University of Florida Thesis Or Dissertation

USE OF BOVINE SOMATOTROPIN TO HASTEN PUBERTY ACHIEVEMENT OF BOS INDICUS-INFLUENCED BEEF HEIFERS

MATHEUS BETELLI PICCOLO

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2018

To my loving parents, Teresa e José Alberto

ACKNOWLEDGMENTS

First, I would like to thank God, without him nothing of this would be possible and to my parents, Teresa Cristina Betelli Piccolo and José Alberto Piccolo; all my grandparents, which I am blessed to have present in my life. To my brothers, Thomas e Rafael Betelli Piccolo; for all the support, guidance and encouragement, and love given to me throughout my life. Each one of them has a special meaning to this accomplishment in my life, and all are majorly responsible for me being able to fulfill my education goals. My family and in special, my parents, provided all the opportunities for me to become the person I am today.

I would like to thank all my committee members, Dr. Philipe Moriel, Dr. John Arthington and Dr. João Vendramini for the patience, friendship and trust beyond measure that they putted on me. Dr. Philipe Moriel deserves especial thanks, for guiding me since 2015 and giving me so many opportunities and for pushing me to become a better student and person, despite of all the headache that I gave to him. His teachings went beyond the academic and therefore I will always have a great respect for him.

Appreciation is also extended to Dr. Reinaldo Cooke, who always made himself available to help me and in several occasions give me valuable advices, and for that I am extremely thankful.

I would like to thank Dr. Corwin Nelson for the help during the laboratory analysis of my experiment, and Dr. Geoffrey Dahl for the help and patience with me when I needed.

I would like to thank my former advisor Dr. José Luiz Moraes Vasconcelos for introducing me to science, for the career opportunities he provided, for his many teachings and even for his annoying way of doing things but in special for his support. Along with Dr.

Vasconcelos, I am extremely thankful for having made part of the study group that he advises;

Conapec was the best part of my undergraduate carrier in terms of developing skills, acquiring experience in the field and making many friends and important contacts in the agribusiness.

I am also deeply grateful to all the staff at the Range Cattle Research and Education

Center. In particular, Mrs. Julie Warren, for her help on field and on the lab and also for being a friend; Mrs. Andrea Dunlap for always being helpful and available when I needed the most; Mr.

Austin Bateman, Mr. Clay Newman, Mr. Tom Fussell, and Mr. Ryann Nevling for all their help when working with cattle and friendship.

Special thanks for my friends Achilles Vieira Neto, Umberto Pardelli and Miguel

Miranda for being always present when needed, and always pushing me to get my head up.

Great thanks to everybody of my fraternity: “Karca 1 Gole”, for all the friendship and support during the last 8 years of my life, nothing would be the same without them.

I would like to thank Juliana Ranches for all her help, teaching me lab procedures or helping me in the field and support as a friend, becoming her friend was a great surprise that living in Ona provided to me.

Also, I would like to thank all the friends that I made in Ona, João Sanchez, José Dias,

Pedro Mamede, Hiran Marcelo, Gleise Silva, Marcelo Vedovatto, Aline Moraes, Kely Koriaken,

JK, Caio, YanYan, Amanda, Nayara, Jhone, Rhaiza, and others for all the help and friendship during my period here.

Last but not least, to my friends in Gainesville, Luara, Felipe, Hendyel, Paula, Roney,

André, Amanda, Fernanda, Jason, Camilo and everybody else that as them made Gainesville a little happier even if just for a moment.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 8

LIST OF FIGURES ...... 9

LIST OF ABBREVIATIONS ...... 10

ABSTRACT ...... 12

CHAPTER

1 INTRODUCTION ...... 14

2 LITERATURE REVIEW ...... 16

Metabolic Imprinting ...... 16 Potential Mechanisms ...... 16 Organ structure ...... 16 Cell number ...... 17 Clonal selection ...... 17 Epigenetics ...... 18 Evidences of Metabolic Imprinting in Beef Heifers ...... 18 Somatotropic Axis ...... 19 Nutrition vs. Somatotropic Axis vs. Puberty Achievement ...... 24 Exogenous Bovine Somatotropin – Structure, Synthesis and Secretion ...... 26 Strategies to Explore the Effects of Metabolic Imprinting in Beef Cattle Production Systems ...... 31

3 PRE-WEANING INJECTIONS OF BOVINE ST ENHANCED REPRODUCTIVE PERFORMANCE OF BOS INDICUS-INFLUENCED REPLACEMENT BEEF HEIFERS...... 32

Introduction ...... 32 Material and Methods ...... 33 Animals and Diets ...... 33 Sample and data collection ...... 35 Laboratory analyses ...... 37 Statistical analyses ...... 39 Results...... 40 Discussion ...... 42 Conclusion ...... 48

LIST OF REFERENCES ...... 60

BIOGRAPHICAL SKETCH ...... 75

LIST OF TABLES

Table page

3-1 Average nutritional composition of concentrate offered during the post-weaning phase (d 127 to 346) to beef heifers ...... 50

3-2 Primer sequences and accession number for all gene transcripts analyzed by quantitative real-time PCR1...... 51

3-3 Pre- and post-weaning growth performance of beef heifers ...... 52

3-4 Pre- and post-weaning plasma IGF-1 concentrations of beef heifers ...... 53

3-5 Liver mRNA expression (fold increase1; yr 3 only) of beef heifers ...... 54

3-6 Reproductive performance of beef heifers ...... 55

LIST OF FIGURES

Figure page

3-1 Pre- (A) and post-weaning (B) body weight of beef heifers that received a s.c. injection of saline solution (SAL; 5 mL; 0.9% NaCl) or 250 mg of sometribove zinc (BST; Posilac, Elanco) on d 0, 14, and 28 (n = 15 heifers/treatment annually; 3 yr)...... 56

3-2 Percentage of pubertal beef heifers during the post-weaning phase...... 58

3-3 Calving distribution (% of heifers that calved) of beef heifers ...... 59

LIST OF ABBREVIATIONS

ADG Average daily gain

AI Artificial insemination

AKT Protein kinase B or PKB bST Bovine somatotropin

BW Body weight

CP Crude protein

CV Coefficient of variance

DM Dry matter

DNA Deoxyribonucleic acid

EW Early-weaned

FSH Follicle stimulating hormone

GH Growth hormone

GHR Growth hormone receptor

GHRH Growth hormone-releasing hormone

IFAS Institute of Food and Agriculture Sciences

IGF-1 Insulin-like growth factor 1

IGF1R Insulin-like growth factor 1 receptor

IGFBP Insulin-like growth factor binding protein

IVDOM In-vitro digestible organic matter

JAK Janus kinase mRNA Messenger Ribonucleic acid

N Nitrogen

NaCl Sodium chloride

NDF Neutral detergent fiber

NEFA Nonesterified fatty acids

NEg Net energy for growth

NEm Net energy for maintenance

NW Normally-weaned

P4 Progesterone

PI3K Phosphatidylinositol 3-kinase

RNA Ribonucleic acid

SAL Saline

ST Somatotropin

STAT Signal transducer and activator of transcription

TDN Total digestible nutrients

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

USE OF BOVINE SOMATOTROPIN TO HASTEN PUBERTY ACHIEVEMENT OF BOS INDICUS-INFLUENCED BEEF HEIFERS

Matheus Betelli Piccolo

August 2018

Chair: Philipe Moriel Major: Animal Sciences

A 3-yr study evaluated the effects of three pre-weaning 14-d apart injections of bovine somatotropin (bST) on growth and reproductive performance of beef heifers. On d 0 of each yr,

