pretranslational stage because changes in IGF-I concentrations

are typically preceded by changes in hepatic IGF-I mRNA expression (Thissen et al., 1994).

Recent studies indicated that IGF-I is a major metabolic signal regulating reproduction in

cattle (Wettemann and Bossis, 2000). Research with developing beef heifers reported that IGF-I,

but not glucose and insulin, was positively associated with ADG and reproductive performance

(Cooke et al. 2007a; Cooke et al., 2008a). Granger et al. (1989) reported that plasma IGF-I

concentrations were negatively associated with age at puberty, and Bossis et al. (1999) reported

that feed restriction of cycling heifers leads to significant decreases in plasma IGF-I and

culminates with onset of anestrous. Cooke et al. (2007a) reported similar ADG, but greater mean

IGF-I concentrations for beef heifers that reach puberty (120 vs. 99 ng/mL) and conceive (123

vs. 111 ng/mL) as yearlings compared those that remain pre-pubertal and/or non-pregnant. In

mature beef cows, blood concentrations of IGF-I were increased for those that resumed estrous

cycles within 20 wk postpartum compared to cows that remained in anestrous (Roberts et al.,

1997). Because of their close association with IGF-I, the IGFBPs have also been shown to

influence performance and reproduction of cattle. Blood concentrations and mRNA expression

of IGFBP3 are associated positively with nutrient intake and rates of BW gain in cattle (Thissen

et al., 1994; Vestergaard et al., 1995; Rausch et al., 2002). On the other hand, IGFBP2 is

believed to decrease the bioavailability and consequently conserve IGF-I when the hormone

synthesis is reduced (Jones and Clemmons, 1995; Armstrong et al., 2001). Ruminants in poor

nutritional status have increased blood concentrations and mRNA expression of IGFBP2 in

several tissues (Vandehaar et al., 1995; Armstrong and Benoit, 1996; Armstrong et al., 2001).

Roberts et al. (1997) reported that serum concentrations of IGFBP-2 were decreased whereas

serum concentrations of IGFBP-3 were increased in beef cows that resumed estrus by 20 wk

33

postpartum compared with anestrous cows. Based on these and other observations, Hess et al.

(2005) postulated that IGF-I and IGFBP3 are positive signals and nutritional mediators into the

reproductive axis.

The effects of IGF-I on cattle reproduction, more specifically within the hypothalamus-

pituitary-ovarian axis, are believed to be via autocrine, paracrine and endocrine mechanisms.

Wettemann et al. (2003) complied results from studies conducted with different mammals, and

reported that IGF-I receptors were detected in the hypothalamus and pituitary, indicating that

GnRH and gonadotropin secretion are potentially modulated by IGF-I. In their review, Diskin et

al. (2003) also cited studies indicating that IGF-I influences hypothalamic and pituitary functions

of mammals. Conversely, Rutter et al. (1989) reported that increases in IGF-I via glucose

supplementation to beef cows at adequate planes of nutrition did not influence LH pulsatile

pattern. Consequently, a large portion of IGF-I effects on reproduction of cattle may rely on its

effects within the ovary. Receptors for IGF-I and also IGF-I mRNA were detected in several

ovarian cells, such as granulosa, thecal, and luteal cells (Spicer and Echternkamp, 1995;

Armstrong and Benoit, 1996; Bao and Garverick, 1998). Previous research demonstrated that

IGF-I enhances the responsiveness of ovarian cells to FSH and LH, stimulating cell mitosis,

follicle growth, and steroidogenesis (Spicer and Stewart, 1996; Stewart et al., 1995; Armstrong

et al. 2001). The ovulation process appears to also be dependent on IGF-I stimulus. Roche (2006)

indicated that follicle size, LH pulsatility, and systemic concentrations of IGF-I are the factors

responsible for the successful ovulation of the dominant follicle. Recent data by Ginther et al.

(2001) indicated that the dominant follicle contains greater free IGF-I concentrations (12 vs. 4

ng/mL) and reduced IGFBP-2 concentration (492 vs. 648 ng/mL) within its fluid compared to

the second-largest follicle after deviation. Further, follicle diameter, estradiol and free IGF-I

34

concentrations were significantly and positively correlated with each other, and negatively

correlated with IGFBP2 concentrations within the dominant follicle postdeviation (Ginther et al.,

2001). Others also reported greater concentrations of total IGF-I and decreased or undetectable

concentrations of IGFBP2, -4, and -5 in dominant follicles compared to subordinate follicles

(Echternkamp et al., 1994; de la Sota et al., 1996; Mihm et al., 1997). Therefore, IGF-I seems to

play an important role in follicle responsiveness to gonadotropins and consequently in follicle

growth and steroid synthesis, which are requisites for successful ovulation and consequent

fertility of cattle.

Recent data from dairy cattle indicated beneficial effects of IGF-I on early pregnancy.

Moreira et al. (2002) reported that addition of IGF-I to in vitro embryo cultures increased the rate

of embryo development to the blastocyst stage and increased the number of blastocyst cells.

Further, Jousan and Hansen (2004) reported that addition of IGF-I to in vitro cultures of embryos

exposed to heat shock increased total embryo cell number and alleviated cell apoptosis. These

positive effects of IGF-I on embryonic development allows for greater secretion of interferon tau

by the embryo (Bilby et al., 2006), and this protein has been shown to regulate secretion of PG

by the uterine endometrium and influence positively the establishment and maintenance of early

pregnancy in cattle (Thatcher et al., 2001).

Progesterone

Another mechanism by which nutrition affects reproduction in cattle is by altering

hepatic P4 metabolism. As commented previously, P4 is required for adequate attainment of

puberty, resumption of estrous cycles, and also establishment and maintenance of pregnancy

(Gonzalez-Padilla et al., 1975; Spencer and Bazer, 2002; Looper et al., 2003). Research has

shown that infrequent intake of large amounts of feed decreases circulating P4 concentrations in

35

beef and dairy cattle. Vasconcelos et al. (2003) reported that P4 concentrations of lactating

pregnant Holstein cows provided 100 % of daily diet at once or half of the diet every 12 h were

decreased by 1 h and remained depressed until 8 to 9 h after feeding compared to P4

concentrations of cows fed a quarter of diet every 6 h or unfed cows. Cooke et al. (2007a)

reported that plasma P4 concentrations of grazing pubertal heifers offered energy-based

supplements 3 times weekly decreased by approximately 11% on supplementation days

compared to non-supplementation days, whereas forage-fed cows similarly supplemented had

decreased P4 concentrations 4 h after supplements were offered. These reductions in circulating

P4 concentrations after large meal consumption can be attributed to increases in hepatic blood

flow and consequent steroid metabolism. After feed intake, liver blood flow is intensified to

transport digested nutrients from the gut through the liver and on to the rest of the body

(Huntington, 1990; Sangsritavong et al., 2002). In the liver, P4 is inactivated or catabolized

mainly by enzymes of the subfamilies cytochrome P450 2C and cytochrome P450 3A, and the

resultant metabolites (21-hydroxyprogesterone and 6β-hydroxyprogesterone, respectively;

Murray 1991, 1992) are eliminated primarily in the urine (Steimer, 2003). Therefore, infrequent

offering of large amounts of feed to cattle may be detrimental to their reproductive function.

During periods when P4 is fundamental, feed intake should be managed properly to avoid

sudden decreases in circulating P4 concentrations.

Temperament, Acclimation, and Reproduction of Cattle

Assessment of Temperament in Beef Cattle

For over a century, the word temperament has been used to define the behavioral responses

of cattle when exposed to human handling (Pott, 1918; Fordyce et al., 1988). As cattle

temperament worsens, their response to human contact or any other handling procedures

36

becomes more agitated and/or aggressive. Within the beef cattle industry, some producers do

select cattle for temperament mainly for safety reasons, whereas the productive and economic

implications of this trait are still not well established (Voisinet et al., 1997a).

Cattle temperament can be assessed and evaluated by diverse techniques. Burrow and

Corbet (2000) described and categorized these into non-restrained and restrained techniques.

Within the non-restrained techniques, cattle temperament is evaluated by their fear or aggressive

response to man when they are free to move within the evaluation area. Examples of these

techniques are the chute exit velocity and the pen score. Exit velocity quantifies the speed of an

individual animal immediately after it leaves the squeeze chute by measuring the time required

for the animal to travel a pre-determined distance. The pen score evaluates the behavioral

response of an individual animal when it enters a small pen and interacts with a person standing

inside the pen. Typically in a 1-5 scale, the pen score increases as the animal response becomes

more aggressive toward the person. The restrained techniques evaluate cattle temperament when

these are physically restricted, such as in the squeeze chute (Burrow and Corbet, 2000). The

major problem with these techniques is that cattle with excitable temperament may “freeze”

when restrained, and consequently not express their true behavior during these assessments

(Burrow and Corbet, 2000). An example of the restrained techniques is the chute score, also

denominated crush score. Cattle are individually restrained in the chute and scored in a 1-5 or

other scale according to its behavior, where chute score increases as shaking and struggling

intensifies (Voisinet et al., 1997a; Arthington et al., 2008). Other methods to assess cattle

temperament have also been reported, such as evaluation of the behavioral responses of small

groups of cattle confined to a pen (Hammond et al., 1996), evaluation of chute score without

squeezing the animal in the chute (Curley et al., 2006), assessment of the amount of human force

37

required to move the animal into the chute, and also the estimation of the animal speed when it

enters the chute (Baszczak et al., 2006). However, the first three methods reported herein (chute

score, pen score, and exit velocity) have been shown to be repeatable within animals (Boivin et

al., 1992; Grandin, 1993; Curley et al., 2006); therefore reliable to quantify cattle temperament,

and also relatively simple to carry out during cattle handling procedures.

Cattle temperament is influenced by several factors such as sex, age, and horn status

(Fordyce et al., 1988; Voisinet et al., 1997a), but none of these characteristics has been shown to

affect cattle temperament as much as breed type. Several studies indicated that cattle with high

Brahman influence have more excitable temperament compared to B. taurus cattle. Hearnshaw

and Morris (1984) evaluated behavioral responses similar to the chute score method of calves

sired by B. indicus (Brahman, Braford, and Africander) or by B. taurus (Hereford, Simmental,

and Friesian), and reported that the mean temperament score of Brahman-sired calves was

greater than that of B. taurus-sired calves (1.96 vs. 1.05, respectively). Further, Brahman-sired

calves had greater scores compared to Braford- or Africander-sired calves (1.95, 1.38, and 1.25,

respectively), whereas similar temperament scores were detected among B. taurus breeds.

Fordyce et al. (1988) reported that Brahman × Shorthorn (50:50 breed composition) steers and

non-lactating mature cows were more temperamental than Shorthorn cohorts. Voisinet et al.

(1997a) reported that yearling steers and heifers with Brahman breeding (Braford, Red Brangus,

and Simbrah) had greater temperament scores compared to Angus and Angus-crosses (3.46 vs.

1.80 on a 5-point scale, respectively). These data indicate that Brahman-crossbred cattle are

potentially difficult to be controlled and handled, which can bring serious management problems

to cattle operations especially if these are based on extensive grazing systems where cattle have

infrequent interaction with humans, such as the cow-calf operations in Florida.

38

Stress and Excitable Temperament

Stress response can be characterized as the reaction of an animal to factors that

potentially influence its homeostasis (Moberg, 2000), whereas animals that are unable to cope

with these factors are classified as stressed or distressed (Dobson and Smith, 2000; Moberg,

2000). Based on this concept, the agitated and/or aggressive responses expressed by cattle with

excitable temperament when exposed to human interaction and other handling procedures can be

attributed to their inability to cope with these situations and therefore classified as a stress

response. In addition to altered behavior, these animals may also experience changes in their

neuroendocrine and nervous systems (Moberg, 2000). The neuroendocrine response to stress is

mainly mediated by the hypothalamus-pituitary-adrenal axis (Dobson and Smith, 2000), and the

hormones produced within this axis influence several aspects in cattle, such as growth, immune

response, and reproductive function (Fell et al., 1999; Dobson et al., 2001).

The Hypothalamic-Pituitary-Adrenal Axis

The neuroendocrine stress response begins when the central nervous system of animals,

including cattle, perceives a threat to homeostasis and activates the secretion of corticotrophin-

release hormone (CRH) by the neurosecretory cells of the hypothalamus into the pituitary portal

blood system (Matteri et al., 2000; Smagin et al., 2001). The sympathetic nervous system is also

stimulated by stressors, further stimulating CRH secretion by the hypothalamus and culminating

in the synthesis of catecholamines by adrenal glands (O’Connor et al., 2000). In the posterior

pituitary, CRH stimulates vasopressin (VP) release and then acts in synergy with VP to stimulate

ACTH synthesis by corticotrophs in the anterior pituitary (Minton et al., 1994; O’Connor et al.,

2000). Secretion of ACTH via CRH stimulus is also enhanced by the presence of cytokines and

other inflammatory mediators produced during periods of infection or inflammation (Carroll and

39

Forsberg, 2007). Release of ACTH into the peripheral circulation occurs in a pulsatile fashion

with a circadian rhythm and stimulates steroidogenesis in the adrenal glands (O’Connor et al.,

2000; Stewart, 2003). Further, ACTH secretion and consequent adrenal steroidogenesis are the

greatest in the morning, decreases throughout the day, and reach nadir levels in the evening

(Thun et al., 1981; Arthington et al., 1997). The adrenal glands consist of two distinct regions:

the adrenal cortex and adrenal medulla. Three types of steroids are synthesized by the adrenal

cortex: glucocorticoids such as cortisol, mineralocorticoids such as aldosterone, and sexual

steroids such as androgens and P4 (Brown, 1994). The adrenal cortex synthesizes these steroids

from cholesterol mainly in response to ACTH, although research has shown that circulating

forms of CRH and VP also regulate cortisol synthesis (Carroll et al., 1996). Conversely, the

adrenal medulla is not regulated by ACTH, and synthesizes catecholamines (mainly epinephrine

and norepinephrine) directly in response to sympathetic nervous system stimuli (Carroll and

Forsberg, 2007).

Mineralocorticoids, more specifically aldosterone, are required to maintain adequate

mineral balance within the body (Brown, 1994). Glucocorticoids and catecholamines elicit

several biologic effects in the organism of stressed animals in order to prepare and sustain their

behavioral responses to stress, usually designated as the “fight or flight” response.

Cathecolamines are rapidly released from the adrenal medulla when animals are exposed to

stressors because of their nervous stimulus nature. Within seconds, epinephrine increases heart

rate and oxygen flow to tissues, whereas norepinephrine mainly induces vasoconstriction to

increase blood pressure (Brown, 1994; Carroll and Forsberg, 2007). Further, epinephrine

stimulates breakdown of liver and muscle glycogen into glucose and stimulates mobilization of

fatty acids from adipose reserves by increasing enzymatic activity in these tissues (Nelson and

40

Cox, 2005). Both catecholamines regulate CRH, ACTH, and cortisol secretion, whereas their

synthesis is in turn facilitated by cortisol (Plotsky et al., 1989; Carroll and Forsberg, 2007).

Cortisol also degrades liver reserves and muscle and adipose tissues to increase the availability

of energy to the animal. However, cortisol modulates enzyme synthesis instead of enzyme

activity; therefore target cells are impacted in a chronic manner (Nelson and Cox, 2005). Besides

fatty acid mobilization, cortisol stimulates protein breakdown in muscle tissues and enhances the

mobilization of the resultant aminoacids to the liver where they serve as substrate for

gluconeogenesis (Carroll and Forsberg, 2007). Further, cortisol has been shown to suppress the

immune response by decreasing synthesis of cytokines. This process is required to prevent

autoimmune diseases; however, if cortisol concentrations are chronically elevated such as during

prolonged stressful situations, it can lead to cases of immunosuppresion (Kelley, 1988).

As commented previously, the adrenal cortex is capable of producing sexual hormones

because these are intermediates or by-products in the synthesis of mineralocorticoids and

glucocorticoids. According to Stewart (2003), the first step of steroidogenesis in the adrenal

gland is the uptake of cholesterol by the mitochondria of adrenal cortex cells, and this process is

mediated by the steroidogenic acute regulatory protein. In the mitochondria, cholesterol is

converted into pregnolone by the enzyme cholesterol desmolase. Pregnolone can be either

converted into P4 by the enzyme β-hydroxysteroid dehydrogenase, or converted into 17-

hydroxypregnolone by the enzyme 17α-hydroxylase. Both P4 and 17-hydroxypregnolone can be

converted into 17-hydroxyprogesterone and directed towards cortisol or androstenedione

synthesis, whereas P4 only can be directed towards aldosterone synthesis. Further,

androstenedione can be converted into estradiol in tissues by the enzymes aromatase and 17β-

hydroxysteroid (Bulun and Adashi, 2003). Therefore, elevated secretion of ACTH may stimulate

41

accumulation and secretion of sexual steroids by the adrenal gland concomitantly with increased

synthesis of glucocorticoids and mineralocorticoids, and this process may influence the

reproductive functions of animals under stressful situations.

Physiologic Responses to Excitable Temperament in Cattle

Several studies were conducted to evaluate the effects of excitable temperament on the

physiologic responses of livestock, more specifically within the hypothalamic-pituitary-adrenal

axis. Stahringer et al. (1990) reported that temperament scores of Brahman heifers were

positively correlated with mean cortisol concentrations (r = 0.65). Hammond et al. (1996)

reported that Brahman heifers had greater temperament scores (scale 1 to 5) and plasma cortisol

concentrations compared to Angus heifers during the summer (3.0 vs. 2.0 for temperament

scores, and 39.4 vs. 28.4 ng/mL for cortisol) and fall (2.5 vs. 2.2 for temperament scores, and

36.9 vs. 18.7 ng/mL for cortisol). Fell et al. (1999) reported that plasma cortisol concentrations

were greater for beef steers classified as nervous compared to those classified as calm prior to

weaning (156.5 vs. 95.1 nmol/L), after weaning (127.8 vs. 84.3 nmol/L), and at feedlot entry

following truck transportation for 350 km (397.8 vs. 229.7 nmol/L). Curley et al. (2006)

collected blood samples and assessed pen score, exit velocity, and chute score of 66 yearling

Brahman bulls on three different occasions (60 d apart), and reported that only pen score and exit

velocity were positively correlated with plasma cortisol concentrations (r ≥ 0.25 and 0.26,

respectively). Curley et al. (2008) stratified Brahman heifers by exit velocity and classified the

fastest ones as temperamental (mean exit velocity = 3.14 m/s), and the slowest ones as calm

(mean exit velocity = 1.05 m/s). The authors administered heifers from both groups with bovine

CRH, collected blood samples every 15 min from -6 h to 6 h post-challenge, and reported that

temperamental heifers had greater plasma ACTH and cortisol concentrations prior to and

42

following CRH challenge compared to calm heifers. The same authors administered ACTH to

the same heifers 14 d after the CRH challenge, collected blood samples in a similar schedule, and

reported greater serum cortisol concentrations for temperamental heifers prior to and during the

last 2 h following ACTH challenge. These data suggest that cattle with excitable temperament

experience stimulated hypothalamus-pituitary-adrenal axis function when exposed to humans

and handling procedures, resulting in increased production and circulating concentrations of

cortisol. This outcome is one of the reasons why cortisol is commonly considered the paramount

to the physiologic stress response (Sapolsky et al., 2000).