Angus × Brangus heifers (n = 15 heifers/treatment/yr; BW = 147 ± 20 kg; Age = 134 ± 11 d) were stratified by BW and age, and randomly assigned to receive a subcuteaneous injection of saline (SAL; 5 mL; 0.9% NaCl) or 250 mg of sometribove zinc (BST; Posilac, Elanco,

Greenfield, IN) on d 0, 14, and 28. Cow-calf pairs were managed as a single group on bahiagrass

(Paspalum notatum) pastures from d 0 until weaning (d 127). From d 127 to 346, heifers were grouped by treatment, allocated to bahiagrass pastures (1 pasture/treatment/yr), and fed a molasses-based supplement (2.9 kg/heifer daily; DM basis) until d 346. Blood samples were collected on d 0, 14, 28, 42, and then every 9-10 d from d 179 to 346. In yr 3, liver biopsy samples were collected on d 0, 42, and 263. Heifers were exposed to Angus bulls from d 263 to

346. Heifers administered bST injections had greater pre-weaning overall plasma concentrations of IGF-1 and ADG from d 0 to 42 (P ≤ 0.05), but similar BW at weaning and post-weaning ADG

(P ≥ 0.25) compared to SAL heifers. Heifers from bST group tended to achieve puberty 26 d earlier (P = 0.10), had greater percentage of pubertal heifers on d 244, 263, 284, and 296 (P ≤

0.04), tended to have greater overall pregnancy percentages (P = 0.10), and had greater (P ≤

0.05) calving percentages in yr 1 and 2 (but not yr 3; P = 0.68) compared to SAL heifers. Liver mRNA expression of GHR-1B and IGF-1 were greater for BST vs. SAL heifers on d 263 (P ≤

0.02). Hence, three half-dose injections of bST administered to suckling beef heifers at 14-d intervals (between 135 and 163 d of age) induced long-term impacts on liver gene expression and may be a feasible management practice to enhance puberty attainment and pregnancy percentages of Bos indicus-influenced beef heifers.

CHAPTER 1 INTRODUCTION

Improving the reproductive performance of replacement beef heifers is one of the major factors affecting overall efficiency and productivity of cow-calf operations (Bagley, 1993). A major determinant of lifetime productivity of beef heifers is the age at puberty attainment and conception relative to the initiation of their first breeding season (Day and Nogueira, 2013).

Additionally, overall cattle herd productivity is positively correlated with percentage of heifers becoming pregnant early in their first breeding season (Lesmeister et al., 1973), which also leads to improved reproductive performance over the next 6 parturitions (Cushman et al., 2013).

Due to their tolerance to elevated environmental heat and humidity conditions, Bos indicus-influenced cattle are common across southeastern United States (Turner, 1980). Growing

Bos indicus cattle require approximately 10% less net energy for maintenance compared to Bos taurus breeds (NRC, 1996). However, heifers with B. indicus influence typically attain puberty at older ages, which reduces their reproductive performance compared to B. taurus heifers (Short et al., 1994). An extensive literature is available regarding post-weaning development of beef heifers (Wiltbank et al., 1969; Short and Bellows, 1971; Ferrell, 1982; Warnick et al., 1991;

Cooke et al., 2008; Moriel et al., 2017). Nonetheless, pre-weaning strategies may have a greater impact on puberty attainment of beef heifers compared to post-weaning management practices

(Roberts et al., 2007). This greater impact of pre-weaning strategies may be attributed to metabolic imprinting effects, which is the concept that physiological outcomes to nutritional/stress challenges occurring during a critical window of early-life can persist for long periods, even after the removal of such challenges (Lucas, 1991). For instance, beef heifers early-weaned (EW) at 70 d of age and limit-fed a high concentrate diet for 90 d after weaning had similar body weight (BW) and average daily gain (ADG) during the breeding season, but

hastened puberty attainment compared to heifers that were weaned at 270 d of age and provided similar post-weaning management (Moriel et al., 2014). In addition, circulating insulin-like growth factor 1 (IGF-1) impacts gonadotropin activity required for the first ovulation in beef heifers by influencing hypothalamic–pituitary secretory activity (Schillo et al., 1992) and augmenting the effects of gonadotropins in ovarian follicular cells (Spicer and Echternkamp,

1995). In agreement, heifer ADG and plasma IGF-1 concentrations from 70 to 160 d of age explained approximately 34% of the variability in age at puberty (Moriel et al., 2014). Thus, metabolic imprinting may be explored to optimize reproductive performance of beef heifers.

Post-weaning injections of exogenous bovine somatotropin (bST) increased plasma concentrations of IGF-1 and hastened puberty attainment in B. taurus heifers (Cooke et al.,

2013). Potential impacts of pre-weaning injections of bST on puberty attainment of beef heifers, particularly B. indicus-influenced heifers remains unknown. It was hypothesized that preweaning injections of bST would enhance growth and percentage of pubertal and pregnant beef heifers compared to saline injections. Hence, a 3 yr study evaluated the effects of pre-weaning injections of bST on blood parameters and liver gene expression measurements associated with somatotropic axis, growth, and reproduction of B. indicus-influenced beef heifers.

CHAPTER 2 LITERATURE REVIEW

Metabolic Imprinting

Fetal programming, also termed “developmental programming”, “the barker hypothesis”, or “developmental origins of health and disease” is the concept that perturbations (e.g. nutrition), during critical prenatal development stages, may have lasting impacts on growth and adult function (Caton and Hess, 2010). However, in most mammalian species, organ development is not complete at birth and continues during the immediate postnatal period. For example, maturation of pancreatic islets and development of neuronal systems in the hypothalamus of rats continue during the suckling period (Kaung, 1994). Early in postnatal life, organisms have the ability to respond to environmental conditions that are unknown to normal development, through adaptations at the cellular, molecular, and biochemical levels (Patel and Srinivasan, 2002).

Consequently, the term “metabolic imprinting” was created to incorporate the adaptive body responses to specific nutritional conditions occurring during a limited stage of susceptibility in early postnatal life. These responses may permanently affect the physiology and metabolism of the organism (Lucas, 1991; Waterland and Garza, 1999; Patel and Srinivasan, 2011).

Potential Mechanisms

Potential mechanisms by which perinatal nutrition may persistently affect an organism’s structure or function includes: induced variations in organ structure, alterations in cell number, clonal selection, and epigenetics (Waterland and Garza, 1999).

Organ Structure

Morphologic modifications occurring during organogenesis may affect the ability of each cell to generate and respond to external signals within the organism. For example, nutrition- induced alterations on organ vascularization may affect the cellular responses to nutrients or

hormonal signals. During limited periods of organogenesis, the fate of cells depends on externally-derived signals from adjacent and distant cells. Therefore, it is plausible that local concentrations of nutrients and metabolites may modulate the end result of organogenesis

(Waterland and Garza, 1999).

Cell Number

During development, organ mass increases either by increasing the number of cells

(hyperplasia) or cell size (hypertrophy). However, different tissues experience distinct and limited periods of hyperplastic and hypertrophic growth. Cell growth rate is nutrient-dependent, and hence, nutritional deprivation or surplus, during critical periods of cell division, may lead to permanent changes in cell number, regardless of subsequent nutrient surplus (Waterland and

Garza, 1999). For instance, offspring born from ewes fed 50% of their total digestible nutrients

(TDN) requirements from d 28 to 78 of gestation had lesser secondary muscle fibers compared to offspring born from nutrient-unrestricted ewes (Zhu et al., 2004). The number of muscle fibers is determined during the prenatal muscle development, and does not increase during the postnatal life. Thus, prenatal nutrition has profound effects on muscle growth and development during the later postnatal life (Zhu et al., 2004).

Clonal Selection

Cellular proliferation of all organs involves the proliferation of a finite population of founder cells. As cell proliferation proceeds, early genetic and epigenetic modifications that occur within individual cells distinguish them from others in subpopulations of rapidly dividing cells. Thus, the nutrient environment may induce an incorrect base pairing during deoxyribonucleic acid (DNA) replication, and result in subtle effects on cellular metabolism that may be transmitted to daughter cells (Waterland and Garza, 1999; Fenech, 2010). Vitamins and minerals serve as cofactors for enzymes and protein structures involved in DNA synthesis, repair

and maintenance of genome integrity (Neibergs and Johnson, 2012). Hence, suboptimal intake of vitamins and minerals may permanently damage the DNA and alter the genomic stability

(Fenech, 2010).

Epigenetics

The epigenetic process is a genetic modification not explained by changes in DNA sequence (Riggs et al., 1996) occuring during periods of genome reprogramming, such as embryogenesis and gestation (Jirtle and Skinner, 2007). Methylation of DNA and histone modifications are the major mechanisms of epigenetics (Thiagalingam et al., 2003). Methylation of DNA molecules is highly correlated with gene expression and consists of DNA methyltransferases adding methyl groups at cytosine-purine-guanine (CpG) islands that are often associated with the promoter region of genes (Simmons, 2011). Hypomethylation at the promoter regions of DNA enhances messenger RNA (mRNA) transcription, whereas hypermethylation is associated with suppressed mRNA transcription (Simmons, 2011). The methylation pattern varies among cells in different tissues (i.e. oocytes and sperm DNA are less methylated compared to cells in somatic tissues, such as muscle), and is maintained during DNA replication, which allows the specific methylation pattern to be transmitted to progeny cells (Waterland and

Garza, 1999). In mice, dietary restriction of methyl donor molecules, such as folic acid, methionine, vitamin B12, and choline were associated with DNA hypomethylation, whereas postweaning supplementation of such methyl donors increased methylation of a wide-variety of genes (Neibergs and Johnson, 2012; Bermingham et al., 2013).

Evidences of Metabolic Imprinting in Beef Heifers

Gasser et al. (2006) demonstrated that beef heifers EW at 3 mo of age and fed to achieve greater ADG (1.27 vs. 0.85 kg/d) until 9 mo of age attained puberty 100 d sooner than heifers normally weaned (NW) at 9 mo of age. Later, Gasser et al. (2006) reported that the hastened

puberty achievement in EW heifers fed high-concentrate diet was attributed to the high-energy consumption, and not due to a direct effect of EW, because NW heifers and a second group of

EW heifers fed to achieve growth rates similar to NW heifers achieved puberty at the same age

(308 ± 26 and 330 ± 25 d of age). Such early activations of puberty attainment cannot be attributed solely to greater ADG and BW of EW vs. NW heifers, but rather to metabolic imprinting-induced effects. For instance, Moriel et al. (2014) observed that beef heifers EW at 70 d of age and limit-fed a high concentrate diet for 90 d after weaning had similar BW and ADG during breeding season, but hastened puberty attainment compared to heifers that were weaned at

270 d of age and provided similar post-weaning management (Moriel et al., 2014). Therefore, those results demonstrate the existence of a critical moment (3 to 6 mo of age) for nutritional stimuli to induce early-activation of the reproductive axis, decrease age at puberty achievement, and increase reproductive success of beef heifers (Gasser et al., 2006a). In addition, altering the circulating concentrations of components of the somatotropic axis through enhanced plane of nutrition seems to be involved in this early activation of puberty (Moriel et al., 2014).

Somatotropic Axis

The somatotropin axis is an essential constituent of multiple systems controlling growth

(Leroith et al., 2001) and reproduction (Hess et al., 2005); connecting nutrition to liver function, gene expression and secretion of gonadotropins in the brain, and a wide variety of tissue metabolism (Thakur et al., 1993; Jiang et al., 2007; Rhoads et al., 2007; Wathes et al., 2007;

Allen et al., 2012). A considerable part of metabolism development and somatotropic axis regulation directly derives from liver function. The liver occupies a unique an vital role in nutritional physiology by being distinctively positioned to respond to nutrients absorbed across the gastrointestinal tract and rumen and to modulate the profile of nutrients available to the rest of the body (Donkin, 2012). The liver also performs essential functions in the body through the

expression of genes encoding plasma proteins, clotting factors, glucose and lipids metabolism, secretion of IGF-1 and others (Jungermann and Katz, 1989). Furthermore, liver gene expression is influenced by transcription factors related to environment and autocrine or paracrine signal responses (Costa et al., 2003), and may have age-related variations on expression due to epigenetic control, altered sensibility and health status (Slagboom and Vijg, 1989).

The somatotropic axis consists primarily of growth hormone (GH), GH receptor (GHR-

1A, -1B, -1C), IGF-1, IGF binding proteins (IGFBP-1, -2, -3, -4, -5, and -6), and IGF receptors

(IGF-1R), which are essential for growth and mammary development, and involved in mediating tissue responses to energy intake (Leroith et al., 2001; Radcliff et al., 2004). At the hypothalamic level, the somatotropic axis comprises of two sets of neurons that synthesize and release growth hormone-releasing hormone (GHRH) or somatostatin, the excitatory and inhibitory regulators of

GH release from the pituitary, respectively (Daftary and Gore, 2005). Growth hormone stimulates hepatic synthesis of IGF-1, and is stimulated by ghrelin and inhibited by IGF-1 via a negative feedback loop (Kojima et al., 1999). In addition, GH antagonizes insulin actions, leading to nutrient partitioning effects (Lucy, 2008), decreased lipogenesis and enhanced lipolysis in adipose tissue, and increased muscle protein accretion in growing animals and milk protein synthesis in lactating cows (Etherton and Bauman, 1998).

Physiological actions of GH are initiated by the binding to GHR, activating the JAK-

STAT transduction pathway. Activated GHR associates with Janus kinase-2, which when activated, phosphorylates STATs on tyrosines that bind to specific DNA sequences and activate gene transcription (Leroith et al., 2001; Carter-Su et al., 2016). There are three different GHR promoters in cattle; each one transcribes different exon 1 sequences (GHR-1A, -1B, and -1C;

Kim, 2014). Although the mRNA is different in exon 1, the receptor protein is the same, because

the GHR protein is encoded in exons 2 through 10 of the mRNA (Edens and Talamantes, 1998).

Additionally, each GHR is expressed differently, as to location and concentration in the body;

GHR-1A mRNA is solely present in the liver, where it represents the bulk of liver GHR mRNA, whereas GHR-1B and -1C mRNA are expressed in a wide variety of tissues, including muscle and adipose tissue (Lucy et al., 2001). Furthermore, age-related differences also affect expression of the exon 1 sequences (Lucy et al., 1998), whereas the binding of GH to GHR-1A increased with calf age, leading to enhanced synthesis of IGF-1 and declining serum concentrations of GH as calves developed (Badinga et al., 1991). Transcripts belonging to the GHR-1A are expressed exclusively on liver tissue, where it accounts for approximately 50% hepatic GHR transcripts, whereas GHR-1B and -1C account for approximately 35 and 15%, respectively (Kim, 2014).

However, GHR-1B and -1C transcripts are expressed to a higher degree on overall body tissues, accounting for approximately 70 and 30% of total GHR transcripts, respectively (Jiang and

Lucy, 2001; Kim, 2014). Growth hormone dissociates slowly from hepatic membrane, which may account for some long-term actions of GH (Badinga et al., 1991).