The effects of excitable temperament on the synthesis of sexual steroids by the adrenal

gland have been poorly evaluated thus far. Nevertheless, a fair amount of studies have reported

that stressors such as chute restraining, heat, and ACTH challenge stimulate the synthesis of P4

by the adrenal gland. Roman-Ponce et al. (1981) reported that lactating dairy cows with

restricted access to shade during the summer in Florida had greater P4 concentrations during the

estrous cycle in addition to increased mean cortisol concentrations compared to cows with free

access to shade. Hollenstein et al. (2005) restrained or not (control) ovariectomized Brown Swiss

cows in a squeeze chute for 2 h, and reported that restrained cows had greater mean cortisol

(33.8 vs. 3.9 ng/mL, respectively) and mean P4 concentrations (1.2 vs. 0.3 ng/mL, respectively)

compared to control cows during the restraining period. Similarly, Thun et al. (1998) reported

that Brown Swiss cows in estrus experienced consistent increases in plasma P4 and cortisol

concentrations during chute restraining for an 8-h period, whereas the effects of restraining on

plasma concentrations of epinephrine and norepinephrine were inconsistent, indicating that

plasma cortisol concentrations may be a more reliable indicator of stress in cattle than

cathecolamines. Yoshida and Nakao (2006) reported that administration of ACTH to

43

ovariectomized Friesian cows in 4 different doses (3, 6, 12, and 25 UI) linearly increased plasma

P4 and cortisol concentrations within 6 h after challenge, whereas peak concentrations of P4 and

cortisol across treatments were positively correlated (r = 0.70). Research by Willard et al. (2005)

reported that administration of ACTH (1.0 IU per kg of BW) to pregnant Brahman heifers

resulted in a 1.5 fold-increase in plasma P4 concentrations 15 min after challenge, whereas P4

concentrations remained constant for heifers administered saline. Conversely, other studies

indicated that administration of ACTH may decrease circulating P4 concentrations of cattle by

impairing corpus luteum function. Da Rosa and Wagner (1981) reported that heifers

administered ACTH daily had greater plasma P4 concentrations on d 3 and 4 after estrus

detection, reduced P4 concentrations during mid-cycle (d 8 to 19 after estrus detection), and

greater plasma corticoid concentrations throughout the estrous cycle compared to cows

administered saline. Da Rosa and Wagner (1981) also reported that administration of corticoids

to adrenalectomized heifers increased plasma corticoid concentrations and decreased plasma P4

concentrations during the estrous cycle compared to ACTH or saline administration. Similar

results were reported by Wagner et al. (1972), indicating that ACTH challenge increased plasma

P4 concentrations early in the estrous cycle when the corpus luteum is not fully functional by

stimulating adrenal P4 production, whereas elevated cortisol through ACTH treatment decreased

corpus luteum function and consequent luteal P4 synthesis during mid-estrous cycle. In

conclusion, research efforts indicate that stimulation of the hypothalamus-pituitary-adrenal axis

increases substantially the production of P4 by the adrenal gland but decreases luteal P4

synthesis, and these events may impact the reproductive function of cattle.

Fear responses in cattle with excitable temperament may also trigger their acute phase

response, although no research thus far associated temperament scores directly with circulating

44

concentrations of acute phase proteins. The acute phase response is a component of the innate

immune system and is stimulated by the proinflammatory cytokines IL-1, IL-6, and tumor

necrosis factor - α, which are secreted by macrophages and other cells in response to external and

internal stimuli such as infections, inflammations, and trauma (Murata et al., 2004; Petersen et

al., 2004). Upon stimulation, these cytokines elicit two major acute responses: increased body

temperature (fever) and hepatic synthesis of the acute phase proteins (Carroll and Forsberg,

2007). Increases in body temperature, which is regulated by proinflammatory cytokines through

the stimulation of PGE2, serve mainly to accelerate enzymatic and cellular processes associated

with the immune response, and also to decrease the survival rates of pathogens (Carroll and

Forsberg, 2007). In the liver parenchyma, the proinflammatory cytokines stimulate hepatocytes

to produce acute phase proteins, such as ceruloplasmin and haptoglobin, which play important

roles in tissue protection, repair, and reestablishment of homeostasis (Murata et al., 2004; Tizard,

2004; Carroll and Forsberg, 2007). In cattle, the acute phase response is stimulated by stressful

management procedures such as transportation, vaccination, and weaning (Arthington et al.,

2003; Arthington et al., 2005; Cooke et al., 2007c). Recent studies have also indicated that

animals subjected to physical or physiologic stressors such as exposure to novel environments

and body restraint experience substantial increases in IL-1 and IL-6 synthesis, and consequent

stimulation of the acute phase response and hypothalamic-pituitary-adrenal axis (Turnbull and

Rivier, 1999). More specifically, IL-1 may cross the blood brain barrier and stimulate CRH

secretion by the hypothalamus, whereas IL-1 and IL-6 are potent stimulators of ACTH synthesis

by the pituitary and consequent cortisol production by the adrenal cortex. In turn, cortisol exerts

a negative feedback onto cytokine activity to prevent unnecessary inflammation, as commented

previously (Kelley, 1988; O’Connor et al., 2000; Carroll and Forsberg, 2007). Still, hepatic

45

synthesis of acute phase proteins was stimulated by administration of glucocorticoids to mature

cows and ACTH to piglets (Yoshino et al., 1993; Burger et al., 1998), and in vitro by addition of

glucocorticoids to cultures of bovine liver slices (Higuchi et al., 1994). Therefore, cattle

exhibiting excitable temperament when exposed to humans and handling procedures may also

experience elevated concentrations of acute phase proteins because of the close association

between the hypothalamic-pituitary-adrenal axis and the acute phase response.

Temperament and Performance of Beef Cattle

The effect of temperament on BW gain of feedlot cattle has been evaluated. Burrow and

Dillon (1997) detected a negative association between daily BW gain, final liveweight, and

carcass weight with exit velocity of Brahman × Shorthorn steers and heifers. Voisinet et al.

(1997) classified B. taurus and B. indicus × B. taurus steers and heifers by temperament (scale 1

to 5), and reported that B. taurus cattle with the calmest temperament (score 1) had a 0.19 kg/d

increase in ADG compared to cohorts with the greatest temperament scores (score 3), whereas B.

indicus-crosses with greater temperament scores (4 and 5) had an approximate decrease of 0.10

kg/d in ADG compared to cohorts with low temperament scores (1 and 2). Fell et al. (1999)

reported greater ADG for Angus × Hereford steers with moderate to low chute scores and exit

velocity (1.04 kg/d) compared to cohorts with high chute scores and exit velocity (1.46 kg/d).

Nkrumah et al. (2007) indicated that chute exit velocity of B. taurus-crosses was negatively

correlated with ADG (r = -0.26) and DMI (r = -0.35), but not with feed conversion (r = 0.03).

Similarly, Brown et al. (2004) reported that chute exit velocity of Bonsmara bulls was negatively

correlated with ADG (r = -0.25) and DMI (r = -0.34), but not with feed efficiency measurements.

These data suggest that cattle with excitable temperament experience reduced performance

compared to cattle with good temperament, and this effect can be attributed to reduced feed

46

intake in temperamental animals. Further, cattle with excitable temperament may also have

altered metabolism and greater partitioning of nutrients towards physiologic and behavioral

responses to stressors compared to calm animals, which results in further decreases in nutrient

availability to sustain their maintenance and growth requirements.

There is a nutrient requirement for the synthesis and release of hormones and metabolites

within the hypothalamus-pituitary-adrenal axis and acute phase response (Carroll and Forsberg,

2007). Additionally, corticoids, cathecolamines, and proinflammatory cytokines stimulate

degradation of muscle and adipose tissues to release energy and also aminoacids that are directed

towards gluconeogenesis or synthesis of the acute phase proteins by the liver (Johnson et al.,

1997; Nelson and Cox, 2005; Carroll and Forsberg, 2007), which may further contribute to the

decreased growth rates of temperamental cattle. Elevated circulating concentrations of

glucocorticoids and proinflammatory cytokines also reduces circulating IGF-I concentrations,

perhaps to shift nutrients from growth to the control of infection and/or inflammation, and also to

support the “fight or flight” response. Studies have shown that cattle receiving endotoxin or

corticoid challenges have decreased pituitary GH synthesis because of stimulated somatostatin

release by the hypothalamus, in addition to a reduced number of tissue receptors for GH, causing

impaired IGF-I synthesis in the liver and many other parts of the body (Elsasser et al., 1997;

Maciel et al., 2001). Associating these physiological events with animal performance, studies

conducted by Fell et al. (1999) and Qiu et al. (2007) indicated that plasma concentrations of

cortisol and acute phase proteins, respectively, were negatively correlated with BW gain of

cattle. Therefore, the physiological and behavioral responses expressed by temperamental cattle

when exposed to human or handling procedures may act as a nutrient sink and impair the

47

growth-promoting effects of IGF-I, resulting in decreased performance compared to cattle with

good temperament.

Temperament and Reproduction of Beef Cattle

Because of the proximity in the endocrine control of both stress and reproductive axis

(hypothalamic-pituitary-adrenal vs. hypothalamic-pituitary-ovarian, respectively), the effects of

several types of stressors on cattle reproduction have been investigated. Besides the deleterious

effects on nutritional status and circulating concentrations of IGF-I, the hormones and

metabolites associated with stress directly affect the hypothalamic-pituitary-ovarian axis.

Diseases and infections (Dobson et al., 2001), excessive heat (Hansen and Arechiga, 1999), and

even transportation (Edwards et al., 1987; Harrington et al., 1995) were reported to decrease

reproductive performance of cattle. Plasse et al. (1970) classified 2-yr-old Brahman heifers

according to temperament (1 = calm, 2 = moderate, and 3 = nervous) and reproductive

performance (heifers with poor reproductive performance received the greatest scores), and

reported that temperament and reproductive scores were positively correlated (r = 0.40), whereas

temperament score was negatively correlated with length of estrus (r = -0.33). The authors

suggested that consideration of temperament in selection programs might have a positive

influence on the reproductive efficiency of the cowherd. Nevertheless, the detrimental effects of

excitable temperament on reproductive performance and function of cattle still require further

investigation.

Within the hypothalamus, elevated concentrations of CRH appear to inhibit GnRH

secretion by neurosecretory cells in mammals (Moberg et al., 1991). These events were further

verified in livestock by Battaglia et al. (1998). These authors evaluated plasma concentrations of

CRH, vasopressin, and GnRH in pituitary portal blood of ewes administered endotoxin or saline,

48

and reported that endotoxin challenge potently stimulated CRH and vasopressin secretion and

suppressed GnRH release into the pituitary portal blood system. Additionally, the authors

reported decreased LH pulsatile secretion in ewes administered endotoxin as a consequence of

suppressed GnRH secretion. Other studies reported that ACTH and cortisol may decrease LH

synthesis by the pituitary even when GnRH secretion is adequate. Li and Wagner (1983)

reported that continuous administration of ACTH to intact heifers, or glucocorticoids to

adrenalectomized heifers, decreased LH release following GnRH challenge compared to heifers

infused with saline. The same authors reported that addition of glucocorticoids to in vitro

cultures of pituitary cells decreased basal or GnRH-stimulated LH secretion compared to cultures

containing no glucocorticoids, and attributed these outcomes to deleterious effects of

glucocorticoids on cellular mechanisms required for adequate LH synthesis and release. Dobson

et al. (2000) reported that daily ACTH administration to dairy and beef heifers beginning of d 15

of the estrous cycle increased plasma cortisol and disrupted LH pulsatile secretion, resulting in

decreased estradiol concentrations, delayed LH surge, and consequent late or failed ovulation.

The negative effects of stress hormones on follicular estradiol synthesis are associated to

impaired LH pulsatility and consequent retarded follicular growth, and may further compromise

the estradiol signaling in the hypothalamus-pituitary for onset of estrus, LH surge, and

consequent ovulation (Smith and Dobson, 2002). Hein and Allrich (1992) administered ACTH or

vehicle to dairy heifers 30 h following corpus luteum regression by PGF2α treatment and reported

that ACTH-treated heifers experienced delayed peak of estradiol, delayed onset of estrus (63 vs.

44 h, respectively), and greater peak concentrations of serum cortisol (91.0 vs. 23.5 ng/mL,

respectively) and P4 (4.1 vs. 1.0 ng/mL, respectively) compared with vehicle-treated heifers. The

authors suggested that elevated P4 concentrations likely originated from the adrenal gland

49

delayed the endocrine signaling for onset of estrus and ovulation. The same authors administered

ACTH or vehicle to ovariectomized dairy cows 10 h following estradiol benzoate treatment to

induce estrus, and reported that ACTH-treated cows experienced decreased occurrence of estrus

(16% vs. 91% of cows exhibiting estrus, respectively), reduced peak concentrations of estradiol

(36.9 vs. 47.5 pg/mL, respectively), and increased peak concentrations of serum cortisol (80.5 vs.

43.4 ng/mL, respectively) and P4 (3.0 vs. 0.7 ng/mL), indicating that stress hormones may also

affect the sensitivity of the brain to estrogens (Battaglia et al. 1999).

Lastly, glucocorticoids are highly involved in parturition. The fetus produces significant

amounts of cortisol when uterine space becomes limited, stimulating the conversion of maternal

circulating P4 to estradiol and also synthesis of PGF2α by the placenta, which triggers a cascade

of events that lead to pregnancy termination (Senger, 2003). Therefore, elevated levels of

glucocorticoids during pregnancy may cause abortions in cattle. Administration of exogenous

glucocorticoids to pregnant beef cows during late-gestation terminated pregnancy mainly by

reducing adrenal and placental P4 synthesis (Johnson et al. 1981). Giri et al. (1990) reported that

beef and dairy cows at different stages of gestation (first, second, and third trimesters) and

administered endotoxin challenges for a 6 h period experienced significant increases in plasma

cortisol and PGF2α concentrations, resulting in a 55% abortion rate within 96 h post-challenge.

Focusing on early pregnancy loss, Geary et al. (2005) reported that processing beef heifers, but

not cows, through an animal handling facility 13 d after AI was detrimental to pregnancy rates,

and attributed these outcomes to deleterious effects of handling stress on maintenance of the

early pregnancy. Merrill et al. (2003) reported that beef cows transported via semi-truck for 4 h

at moderate air temperature 14 d after AI experienced greater plasma cortisol concentrations

immediately after transporting (29 vs. 18 ng/mL, respectively), and also numerically decreased

50

pregnancy rates to AI (69 vs. 76 %, respectively) compared to non-transported cows.

Additionally, cattle exposed to heat stress have increased incidence of early pregnancy loss, and

this effect may be attributed to the elevated temperatures that the conceptus is exposed to, and/or

to the maternal neuroendocrine responses to heat stress (Hansen and Arechiga, 1999).

Nevertheless, further research is required to address the effects of stress responses on pregnancy

maintenance of cattle, especially because the mechanisms by which increased stress, cortisol, or

both, might affect PGF2α or initiate pregnancy loss are yet to be established (Merrill et al. 2007).

In conclusion, excitable temperament may affect negatively the attainment of puberty, fertility,

and maintenance of pregnancy in cattle by impairing synthesis and secretion of hormones

associated with reproductive function, such as GnRH, gonadotropins, and steroids.

Cattle Acclimation

One alternative to alleviate the detrimental effects of excitable temperament on

productive traits of cattle is to adapt them to humans and handling procedures. However, a

limited amount of research has been conducted thus far to address this question. Fordyce et al.

(1987) indicated that Brahman-crossbred steers frequently exposed to handling procedures

exhibited calmer temperament compared with cohorts with no handling experiences. Crookshank

et al. (1979) reported that serum cortisol concentrations decreased significantly in calves

(transported or not) that were handled and bled several times instead of only once within 16 d

after transportation, indicating that calves became more familiarized and hence less excited

during handling procedures. Andrade et al. (2001) subjected Brahman cows to an acclimation

process which consisted of restraining cows twice weekly during a 19-d period in the squeeze

chute for 10 min, and reported that plasma cortisol concentrations decreased from d 1 to d 19

(3.92 vs. 0.99 ng/mL, respectively), indicating that cows became acclimated to chute restraining.

51

Curley et al. (2006) assessed exit velocity and cortisol concentrations of yearling Brahman bulls

on three separate occasions (d 0, 60, and 120), and reported that mean exit velocity and plasma

cortisol concentrations decreased from d 0 to d 120, indicating that cattle became acclimated

with humans over the data collection period as reflected by both temperament and endocrine

measurements. Echternkamp (1984) compared plasma concentrations of cortisol, LH, and P4 of

ovariectomized Hereford cows acclimated to physical restrain for blood collection with those of

cohorts with no previous acclimation. The author reported that acclimated cows had decreased

mean concentration of cortisol (5.7 vs. 66.1 ng/mL, respectively) and increased mean LH

concentration (8.1 vs. 4.1 ng/mL, respectively) and pulsatility (4.3 vs. 1.3 pulses/4 h,

respectively) compared to non-acclimated cows. Further, cortisol and P4 concentrations were

negatively correlated with LH concentrations (r = -83 and -35, respectively) within cows,

whereas cortisol and P4 were positively correlated (r = 0.62), suggesting that cattle acclimation

may alleviate their neuroendocrine response to handling and consequently enhance their

gonadotropin secretory activity and subsequent reproductive response. In conclusion,

acclimation procedures are options to improve endocrine and behavioral responses of cattle with

excitable temperament to management events involving human interaction and handling, which

may result in beneficial effects on production traits such as BW gain and reproductive

performance.