Bovine IGF-1 is a single-chain, polypeptide hormone with a molecular weight of approximately 7.6 kDa, and involved in carbohydrate, protein and fat metabolism, and cell proliferation and differentiation (Leroith et al., 2001; Daftary and Gore, 2005). The IGF-1 receptor is widely expressed in the body tissues, including muscle, adipose tissue, hypothalamus, pituitary, gonads and reproductive tract (Lui, 2017). The binding of IGF-1 to IGF1R and one of the six IGF binding proteins modulate the activity of IGF-1 within target tissues (Leroith et al.,

2001; Daftary and Gore, 2005; Hess et al., 2005). Over 90% of IGF-1 in blood is bound to one type of IGFBP (Hess et al., 2005). Potentiation of IGF-1 actions involves interaction of individual binding proteins (i.e., IGFBP-1, -3, and -5) with the cell surface, thereby increasing

the bioavailability of IGF-1 in the microenvironment surrounding cells and enhancing ligand- receptor association (Roberts et al., 2001). The IGFBP binds to IGF-1 with high affinity, transporting IGF-1 among body tissues, regulating their metabolic clearance, enhancing or blocking its binding to IGF1R and providing cell type-specific targeting (Le Roith et al., 2001), thereby indirectly participating in control of IGF-1 bioavailability and bioreactivity (Silva et al.,

2009). Additionally, IGFBPs have a conserved structure which can be divided in three domains of similar size: cysteine-rich amino- and carboxy-terminal domains, joined by an unconserved central, linker domain (Baxter, 2013).

Several factors are known to affect IGF-1 production, such as age (Badinga et al., 1991), gender, nutrition, and physiologic state (Lucy, 2008). Anabolic effects of systemic IGF-1 are related to relative abundance of IGFBP-3 (Armstrong and Benoit, 1996), whereas IGFBP-2 is associated with poor nutritional status (Armstrong and Benoit, 1996). In agreement, Roberts et al. (1997) reported that serum concentrations of IGFBP-2 of beef cows at 2 wk postpartum diminished, whereas serum concentrations of IGFBP-3 increased in cows that resumed estrus by

20 wk postpartum compared to anestrous cows. However, age and nutrition had little or no effect on GHR-1B mRNA expression in the semitendinosus muscle and subcutaneous adipose tissue

(Lucy et al., 2001). In contrast, GHR-1A mRNA expression increased with age (Lucy et al.,

2001) and growth rate of calves (Radcliff et al., 2004). This is attributed to GHR-1B having characteristics of housekeeping gene promoters, while GHR-1A is different from -1B or -1C because it is liver specific and controlled by a variety of developmental and metabolic signals

(Kobayashi et al., 1999).

High-feeding levels (Radcliff et al., 2004) has been shown to affect multiple components of the somatotropic axis of cattle (Thissen et al., 1994). Growth hormone binding to hepatic

membrane is highly correlated with GHR-1A mRNA expression (Radcliff et al., 2003), which is also highly correlated with hepatic expression of IGF-1 mRNA (Lucy et al., 2001). Thus, an increased hepatic expression of GHR-1A consequently enhances IGF-1 synthesis (Radcliff et al.,

2004). Conversely, Smith et al. (2002) reported that newborn calves with increased nutrient intake had greater plasma concentrations of insulin and IGF-1, but had no effects on semitendinosus muscle expression of GHR and IGF-1 mRNA.

Furthermore, heifers fed to achieve greater ADG had less serum concentrations of

IGFBP-2 and greater serum concentrations of IGF-1 as animals approached puberty, which were correlated with increased liver mRNA expression of GHR-1A and IGF-1 shortly after first estrus

(Radcliff et al., 2004). Weller et al. (2016) fed prepubertal heifers at 100 d of age to achieve high, low or maintenance gains during an 84-d feeding period. Heifers from high and low gain groups achieved similar body fat mass, but both had greater body fat mass compared to the maintenance group. These authors observed that high gain heifers had greater plasma IGF-1 concentrations and liver mRNA abundance of GHR, IGF-1, and IGFBP-3 compared to maintenance and low gain heifers. In contrast, IGFBP-2 liver mRNA abundance was lower in high gain heifers than in maintenance heifers (Weller et al., 2016). Allen et al., (2012) evaluated the metabolic regulation of neuroendocrine function of major metabolic-sensing neurons located in the arcuate nucleus of the hypothalamus, by enhancing feed intake and ADG from 3 to 6 mo of age. Authors reported greater ADG, serum concentrations of insulin and IGF-1 and liver weights in heifers fed to achieve higher ADG, and these findings were correlated with 346 differently expressed genes. Furthermore, enhanced nutrition decreased mRNA expression of

GHR and neuropeptide Y in the arcuate nucleus, which were suggested to participate in the metabolic status permitting reproductive maturation (Allen et al., 2012).

Nutrition vs. Somatotropic Axis vs. Puberty Achievement

Day and Anderson (1998) proposed that the period from birth to puberty in beef heifers could be divided into infantile (birth to 2 mo of age), developmental (2 to 6 mo of age), static (6 to 10 mo of age), and peripubertal periods (10 to 12 mo of age). During the infantile period, pulsatile secretion and ovarian inhibition of LH secretion increase at approximately 6 wk of age and is fully established by 8 wk of age. After this stage, heifers enter the developmental period, when there is a greater GnRH secretion by the hypothalamus, which stimulates follicular growth and estradiol concentrations, and hence, the number of follicles peaks at approximately 14 wk of age (Dodson et al., 1988; Evans et al., 1994; Rawlings et al., 2003). However, LH secretion effects are inhibited due to a strong negative feedback caused by enhanced estradiol concentrations stimulated by the higher GnRH production, leading to a decrease in follicle numbers, which will remain at low levels throughout the static phase (Schillo et al., 1982; Day et al., 1987; Day and Anderson, 1998). In summary, each period has specific and important steps that contribute to the successful reproductive development of beef heifers (Gasser, 2013).

Nonetheless, as discussed below, later evidences demonstrated that the developmental phase may have the greatest impact on puberty attainment of beef heifers.

Accelerated growth rate during the pre-weaning phase has been shown to decrease age at puberty attainment of beef heifers (Gasser et al., 2006a; Gasser et al., 2006b; Gasser et al.,

2006c; Gasser et al., 2006d; Moriel et al., 2014). In an sequence of experiments, Gasser et al.,

(2006a,b,c,d) reported greater frequency of LH pulses (Gasser et al., 2006c), mean LH concentrations (Gasser et al., 2006b), follicular growth wave, and accelerated decrease in the negative feedback of estradiol on LH secretion (Gasser et al., 2006b,d) for EW heifers experiencing greater ADG compared to NW heifers.

Nutrition-induced metabolic signals leading to early activation of the reproductive axis in heifers are not fully understand yet. However, heifers approaching puberty experience an increase in serum concentrations of IGF-1 and LH (Yelich et al., 1996). Cooke et al. (2013) concluded that heifers with elevated circulating IGF-1 concentrations experienced hastened puberty establishment independently of growth rate, nutritional plane, body fat content, and circulating leptin concentrations. Therefore, adequate circulating concentrations of nutrition- related hormones, such as IGF-1, may be needed to achieve ovulation (Velazquez et al., 2008).

During periods of positive nutritional status, animals experience greater circulating concentrations of IGF-1 and GH, enhanced liver mRNA expression of IGF-1 and GHR-1A, and consequently, higher binding capacity of IGF-1 and GH to its receptors (Breier, 1999), leading to positive influence on hypothalamic control of LH secretion and reproductive development of prepubertal heifers (Schillo et al., 1992).

Local and systemic GH and IGF-1 exert stimulatory or permissive roles at each level of the hypothalamic-pituitary-gonadal axis and follicular development and maturation (Schams et al., 1999; Silva et al., 2009). For instance, circulating IGF-1 participates on gonadotropin secretion and activity required for the first ovulation and puberty establishment in heifers by influencing hypothalamic-pituitary secretory activity (Schillo et al., 1992) while amplifying gonadotropins effects in follicular cells (Spicer and Echternkamp, 1995). Additionally, local and systemic IGF-1 stimulate cell proliferation, mitogenesis and steroidogenesis of granulosa cells

(Mani et al., 2010), stimulating FSH action and promoting follicular growth, differentiation and maturation (Mazerbourg et al., 2003; Silva et al., 2009). Furthermore, in vivo dose–response studies demonstrated that bovine recombinant GH acted through increased peripheral concentrations of insulin and IGF-1 to positively affect follicle development in heifers (Gong et

al., 1997; Silva et al., 2009). Insulin play a more important role as estradiol stimulator than IGF-

1, however, IGF-1 and -2 can inhibit insulin-stimulated estradiol secretion by granulosa cells of follicles through competition for binding sites (Spicer and Echternkamp, 1995).

Insulin-like growth fator-1 receptor, IGF-1 and IGFBPs mRNA are present in the brain, pituitary, gonads and reproductive tract (Daftary and Gore, 2005). Daftary and Gore (2003) also observed that in vitro IGF-1 treatment of explanted preoptic area-anterior hypothalamuses of peripubertal mice indicated that IGF-1 had stimulatory effects on GnRH gene expression.

Additionally, the pattern of hypothalamic IGF-1 mRNA expression changes with time, increasing during neonatal phase, decreasing during prepubertal hiatus, and then increasing as it approaches pubertal maturity (Daftary and Gore, 2003).

Hence, management and nutritional strategies that enhance circulating IGF-1 concentrations are expected to hasten puberty achievement of beef heifers. One strategy capable of increasing plasma IGF-1 concentrations is the use of injections of bovine somatotropin

(Buskirk et al., 1996).

Exogenous Bovine Somatotropin – Structure, Synthesis and Secretion

Exogenous bovine ST (bST) is available as a recombinant protein in a sustained release formulation (Posilac, Monsanto). Commercial use of bST to increase milk production in lactating dairy cows was approved by the U.S. Food and Drug Administration (FDA) in 1993. Since then multiple dairy herds in US have used bST, bringing productive and economic benefits to producers (Raymond et al., 2009). Great research efforts has been targeted to its use (Etherton and Bauman, 1998; Tarazon-Herrera et al., 2000; Santos et al., 2004; Carriquiry et al., 2008;

Carstens et al., 2010), ranging from enhancement of milk yield (Binelli et al., 1995; Bauman,

1999; Tarazon-Herrera et al., 2000), growth (Bass et al., 1992; Moallem et al., 2004), reproduction performance (Santos et al., 2004; Cooke et al., 2013), and production of leaner

carcasses (Nanke et al., 1993; Binelli et al., 1995; Schlegel et al., 2006) to increase in the productive efficiency while diminishing negative environmental impacts (Capper et al., 2008).

Bovine somatotropins are known to affect nutrient partitioning between muscle and adipose tissue, leading to alterations in growth (Bauman and Vernon, 1993; Bilby, 2005).

Injections of bST stimulate the production of IGF-1 in the same manner as endogenous GH, with circulating concentrations of IGF-1 increasing shortly after bST treatment (Gong et al., 1993;

Bilby et al., 1999; Cooke et al., 2013). Furthermore, bST applications decrease plasma leptin and lipid synthesis (Bauman et al., 1994; Etherton and Bauman, 1998). As a result, these coordinated changes in physiological pathways alter the partitioning of absorbed nutrients, involving a variety of tissues, and affecting the metabolism of all nutrient classes (carbohydrate, lipid, protein, and minerals), modulating plasma concentrations of other metabolites and hormones such as NEFA, glucose and insulin (Hess et al., 2005). In lactating dairy cows, bST affects milk production by partitioning nutrients towards milk synthesis (Etherton and Bauman, 1998).

Adiposity is negatively correlated with GH concentrations (Gluckman et al., 1987). In adipose tissue, ST treatment decreases glucose uptake and stimulates insulin uptake of glucose by adipocytes (Etherton and Louveau, 1992). For instance, glucose utilization by adipose tissue of ST-treated pigs is reduced in approximately 30% of whole-body glucose turnover (Dunshea et al., 1992), thereby decreasing lipid synthesis while possibly increasing lipolysis in adipose tissue

(van der Walt, 1994). These responses when combined enable nutrient partitioning to other tissues. Cows treated with bST had increased hepatic gluconeogenesis and reduced whole-body glucose oxidation; therefore, enhancing glucose hepatic output and glucose availability for other tissues, such as the mammary parenchyma. Additionally, ST also reduces hepatic response to

insulin allowing the liver to sustain increased rates of gluconeogenesis that are critical to support the increase in milk synthesis (Bauman et al., 1994).

Responses of the somatotropic axis to ST treatment are dependable of somatotropic axis maturation and ability to respond to available somatotropin. Animals are born with functional somatotropic axis (Dunshea et al., 1992), and exogenous ST triggers body responses as early as 1 d of age in beef cattle (Govoni et al., 2004). However, maturation is of great importance in the ontogeny of growth regulation, and therefore ST might have diminished effects during early stages of life (Campbell et al., 1991). Badinga et al., (1991) reported that ST receptors in bovine hepatocytes increase with age, peaking with 6 mo of age and declining thereafter. In rats, ST receptor mRNA in the brain decrease with age and increases with age in peripheral tissues (van der Walt, 1994; Lobie et al., 1993). In humans, serum concentrations of IGF-1 and IGFBP-3 increase with age and pubertal stage (Juul et al., 1994; Jull et al., 1995). In agreement,

Velayudhan et al. (2007) observed delayed responses on serum IGFBP-3 concentrations coupled with relatively slower growth rates to exogenous bST treatment in animals starting bST treatment with 200 d of age compared to 250 and 300 d. Furthermore, heifers under positive energy balance experienced enhanced serum IGF-1 concentrations after bST treatment compared to heifers under negative energy balance, suggesting a possible uncoupling of the somatotropic axis during negative energy balance (Yung et al., 1996).

Due to its lipolytic effects, bST has been used for the production of leaner carcasses.

Holstein steers administered bST injections experienced greater G:F and ADG compared to non- treated control steers, independently if treatments were administered during the growing or finishing phases. Additionally, animals that received bST injections during growing and finishing phases had reduced carcass quality grade and lipid accretion, but increased lean muscle

accretion (Schlegel et al., 2006). Moreover, animals treated with bST throughout the study had spleen and kidney weights 7.5 and 23% greater than non-treated cohorts, respectively (Schlegel et al., 2006). Beef cattle treated daily with 33 µg of bST/kg of BW from 200, 250 or 300 d of age until 400 d of age, experienced greater ADG, G:F and longissimus muscle area compared to untreated cattle. However, growth responses and serum concentrations of IGF-1 and IGFBP-3 were more pronounced when treatment started at older ages (Velayudhan et al., 2007). Likewise, serum concentrations of IGFBP-3 increased only after 100 d of bST treatment (starting at 200 d of age) indicating that the responses in serum concentrations of IGFBP-3 to exogenous bST were delayed in younger animals (Velayudhan et al., 2007).