Novel Strategies to Enhance the Reproductive Efficiency of Cow-Calf Operations

Energy supplementation is often required in Florida grazing cow-calf operations to

compensate for the reduced nutrient density of forages, and thus maintain cattle at adequate

levels of nutrition. These supplements are usually offered 3 times or once weekly in order to

reduce labor costs. However, recent data indicate that nutritional status of cattle is improved

52

when supplements are offered daily instead of 3 times weekly. Therefore, offering energy-based

supplements to developing heifers or mature cows on a daily basis may be an alternative to

enhance their feed efficiency and reproductive performance, whereas the positive outcomes from

daily feeding may at least compensate for the extra expenses associated with labor.

Another disadvantage encountered in typical Florida cow-calf operations is the excitable

temperament of cattle because of their significant Brahman breeding. Alternatives to ameliorate

cattle temperament will likely bring beneficial effects to productive and reproductive traits of

these animals. Acclimation of cattle to human interaction and handling procedures alleviate their

behavioral and neuroendocrine responses to these practices, and are expected to allow for

hastened development of heifers and enhanced reproductive efficiency of mature cows.

To address these nutritional and management assumptions, four experiments were

conducted to evaluate the effects of supplementation frequency and acclimation to human

handling on performance, physiological responses, and reproductive performance of Brahman-

crossbred females. Results from the experiments are reported and discussed in the following

chapters.

53

CHAPTER 3 EFFECTS OF SUPPLEMENTATION FREQUENCY ON PERFORMANCE,

REPRODUCTIVE, AND METABOLIC RESPONSES OF BRAHMAN-CROSSBRED FEMALES

Introduction

Energy supplementation is a common practice in cow-calf systems because BW gain and

reproductive function of beef females are influenced positively by energy intake (Schillo et al.,

1992; Roberts et al., 1997). The labor expenses associated with supplement feeding contribute

significantly to the fixed costs of cattle operations (Miller et al., 2001); therefore offering

supplements 3 times or once weekly instead of daily are typical strategies to decrease costs of

production. However, reducing the supplementation frequency of energy feeds to cattle

consuming low-quality forages can be detrimental to their performance (Kunkle et al., 2000).

Supplementation frequency can affect performance of beef females by many

mechanisms, including the modulation of blood concentrations of hormones and metabolites.

Infrequent feed intake reduces circulating progesterone (P4) concentrations (Vasconcelos et al.,

2003) and consequently may be detrimental to puberty attainment and pregnancy establishment

(Gonzalez-Padilla et al., 1975; Spencer and Bazer, 2002). Blood concentrations of glucose,

insulin, and IGF-I are affected positively by increased supplementation frequency (Cooke et al.,

2007b), and these substances are associated with BW gain and reproductive function of cattle

(Schillo et al., 1992; Spicer and Echternkamp, 1995). Based on these observations, we

hypothesized that beef females consuming low-quality forages would benefit if supplemented

daily instead of 3 times weekly with an energy supplement.

Two experiments were conducted to investigate the effects of supplementation frequency

on Brahman-crossbred females. Experiment 1 evaluated BW gain, concentrations of plasma

Reprinted with permission from the Journal of Animal Science (2008, 86:2296-2309)

54

metabolites and hormones, mRNA expression of liver and muscle genes associated with

metabolism and growth, and reproductive performance of heifers. Experiment 2 assessed plasma

metabolites and hormone concentrations, and mRNA expression of hepatic genes associated with

nutritional metabolism of mature cows.

Materials and Methods

Both experiments were conducted at the University of Florida – IFAS, Range Cattle

Research and Education Center, Ona. The first experiment was conducted from September to

December, 2006 and was divided into a sampling phase (September and October) and a breeding

phase (November and December). The second experiment was conducted during the months of

October and November, 2006. The animals utilized in these experiments were cared for in

accordance with acceptable practices as outlined in the Guide for the Care and Use of

Agricultural Animals in Agricultural Research and Teaching (FASS, 1999), and the experimental

protocols were reviewed and approved by the University of Florida, Institutional Animal Care

and Use Committee (No. E659 and SOP0064).

Animals

Experiment 1. Fifty-six Brahman × Angus heifers (initial BW ± SD = 228 ± 28 kg;

initial age ± SD = 293 ± 29 d) were utilized in this experiment. For the sampling phase (d 0 to d

45), heifers were stratified by initial BW and age, and randomly allocated to 14 pens (4

heifers/pen) on d -11. Pens were assigned randomly to receive an energy supplement based on

fibrous byproducts daily (S7) or 3 times weekly (S3), at a weekly rate of 18.2 kg of DM per

heifer. Pen was considered the experimental unit (7 pens/treatment), and each pen consisted of 2

ha of bahiagrass (Paspalum notatum) pasture. Heifers were adapted to assigned treatments from

d -11 to d -1. For the breeding phase (d 46 to d 107), heifers were re-allocated by treatment into 2

55

bahiagrass pastures and exposed to Angus bulls. During both sampling and breeding phase,

heifers were not rotated among pastures.

Experiment 2. Twelve non-lactating, non-pregnant multiparous Brahman × British cows

(BW ± SD = 553 ± 50 kg; average age = 6 ± 2 yr) were stratified by BW and age, housed in

individual pens, and randomly assigned to receive S3 or S7, at a weekly rate of 20.3 kg of DM

per cow. Cow was considered the experimental unit (6 cows/treatment). Prior to the beginning of

the experiment, with the purpose of acquiring cows with similar and substantial plasma P4

concentrations on d 0 of the study, cows received a 100 μg treatment of GnRH (Cystorelin;

Merial Ltd., Duluth, GA) and received a controlled internal drug releasing device containing

1.38 g of P4 (CIDR; Pfizer Animal Health, New York, NY) on d -18, PGF2α treatment (25 mg;

Lutalyse; Pfizer Animal Health, New York, NY) and CIDR removal on d -12, and a second

GnRH treatment (100 μg) on d -10. On d -4, cows received another PGF2α treatment (25 mg)

and received 2 CIDR that remained in cows throughout the experimental period (d 0 to 17).

Transrectal ultrasonography examinations (7.5 MHz transducer, Aloka 500V, Wallingford, CT)

were performed immediately and 48 h after second GnRH (d -8) and PGF2α (d -4) treatments to

verify ovulation and corpus luteum regression, respectively. All cows utilized in this experiment

responded to the hormonal treatment, and were adapted to assigned treatments from d -8 to d 0.

Diets

Experiment 1. Forage and supplement samples were analyzed for nutrient content by a

commercial laboratory (Dairy One Forage Laboratory, Ithaca, NY). All samples were analyzed

by wet chemistry procedures for concentrations of CP, ADF, and NDF, whereas TDN was

calculated using the equation proposed by Weiss et al. (1992). Pasture quality was estimated at

54% TDN and 8.8% CP (DM basis) from samples collected at the beginning and during the

56

experiment. The pastures utilized in this experiment were not fertilized prior to or during the

experimental period. Stargrass (Cynodon nlemfuensis) hay was offered in amounts to ensure ad

libitum access when pasture availability was limited. Hay quality was estimated at 53% TDN

and 7.7% CP (DM basis) from samples collected at the beginning of the experiment. A complete

commercial mineral/vitamin mix (14% Ca, 9% P, 24% NaCl, 0.20% K, 0.30% Mg, 0.20% S,

0.005% Co, 0.15% Cu, 0.02% I, 0.05% Mn, 0.004% Se, 0.3% Zn, 0.08% F, and 82 IU/g of

vitamin A) and water were offered for ad libitum consumption throughout the experiment.

Random samples of the supplement were also collected during the experiment. Composition and

nutritional profile of the supplement are described in Table 3-1. Heifers were offered supplement

at 0700, daily (S7) or on Mondays, Wednesdays, and Fridays (S3).

Experiment 2. Forage and supplement samples were analyzed for nutrient content

similarly as in Exp. 1. Stargrass hay was offered for ad libitum consumption throughout the

entire experiment, and hay quality was estimated at 51% TDN and 6.0% CP (DM basis) from

samples collected at the beginning of the experiment. Cows had free access to a complete

mineral mix (similar to Exp. 1) and water. A random sample of the supplement was also

collected at the beginning of the experiment. Composition and nutritional profile of the

supplement are described in Table 3-1. Cows were offered supplement at 0800, daily (S7) or on

Mondays, Wednesdays, and Fridays (S3).

Sampling

Experiment 1. Heifers were weighed on 2 consecutive days to determine both full and

shrunk (after 16 h of feed and water restriction) BW before the start (d -12 and d -11) and at the

end of the experiment (d 107 and d 108). Blood samples were collected weekly (Wednesday)

throughout the entire experiment to determine onset of puberty using plasma P4 concentrations.

57

Heifers were considered pubertal once plasma P4 concentrations were greater than 1.5 ng/mL for

2 consecutive weeks (Cooke et al., 2007a).

During the sampling phase, in addition to the weekly collections, blood samples were

obtained once per day during 4 consecutive days, every other week, starting at 4 h after

supplement was offered to determine concentrations of glucose, blood urea nitrogen (BUN),

insulin, IGF-I, and P4. These samples were collected from d 0 to d 3, d 14 to d 17, d 28 to d 31,

and d 42 to d 45, which were classified as periods (PR1, PR2, PR3 and PR4, respectively).

Periods began on Monday and ended on Thursday.

Heifers within pen were assigned randomly for either muscle or liver biopsying on d 35

or 36 of the experiment (Monday or Tuesday). Biopsy procedures began 4 h after supplement

was offered. As a result, 2 liver and 2 muscle samples were obtained from each pen for

quantitative real-time RT-PCR assessment of IGF-I, IGFBP-3, pyruvate carboxylase (PC),

cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), mitochondrial PEPCK (PEPCK-M),

and cyclophilin mRNA expression in liver samples, and IGF-I, IGFBP-3, IGFBP-5, myostatin,

and cyclophilin mRNA expression in muscle samples.

During the breeding phase, each treatment group was exposed to 2 mature Angus bulls at

the same time (1:14 bull:heifer ratio), and bulls were rotated weekly between groups to account

for potential bull effects. Heifer pregnancy status was verified by detecting a fetus with

transrectal ultrasonography (5.0 MHz transducer, Aloka 500V) 70 d after the end of the

experiment. Date of conception was estimated retrospectively by subtracting gestation length

(286 d; Reynolds et al., 1980) from the calving date.

Experiment 2. During a 3-wk period, blood samples were collected immediately prior to

and 4, 8, 24, 28, and 32 h after the first supplement feeding of the week in which cows from both

58

treatments were offered supplements (Mondays; d 1, 8, and 15). Blood samples were analyzed

for concentrations of glucose, BUN, insulin, IGF-I, and P4.

Liver samples were collected on d 15 and 16 via needle biopsy, concurrently with blood

samplings at 4 and 28 h after first supplement feeding of wk 3, to determine the mRNA

expression of IGF-I, IGFBP-3, PC, PEPCK-C, PEPCK-M, and cyclophilin via quantitative real-

time RT-PCR.

Blood Analysis

Blood samples were collected via jugular venipuncture during Exp. 1 and from coccygeal

vein or artery during Exp. 2 into commercial blood collection tubes (Vacutainer, 10 mL; Becton

Dickinson, Franklin Lakes, NJ) containing sodium heparin, placed on ice immediately, and

centrifuged at 2,400 × g for 30 min for plasma collection. Plasma was frozen at -20°C on the

same day of collection.

Glucose and BUN concentrations were determined using quantitative colorimetric kits

G7521 and B7551, respectively (Pointe Scientific, Inc., Canton, MI). A double antibody RIA

was used to determine concentrations of insulin (Malven et al., 1987; Badinga et al., 1991), and

IGF-I (Badinga et al., 1991). The extraction procedure used in the IGF-I assay was modified

from Badinga et al. (1991) by using an ethanol:acetone:acetate ratio of 6:3:1. Concentrations of

P4 were determined using Coat-A-Count solid phase 125I RIA kit (DPC Diagnostic Products Inc.,

Los Angeles, CA). The intra- and inter-assay CV for Exp. 1 were, respectively, 4.1 and 2.7% for

glucose, 3.8 and 5.3% for BUN, 8.8 and 7.9% for insulin, 8.9 and 11.8% for IGF-I, and 4.8 and

5.9% for P4. The intra- and inter-assay CV for Exp. 2 were, respectively, 4.8 and 6.4% for

glucose, 3.8 and 9.5% for BUN, 12.3 and 12.0% for insulin, 8.6 and 5.1% for IGF-I, and 6.7 and

6.1% for P4.

59

Tissue Analysis

Tissue Collection and RNA Extraction. Liver and LM biopsies were performed by

trained personnel following the techniques described by Arthington and Corah (1995).

Immediately after collection, liver and muscle samples (average 100 mg of tissue; wet weight)

were placed in 1 mL of RNA stabilization solution (RNAlater; Ambion, Inc., Austin, TX),

maintained at 4°C for 24 h, and stored at -20°C.

Total RNA was extracted from tissue samples using TRIzol Plus RNA Purification Kit

(Invitrogen, Carlsbad, CA). Quantity and quality of isolated RNA were assessed via UV

absorbance at 260 nm and 260/280 nm ratio, respectively (GeneQuant spectrophotometer;

Amersham Pharmacia Biotech, Cambridge, UK). Extracted RNA was stored at -80°C until

further processing.

Real-Time RT-PCR. Extracted RNA from liver and muscle samples (2.5 and 1.0 μg,

respectively) were incubated at 37oC for 30 min in the presence of RNase-free DNase (New

England Biolabs, Inc., Ipswich, MA) to remove contaminant genomic DNA. After inactivating

the DNase (75oC for 15 min), samples were reverse-transcribed using the High Capacity cDNA

Reverse Transcription Kit with random hexamers (Applied Biosystems; Foster City, CA). Real-

time PCR was completed using the SYBR Green PCR Master Mix (Applied Biosystems) and

specific primer sets (25 ng/mL; Table 3-2), with a 7300 Real-Time PCR System (Applied

Biosystems). Following incubation at 95°C for 10 min, 40 cycles of denaturation (95oC for 15

sec) and annealing/synthesis (60oC for 2 min) were completed. Each RNA sample was analyzed

in triplicate, and the absence of genomic contamination was verified by including a fourth

reaction lacking exposure to the reverse transcriptase. At the end of each PCR, amplified

products were subjected to a dissociation gradient (95°C for 15 sec, 60°C for 30 sec, and 95°C

60

for 15 sec) to verify the amplification of a single product by denaturation at the anticipated

temperature. A portion of the amplified products were purified with the QIAquick PCR

purification kit (Qiagen Inc.; Valencia, CA) and sequenced at the University of Florida DNA

Sequencing Core Facility to verify the specificity of amplification. All amplified products

represented only the genes of interest.

Responses were quantified based on the threshold cycle (CT), the number of PCR cycles

required for target amplification to reach a pre-determined threshold. All CT responses from

genes of interest were normalized to cyclophilin CT examined in the same sample and run at the

same time as the targets. Results are expressed as relative fold change (2-ΔΔCT), as described by

Ocón-Grove et al. (2008).

Internal RNA standard samples were included in each assay (PCR plate). The inter-assay

CV for Exp. 1 was 0.7% for liver IGF-I, 0.6% for liver IGFBP-3, 0.6% for PC, 0.6% for PEPCK-

C, 0.8% for PEPCK-M, 0.7% for muscle IGF-I, 0.5% for muscle IGFBP-3, 0.9% for IGFBP-5,

and 0.7% for myostatin. The inter-assay CV for Exp. 2 was 0.9% for liver IGF-I, 1.0% for liver

IGFBP-3, 0.9% for PC, 1.1% for PEPCK-C, and 1.2% for PEPCK-M.

Statistical Analysis

Experiment 1. Performance, physiological, and gene expression data were analyzed

using the MIXED procedure of SAS (SAS Inst., Inc., Cary, NC) and Satterthwaite approximation

to determine the denominator df for the tests of fixed effects. Gene expression data were further

tested for normality with the Shapiro-Wilk test from the UNIVARIATE procedure of SAS, and

results indicated that all data were distributed normally (W ≥ 0.90). The model statement used

for hormone and metabolite analysis contained the effects of treatment, period, day(period), and

the interactions of treatment × period and treatment × day(period). Data were analyzed using

61

heifer(pen) and pen(treatment) as random variables. The model statement used for ADG

contained only the effect of treatment. Data were analyzed using pen(treatment) as the random

variable. The model statement used for gene expression analysis contained the effects of

treatment, day, and the interaction. Results are reported as least square means and were separated

using LSD. Puberty and pregnancy data were analyzed with survival analysis (LIFETEST

procedure of SAS) by regressing the proportion of prepubertal or non-pregnant heifers on week

of the study or breeding season, respectively. Differences between treatment survival curves

were determined by the Wilcoxon test. For all analysis, significance was set at P ≤ 0.05,

tendencies were determined if P > 0.05 and ≤ 0.10, and results are reported according to

treatment effects if no interactions were significant, or according to the highest-order interaction

detected.

Experiment 2. Data were analyzed using the PROC MIXED procedure of SAS and

Satterthwaite approximation to determine the denominator df for the tests of fixed effects. Gene

expression data also were tested for normality with the Shapiro-Wilk test from the

UNIVARIATE procedure of SAS, and results indicated that all data were distributed normally

(W ≥ 0.90). The model statement used for hormone and metabolite analysis contained the effects

of treatment, week, time(week), and the interactions of treatment × week and treatment ×

time(week). Data were analyzed using cow(treatment) and cow(treatment) × week as random

variables. The model statement used for gene expression analysis contained the effects of

treatment, day, and the interaction. The random variable was cow(treatment). Additionally,

mRNA expression of PEPCK-C, PEPCK-M, and PC were analyzed jointly. This model

statement contained the effects of treatment, day, enzyme, and the resultant interactions, whereas

cow(treatment) was the random variable. Significance was set at P ≤ 0.05, tendencies were

62

determined if P > 0.05 and ≤ 0.10. Results reported are least square means, were separated using

LSD, and are reported according to treatment effects if no interactions were significant, or

according to the highest-order interaction detected.

Results and Discussion

Experiment 1

Heifers provided S7 had greater (P = 0.03) ADG compared to S3 heifers (0.41 vs. 0.33

kg/d, respectively; SEM = 0.02), concurring with studies reporting increased BW gain of cattle

fed low-quality forages and offered energy supplements daily vs. 3 times weekly (Kunkle et al.,

2000; Cooke et al., 2007b). Reproductive performance was also affected by treatments.