Zulu et al. (2002) suggested that steroidogenesis effects of IGF-1 on follicular cells occur through stimulation of FSH, LH and LH receptors. Gong et al. (1993) reported increased number of small follicles and greater peripheral insulin concentrations in Hereford x Friesian heifers receiving 25 mg of bST daily compared to non-treated heifers. In agreement, bST administration at the beginning of timed-AI protocols or at the time of AI improved pregnancy rates and stimulated embryo development in lactating dairy cows (Moreira et al., 2001; Moreira et al.,

2002). Santos et al. (2004) reported a decreased embryo mortality for bST vs. non-treated dairy cows (6.7 vs. 14.0%). Together, these results indicate that bST treatment may increase fertility through effects on oocyte maturation, embryonic development, and altered oviduct/uterine functions (Moreira et al., 2001). Conversely, Oosthuizen et al. (2017) reported negative effects of bST application at the beginning of timed AI protocol on reproductive performance of beef heifers. Application of 650 mg of bST at the start of a timed AI protocol decreased the percentage of heifers pregnat to AI (42.5 vs. 29.9% for control vs. bST, respectively), but not on final percentage of pregnant heifers. These results are in agreement with Bilby et al. (2004) that

reported negative effects on embryo development when cows treated with bST achieved plasma concentrations of IGF-1 above 600 ng/ml. These results indicate the existence of a threshold where IGF-1 has stimulating effects on reproductive performance, but exceeding this threshold may have deleterious effects. Additionally, lactating dairy cows treated with two sequential injections of bST (325 mg at AI and 14 d after AI) had 27% greater pregnancy rate at 66 d post-

AI, whereas a single treatment of 325 mg of bST at the time of AI had no impact on any reproductive measures compared to non-treated cows (Ribeiro et al., 2014). Nonetheless, bST doses of 200 and 500 mg generated similar responses on serum concentrations of IGF-1 (Bilby et al., 1999). However, 500 mg of bST decreased conception and pregnancy rates within the first month of treatment (Cole et al., 1991). Also, daily applications of bST (25 mg/d) during synchronization protocol of dairy heifers decreased plasma estradiol concentrations by 30% while inhibiting development of preovulatory follicles and enhancing development of second follicle wave (Lucy et al., 1994).

Post-weaning bST treatment also has effects on puberty achievement of heifers. Age at puberty in heifers is negatively correlated with ADG, and bST treatment has enhanced growth performance of prepubertal animals (Groenewegen et al., 1990; Bauman et al., 1994;

Velayudhan et al., 2007). In contrast, Moallem et al. (2004) reported no reduction in age at puberty despite of enhanced growth performance of dairy heifers treated daily with 0.1 mg/kg

BW of bST from 90 to 314 d of age. Similarly, Buskirk et al. (1996) reported that bST treated heifers experienced enhanced ADG but no differences in age at puberty compared to non-treated cohorts. Moreover, dairy heifers fed a high-energy diet and receiving daily bST injections (25

µg/kg of BW) tended to have a greater BW gain compared to heifers offered the same diet without bST injections. However, pregnancy and calving percentages of bST-treated heifers did

not differ compared with non-treated heifers under the same growth rate (Radcliff et al., 2000).

Beef heifers receiving 250 mg of bST every 14 d for 210 d after weaning had similar growth performance, decreased circulating leptin concentration and backfat thickness, but attained puberty sooner compared to non-treated heifers (Cooke et al., 2013). In contrast, Buskirk et al.

(1996) applied 250 mg of bST to beef heifers every 14 d for 112 d, starting at 120 d of age, and observed no significant effects on puberty achievement.

Strategies to Explore the Effects of Metabolic Imprinting in Beef Cattle Production Systems

Metabolic imprinting effects are associated with critical and transitory windows in which early nutritional interventions may result in long-term consequences to animal metabolism.

Therefore, identifying strategies that are able to explore those critical periods of development and enhance calf performance may provide unique opportunities to optimize feed resources and increase the profitability of beef cattle management systems. Early-weaning beef calves prior to the breeding season is a strategy that was capable of eliciting metabolic imprinting effects and hastening puberty attainment of beef heifers (Moriel et al., 2014). However, few beef producers are willing to adopt this management practice due to the lack of information on calf management following EW and the high-costs associated with feeding concentrate-based diets throughout the entire period of calf development. Therefore, the evaluation of alternative management systems for beef heifers is required.

It was hypothesized that preweaning injections of bST would enhance growth and percentage of pubertal and pregnant beef heifers compared to saline injections. Hence, this 3 yr study evaluated the effects of preweaning injections of bST on blood parameters and liver gene expression measurements associated with somatotropic axis, growth, and reproduction of Bos indicus-influenced beef heifers.

31 CHAPTER 3 PRE-WEANING INJECTIONS OF BOVINE ST ENHANCED REPRODUCTIVE PERFORMANCE OF BOS INDICUS-INFLUENCED REPLACEMENT BEEF HEIFERS*

Introduction

A major determinant of lifetime productivity of beef heifers is the age at attainment of puberty (Lesmeister et al., 1973; Day and Nogueira, 2013). Day and Anderson (1998) proposed that the period from birth to puberty in beef heifers could be divided into infantile, developmental, static, and peripubertal periods. Enhancing the ADG and nutrient intake of beef heifers during the developmental phase (60 to 180 d of age) hastened follicle size (Gasser et al.,

2006c, 2006d) and puberty attainment (Moriel et al., 2014). Circulating IGF-I impacts the gonadotropin activity required for the first ovulation in beef heifers by influencing hypothalamic–pituitary secretory activity (Schillo et al., 1992) and augmenting the effects of gonadotropins in ovarian follicular cells (Spicer and Echternkamp, 1995). In agreement, heifer

ADG and plasma IGF-1 concentrations from 70 to 160 d of age explained approximately 34% of the variability of age at puberty (Moriel et al., 2014). These responses may be attributed to metabolic imprinting, which is the concept that body physiological responses to early-life nutritional challenges can persist for long periods, even after the removal of such challenges

(Lucas, 1991). Thus, metabolic imprinting may be explored to optimize reproductive performance of beef heifers.

Postweaning injections of bovine ST hastened puberty attainment of Bos taurus heifers

(Cooke et al., 2013). However, less emphasis has been placed on pre- vs. post-weaning

*Reprinted with permission from the Journal of Animal Science;

Piccolo, M. B., J. D. Arthington, G. M. Silva, G. C. Lamb, R. F. Cooke, and P. Moriel.

2018. Preweaning injections of bovine somatotropin enhanced reproductive performance of Bos indicus-influenced replacement beef heifers. J. Anim. Sci. 96:618–631. doi:10.1093/jas/sky016.

Available from: http://dx.doi.org/10.1093/jas/sky016

management strategies, despite their greater impact on attainment of puberty in beef heifers

(Roberts et al., 2007). It was hypothesized that preweaning injections of bovine ST would enhance growth and percentage of pubertal and pregnant beef heifers compared to saline injections. Hence, this 3 yr study evaluated the effects of preweaning injections of bovine ST on blood parameters and liver gene expression measurements associated with somatotropic axis, growth, and reproduction of Bos indicus-influenced beef heifers

Material and Methods

The 3 yr experiment described herein was conducted at the University of Florida,

Institute of Food and Agricultural Sciences, Range Cattle Research and Education Center

(RCREC), Ona, Florida (27°23′N and 81°56′W) from March 2014 to December 2017. Heifers used in these experiments were cared for by acceptable practices as outlined in the Guide for the

Care and Use of Agricultural Animals in Research and Teaching (FASS, 2010) and approved by the IFAS-Animal Research Committee.