Attainment of puberty (Figure 3-1) and pregnancy (Figure 3-2) were hastened (P = 0.03 and

0.02, respectively) in S7 heifers compared to S3 heifers. In a review article, Kunkle et al. (2000)

indicated that decreased supplementation frequency of energy supplements containing high-

starch ingredients is detrimental to cattle performance because of impaired rumen function,

forage intake, and digestibility. However, different outcomes were observed in cattle

supplemented with low-starch energy byproducts. Loy et al. (2007) reported similar mean forage

intake, rumen pH, and in situ forage NDF disappearance of beef heifers supplemented with

distillers grains daily or on alternate days. Cooke et al. (2007b) reported that beef steers offered

citrus pulp-based supplements daily had similar mean forage DMI but improved ADG compared

to steers offered the same supplement 3 times weekly, and attributed the differences in ADG to

beneficial effects of daily supplementation on concentrations of hormones and metabolites

associated with energy intake.

A treatment x day(period) interaction was detected (P < 0.01) for BUN analysis (Table 3-

3). During the initial half of PR2, and throughout PR3 and PR4, S3 heifers had greater (P ≤ 0.05)

63

BUN concentrations compared to S7 heifers during the days that only S7 heifers were offered

supplements, but decreased (P < 0.01) BUN concentrations during the days that both treatment

groups were offered supplements. Concentrations of BUN are positively associated with intake

of rumen-degradable protein, levels of ruminal ammonia, and ruminal protein:energy ratio

(Hammond, 1997). The reductions in BUN concentrations detected in S3 heifers on days that

both treatment groups were supplemented reflected a decreased ruminal protein:energy ratio

created in these heifers because they were offered a greater amount of supplemental energy

compared to S7 heifers. Conversely, increased BUN concentrations of S3 heifers during days

that only S7 heifers were supplemented reflected an increased ruminal protein:energy ratio

because S3 heifers did not receive any supplemental energy but likely maintained a significant

ruminal N supply due to urea-recycling (Lapierre and Lobley, 2001). This biphasic pattern of

BUN concentrations detected in S3 heifers was also reported by others (Bohnert et al., 2002) and

illustrates the greater daily variation of energy and protein intake of S3 heifers compared to S7

heifers. Heifers from both treatments frequently had BUN concentrations above the optimal level

(11 and 15 mg/dL; Byers and Moxon, 1980), indicating that rumen-degradable protein and thus

CP were consumed in excess by these animals. Further, the sharp increases in BUN detected for

S3 heifers during non-supplementation days may have influenced negatively their performance

because excessive rumen ammonia concentrations requires additional energy to be metabolized

into urea by the liver (Reynolds, 1992).

A treatment x day(period) interaction was detected (P < 0.01) for glucose and insulin

analysis (Table 3-3 and 3-4, respectively). A day(period) effect was detected (P < 0.01) for

glucose concentrations of S3 heifers throughout the experimental period, whereas a tendency (P

= 0.06) for a day(effect) in glucose concentrations of S7 heifers was detected only during PR4.

64

Similarly, a day(period) effect was detected (P < 0.01) for insulin concentrations of S3 heifers

throughout the experimental period, whereas it was significant (P = 0.02) for S7 heifers only

during PR4. In addition, glucose and insulin concentrations were typically greater for S3 heifers

compared to S7 heifers on the days that only S7 heifers were supplemented, but not on days that

both treatment groups were supplemented (Table 3-3 and 3-4). Significant differences (P ≤ 0.05)

were detected on Wednesday during PR1, Tuesday during PR2, Tuesday and Thursday during

PR3, and Tuesday during PR4 for glucose, and on Tuesday during PR2, PR3, and PR4 for

insulin. Concentrations of plasma glucose and insulin are influenced positively by rate of nutrient

intake (Vizcarra et al., 1998), therefore the day(period) effects observed in this experiment

reflected the differences in nutrient intake pattern between treatments. During the experimental

period, S3 heifers received and readily consumed (within 2 h) bolus amounts of supplements on

3 d of the week, but had low-quality forage as the only source of feed available on the remaining

days. Conversely, S7 heifers received and consumed within 1 h smaller portions of supplements

on a daily basis.

A treatment x day interaction was detected for mRNA expression of liver PC (P = 0.02)

and PEPCK-C (P < 0.01; Table 3-5). These interactions were detected because a day effect (P <

0.01) was significant for these transcripts in S3 heifers but not in S7 heifers. Additionally, the

mRNA expressions of PC and PEPCK-C were greater (P < 0.01 and P = 0.02, respectively) for

S3 heifers compared to S7 heifers when both treatment groups were supplemented (d 35), but

were similar or greater (PEPCK-C; P = 0.04) for S7 heifers when only these were supplemented

(d 36). Treatment effects on liver PEPCK-M mRNA expression differed from the other

gluconeogenic transcripts because S7 heifers tended (P = 0.08) to have a greater mRNA

expression of liver PEPCK-M compared to S3 heifers (Table 3-5). The treatment effects detected

65

on mRNA expression of liver PC and PEPCK-C also reflect differences in the nutrient intake

pattern between S3 and S7 heifers. Expression of these enzymes was associated positively with

enzymatic activity and consequent glucose synthesis in cattle (Greenfield et al., 2000; Agca et

al., 2002; Bradford and Allen, 2005). Given that the availability of nutrients originating from

ruminal fermentation rapidly increase in forage-fed ruminants after supplementation (Seoane and

Moore, 1969; Rihani et al., 1993; Farmer et al., 2001) and that expression of liver enzymes

associated with gluconeogenesis are quickly altered (She et al., 1999; Massillon et al., 2003) and

increased when precursors are available (Greenfield et al., 2000; Karcher et al., 2007), the

greater mRNA expression of PC and PEPCK-C detected in S3 heifers compared to S7 heifers

when both treatment groups were offered supplements can be attributed to their greater

supplement consumption on that day. Accordingly, mRNA expression of these enzymes was

significantly reduced in S3 heifers but not in S7 heifers when only S7 heifers received

supplements. The reason to why PC and PEPCK-C expression was altered by 4 h post-

supplementation whereas plasma glucose and insulin responses to supplement consumption were

not detected until the next day in S3 heifers are likely due to the time required for PC and

PEPCK-C mRNA to be translated into the active enzymes and substantially change the

magnitude of glucose synthesis and release by the liver. Previous studies also reported that

plasma glucose concentrations of forage-fed developing heifers (Cooke et al., 2007a) and

yearling steers (Cooke et al., 2007b) offered supplements based on low-starch energy byproducts

3 times weekly were greater at 28 vs. 4 h after supplementation. Liver PEPCK-M may be

responsible for as much as 61% of glucose synthesis in ruminant hepatocytes (Aiello and

Armentano, 1987), although it is considered constitutive and not responsive to hormones and

nutritional state (Greenfield et al., 2000; Agca et al., 2002). Nevertheless, the tendency for

66

greater mRNA expression of liver PEPCK-M in S7 heifers compared to S3 heifers may be an

indicator of their improved energy status and contributed to their increased performance.

Concentrations of IGF-I were typically increased for S7 heifers compared to S3 heifers,

and there was a treatment x day(period) interaction (P < 0.01; Table 3-4). Significant differences

(P ≤ 0.05) were detected on Mondays (PR1, PR2, PR3, and PR4), and Wednesday of PR4.

Furthermore, mean IGF-I concentrations tended (P = 0.10) to be greater for S7 vs. S3 heifers

(190 and 161 ng/mL, respectively; SEM = 12). Nutritional status, growth, and reproductive

performance of beef cattle are influenced positively by circulating IGF-I (Roberts et al., 1997;

Diskin et al., 2003; Cooke et al., 2007a). Therefore, the treatment effects detected for ADG and

reproductive performance in this experiment can be attributed, at least to some degree, to the

greater concentrations of IGF-I frequently detected for S7 compared to S3 heifers. Although

several studies reported a positive association between IGF-I concentrations and nutrient intake

(Ellenberger et al., 1989; Bossis et al., 1999; Lapierre et al., 2000), treatment differences in IGF-I

concentrations were detected despite the similarity in overall supplement intake between S3 and

S7 heifers. Thus, it remains possible that such differences resulted from treatment effects on

forage intake, but this explanation is unlikely because previous research efforts reported similar

mean forage intake of cattle offered low-starch supplements daily or 3 times weekly (Cooke et

al., 2007b; Loy et al., 2007). In addition, increases in plasma IGF-I concentrations due to

enhanced nutrient or feed intake are usually accompanied by increased mean glucose and insulin

concentrations (Bossis et al., 1999; Lapierre et al., 2000; Hersom et al., 2004), and nutrients

originated from low-quality forages do not contribute significantly to hepatic IGF-I synthesis and

release (Cooke et al., 2007a).

67

Heifers provided S7 had greater (P = 0.04) expression of liver IGF-I mRNA compared to

S3 heifers (Table 3-5). A treatment x day interaction was detected (P = 0.04) for liver IGFBP-3

because a day effect (P < 0.01) was detected for IGFBP3 mRNA expression in S3 but not in S7

heifers, whereas IGFBP3 mRNA expression was greater (P = 0.03) for S3 heifers compared to

S7 heifers when both treatment groups were supplemented (d 35) but similar when only S7

heifers were supplemented (d 36). The major source of circulating IGF-I synthesis is the liver

(D’Ercole et al., 1984). Blood concentrations of IGF-I are associated with IGF-I mRNA

expression in hepatic cells (Thissen et al., 1994), and this relationship is supported by the present

experiment. The availability of energy substrates has a positive influence on the expression of

liver IGF-I mRNA and consequent translation into the circulating protein (McGuire et al., 1992;

Thissen et al., 1994). Increased hepatic IGF-I mRNA expression and plasma IGF-I

concentrations detected in S7 heifers may be attributed to a greater availability of substrates for

metabolic and physiologic processes in these heifers due to an improved efficiency of nutrient

metabolism. By consuming small quantities of supplement on a daily basis, S7 heifers were

perhaps capable of retaining and processing these nutrients more efficiently than S3 heifers,

although further research efforts are required to evaluate this assumption. Treatment effects on

liver IGFBP3 mRNA expression differed from those detected for IGF-I, although IGFBP3

expression and synthesis are stimulated by circulating IGF-I (Thissen et al., 1994). Nevertheless,

the day effect detected for expression of liver IGFBP-3 mRNA only in S3 heifers may be an

additional indicator of the daily variation of nutrient intake and consequent availability of

substrates for metabolic processes in these heifers.

A treatment x day interaction was detected (P = 0.05) for muscle myostatin mRNA

expression (Figure 3-3). A day effect (P < 0.01) was detected in S3 heifers but not in S7 heifers.

68

Further, myostatin mRNA expression was greater (P = 0.04) for S3 heifers compared to S7

heifers when both treatment groups were supplemented (d 35) but similar when only S7 heifers

were supplemented (d 36). Myostatin is a growth factor expressed in skeletal muscle of

developing and mature animals that negatively influences muscle growth (Lee and McPherron,

2001; Dayton and White, 2008). Myostatin is believed to impair glucose uptake in muscle tissues

by decreasing the activity of insulin-dependent GLUT4, resulting in insulin resistance and

impaired muscle tissue development (Strassman et al., 2002; Antony et al., 2007). The treatment

effects detected for myostatin mRNA expression indicate that muscle tissues of S7 heifers were

developing in a more constant pattern compared to those of S3 heifers, possibly due to treatment

differences on nutrient intake and availability. No further treatment differences were detected

within mRNA expression of muscle genes (data not shown). Nevertheless, the lack of treatment

effects on muscle IGF-I and IGFBP may indicate that within the IGF-I sources, circulating IGF-I

was likely the major contributor for heifer body growth. Supporting our findings, Johnson et al.

(1996) reported that muscle growth is significantly stimulated by circulating IGF-I. Conversely,

IGF-I synthesized by skeletal muscle in growing cattle is implicated as an important and often

essential autocrine and paracrine mediator of tissue development and differentiation (McGuire et

al., 1992; Johnson et al., 1998), and research with mice suggested that IGF-I synthesized in

muscle tissues exerts a more important role in muscle growth compared to hepatic-originated

IGF-I (Sjogren et al., 1999; Yakar et al., 1999).

In summary, offering an energy supplement based on fibrous byproducts daily instead of

3 times weekly to developing heifers resulted in a normalized mRNA expression pattern of genes

associated with nutritional metabolism and growth, reduced daily variation in plasma

concentrations of BUN, glucose, and insulin, and improved heifer nutritional status as reflected

69

by increased hepatic mRNA expression and elevated plasma concentrations of IGF-I. These

beneficial metabolic effects were translated into greater BW gain and hastened attainment of

puberty and pregnancy; therefore, daily supplementation of high-energy byproducts enhances

development and performance of Brahman-crossbred heifers consuming low-quality forages.

Experiment 2

A treatment effect and a treatment x time(week) interaction were detected (P = 0.03 and

P < 0.01, respectively) for BUN (Table 3-6). Cows receiving S7 had increased BUN

concentrations compared to S3 cows during wk 2 and 3 (Table 3-6), with significant differences

(P < 0.05) detected at 4 and 8 h after the first supplement feeding of wk 2, and at 0, 4, 8, 24 and

32 h after the first supplement feeding of wk 3. Additionally, S7 cows had greater mean BUN

concentrations compared to S3 cows (9.2 vs. 7.9 mg/dL, respectively; SEM = 0.36). These

results differed from Exp. 1 because S7 cows likely had greater mean ruminal ammonia

concentrations compared to S3 cows, BUN concentrations increased in a similar pattern for S3

and S7 cows after supplement consumption, and were typically at adequate levels for both

treatments (8 mg/dL; Hammond, 1997). The differences in BUN responses between Exp. 1 and 2

may be explained by the reduced amount of supplement and thus rumen-degradable protein

consumed by cows vs. heifers in relation to their BW (0.5 and 1.0% of BW on a daily basis,

respectively). Cows were offered supplements at a daily rate of 0.5% of BW to avoid energy

overfeeding that may hinder the detection of treatment effects (Cooke et al., 2007a), whereas this

rate is adequate to maintain brood cows at a moderate positive energy balance (NRC, 1996).

A treatment x time(week) interaction was detected (P = 0.01) for insulin (Table 3-6)

because a time(week) effect was detected for insulin concentrations of S3 cows (P < 0.01) but

not S7 cows, and treatment differences were detected at 0 h (P = 0.03) and 48 h (P = 0.05) after

70

the first supplement feeding of wk 2 (Table 3-6). Similarly, a significant time(week) effect was

detected for glucose concentrations of S3 cows (P < 0.01) but not S7 cows, although a treatment

x time(week) interaction was not significant (P = 0.12; data not shown). Within the

gluconeogenic enzymes, a treatment x day interaction was detected (P < 0.01) only for PC

mRNA expression. Similar to Exp. 1, PC mRNA expression was numerically greater for S3

cows compared to S7 cows when both treatment groups were supplemented (d 15), but

numerically greater for S7 cows when only these were offered supplements (d 16; Table 3-7).

When data from all 3 gluconeogenic enzymes are analyzed jointly, S3 cows tended (P = 0.09) to

have increased mRNA expression of these transcripts on d 15 (2.49 vs. 2.07 relative fold change,

respectively; SEM = 0.17), but reduced (P = 0.03) mRNA expression of these transcripts on d 16

(1.48 vs. 2.03 relative fold change, respectively; SEM = 0.17) compared to S7 cows (treatment x

day interaction, P < 0.01). Further, a day effect was observed in the combined mRNA expression

of gluconeogenic enzymes for S3 cows (P < 0.01), but not for S7 cows. These results support

those reported in Exp. 1 particularly because supplement intake behavior was similar in both

experiments, and indicate that concentrations of glucose and insulin, and expression of

gluconeogenic enzymes transcripts varied significantly within 32 h after supplementation in S3

cows, but remained generally constant for S7 cows during the same time period due to treatment

differences in nutrient intake pattern. Different from Exp. 1, treatment effects detected for

PEPCK-M mRNA expression were similar to those detected for PC and PEPCK-C, indicating

that mRNA expression of this transcript was also influenced by nutrient intake pattern.

A treatment x week interaction was detected (P = 0.02) for IGF-I (Table 3-8) because

concentrations of this hormone increased for S7 cows (P < 0.01) but not for S3 cows with the

advance of the experiment. As a consequence, S7 cows tended (P = 0.09) to have greater IGF-I

71

concentrations compared to S3 cows on wk 3 (Table 3-8), indicating that nutritional status was

improving for S7 cows compared to S3 cows with the advance of the experiment. No treatment

effects were detected in the expression of liver IGF-I and IGFBP-3 mRNA (Table 3-7).

However, liver IGF-I mRNA expression was numerically increased for S7 cows compared to S3

cows, and the lack of statistical significance is most likely related to the reduced number of

animals utilized in this experiment.

No differences were detected for plasma P4 concentrations (data not shown) between

treatments. A week effect was detected (P < 0.01) because P4 concentrations decreased linearly

for both treatments from the first week to the last week of the experiment, and this effect can be

associated with the decrease in P4 release from CIDR with advancing time (6.62, 4.54, and 2.68

ng/mL for wk 1, 2, and 3, respectively; P < 0.01; SEM = 0.37). Infrequent intake of large

amounts of feed decreases circulating concentrations of P4 in cattle (Vasconcelos, et al., 2003,

Cooke et al., 2007a), and therefore may impair reproductive performance because P4 is required

for adequate attainment of puberty (Gonzalez-Padilla et al., 1975) and establishment of

pregnancy (Spencer and Bazer, 2002). Cooke et al. (2007a) reported that beef heifers and cows

consuming low-quality forages and offered supplements based on low-starch energy byproducts

3 times weekly at a daily rate of 1.0% of BW had reduced plasma P4 concentrations on days that

supplements were offered vs. days that supplements were not offered. In the present experiment,

cows were offered moderate amounts of supplement (0.5% of BW on a daily basis) to avoid

energy overfeeding; therefore this lower level of supplementation likely prevented plasma P4

concentrations from changing significantly. Conversely, heifers from Exp. 1 were supplemented

at 1.0% of BW on a daily basis and treatment effects on P4 concentrations were not evaluated

due to the reduced number of pubertal heifers during the sampling phase. Consequently, further

72

research is required to address the effects of supplementation frequency on circulating P4

metabolism of developing heifers.

In summary, offering an energy supplement based on fibrous byproducts daily instead of

3 times weekly to mature cows resulted in a normalized pattern of gluconeogenic enzymes

mRNA expression, reduced variation in plasma concentrations of glucose and insulin, and

improved cow nutritional status as observed by increasing plasma IGF-I concentrations.

Therefore, it could be postulated that Brahman-crossbred cows consuming low-quality forages

and supplemented daily with high-energy byproducts would experience improved performance

and reproductive efficiency compared to cows supplemented 3 times weekly, although further

research is required to address this matter.

73

Table 3-1. Ingredient composition and nutrient profile of the supplement offered in Exp. 1 and 2.

Item Concentration

Ingredients, as-fed basis

Wheat middlings, % 66.67

Soybean hulls, % 26.93

Blackstrap molasses, % 3.75

Cottonseed meal, % 2.65

Nutrient profile1, DM basis

DM, % 88.9

TDN, % 62.5

NEm, Mcal/kg2 1.35

NEg, Mcal/kg2 0.77

CP, % 22.2

Rumen degradable protein3, % 16.6

Non-fiber carbohydrates, % 32.1

NDF, % 39.4

ADF, % 18.5

Ca, % 0.31

P, % 0.94 1 Values obtained from a commercial laboratory wet chemistry analysis (Dairy One Forage Laboratory, Ithaca, NY); TDN calculated as described by Weiss et al. (1992). 2 Calculated with the equations proposed by the NRC (1988). 3 Estimated using the NRC model (2001).

74

Table 3-2. Primer sets used for quantitative real-time RT-PCR.

Target gene Primer sequence1 Product size Accession no

IGF-I

Forward 5’- CTC CTC GCA TCT CTT CTA TCT -3’ 236 bp NM_001077828

Reverse 5’- ACT CAT CCA CGA TTC CTG TCT -3’

IGFBP-3

Forward 5’- AAT GGC AGT GAG TCG GAA GA -3’ 194 bp NM_174556.1

Reverse 5’- AAG TTC TGG GTG TCT GTG CT -3’

IGFBP-5

Forward 5’- TCC GAG ATG GCA GAG GAG -3’ 289 bp XM_600908.3

Reverse 5’- GGT CAC AGT TGG GCA GGT -3’

Pyruvate carboxylase

Forward 5’- CCA ACG GGT TTC AGA GAC AT -3’ 184 bp NM_177946.3

Reverse 5’- TGA AGC TGT GGG CAA CAT AG -3’

Cytosolic phosphoenolpyruvate carboxykinase

Forward 5’- CAA CTA CTC AGC CAA AAT CG -3’ 115 bp NM_174737.2

Reverse 5’- ATC GCA GAT GTG GAC TTG -3’

Mitochondrial phosphoenolpyruvate carboxykinase

Forward 5’- GCT ACA ACT TTG GGC GCT AC -3’ 168 bp XM_583200

Reverse 5’- GTC GGC AGA TCC AGT CTA GC -3’

Myostatin

Forward 5’- GTG TTG CAG AAC TGG CTC AA -3’ 215 bp NM_001001525

Reverse 5’- CAG CAT CGA GAT TCT GTG GA -3’

Cyclophilin

Forward 5’- GGT ACT GGT GGC AAG TCC AT -3’ 259 bp NM_178320.2

Reverse 5’- GCC ATC CAA CCA CTC AGT CT -5’

1 Primers sequences obtained from: IGF-I and IGFBP-5, Wall et al. (2005); IGFBP-3, Li et al. (2007); PC and PEPCK-C, Dr. Erin Conner, BARC/USDA; PEPCK-M, myostatin and cyclophilin, Jellyfish Sequence Analysis Software (Field Scientific LLC, Lewisburg, PA).

75

1 Day

s at w

hich

hei

fers

from

bot

h tre

atm

ents

wer

e of

fere

d su

pple

men

ts a

re u

nder

lined

. Sup

plem

ents

wer

e of

fere

d at

070

0 h.

2 S

ampl

es w

ere

colle

cted

from

d 0

to d

3, d

14

to d

17,

d 2

8 to

d 3

1, a

nd d

42

to d

45

of th

e st

udy,

whi

ch w

ere

clas

sifie

d as

per

iods

1

to

4, re

spec

tivel

y. P

erio

ds b

egan

on

Mon

day

and

ende

d on

Thu

rsda

y.

3Tr

eatm

entc

ompa

rison

with

inpe

riod

and

day.

Tabl

e 3-

3. B

lood

ure

a ni

troge

n (B

UN

) and

pla

sma

gluc

ose

conc

entra

tions

(mg/

dL) o

f hei

fers

(Exp

. 1) o

ffer

ed a

n en

ergy

supp

lem

ent

base

d on

fibr

ous b

ypro

duct

s dai

ly (S

7) o

r 3 ti

mes

wee

kly

(S3)

, at a

wee

kly

rate

of 1

8.2

kg o

f DM

per

hei

fer.

1,2

76

1 Day

s at w

hich

hei

fers

from

bot

h tre

atm

ents

wer

e of

fere

d su

pple

men

ts a

re u

nder

lined

. Sup

plem

ents

wer

e of

fere

d at

070

0 h.

2 S

ampl

es w

ere

colle

cted

from

d 0

to d

3, d

14

to d

17,

d 2

8 to

d 3

1, a

nd d

42

to d

45

of th

e st

udy,

whi

ch w

ere

clas

sifie

d as

per

iods

1

to

4, r

espe

ctiv

ely.

Per

iods

beg

an o

n M

onda

y an

d en

ded

on T

hurs

day.

3

Trea

tmen

tcom

paris

onw

ithin

perio

dan

dda

y.

Tabl

e 3-

4. P

lasm

a co

ncen

tratio

ns (n

g/m

L) o

f ins

ulin

and

IGF-

I of h

eife

rs (E

xp. 1

) off

ered

an

ener

gy su

pple

men

t bas

ed o

n fib

rous

by

prod

ucts

dai

ly (S

7) o

r 3 ti

mes

wee

kly

(S3)

, at a

wee

kly

rate

of 1

8.2

kg o

f DM

per

hei

fer.

1,2

77

78

1 H

eife

rs fr

om b

oth

S3 a

nd S

7 gr

oups

wer

e su

pple

men

ted

on d

35,

whe

reas

onl

y S7

hei

fers

wer

e su

pple

men

ted

on d

36

o

f the

stud

y. T

issu

e co

llect

ion

star

ted

4 h

afte

r sup

plem

ent f

eedi

ng ti

me.

2 V

alue

s are

exp

ress

ed a

s rel

ativ

e fo

ld c

hang

e (O

cón-

Gro

ve e

t al.,

200

8).

3 PC

= P

yruv

ate

carb

oxyl

ase;

PEC

K-C

= c

ytos

olic

pho

spho

enol

pyru

vate

car

boxy

kina

se; P

EPC

K-M

= m

itoch

ondr

ial

PEP

CK

. 4 T

reat

men

t com

paris

on w

ithin

sam

plin

g da

y.

Tabl

e 3-

5. E

xpre

ssio

n of

hep

atic

gen

es a

ssoc

iate

d w

ith n

utrit

iona

l met

abol

ism

and

gro

wth

of h

eife

rs (E

xp. 1

) off

ered

an

en

ergy

supp

lem

ent b

ased

on

fibro

us b

ypro

duct

s dai

ly (S

7) o

r 3 ti

mes

wee

kly

(S3)

, at a

wee

kly

rate

of

18.2

kg

of D

M p

er h

eife

r. 1,

2

79

Tabl

e 3-

6. B

lood

ure

a ni

troge

n (B

UN

) and

pla

sma

insu

lin c

once

ntra

tions

of c

ows (

Exp.

2) o

ffer

ed a

n en

ergy

supp

lem

ent b

ased

on

fibro

us b

ypro

duct

s dai

ly (S

7) o

r 3 ti

mes

wee

kly

(S3)

, at a

wee

kly

rate

of 2

0.3

kg o

f DM

per

cow

. 1

1 Cow

s fro

m b

oth

treat

men

ts w

ere

offe

red

supp

lem

ents

afte

r blo

od w

as sa

mpl

ed a

t 0 h

. 2 Tr

eatm

entc

ompa

rison

with

inw

eek

and

hour

.

Tabl

e 3-

7. E

xpre

ssio

n of

hep

atic

gen

es a

ssoc

iate

d w

ith n

utrit

iona

l met

abol

ism

and

stat

us o

f cow

s (Ex

p. 2

) off

ered

an

ene

rgy

supp

lem

ent b

ased

on

fibro

us b

ypro

duct

s dai

ly (S

7) o

r 3 ti

mes

wee

kly

(S3)

, at a

wee

kly

rate

of

20.

3 kg

of D

M p

er c

ow. 1,

2

1 C

ows f

rom

bot

h S3

and

S7

grou

ps w

ere

supp

lem

ente

d on

d 1

5, w

here

as o

nly

S7 c

ows w

ere

supp

lem

ente

d on

d

16

of th

e st

udy.

Liv

er sa

mpl

es w

ere

colle

cted

4 h

afte

r sup

plem

ent f

eedi

ng ti

me.

2 V

alue

s are

exp

ress

ed a

s rel

ativ

e fo

ld c

hang

e (O

cón-

Gro

ve e

t al.,

200

8).

3 PC

= P

yruv

ate

carb

oxyl

ase;

PEC

K-C

= c

ytos

olic

pho

spho

enol

pyru

vate

car

boxy

kina

se; P

EPC

K-M

=

m

itoch

ondr

ial P

EPC

K

4

80

Table 3-8. Plasma IGF-I concentrations (ng/mL) of cows (Exp. 2) offered an energy supplement based on fibrous byproducts daily (S7) or 3 times weekly (S3), at a weekly rate of 20.3 kg of DM per cow. 1

Item Wk 1 Wk 2 Wk 3 SEM

S7 196 201 244 18

S3

203

18 191 196

P1

0.79

0.71 0.09

1 Treatment comparison within week.

81

0.000.100.200.300.400.500.600.700.800.901.00

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Prop

ortio

n of

pre

pube

rtal

hei

fers

Week of study

S7 S3

Figure 3-1. Proportion of prepubertal heifers by week during Exp. 1. Survival curves represent heifers offered an energy supplement based on fibrous byproducts daily (S7) or 3 times weekly (S3), at a weekly rate of 18.2 kg of DM per heifer. Heifers were considered pubertal once plasma progesterone concentrations were greater than 1.5 ng/mL for 2 consecutive weeks, and puberty attainment was declared at the first week of elevated progesterone. A treatment effect was detected (P = 0.03).

82

0.000.100.200.300.400.500.600.700.800.901.00

8 9 10 11 12 13 14 15 16

Prop

ortio

n of

non

-pre

gnan

t hei

fers

Week of study

S7 S3

Figure 3-2. Proportion of non-pregnant heifers by week during the 60-d breeding season of Exp. 1. Survival curves represent heifers offered an energy supplement based on fibrous byproducts daily (S7) or 3 times weekly (S3), at a weekly rate of 18.2 kg of DM per heifer. Date of conception was estimated retrospectively by subtracting gestation length (286 d; Reynolds et al., 1980) from the calving date. A treatment effect was detected (P = 0.02).

83

0

1

2

3

4

5

6

7

S3 and S7 supplemented (Day 35) Only S7 supplemented (Day 36)

Myo

stat

in e

xpre

ssio

n, r

elat

ive

fold

cha

nge

S7 S3*

Figure 3-3. Expression of muscle myostatin, as relative fold change (Ocón-Grove et al., 2008), of heifers (Exp. 1) offered an energy supplement based on fibrous byproducts daily (S7) or 3 times weekly (S3), at a weekly rate of 18.2 kg of DM per heifer. Heifers from both S3 and S7 groups were supplemented on d 35, whereas only S7 heifers were supplemented on d 36 of the study. Tissue collection started 4 h after supplement feeding time. A treatment x day interaction was detected (P = 0.05). A day effect was detected for S3 heifers (P < 0.01), but nor for S7 heifers. Treatment comparison within days: * P = 0.04.

84

CHAPTER 4 EFFECTS OF ACCLIMATION TO HANDLING ON PERFORMANCE, REPRODUCTIVE,

AND PHYSIOLOGICAL RESPONSES OF BRAHMAN-CROSSBRED HEIFERS

Introduction

Development of replacement heifers is one of the most important components of the cow-

calf production system (Bagley, 1993). Management strategies that maximize the number of

heifers conceiving by 15 mo of age improve the profitability of cow-calf operations because

heifers that calve as 2-yr olds wean more and heavier calves during their productive lives

(Lesmeister et al., 1973). Because conception rates are greater during the third estrus compared

with the pubertal estrus (Byerley et al., 1987), replacement heifers should be managed to attain

puberty at 12 mo of age so they can conceive by 15 mo of age (Schillo et al., 1992; Bagley,

1993).

Age at puberty is influenced by breed type, and heifers containing Brahman breeding

typically reach puberty after 15 mo of age (Plasse et al., 1968; Rodrigues et al., 2002). In

addition to this genetic effect, Brahman-crossbred heifers are often described as temperamental,

and this trait is expected to further negatively influence their reproductive function (Plasse et al.,

1970). Cattle with excitable temperament experience stimulated secretion and circulating

concentrations of ACTH and cortisol (Curley et al., 2008). These hormones directly impair the

mechanisms responsible for puberty establishment and fertility of heifers, such as synthesis and

release of GnRH and gonadotropins (Li and Wagner, 1983; Dobson et al., 2000). However,

acclimation of beef females to handling has been reported to alleviate these negative

physiological effects of temperament on reproduction (Echternkamp, 1984). Based on these

previous observations, we hypothesized that Brahman-crossbred heifers exposed to handling

acclimation procedures after weaning would experience improved temperament, alleviated

adrenal steroidogenesis, and enhanced reproductive performance. The objectives of the present

85

experiment were to compare growth, temperament, plasma measurements associated with stress

response, puberty attainment and pregnancy rates of Brahman × Angus and Braford heifers

exposed or not to acclimation procedures.

Materials and Methods

This experiment was conducted over 2 yr (2006 and 2007) at the University of Florida –

IFAS, Range Cattle Research and Education Center, Ona. Each experimental period was divided

into a sampling phase (d 0 to 130) and a breeding phase (d 131 to 191). The animals utilized

were cared for in accordance with acceptable practices as outlined in the Guide for the Care and

Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 1999).

Animals

A total of 37 Braford (37.5% Brahman + 62.5% Hereford) and 43 Brahman × Angus

(approximately 25% Brahman) heifers weaned at approximately 7 mo of age were assigned to

the experiment. Twenty heifers from each breed type were utilized in yr 1, whereas 23 Brahman

× Angus and 17 Braford heifers were utilized in yr 2. Within 30 d after weaning, heifers were

initially evaluated (morning of d 0 and 10) for BW, puberty status, and temperament score (d 10

only). Puberty was assessed via trans-rectal ultrasonography (7.5 MHz transducer, Aloka 500V,

Wallingford, CT) to verify ovarian activity and plasma progesterone (P4) concentrations. Heifers

were considered pubertal if a corpus luteum with volume greater than 2.15 cm3, and plasma P4

concentrations greater than 1.5 ng/mL (Cooke et al., 2007a) were concurrently detected in one or

both evaluations. Corpus luteum volume was calculated using the formula for volume of a sphere

(V = 4/3π × [D/2]3, where D is the maximum luteal diameter). On d 11, heifers were stratified by

breed, puberty status, temperament score, BW and age, and randomly assigned to receive or not

(control) the acclimation treatment. Heifer was considered the experimental unit (40

86

heifers/treatment). Across years and breeds, heifer mean BW and age at the beginning of the

experiment were (± SD), respectively, 253 ± 27 kg and 269 ± 26 d.

Diets

During both sampling and breeding phases, heifers were be maintained on separate 6 ha

limpograss (Hemarthria altissima) pastures according to treatment. The pastures utilized in this

experiment were not fertilized prior to or during the experimental period, and heifers were

rotated among pastures every 2 wk. Heifers received a blend of soybean hulls and cottonseed

meal (75:25 as fed-basis) 3 times weekly, at a rate to provide a daily amount of 2.7 kg of DM per

heifer. Stargrass (Cynodon nlemfuensis) hay was offered in amounts to ensure ad libitum access

when pasture availability was limited. A complete commercial mineral/vitamin mix (14% Ca,

9% P, 24% NaCl, 0.20% K, 0.30% Mg, 0.20% S, 0.005% Co, 0.15% Cu, 0.02% I, 0.05% Mn,

0.004% Se, 0.3% Zn, 0.08% F, and 82 IU/g of vitamin A) and water were offered for ad libitum

consumption throughout the experiment. Forage and supplement samples were collected at the

beginning and during the experiment, and analyzed for nutrient content by a commercial

laboratory (Dairy One Forage Laboratory, Ithaca, NY). All samples were analyzed by wet

chemistry procedures for concentrations of CP, ADF, and NDF, whereas TDN was calculated

using the equation proposed by Weiss et al. (1992). During yr 1, nutrient composition (DM

basis) was estimated to be 54% TDN and 9.3% CP in pasture samples, 52% TDN and 11.9% CP

in hay samples, 60% TDN and 11.3% CP in soybean hull samples, and 69% TDN and 49.4% CP

in cottonseed meal samples. During yr 2, nutrient composition (DM basis) was estimated to be

54% TDN and 9.6% CP in pasture samples, 54% TDN and 13.6% CP in hay samples, 60% TDN

and 11.7% CP in soybean hull samples, and 67% TDN and 47.0% CP in cottonseed meal

samples.

87

Acclimation Procedure

Acclimated heifers were exposed to a handling process for 4 wk (d 11 to 39 of the

experiment). During this period, heifers were gathered in the pasture and obliged to walk to the

handling facility 3 times weekly (Mondays, Wednesdays, and Fridays). The total distance

traveled by heifers during each of the acclimation events was approximately 2 km (round-trip).

During the first wk of acclimation, heifers were processed through the handling facility but not

restrained in the squeeze chute. During the second wk, heifers were processed through the

handling facility and experienced a rapid chute restraining (5 s). On the third and fourth wk,

heifers were similarly processed as in wk 2 but were restrained in the squeeze chute for longer

periods (30 s). During the initial three wks, heifers were allowed to return to the pastures

immediately after processing, whereas during the fourth wk heifers remained in the handling

facility for 1 h and then returned to pasture.