Animals and Diets

On d 0 of each year (n = 3 yr), 30 cow–calf pairs were selected from four herds of mature, lactating, Angus × Brahman crossbred beef cows (10 ± 3 yr of age). Only cow–calf pairs with heifer calves of similar BW and approximately 120 to 150 d of age (147 ± 20 kg; 134 ± 11 d) were selected for the study. The age criterion was based on previous results indicating that beef heifers of approximately 70 to 180 d of age were susceptible to long-term, nutrition-induced impacts on postweaning puberty attainment (Moriel et al., 2014). Immediately after selection, cow–calf pairs were stratified by heifer BW and age, and heifers were randomly assigned to receive an s.c. injection of a saline solution (SAL; 5 mL; 0.9% NaCl) or 250 mg of sometribove zinc (BST; Posilac, Elanco, Greenfield, IN) on d 0, 14, and 28. Injections were always administered in the neck, alternating between the right and left side of the heifer. The interval

between injections (every 14 d) and dosage (250 mg) of sometribove zinc was chosen according to Buskirk et al. (1996) who successfully reported an increase in plasma IGF-1 concentrations after similar dosage and interval between bovine ST injections, without any detrimental effects to heifer growth and physiological parameters. The injections containing 250 mg of sometribove zinc were prepared by transferring the contents of a standard, commercially available injection of

Posilac (500 mg of sometribove zinc) into a sterile container and determining its total volume, and then, the total volume was split in half to achieve the 250 mg dosage, which was administered to each heifer using sterile syringes. The number of injections (n = 3 injections) was selected so that the plasma IGF-1 concentrations remained increased for a total period of 42 d after the first bovine ST injection, which corresponds to the age window (70 to 180 d) that heifers were susceptible to nutrition-induced impacts on puberty (Moriel et al., 2014).

All cow–calf pairs were managed as a single group, without access to concentrate, and grazed the same bahiagrass (Paspalum notatum) pastures (4 pastures/yr; 4 ha/pasture) from d 0 until weaning (d 127). Immediately after weaning, cows returned to their original herds, whereas heifers were sorted by treatment, transferred into one of eight bahiagrass pastures (one pasture per treatment; 0.8 ha/pasture), and offered the same concentrate supplementation strategy until the end of the study (d 346). Treatment groups were rotated among the eight bahiagrass pastures every 9 to 10 d throughout the study to prevent any potential confounding effects of pasture on the variables investigated herein. Postweaning concentrate was formulated using NRC (2000) and designed to allow heifers to achieve 60% of their mature BW at the initiation of breeding season (assuming a mature BW of 499 kg; based on average BW of mature cows in the same location; Moriel et al., 2017). Concentrate supplementation was offered three times weekly

(Monday, Wednesday, and Friday) at 0800 h to achieve an average daily intake of 2.9 kg of

supplement DM per heifer daily from d 127 to 346. Nutritional composition of concentrate supplement is shown in Table 1. All cows and heifers were provided free-choice access to water and a salt-based trace mineral and vitamin mix during the entire study (University of Florida,

Institute of Food and Agricultural Sciences, Cattle Research Mineral, Brookville, OH; 16.8% Ca,

4% P, 20.7% NaCl, 1.0% Mg, 60 mg/kg Co, 1,750 mg/kg Cu, 350 mg/kg I, 60 mg/kg Se, 5,000 mg/kg Zn, 441 IU/g of vitamin A, 33 IU/g of vitamin D3, and 0.44 IU/g of vitamin E). In each year, free-choice access to long-stem stargrass (Cynodon nlemfuensis) hay was offered when pasture availability was limited (d 263 to 346). Heifers were exposed to mature Angus bulls from d 263 to 346 (one bull per group). Bulls passed the breeding soundness exam 90 d before the start of the study and were rotated between treatment groups every 9 to 10 d during the breeding season to remove any potential effects of bull on the variables investigated herein.

Sample and Data Collection

Individual heifer shrunk BW was recorded on d 0 and 42, after 6 h of feed and water withdrawal, and then approximately every 28 d from d 127 to 346, after 16 h of feed and water withdrawal. Full BW of heifers were recorded on d 14 and 28 to avoid any potential impacts of shrink-induced stress on blood metabolites and hormones during the period of treatment injections. Hip height of heifers was assessed on d 179 and 346.

Blood samples (10 mL) were collected from all heifers via jugular venipuncture into tubes (Vacutainer, Becton Dickinson) containing sodium-heparin (158 United States

Pharmacopeia units) for plasma harvest on d 0, 14, 28, 42, 127, 234, 263, and 296 to determine the plasma concentrations of IGF-1. Blood samples (10 mL) also were collected from all heifers via jugular venipuncture into tubes (Vacutainer, Becton Dickinson) containing no additives for serum harvest at 9 to 10 d intervals from d 179 to 346 to determine the serum concentrations of progesterone (P4). Blood samples were immediately placed on ice following collection and then

centrifuged at 1,200 × g for 25 min at 4 °C. Plasma and serum samples were stored frozen at

−20°C until later laboratory analysis. Onset of puberty in this study was defined as the first increase in concentrations of P4 greater than 1.0 ng/mL. The first increase in concentrations of

P4 that exceeds 1.0 ng/mL is associated with females containing a luteal structure because of ovulation or luteinization suggesting onset of puberty (Perry et al., 1991). Body weight at puberty was determined using the ADG, and initial and final BW measurements of the respective

28 d interval when puberty was attained (BW at puberty = initial BW of the respective 28 d interval + [ADG of the respective 28 d interval × number of days between the day at puberty attainment and initial BW collection]).

Percentages of pregnant heifers were determined by palpation of the uterus and its contents per rectum by a trained veterinarian approximately 45 d after the end of the breeding season. Heifers were checked twice daily for calving. Calving date was converted to Julian date, and calf birth BW was obtained within 12 h of birth. Pregnancy loss was calculated as percentage of heifers diagnosed pregnant at approximately 45 d after the end of breeding season, but failed to calve. Calving distribution was reported as the percentage of heifers that calved weekly relative to the total number of heifers per treatment.

Hand-plucked samples of pastures, concentrate, and hay were collected every 56 d from d

0 to 346, dried in a forced-air oven at 56 °C for 72 h, ground in a Wiley mill (Model 4, Thomas-

Wiley Laboratory Mill, Thomas Scientific, Swedesboro, NJ) to pass a 4 mm stainless steel screen, and pooled across month within each year. The pooled concentrate samples were analyzed in duplicates by a commercial laboratory (Dairy One Forage Laboratory, Ithaca, NY) for concentrations of CP (method 984.13; AOAC, 2006), TDN (Weiss et al., 1992), and NEm and NEg (NRC, 2000). Nutritive analyses of pooled samples of pastures and hay were performed

at the University of Florida Forage Evaluation Support Laboratory using the micro-Kjeldahl technique for N (Gallaher et al., 1975) and the two-stage technique for in vitro organic matter digestibility (IVDOM; Moore and Mott, 1974). Average nutritional composition of pastures during the preweaning phase was 42.0% IVDOM and 10.9% CP (DM basis), whereas the average nutritional composition (DM basis) of pastures and hay during the postweaning phase was 35.3% and 46.3% IVDOM and 9.6% and 7.2% CP NDF, respectively.