Sampling

During the sampling phase, heifer puberty status and BW were assessed in the morning

of d 40 and 50, 80 and 90, and 120 and 130, in addition to the initial evaluation (d 0 and 10).

Heifer puberty status was determined by trans-rectal ultrasonography (7.5 MHz transducer,

Aloka 500V) to verify ovarian activity and blood samples collected for analysis of P4

concentrations. Heifers were considered pubertal once a corpus luteum with volume greater than

2.15 cm3, and plasma P4 concentrations greater than 1.5 ng/mL were concurrently detected in

one or both evaluations performed on a 10-d interval. Corpus luteum volume was calculated

using the formula for volume of a sphere (V = 4/3π × [D/2]3, where D is the maximum luteal

diameter). Heifer BW gain during the sampling phase was calculated by averaging the values

88

obtained in both 10-d interval assessments. Further, heifer shrunk (after 16 h of feed and water

restriction) BW was collected on d 1 and 192 for calculation of heifer ADG during the study.

Blood samples collected throughout the sampling phase for puberty assessment were also

analyzed for plasma IGF-I concentrations to evaluate heifer energy status. On d 10 and 40, 2

extra blood samples were collected from each heifer in addition to the morning collection. These

samples were collected in 3 h-intervals to account for the circadian rhythm of adrenal

steroidogenesis (Thun et al., 1981; Arthington et al., 1997), and analyzed for plasma

concentrations of cortisol, P4, ceruloplasmin and haptoglobin. Heifers remained in the handling

facility during the intervals between blood collections.

Heifer temperament scores were obtained in the morning of d 40, following blood

collection and ultrasonography exam, to evaluate treatment effects. Heifer temperament was

assessed by pen score, chute score, and exit velocity. Chute score was assessed by a single

technician based on a 5-point scale, where 1 = calm, no movement, and 5 = violent and

continuous struggling. For pen score assessment, heifers exited the chute and entered a pen

containing a single technician, and were assigned a score on a 5-point scale, where 1 =

unalarmed and unexcited, and 5 = very excited and aggressive toward the technician in a manner

that requires evasive action to avoid contact between the technician and heifer. Exit velocity was

assessed by determining the speed of the heifer exiting the squeeze chute by measuring rate of

travel over a 1.5-m distance with an infrared sensor (FarmTek Inc., North Wylie, TX). Further,

within each assessment day (d 10 and 40), heifers were divided in quintiles according to their

chute exit velocity, and assigned a score from 1 to 5 (exit score; 1 = slowest heifers; 5 = fastest

heifers). Individual temperament scores were calculated by averaging heifer chute score, pen

score, and exit score.

89

During the breeding phase (d 131 to 191), each treatment group was exposed to one

mature Angus bull (1:20 bull to heifer ratio), and bulls were rotated between groups every other

wk to account for potential bull effects. Heifer pregnancy status was verified by detecting a fetus

with transrectal ultrasonography (5.0 MHz transducer, Aloka 500V) 70 d after the end of the

breeding season.

Blood Analysis

Blood samples were collected via jugular venipuncture into commercial blood collection

tubes (Vacutainer, 10 mL; Becton Dickinson, Franklin Lakes, NJ) containing sodium heparin,

placed on ice immediately, and centrifuged at 2,400 × g for 30 min for plasma collection. Plasma

was frozen at -20°C on the same day of collection.

Concentrations of P4 and cortisol were determined using Coat-A-Count solid phase 125I

RIA kits (DPC Diagnostic Products Inc., Los Angeles, CA). A double antibody RIA was used to

determine concentrations and IGF-I (Badinga et al., 1991; Cooke et al., 2007a). Concentrations

of ceruloplasmin were determined according to procedures described by Demetriou et al. (1974).

Concentrations of haptoglobin were determined by measuring haptoglobin/hemoglobin complex

by the estimation of differences in peroxidase activity (Makimura and Suzuki, 1982), and results

are expressed as arbitrary units from the absorption reading at 450 nm × 100. All samples were

analyzed in duplicates. Across years, the intra- and interassay CV were, respectively, 9.2 and

6.9% for cortisol, 6.3 and 7.3% for P4, 5.1 and 3.5% for ceruloplasmin, 8.8 and 9.8% for

haptoglobin, and 4.5 and 4.6% for IGF-I. Assay sensitivity was 0.1 ng/mL for P4, 0.5 μg/dL for

cortisol, and 10 ng/mL for IGF-I.

90

Statistical Analysis

Growth, temperament, and physiological data were analyzed using the MIXED procedure

of SAS (SAS Inst., Inc., Cary, NC) and Satterthwaite approximation to determine the

denominator df for the tests of fixed effects. The model statement used for analysis of hormones

and metabolites, heifer BW, and temperament contained the effects of treatment, breed, time

variables, and appropriate interactions. Data were analyzed using heifer(breed × treatment × yr)

as random variable. Further, temperament and physiological data were adjusted covariately to

measurement obtained prior to acclimation period (d 10). The model statement used for ADG

contained the effect of treatment, breed, and yr. Data were also analyzed using heifer(breed ×

treatment × yr) as random variable. Results are reported as least square means and were

separated using LSD. Puberty data were analyzed with the GLM and LOGISTIC procedure of

SAS. The model statement contained the effects of treatment, breed, time of estimated puberty

establishment, year, and the appropriate interactions. Pregnancy data were analyzed with the

GLM procedure of SAS, and the model statement contained the effects of treatment, breed, year,

and the appropriate interactions. Pearson correlations were calculated among ADG, mean

concentrations of hormones and metabolites, and mean temperament measurements of heifers

during the study. These correlation coefficients were determined across years, breeds, and

treatments with the CORR procedure of SAS. For all analysis, significance was set at P ≤ 0.05

and tendencies were determined if P > 0.05 and ≤ 0.10. Results are reported according to

treatment effects if no interactions were significant, or according to the highest-order interaction

detected.

91

Results and Discussion

No interactions containing the effects of treatment, breed, and year were detected for

measurements of heifer growth, plasma measurements, temperament, and reproductive

performance; therefore results were combined between breeds and year. Further, no treatment ×

time(day) interaction was detected for the analysis of plasma measurements on d 10 and 40;

therefore results regarding treatment effects were combined among samples collected within

each day.

Acclimated heifers had reduced (P < 0.01) ADG compared with control heifers (0.50 vs.

0.58 kg/d respectively; SEM = 0.02; Table 4-1). Given that both treatment groups were provided

similar pastures and supplements during the study, treatment effects on ADG can be attributed to

the additional exercise that acclimated heifers were exposed to during the acclimation period.

During each acclimation event, heifers had to walk nearly 2 km in addition to the activity inside

the handling facility, whereas control heifers remained on their pasture. According to the Cornell

Net Carbohydrate and Protein System for Cattle model (ver. 5.0.40), acclimated heifers required

approximately 0.55 Mcal of ME during each of the acclimation events. Supporting this rationale,

heifers from both treatments had similar BW prior to the acclimation period, but control heifers

had numerically greater BW compared with acclimated heifers immediately following the

acclimation period (treatment × day interaction; P < 0.01; Figure 4-1).

A treatment effect was detected (P < 0.01) for puberty attainment. Although age at

puberty in cattle is highly determined by BW and growth rate (Schillo et al., 1992), heifers

exposed to acclimation procedures reached puberty sooner than control heifers despite their

reduced ADG (Figure 4-2). Research with humans and rodents reported that prepubertal females

exposed to exercise regimens attained puberty at older ages compared with control cohorts

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(Warren, 1980; Manning and Bronson, 1991). These authors attributed the delayed onset of

puberty to suppressive effects of exercise and consequent additional energy consumption on

GnRH and gonadotropin synthesis and secretion. Pregnancy rates, however, were similar

between treatments (Table 4-1). Nevertheless, treatment effects on pregnancy reported in this

document were evaluated using heifer pregnancy status obtained via transrectal ultrasonography

70 d after the end of the breeding season. This analysis did not account for the time during the

breeding season in which heifers became pregnant, whereas it is expected that acclimated heifers

conceived earlier compared with control heifers, particularly because of treatment effects on

puberty attainment. As soon as the calving season for heifers utilized in yr 2 is concluded, date of

conception will be estimated retrospectively by subtracting gestation length (286 d; Reynolds et

al., 1980) from the calving date, and treatment effects on pregnancy will be re-analyzed and

reported as pregnancy attainment over time during the breeding season.

Treatment effects were detected (P < 0.05) for concentrations of cortisol and P4.

Acclimated heifers had reduced (P < 0.01) cortisol concentrations compared with control heifers

after the acclimation period (3.8 vs. 5.1 μg/dL; SEM = 0.17; Figure 4-3). Within prepubertal

heifers, P4 concentrations were reduced (P = 0.03) in acclimated heifers compared with control

heifers after the acclimation period (0.52 vs. 0.78 ng/mL; SEM = 0.08; Figure 4-4). Previous

research indicated that acclimation of cattle to handling procedures was an alternative to prevent

elevated concentrations of cortisol in response to handling stress (Crookshank et al., 1979;

Andrade et al., 2001; Curley et al., 2006). The same rationale can be extrapolated to treatment

effects on prepubertal P4 concentrations. Although the major sources of P4 synthesis in cattle -

the corpus luteum and the pregnant placenta (Hoffmann and Schuler, 2002) – are absent in

prepubertal heifers, their adrenal gland is also capable of producing significant amounts of P4 as

93

an intermediate of cortisol synthesis via ACTH stimuli (Gonzalez-Padilla et al., 1975; Brown,

1994). Supporting this rationale, concentrations of cortisol and P4 were positively correlated in

prepubertal heifers herein (Table 4-2), and also in previous research efforts evaluating the effects

of ACTH administration to ovariectomized heifers and cows (Bage et al., 2000; Yoshida and

Nakao, 2005). In accordance with previous studies indicating that the adrenal gland synthesizes

steroids in a circadian rhythm (Thun et al., 1981; Arthington et al., 1997), a time effect was

detected (P < 0.01) for the analysis of both cortisol and prepubertal P4. Concentrations of

cortisol were the greatest (P < 0.01) in the morning collection for heifers from both treatments

(4.9 vs. 3.74 and 4.06 μg/dL, respectively; SEM = 0.16). Similarly, P4 concentrations of

prepubertal heifers were greater (P < 0.01) in the morning collection compared with the

subsequent collections (1.01 vs. 0.54 and 0.53 ng/mL, respectively; SEM = 0.08).

No treatment effects were detected for concentrations of IGF-I, ceruloplasmin, and

haptoglobin (Table 4-1), although previous studies reported that elevated concentrations of

corticoids can stimulate synthesis of acute phase proteins (Yoshino et al., 1993; Higuchi et al.,

1994) and reduce circulating concentrations of IGF-I (Maciel et al., 2001) in cattle. Nevertheless,

positive correlations were detected (P < 0.05; Table 4-2) between concentrations of IGF-I and

heifer ADG, concentrations of ceruloplasmin and haptoglobin, and heifer temperament and

concentrations of ceruloplasmin (Table 4-2). Additionally, both heifer ADG and IGF-I

concentrations were negatively correlated with heifer temperament and ceruloplasmin

concentrations (Table 4-2). These results support previous studies reporting that IGF-I is a key

factor for heifer growth and its synthesis can be reduced during the acute phase response

(Elsasser et al., 1997; Cooke et al., 2007a), and also suggests that cattle with excitable

temperament experience reduced concentrations of IGF-I and stimulated acute phase response,

94

which contributes to their decreased BW gain compared to cattle with calmer temperament

(Voisinet et al., 1997; Fell et al., 1999; Qiu et al., 2007).

No treatment effects were detected for temperament scores (Table 4-3), but acclimated

heifers had reduced chute score (P < 0.01) compared with control heifers after the acclimation

period (Table 4-3). Further, all measurements of temperament were positively correlated to each

other, and also to cortisol concentrations (P < 0.01; Table 4-4). Fordyce et al. (1987) indicated

that Brahman-crossbred steers frequently exposed to handling procedures exhibited calmer

temperament compared with cohorts with no handling experiences. Curley et al. (2006) reported

that frequent handling of Brahman bulls decreased their exit velocity, but not their pen and chute

score. The positive correlations detected among measurements of temperament and cortisol

concentrations reported herein were also described by others (Stahringer et al. 1990; Fell et al.,

1999; Curley et al., 2006), indicating that these three measurements of cattle behavior during

handling can be used as indicators of temperament and also denote the amount of stress that the

animal is experiencing (Thun et al., 1998; Sapolsky et al., 2000). However, pen score and exit

velocity are classified as non-restrained techniques to evaluate cattle temperament, whereas

chute score belongs to the restrained techniques category (Burrow and Corbet, 2000). An

important flaw within restrained techniques is that cattle with excitable temperament may

“freeze” when restrained, and consequently not express their true behavior during these

assessments (Burrow and Corbet, 2000). Therefore, treatment effects detected in the present

study for chute score should be interpreted with caution, even though this measurement of

temperament was positively correlated with cortisol and non-restrained assessments (Table 4-4).

Breed effects were detected (P < 0.05; Table 4-5) for ADG and puberty attainment.

Brahman × Angus heifers had greater (P = 0.02) ADG compared with Braford heifers during the

95

study (0.55 vs. 0.49 kg/d, respectively; SEM = 0.02). A greater (P < 0.01) number of Brahman ×

Angus heifers attained puberty during the sampling phase compared with Braford heifers (80.6

vs. 38.8% pubertal heifers, respectively; SEM = 6.9). No further breed effects were detected

(Table 4-5). These results support previous data from our research group (Arthington et al.,

2004) reporting inferior ADG and reproductive performance of Braford heifers compared with

Brahman × Angus heifers. The Braford heifers utilized herein and by Arthington et al. (2004)

were originated from a straightbred Braford cowherd that has been sharing similar genetics for

more than 20 yr, which likely resulted in accumulative inbreeding. Martin et al. (1992) indicated

that inbreeding can be detrimental to cattle growth and age at puberty. Therefore, breed

differences in ADG and puberty attainment reported herein can be attributed, at least in part, to

accumulative inbreeding effects within Braford heifers, and also to the retained heterosis of

Brahman × Angus heifers (Koger, 1980).

The main hypothesis of the present experiment was that acclimation to human handling

would alleviate the detrimental effects of excitable temperament on reproductive function of

Brahman-crossbred heifers, and allow acclimated heifers to reach puberty earlier than non-

acclimated cohorts. Plasse et al. (1970) reported that excitable temperament influences

negatively the reproductive performance of beef females. Cattle with high Brahman influence

typically exhibit excitable temperament (Hearnshaw and Morris, 1984; Fordyce et al., 1988;

Voisinet et al., 1997) and experience stimulated function of the hypothalamic-pituitary-adrenal

axis when exposed to humans and handling procedures, resulting in increased production and

circulating concentrations of ACTH and consequently cortisol (Stahringer et al., 1990; Curley et

al., 2008). These hormones have been shown to directly impair the physiological mechanisms

required for puberty attainment in heifers, particularly synthesis and release of LH by the

96

anterior pituitary (Li and Wagner, 1983; Dobson et al., 2000), whereas methods to acclimate

cattle to handling procedures were reported to be successful in reducing circulating cortisol

concentrations (Crookshank et al., 1979; Andrade et al., 2001) and increasing pulsatility and

mean concentrations of LH (Echternkamp, 1984). Supporting this rationale and our hypothesis,

acclimated heifers in the present study had reduced cortisol concentrations and hastened onset of

puberty compared with non-acclimated cohorts. Nevertheless, the mechanisms by which

acclimation procedures hastened puberty attainment regardless of decreased rates of BW gain

remain unclear. Based on our hypothesis, it can be speculated that reduced cortisol

concentrations in acclimated heifers facilitated the initiation of the physiological events required

for puberty attainment, particularly increased LH pulse frequency and consequent first ovulatory

LH surge (Smith and Dobson, 2002). Although concentrations of cortisol were only evaluated

when heifers were handled and restrained for blood collection, one can speculate that acclimated

heifers also had reduced cortisol concentrations compared to control heifers on a daily basis

given that heifers from both groups were often exposed to brief human interaction, particularly

because of feeding and traffic of personnel/vehicles within the research station.

Another mechanism responsible for treatments effects on puberty attainment can be

attributed to prepubertal P4 synthesis by the adrenal gland. Approximately 55% of the Brahman

× Angus and 86% of the Braford heifers from the present study experienced P4 concentrations

above 1.0 ng/mL before reaching puberty, whereas the greatest prepubertal P4 value detected for

both breeds was 2.8 ng/mL. These results indicate that elevated production of P4 by the adrenal

gland is a common occurrence in prepubertal Brahman-crossbred heifers during handling

practices. This response was likely due to heifer temperament, as positive correlations (P < 0.01;

Table 4-2) were detected between concentrations of P4 and cortisol (r = 0.50), P4 and

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temperament score (r = 0.45), and cortisol and temperament score (r = 0.58). Progesterone

appears to be a required stimulus for puberty establishment in heifers given that it suppresses the

number of estradiol receptors in the hypothalamus and primes the hypothalamic-pituitary-ovarian

axis toward enhanced synthesis and pulsatile secretion of LH (Anderson et al., 1996; Looper et

al., 2003). Transient increases in circulating concentrations of P4 were detected in beef heifers 2

wk prior to the onset of puberty (Gonzalez-Padilla et al., 1975), and the origin of this hormone

was attributed to the adrenal gland and/or luteal structures found within the ovary (Gonzalez-

Padilla et al., 1975; Berardinelli et al., 1979). Based on this information, it can be theorized that

acclimated heifers experienced transient increases in adrenal steroidogenesis and thus P4

synthesis during the acclimation process, particularly during the initial weeks when heifers were

still unfamiliar with the acclimation events. Transient increases in adrenal P4 synthesis during

the early phases of the acclimation process, combined with alleviated cortisol synthesis as

acclimation advanced, may have contributed to hastened puberty attainment of acclimated

heifers compared with control cohorts. Nevertheless, further research is required to properly

address this assumption.

In conclusion, results from this experiment indicate that acclimation of Brahman-

crossbred heifers to handling procedures and human interaction reduced ADG because of the

additional exercise that heifers were exposed to, but alleviated adrenal steroidogenesis and

hastened onset of puberty. Therefore, acclimation of Brahman × Angus and Braford replacement

heifers to human handling after weaning may be an alternative to enhance their reproductive

development, and increase the efficiency of heifer development programs in cow-calf operations

containing Brahman-influenced cattle.

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Table 4-1. Average daily gain, pregnancy rates, and concentrations of plasma ceruloplasmin, haptoglobin, and IGF-I of heifers exposed or not (control) to handling acclimation procedures.

Item Acclimated Control SEM P-value

ADG, kg/d 1 0.50 0.58 0.016 < 0.01

Pregnancy rate, % 2 68 69 7.8 0.47

Ceruloplasmin, mg/dL 3 27.3 25.7 0.69 0.11

Haptoglobin, 450 nm × 100 3 1.52 1.27 0.169 0.29

IGF-I, ng/mL 3 216 215 6.2 0.86

1 Calculated using initial (d 1) and final (d 192) shrunk BW. 2 Pregnant heifers / total heifers during the breeding season. 3 Least squares means adjusted covariately to values obtained prior to acclimation period.

99

100

1 U

pper

row

= c

orre

latio

n co

effic

ient

s. L

ower

row

= P

–val

ues.

2 Pr

epub

erta

l hei

fers

onl

y (n

= 7

1)

Tabl

e 4-

2. P

ears

on c

orre

latio

n co

effic

ient

s am

ong

AD

G, p

lasm

a m

easu

rem

ents

, and

tem

pera

men

t of h

eife

rs. 1

Table 4-3. Temperament measurements of heifers exposed or not (control) to handling acclimation procedures. 1

Item Acclimated Control SEM P-Value

Chute score 2 1.37 1.84 0.091 < 0.01

Pen score 3 2.85 2.72 0.137 0.51

Exit velocity, m/s 4 2.91 2.74 0.148 0.43

Temperament score 5 2.46 2.48 0.096 0.93

1 Least squares means adjusted covariately to values obtained prior to acclimation period. 2 Assessed by a single technician based on a 5-point scale, where 1 = calm, no movement, and 5 = violent and continuous struggling. 3 Heifers exited the chute and entered a pen containing a single technician, and were assigned a score on a 5-point scale, where 1 = unalarmed and unexcited, and 5 = very excited and aggressive toward the technician in a manner that requires evasive action to avoid contact between the technician and heifer. 4 Assessed by determining the speed of the heifer exiting the squeeze chute by measuring rate of travel over a 1.5-m distance with an infrared sensor (FarmTek Inc., North Wylie, TX). 5 Calculated by averaging heifer chute score, exit score, and pen score. Exit score was calculated by dividing chute exit velocity results into quintiles, and assigning heifers with a score from 1 to 5 (exit score; 1 = slowest heifers; 5 = fastest heifers).

101

Table 4-4. Pearson correlation coefficients among measurements of temperament and plasma cortisol concentrations of heifers. 1

Item Cortisol Chute score Exit velocity

Chute score 0.44 < 0.01 Exit velocity 0.55 0.46 < 0.01 < 0.01 Pen score 0.48 0.40 0.69 < 0.01 < 0.01 < 0.01

1 Upper row = correlation coefficients. Lower row = P–values.

102

Table 4-5. Effects of breed on performance, temperament, and physiologic parameters of replacement heifers. 1

Item Brahman × Angus Braford SEM P-Value

ADG, kg/d 2 0.55 0.49 0.016 0.02

Puberty rate, % 3 81 39 6.9 < 0.01

Pregnancy rate, % 4 71 67 7.8 0.39

Temperament score 5 2.47 2.62 0.121 0.50

Cortisol, μg/dL 4.01 4.45 0.200 0.12

IGF-I, ng/mL 200 206 6.4 0.48

Ceruloplasmin, mg/dL 25.2 26.5 0.67 0.18

Haptoglobin , 450 nm × 100 2.14 1.74 0.315 0.35

1 Values reported are least square means from all samples/assessments obtained throughout the study 2 Calculated using initial (d 1) and final (d 192) shrunk BW. 3 Estrous cycling heifers / total heifers during the experimental period. 4 Pregnant heifers / total heifers during the breeding season. 5 Calculated by averaging heifer chute score, exit score, and pen score. Exit score was calculated by dividing exit velocity results into quintiles, and assigning heifers with a score from 1 to 5 (exit score; 1 = slowest heifers; 5 = fastest heifers).

103

200

240

280

320

360

400

d 0 and 10 d 80 and 90 d 120 and 130

BW

, kg

Acclimated Control A

200

240

280

320

360

400

d 0 and 10 d 40 and 50 d 80 and 90 d 120 and 130

BW

, kg

Acclimated Control B

Figure 4-1. Body weight of heifers exposed or not (control) to handling acclimation procedures

(d 11 to 39). Panel A reports results combined from both yr, whereas Panel B reports results from yr 1. In yr 2, the scale was not functional during d 40 and 50; therefore data combined among yr do not include BW values obtained on d 40 and 50 in yr 1. A treatment x day effect was detected in both analyses (P < 0.01; SEM = 4.6 and 6.0 for panels A and B, respectively). Heifers from both treatments had similar BW prior to the acclimation period (d 10 to 40), but control heifer had numerical greater BW compared with acclimated heifers during the remainder of the study.

104

0

10

20

30

40

50

60

70

80

90

100

d 0 and 10 d 40 and 50 d 80 and 90 d 120 and 130

Pube

rtal

hei

fer,

%

Acclimated Control

Figure 4-2. Puberty attainment of heifers exposed or not (control) to handling acclimation procedures (d 11 to 39). Heifers were considered pubertal once a corpus luteum and plasma P4 concentrations greater than 1.5 ng/mL were concurrently detected in one or both evaluations performed on a 10-d interval. A treatment effect was detected (P = 0.02; SEM = 6.5).

105

0

1

2

3

4

5

6

7

Pre-acclimation (d 10) Post-acclimation (d 40)

Plas

ma

cort

isol

, μg/

dL

Acclimated Control

Figure 4-3. Plasma cortisol concentrations of heifers exposed or not (control) to handling acclimation procedures (d 11 to 39). Samples collected on d 10 served as covariate, therefore results reported for d 40 are covariately adjusted least square means. Acclimated heifers had reduced (P < 0.01; SEM = 0.17) concentrations of cortisol compared to control heifers on d 40.

106

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Pre-acclimation (d 10) Post-acclimation (d 40)

Plas

ma

prog

este

rone

, ng/

mL

Acclimated Control

Figure 4-4. Plasma progesterone concentrations of prepubertal heifers exposed or not (control) to

handling acclimation procedures (d 11 to 39). Samples collected on d 10 served as covariate, therefore results reported for d 40 are covariately adjusted least square means. Acclimated heifers had reduced (P = 0.03; SEM = 0.08) concentrations of progesterone compared to control heifers on d 40.

107

CHAPTER 5 EFFECTS OF TEMPERAMENT AND ACCLIMATION ON PERFORMANCE,

PHYSIOLOGICAL RESPONSES, AND PREGNANCY RATES OF BRAHMAN-CROSSBRED COWS

Introduction

The major objective of cow-calf production systems is to produce one calf per cow

annually. Ovulation of a competent oocyte determines the length of the postpartum interval and

also the fertility of beef cows during the breeding season (Short et al., 1990). Follicle size and

LH pulsatility are major factors responsible for a successful ovulation (Roche, 2006); therefore,

alternatives to stimulate GnRH delivery to the pituitary, and anticipate/enhance the ovulatory LH

surge are options to maximize reproductive performance of beef cows (Day, 1994).

Brahman-crossbred cows, however, may experience impaired pituitary sensitivity to GnRH

(Griffin and Randel, 1978), which may compromise their ability to resume estrous cycles and

conceive. In addition, Brahman-crossbred cattle are often described as temperamental

(Hearnshaw and Morris, 1984; Fordyce et al., 1988; Voisinet et al., 1997), and this trait is

expected to further negatively influence their reproductive function (Plasse et al., 1970). Cattle

with excitable temperament often experience stimulated secretion and circulating concentrations

of ACTH and cortisol (Curley et al., 2008). These hormones directly impair synthesis and release

of GnRH and gonadotropins (Li and Wagner, 1983; Dobson et al., 2000). Nevertheless,

acclimation of beef females to human interaction and handling has been reported to alleviate

these negative physiological effects of excitable temperament on reproduction (Echternkamp,

1984). Based on this information, we hypothesized that Brahman-crossbred cows exposed to

acclimation procedures would experience improved temperament, reduced circulating

concentrations of cortisol, and increased reproductive performance compared to non-acclimated

cohorts. The objectives of the present experiment were to compare BW and BCS, measurements

108

of temperament, concentrations of plasma hormones and metabolites, and pregnancy attainment

in Brahman × British and Braford cows exposed or not to acclimation procedures.

Materials and Methods

This experiment was conducted over 2 yr (2006 and 2007) at the University of Florida –

IFAS, Range Cattle Research and Education Center, Ona. Each experimental period was divided

into a acclimation phase (August to January) and a breeding season (January to April). The

animals utilized were cared for in accordance with acceptable practices as outlined in the Guide

for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS,

1999).

Animals and Diets

A total of 160 Braford (37.5% Brahman + 62.5% Hereford) and 235 Brahman × British

(mostly Angus, and containing approximately 25% of Brahman breeding) cows were assigned to

the experiment. Initial mean BW and age across breeds and yr were (± SD), respectively, 545 ±

71 kg and 6 ± 3 yr. Approximately 45 d after weaning in yr 1 (August 2006), cows were

evaluated for BW, BCS (Wagner et al., 1988), and temperament, stratified by these

measurements in addition to breed and age, and randomly allocated to 14 groups. Braford cows

were divided into 8 groups of approximately 20 cows each, whereas Brahman × British cows

were divided into 6 groups of approximately 40 cows each. Groups were assigned randomly to

receive or not (control) the acclimation treatment (7 groups/treatment). In yr 2, cows were re-

evaluated within 45 d after weaning (August 2007) for BW, BCS, and temperament, and were

stratified and divided into 14 groups similarly as in yr 1. However, cows were re-allocated into

groups in order that they received the same treatment assigned in yr 1. Within acclimated cows, 3

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Braford and 19 Brahman × British cows were culled prior to the beginning of yr 2 and replaced

by cohorts not previously assigned to the experiment.

During both years, groups were maintained on separate bahiagrass (Paspalum notatum)

pastures and were rotated among pastures weekly. Stargrass (Cynodon nlemfuensis) was offered

in amounts to ensure ad libitum access when pasture availability was limited. From early

December to the end of the breeding season, cows received a blend of sugarcane molasses and

urea (97:3 as fed-basis) 3 times weekly, at a rate to provide a daily amount of 1.8 kg of DM per

cow. Nutrient composition of sugarcane molasses was estimated to be (DM basis) 79.2% of TDN

and 12.3% of CP (SDK Laboratories, Hutchinson, KS). A complete commercial mineral/vitamin

mix (14% Ca, 9% P, 24% NaCl, 0.20% K, 0.30% Mg, 0.20% S, 0.005% Co, 0.15% Cu, 0.02% I,

0.05% Mn, 0.004% Se, 0.3% Zn, 0.08% F, and 82 IU/g of vitamin A) and water were offered for

ad libitum consumption throughout the experiment.

Acclimation Procedure

The acclimation process was applied from August to January, and cows for both

treatment groups calved during this period (from October to December of each yr). During the

acclimation period, acclimated groups were visited twice weekly. During each visit, a person

walked among cows for 15 min, and hand-offered approximately 50 g of range cubes per cow.

Range cubes consisted of (as-fed basis) 66.7% of wheat middlings, 27.0% of soybean hulls,

3.7% of blackstrap molasses, and 2.6% of cottonseed meal. Nutrient composition of range cubes

was estimated to be (DM basis) 62.5% of TDN and 22.2% of CP (Dairy One Forage Laboratory,

Ithaca, NY). The amount of range cubes offered during each acclimation visit provided no

meaningful nutritional contribution to cows (approximately 0.5 and 1.0 % of TDN and CP daily

requirements, respectively).

110

Sampling

At the beginning of the acclimation phase (August) and breeding season (January), one

blood sample was collected from each cow in addition to BW, BCS, and temperament

evaluation. Blood samples were analyzed for plasma concentrations of cortisol, ceruloplasmin,

haptoglobin, and IGF-I (pre-breeding samples only). Cow temperament was assessed by pen

score, chute score, and exit velocity. Chute score was assessed by a single technician based on a

5-point scale, where 1 = calm, no movement, and 5 = violent and continuous struggling. For pen

score assessment, cows exited the chute and entered a pen containing a single technician, and

were assigned a score on a 5-point scale, where 1 = unalarmed and unexcited, and 5 = very

excited and aggressive toward the technician in a manner that requires evasive action to avoid

contact between the technician and cow. Exit velocity was assessed by determining the speed of

the cow exiting the squeeze chute by measuring rate of travel over a 1.5-m distance with an

infrared sensor (FarmTek Inc., North Wylie, TX). Further, within each assessment day, cows

were divided in quintiles according to their chute exit velocity, and assigned a score from 1 to 5

(exit score; 1 = slowest cows; 5 = fastest cows). Individual temperament scores were calculated

by averaging cow chute score, pen score, and exit score.

Breeding Season

During yr 1 and 2, each Braford group was exposed to one mature Angus bull (1:20 bull

to cow ratio) for 90 d. Bulls were not rotated among groups, but were submitted to and approved

by a breeding soundness evaluation (Chenoweth and Ball, 1980) before the breeding season.

During yr 1, Brahman × British cows were assigned to an estrus synchronization + fixed-time AI

protocol at the beginning of the breeding season (d 0). Cows received a 100 μg treatment of

GnRH (Cystorelin; Merial Ltd., Duluth, GA) and were inserted with a controlled internal drug

111

releasing device containing 1.38 g of progesterone (CIDR; Pfizer Animal Health, New York,

NY) on d 0, received a PGF2α treatment (25 mg; Lutalyse; Pfizer Animal Health, New York,

NY), CIDR and calf removal on d 6, followed by calf return and fixed-time AI on d 8. Fifteen

days after AI, Brahman × British groups were exposed to two mature Angus or Brangus bulls

(1:20 bull to cow ratio) for 90 d. Bulls were not rotated among groups, but were submitted to and

approved by a breeding soundness evaluation (Chenoweth and Ball, 1980) before the breeding

season. During yr 2, Brahman × British cows were only exposed to natural breeding by mature

bulls similarly as in yr 1. Mean days post-partum at the beginning of the breeding season were (±

SD), respectively, 72 ± 23.8 and 98 ± 22.6 d for Braford and Brahman × British cows during yr

1, and 48 ± 24.2 and 22 ± 21.0 d for Braford and Brahman × British cows during yr 2.

Cow pregnancy status was verified by detecting a fetus via rectal palpation 90 d after the

end of both breeding seasons. Pregnancy rates to AI in Brahman × British cows were evaluated

by detecting a fetus with transrectal ultrasonography (5.0 MHz transducer, Aloka 500V,

Wallingford, CT) 50 d after AI. For yr 1 only, date of conception was estimated retrospectively

by subtracting gestation length (286 d; Reynolds et al., 1980) from the calving date.

Blood Analysis

Blood samples were collected via jugular venipuncture into commercial blood collection

tubes (Vacutainer, 10 mL; Becton Dickinson, Franklin Lakes, NJ) containing sodium heparin,

placed on ice immediately, and centrifuged at 2,400 × g for 30 min for plasma collection. Plasma

was frozen at -20°C on the same day of collection.

Concentrations of cortisol were determined using a Coat-A-Count solid phase 125I RIA kit

(DPC Diagnostic Products Inc., Los Angeles, CA). A double antibody RIA was used to

determine concentrations and IGF-I (Badinga et al., 1991; Cooke et al., 2007a). Concentrations

112

of ceruloplasmin were determined according to procedures described by Demetriou et al. (1974).

Concentrations of haptoglobin were determined by measuring haptoglobin/hemoglobin complex

by the estimation of differences in peroxidase activity (Makimura and Suzuki, 1982), and results

are expressed as arbitrary units from the absorption reading at 450 nm × 100. Across years, the

intra- and interassay CV were, respectively, 9.5 and 7.2% for cortisol, 5.6 and 6.3% for

ceruloplasmin, 8.4 and 9.7% for haptoglobin, and 5.8 and 6.3% for IGF-I. Assay sensitivity was

0.5 μg/dL for cortisol and 10 ng/mL for IGF-I.

Statistical Analysis

Performance, temperament, and physiological data were analyzed using the MIXED

procedure of SAS (SAS Inst., Inc., Cary, NC) and Satterthwaite approximation to determine the

denominator df for the tests of fixed effects. The model statement used for analysis of hormones

and metabolites, BW, BCS, and temperament contained the effects of treatment, breed, time

variables, and appropriate interactions. Data were analyzed using group(breed × treatment × yr)

as random variable. Results are reported as least square means and were separated using LSD.

Pregnancy data for yr 1 were analyzed with the GLM and LOGISTIC procedure of SAS, and the

model statement contained the effects of treatment, breeds, estimated time of conception, and the

appropriate interactions. Further, cow days postpartum at the beginning of the breeding season

was included into the analysis as covariate; therefore, pregnancy results for yr 1 are reported as

covariately adjusted means and were separated using LSD. Pregnancy data for yr 2 were

analyzed with the GLM procedure of SAS. The model statement contained the effects of

treatment, breed, year, and appropriate interactions, and did not include days postpartum as

covariate so potential carry over effects of treatments across yr could be accounted for. Results

are also reported as least square means and were separated using LSD. The probability of cows

113

to become pregnant during the breeding season was evaluated according to BCS, temperament

score, and concentrations of plasma metabolites and hormones obtained at the beginning of

breeding. The GLM procedure of SAS was utilized to determine if each individual measurement

influenced pregnancy rates linearly, quadratically, and/or cubically. If multiple continuous order

effects were significant, the effect with the greatest F-value was selected. The LOGISTIC

procedure was utilized to determine the intercept and slope(s) values according to maximum

likelihood estimates from the significant effect selected, and the probability of pregnancy was

determined according to the equation: Probability = (e logistic equation) / (1 + e logistic equation). Logistic

curves were constructed according to the minimum and maximum values detected for each

individual measurement. Pearson correlations were calculated, within yr and collection day,

among plasma and temperament measurements. These correlation coefficients were determined

across breeds and treatments with the CORR procedure of SAS. For all analysis, significance

was set at P ≤ 0.05 and tendencies were determined if P > 0.05 and ≤ 0.10. Results are reported

according to treatment effects if no interactions were significant, or according to the highest-

order interaction detected.

Results and Discussion

A treatment × breed × yr interaction was detected (P = 0.05) for mean BW (Tables 5-1).

During yr 2, acclimated Braford cows had greater (P = 0.01) mean BW compared to control

cohorts (557 vs. 527 kg of BW, respectively; SEM = 7.8). However, no treatment effects were

detected (P = 0.38) for BW change during the acclimation period of both yr (Table 5-2). A

treatment × breed × yr interaction was also detected (P = 0.02) for mean BCS and BCS change

(Table 5-1). During yr 1, acclimated Braford cows had reduced (P = 0.04) mean BCS compared

to control cohorts (5.71 vs. 6.06 of BCS, respectively; SEM = 0.114). Conversely, acclimated

114

Braford cows had greater (P = 0.05) mean BCS compared to control cohorts during yr 2 (6.04 vs.

5.71 of BCS, respectively; SEM = 0.116). Acclimated Brahman × British cows had reduced (P <

0.01) BCS change compared to control cohorts during the acclimation period in yr 2 (-0.30 vs. -

0.86 of BCS; SEM = 0.119). These results may suggest that the acclimation process influenced

cow nutritional status and resulted in the treatment effects detected for mean BW and BCS.

Nevertheless, the detection of treatment effects was inconsistent between breeds and yr. Further,

mean BCS of cows from both breeds and treatments during yr 1 and yr 2 were always adequate

(Kunkle et al., 1994), and treatment effects detected for mean BCS and BCS changes, although

statistically significant, can be considered marginal and insufficient to affect performance of

cows with BCS greater than 5 (Kunkle et al., 1994; Cooke et al., 2008b). Further supporting this

rationale, no treatment effects were detected for IGF-I concentrations (Table 5-2), given that

circulating concentrations of IGF-I are positively associated with nutritional status of cattle

(Ellenberger et al., 1989; Bossis et al., 1999; Lapierre et al., 2000). To ensure that acclimation

effects were not confounded with nutritional effects, only 50 g of range cubes were offered per

cow during each of the acclimation events.

No treatment effects were detected for temperament score and concentrations of cortisol

and haptoglobin (Table 5-2). Further, no treatment effects were detected for individual

measurements of cow temperament (Table 5-2). A treatment × day interaction was detected (P =

0.04) for ceruloplasmin (Table 5-3). Acclimated cows tended (P = 0.08) to have decreased

ceruloplasmin concentrations compared to control cows at the beginning of the acclimation

period (15.8 vs. 16.6, respectively; SEM = 0.30), but similar (P = 0.82) at the beginning of the

breeding season (14.2 vs. 14.1; SEM = 0.30). Previous research indicated that acclimation of

cattle to human interaction was an alternative to improve temperament and prevent elevated

115

cortisol concentrations in response to excitable temperament and handling stress (Fordyce et al.,

1987; Andrade et al., 2001; Curley et al., 2006). Based on these studies, we hypothesized that

Brahman-crossbred cows would benefit from acclimation particularly because of their common

excitable temperament during handling practices (Hearnshaw and Morris, 1984; Fordyce et al.,

1988; Voisinet et al., 1997). This rationale can be extrapolated to ceruloplasmin and haptoglobin

concentrations because the acute phase response is stimulated by elevated concentrations of

corticoids (Yoshino et al., 1993; Higuchi et al., 1994). However, cows from both treatments had

similar temperament, and concentrations of cortisol and acute phase proteins after the

acclimation period. This lack of treatment effects may indicate that the acclimation process failed

to improve cow temperament and the physiological responses associated with this trait.

However, the sampling schedule of the present experiment perhaps hindered the detection of

treatment effects on cortisol concentrations. Because of the large number of animals utilized, in

addition to the extensive grazing conditions that they were maintained, cows from both

treatments were sampled for blood without any specific order throughout the day during all

collection days. Adrenal steroidogenesis occurs in a circadian rhythm in cattle, being highest in

the morning, decreasing during the day, and reaching nadir levels in the evening (Thun et al.,

1981; Arthington et al., 1997). Therefore, the influence of diurnal variation in cortisol

concentrations may have impaired the detection of treatment effects for this hormone.

Nevertheless, positive correlations were often detected (P < 0.05), across breeds and treatments,

between concentrations of ceruloplasmin and haptoglobin, and between measurements of

temperament and cortisol concentrations (Table 5-4 and 5-5). Ceruloplasmin and haptoglobin are

correlated because both proteins are components of the acute phase response in cattle (Carroll

and Forsberg, 2007). Positive correlations among measurements of temperament and cortisol

116

concentrations were also described by others (Stahringer et al. 1990; Fell et a., 1999; Curley et

al., 2006), suggesting that cattle with excitable temperament experience elevated concentrations

of cortisol, whereas chute score, exit velocity, and pen score can be used as indicators of cattle

temperament and also denote the amount of stress that the animal is experiencing during

handling practices (Thun et al., 1998; Sapolsky et al., 2000).

During yr 1 of the experiment, a treatment × breed interaction was detected for pregnancy

attainment (P < 0.01; Figure 5-1). Acclimated Braford cows became pregnant earlier and at a

greater number (P < 0.01) during the breeding season compared to control cohorts. Conversely,

no treatment effects were detected for Brahman × British cows in pregnancy rates to fixed-time

AI (6.8 and 9.8 % of pregnant cows / total cows for acclimated and control treatments,

respectively; P = 0.45; SEM = 2.85) and also accumulative (AI + bull breeding) pregnancy

during the breeding season (P = 0.40; Figure 5-1). Acclimation increased reproductive

performance of Braford cows during yr 1 of the experiment, but the reasons for this effect cannot

be clarified because of the similar treatment responses observed among the other measurements

evaluated. The lack of similar treatment effects for pregnancy of Brahman × British cows during

yr 1 of the experiment can be attributed, at least in part, to the estrus synchronization + fixed-

time AI protocol, which resulted in unexpected low pregnancy rates and likely influenced

reproductive function of cows during the remainder of the breeding season. Brahman × British

cows were assigned to a fixed-time AI breeding prior to bull exposure to evaluate if acclimation

would alleviate the detrimental effects of excitable temperament on reproduction when cattle had

to be handled frequently. The protocol utilized herein was selected because calf removal is an

alternative to stimulate ovulation instead of GnRH administration (Williams et al., 1983; Pursley

et al., 1995), and exogenous GnRH was expected to bypass the effects of excitable temperament

117

on the physiologic mechanisms associated with fertility, more specifically gonadotropin

secretory activity. The protocol utilized herein was based on a previous study reporting

satisfactory pregnancy rates of B. indicus × B. taurus cows exposed to a 7 d CIDR treatment

followed by 48 h calf removal and fixed-time AI (Vasconcelos et al., 2008); therefore the

reasons for the reduced pregnancy rates to AI obtained herein are unknown, but this outcome

likely influenced treatment effects on reproductive performance of Brahman × British.

In contrast to yr 1, a treatment × breed interaction was detected (P = 0.03) for pregnancy

rates during yr 2 because acclimated Braford cows had reduced (P = 0.04) pregnancy rates

compared to control cohorts (76.3 vs. 88.1 pregnant cows / total cows, respectively; SEM =

4.06), whereas no treatment effects were detected (P = 0.41) for Brahman × British cows (89.3

vs. 85.3 pregnant cows / total cows for acclimated and control treatments, respectively; SEM =

3.39). Nevertheless, treatment effects on pregnancy during yr 2 were evaluated using cow

pregnancy status assessed by rectal palpation 90 d after the end of the breeding season. This

analysis did not account for the time during the breeding season in which cows became pregnant,

whereas cows that become pregnant earlier have greater chance to re-breed during the next

breeding season (Reynolds, 1967; Wiltbank, 1970). Therefore, treatment effects on estimated

pregnancy attainment during yr 2 are required for better evaluation of acclimation effects on

reproductive performance of Brahman-crossbred cows, and as soon as the calving season for

these cows is concluded, date of conception will be estimated and treatment effects on pregnancy

will be re-analyzed and reported similarly as pregnancy rates for yr 1.

Breed comparisons were not performed for physiologic, reproductive, and performance

measurement because Braford and Brahman × British cows were maintained at different

management scenarios, such as grazing systems, stocking rates, and group sizes. Day effects,

118

however, were evaluated (Table 5-3). Cows from both breed types had reduced BW (P < 0.01)

and BCS (P < 0.01) at the beginning of breeding season compared to the beginning of the

acclimation period (Table 5-3). Cows calved during the acclimation period and were nursing

young calves at the onset of breeding, which likely stimulated mobilization of body reserves and

tissues to sustain lactation during this period (NRC, 1996).

The probability of cows to become pregnant, according to measurements obtained at the

beginning of the breeding season, was evaluated within each yr because mean days post-partum

across breeds at the onset of breeding differed (P < 0.01) from yr 1 to yr 2 (88 vs. 34 d,

respectively; SEM = 1.5). Plasma IGF-I concentrations and cow BCS affected quadratically (P <

0.01) the probability of pregnancy during both yr (Figure 5-2). These results indicate that

reduced or excessive energy status is detrimental to reproductive performance of cattle, as

reported by others (Armstrong et al., 2001; Bilby et al., 2006; Cooke et al., 2008b). Cow

temperament score and plasma cortisol concentrations affected the probability of pregnancy

linearly (P = 0.03) during yr 1, and quadratically (P < 0.01) during yr 2 (Figure 5-3). These

results indicate and support our hypothesis that excitable temperament and elevated cortisol

concentrations, partially explained by cow temperament (Table 5-4 and 5-5), are detrimental to

reproductive function of cows. Concurring with our findings, Plasse et al. (1970) reported that

excitable temperament influences negatively the reproductive performance of beef females.

Additionally, as observed in yr 2, reduced cortisol concentrations and temperament score during

the early postpartum period may denote health disorders that negatively affect cattle

reproduction, such as lethargy, lameness (Sprecher et al., 1997), and immunosuppresion (Goff,

2006). Plasma concentrations of ceruloplasmin and haptoglobin affected the probability of

pregnancy linearly during yr 1 (P < 0.01 and = 0.04, respectively) and yr 2 (P = 0.01; Figure 5-

119

4), indicating and supporting previous data reporting that the acute phase response is detrimental

to reproductive function of livestock (Peter et al., 1989; Battaglia et al., 2000; Williams et al.,

2001).

In conclusion, results from this experiment indicate that acclimation of Brahman-

crossbred cows to human interaction did not influence temperament and concentrations of

plasma cortisol and acute phase proteins. Nevertheless, reproductive performance of Braford

cows was enhanced during yr 1. Measurements and physiologic responses associated with cow

temperament, acute phase response, and energy status influenced the probability of cows to

become pregnant during the breeding season. Therefore, management strategies that improve

cow disposition, enhance their immune status, and maintain the cowherd at adequate levels of

nutrition are required for optimal reproductive performance of Brahman-crossbred cows, and

consequent productivity of cow-calf operations containing these types of cattle.

120

Table 5-1. Mean BW and BCS of Brahman-crossbred cows exposed or not (control) to acclimation procedures.

Year 1 Year 2

Item Brahman × British Braford SEM Brahman ×

British Braford SEM

BW, kg

Acclimated 517 519 7.7 514 556 8.0

Control 506 523 7.8 514 526 7.9

P2 0.32 0.72 0.97 0.01

BCS1

Acclimated 5.7 5.7 0.12 5.8 6.0 0.12

Control 5.5 6.1 0.11 5.7 5.7 0.12

P2 0.21 0.04 0.91 0.05

BCS change3

Acclimated -1.07 -0.66 0.116 -0.30 -0.59 0.123

Control -1.09 -0.82 0.115 -0.86 -0.44 0.119

P2 0.89 0.35 < 0.01 0.39

1 Emaciated = 1, obese = 9; Wagner et al., 1998 2 Treatment comparison within breed and year. 3 Calculated using BCS collected at the beginning of acclimation period and breeding season.

121

122

Table 5-2. Body weight change, temperament measurements, and plasma concentrations of cortisol, haptoglobin, and IGF-I of Brahman-crossbred cows exposed or not (control) to acclimation procedures.

Item Acclimated Control SEM P

BW change, kg 1 -43 -50 5.4 0.38

Chute Score 2 1.98 1.96 0.034 0.59

Pen Score 3 2.48 2.58 0.043 0.12

Exit Velocity, m/s 4 2.12 2.08 0.049 0.57

Temperament score 5 2.50 2.49 0.042 0.94

Cortisol, μg/dL 3.33 3.36 0.122 0.88

Haptoglobin, 450 nm × 100 1.96 2.12 0.275 0.68

IGF-I, ng/mL 133 123 5.7 0.21

1 Calculated using BW collected at the beginning of the acclimation period and breeding season. 2 Assessed by a single technician based on a 5-point scale, where 1 = calm, no movement, and 5 = violent and continuous struggling. 3 Cows exited the chute and entered a pen containing a single technician, and were assigned a score on a 5-point scale, where 1 = unalarmed and unexcited, and 5 = very excited and aggressive toward the technician in a manner that requires evasive action to avoid contact between the technician and cow. 4 Assessed by determining the speed of the cow exiting the squeeze chute by measuring rate of travel over a 1.5-m distance with an infrared sensor (FarmTek Inc., North Wylie, TX). 5 Calculated by averaging cow chute score, exit score, and pen score. Exit score was calculated by dividing exit velocity results into quintiles, and assigning cows with a score from 1 to 5 (exit score; 1 = slowest cows; 5 = fastest cows).

Table 5-3. Body weight, BCS, and plasma ceruloplasmin concentrations of Brahman-crossbred cows exposed or not (control) to acclimation procedures.1

Item Beginning of acclimation Beginning of breeding SEM

Treatment effects

Ceruloplasmin, mg/dL

Acclimated 15.8 14.2 0.31

Control 16.6 14.2 0.30

P2 0.08 0.82

Day effects3

BW 546a 498b 3.3

BCS4 6.1b 5.4b 0.05

1 Day effects are reported across treatments (treatment × day effect; P > 0.10) 2 Treatment comparison within day. 3 Values with different superscripts within measurement differ (P < 0.01) 4 Emaciated = 1, obese = 9; Wagner et al., 1998

123

1 U

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row

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ower

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Tabl

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4. P

ears

on c

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n co

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s am

ong

mea

sure

men

ts o

f tem

pera

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124

125

Tabl

e 5-

5. P

ears

on c

orre

latio

n co

effic

ient

s am

ong

mea

sure

men

ts o

f tem

pera

men

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hman

-cro

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urin

g yr

2 o

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stud

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1 U

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= P

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0102030405060708090

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15Week of the breeding season

Acclimated Control Panel A

0102030405060708090

100

1 2 3 4 5 6 7 8 9 10 11 12Week of the breeding season

Acclimated Control

Acc

umul

ativ

e pr

egna

ncy

atta

inm

ent,

%

Panel B

Figure 5-1. Pregnancy attainment (pregnant cows / total cows) during the breeding season of

Brahman x British (Panel A) and Braford (Panel B) cows exposed or not (control) to acclimation procedures during yr 1 of the study. Brahman x British cows were assigned to timed-AI during wk 1, and exposed to bulls for a 90-d breeding season beginning in wk 4. Braford cows were only exposed to a 90-d bull-breeding. Date of conception was estimated retrospectively by subtracting gestation length (286 d; Reynolds et al., 1980) from the calving date. Values reported are least square means adjusted covariately to cow days postpartum at the beginning of the breeding season. A treatment effect was detected for Braford cows (P < 0.01), but not for Brahman x British cows (P = 0.48).

126

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7 8 9BCS

Year 1 Year 2

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200 250 300 350

Plasma IGF-I, ng/mL

Year 1 Year 2

Prob

abili

ty o

f pre

gnan

cy, %

Figure 5-2. Effects of BCS (emaciated = 1, obese = 9; Wagner et al., 1988) and plasma IGF-I concentrations, assessed at the beginning of the breeding season, on the probability of Brahman-crossbred cows to become pregnant. A quadratic effect was detected during yr 1 and 2 for BCS (P < 0.01) and plasma IGF-I (P = 0.02 and < 0.01, respectively).

127

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5Temperament Score

Year 1 Year 2

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9Plasma cortisol, μg/dL

Year 1 Year 2

Prob

abili

ty o

f pre

gnan

cy, %

Figure 5-3. Effects of temperament score and plasma cortisol concentrations, assessed at the

beginning of the breeding season, on the probability of Brahman × British and Braford cows to become pregnant. For temperament score, a linear effect (P = 0.03) and a quadratic effect (P < 0.01) were detected for both breeds during yr 1 and 2, respectively. For plasma cortisol, a linear effect was detected (P = 0.04) for Braford cows during yr 1, whereas a quadratic effect was detected (P = 0.02) for both breeds during yr 2. Temperament score was calculated by averaging cow chute score, exit score, and pen score.

128

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30 35 40Plasma ceruloplasmin, mg/dL

Year 1 Year 2

Figure 5-4. Effects of plasma ceruloplasmin and haptoglobin concentrations, assessed at the beginning of the breeding season, on the probability of Brahman-crossbred cows to become pregnant. A linear effect was detected during yr 1 and 2 for plasma ceruloplasmin (P < 0.01 and = 0.01, respectively) and haptoglobin (P = 0.04 and = 0.01, respectively).

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9 1

Plasma haptoglobin, 450 nm × 100

0

Year 1 Year 2

Prob

abili

ty o

f pre

gnan

cy, %

129

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BIOGRAPHICAL SKETCH

Reinaldo Fernandes Cooke was born in São Paulo, Brazil, in 1979. He is the oldest son of

Nelson de Burgos Cooke and Rosangela C. F. Cooke. Reinaldo obtained his B.S. in animal

sciences from São Paulo State University (UNESP) in 2003. In 2004, Reinaldo moved to

Gainesville, FL, to begin his Master of Science program at the University of Florida in animal

sciences, advised by Dr. John Arthington. His research focused on beef cattle nutrition. In spring

2006, Reinaldo was awarded with the M. S. degree in animal sciences and immediately began his

Ph.D. studies at the same institution. During his Ph.D. training, Reinaldo evaluated nutritional

and management strategies to improve the reproductive performance of beef heifers and cows.

After graduation, Reinaldo hopes to pursue a career in academia, and is currently applying for

beef cattle nutrition positions in different parts of the U.S.