Based on the results obtained in yr 1 and 2, liver biopsy sample collections (six heifers per treatment) were performed on d 0, 42, and 263 of yr 3 to determine the long-term impact of treatments on liver mRNA expression of genes associated with energy metabolism. All liver samples were collected via needle biopsy, following the procedure described by Arthington and

Corah (1995). Immediately following collection, 100 mg of wet liver tissue per heifer was stored into 1.5 mL of RNA stabilization solution (RNAlater, Ambion Inc., Austin, TX), kept on ice for

8 h, and stored at −80 °C until later analyses of mRNA expression of cyclophilin, GH receptor

1A and 1B (GHR-1A and GHR-1B), IGF-1, IGF binding protein 3 (IGFBP-3), and 40S ribosomal protein S9 (RSP9). Primer sequence for each gene is shown in Table 2.

Laboratory Analyses

Plasma concentrations of IGF-I were determined using a human-specific commercial

ELISA kit (SG100; R&D Systems, Inc., Minneapolis, MN) with 100% cross-reactivity with bovine IGF-I and previously validated for bovine samples (Moriel et al., 2012). Intra- and inter- assay CV for IGF-1 assay were 1.60% and 3.65%, respectively. Plasma P4 concentrations were determined using a solid-phase, competitive, chemiluminescent enzyme immunoassay (Immulite

1000, Diagnostics Products Corp.) previously validated for bovine samples (Martin et al., 2007).

Detectable range and intra-assay for plasma P4 concentrations were, respectively, 0.2 to 40 ng/mL and 4.38%.

A detailed description of procedures for mRNA isolation and tissue gene expression was described by Cappellozza et al. (2014). Briefly, total RNA was extracted from liver tissue samples using the TRIzol Plus RNA Purification Kit (Invitrogen, Carlsbad, CA). Extracted RNA was quantified via UV absorbance (UV Mini 1240; Shimadzu Scientific Instruments, Inc.,

Columbia, MD) at 260 nm, incubated (2.5 µg) at 37 °C for 30 min in the presence of RNase-free

(DNase; New England Biolabs Inc., Ipswich, MA) and 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 gene-specific primers (20 pM each) with the StepOne Real-Time PCR system

(Applied Biosystems). At the end of each real-time PCR, amplified products were subjected to a dissociation gradient (95 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s) to verify the amplification of a single product by denaturation at the anticipated temperature. A portion of the amplified products was purified with the QIAquick PCR purification kit (Qiagen Inc., Valencia,

CA) and sequenced at the Oregon State University Center for Genome Research and

Biocomputing (Corvallis, OR), whereas the remaining portion was sequenced at the Department of Animal Science from University of Florida to verify the specificity of amplification. All amplified products represented only the genes of interest. Primer sequence of target genes was validated by previous studies, except for GHR-1B, which was designed based on the bovine gene sequences deposited in the National Center for Biotechnology Information and using the Primer

Express v.3.0.1 software (Applied Biosystems, Foster City, CA). Responses were quantified based on the threshold cycle (CT) and were normalized to geometrical mean of CT values from cyclophilin and RSP9 (∆CT) examined in the same sample and assessed at the same time as the

targets. Within each target gene, results are expressed as relative fold change (2−∆∆CT) using the average ∆CT of all samples (Ocón-Grove et al., 2008).

Statistical Analyses

All data were analyzed as a complete randomized design using SAS (SAS Institute Inc.,

Cary, NC, USA, version 9.4) with Satterthwaite approximation to determine the denominator degrees of freedom for the test of fixed effects. Heifer was the experimental unit, whereas heifer within treatment × year was included as random effect in all analyses. Growth and physiological results were analyzed using the MIXED procedure, whereas reproductive binary data (puberty attainment, percentage of heifers that became pregnant and calved, pregnancy loss, and weekly calving distribution) were analyzed using the GLIMMIX procedure. Heifer ADG, BW and age at puberty, and mature BW on d 263 were tested for fixed effects of preweaning treatment, year, and treatment × year. Heifer BW, plasma IGF-1, liver mRNA expression, puberty attainment, and calving distribution were tested for fixed effects of treatment, day of the study (or week of calving season), year, and all resulting interactions, using heifer within treatment × year as the subject. Results from d 0 were included as covariates in each respective analysis but removed from the model when P > 0.10. Proper covariance structure for each repeated measure analysis was selected based on the lowest Akaike information criterion. Compound symmetry covariance structure was used for statistical analyses of postweaning heifer BW, plasma IGF-1 concentrations, and liver mRNA expression of IGFBP-3. Autoregressive 1 covariance structure was used for the analyses of preweaning heifer BW, liver mRNA expression of IGF-1, calving distribution, and puberty attainment. Unstructured covariance structure was used for the statistical analyses of liver mRNA expression of GHR-1A and GHR-1B. All results are reported as least squares means. Data were separated using PDIFF if a significant F-test was detected.

Significance was set at P ≤ 0.05 and tendencies at 0.05 < P ≤ 0.10.

Results

Heifer BW on d 0 did not differ (P ≥ 0.90) between treatments but was included as covariate (P < 0.0001) in the statistical analyses of preweaning heifer BW. Effects of treatment × year × day of the study, and treatment × year were not detected (P ≥ 0.30) for pre- and postweaning heifer BW. Effects of treatment × day of the study were detected for preweaning (P =

0.01; Figure 3-1a), but not for postweaning BW of heifers (P = 0.50; Figure 3-1b). Heifers administered preweaning injections of bovine ST tended (P = 0.09) to be heavier on d 42, but had BW on d 14 and 28 and from d 127 to 346 that did not differ (P ≥ 0.17) compared with SAL heifers. Effects of year were detected (P = 0.0007) for mean postweaning BW of heifers, which was greatest in yr 2, least in yr 3, and intermediate in yr 1 (P ≤ 0.05; 294, 266, and 279 ± 5.3 kg, respectively).

Effects of treatment × year were not detected (P ≥ 0.14) for ADG of heifers during the pre- and post-weaning phases, age and BW at puberty attainment and percentage of mature BW on d 263 (Table 3-3). Heifers assigned to BST had greater (P = 0.03) ADG from d 14 to 28 and 0 to 42, less ADG from d 127 to 346 (P = 0.04), and ADG from d 0 to 127 and 127 to 346 that did not differ (P ≥ 0.50) compared to SAL heifers (Table 3-3). Hip height on d 179 and 346, and hip height change from d 179 to 346 did not differ (P ≥ 0.41) between treatments (Table 3-3).

Effects of treatment × year and treatment × year × day of the study were not detected (P

≥ 0.14) for pre- and post-weaning plasma concentrations of IGF-1. Effect of treatment × day of the study was not detected (P = 0.83) for preweaning plasma concentrations of IGF-1, but BST heifers had greater (P = 0.05) overall plasma IGF-1 concentrations from d 0 to 42 compared to

SAL heifers, after covariate adjusted for plasma IGF-1 concentrations obtained on d 0 (P <

0.0001; Table 3-4). Effect of treatment × day of the study was detected (P = 0.04) for postweaning plasma concentrations of IGF-1. Heifers treated with BST had greater (P = 0.008)

plasma concentrations of IGF-1 on d 234, but plasma concentrations of IGF-1 on d 263 and 296 did not differ (P ≥ 0.82) compared to SAL heifers (Table 3-4).

Liver mRNA expression of GHR-1A, GHR-1B, and IGF-1, but not IGFBP-3 (P = 0.77), was covariate adjusted (P ≤ 0.06) to respective mRNA expression obtained on d 0. Effects of treatment × day of the study were detected (P ≤ 0.02) for liver mRNA expression of GHR-1B and IGF-1, but not for GHR-1A and IGFBP-3 (P ≥ 0.15; Table 3-5). Liver mRNA expression of

GHR-1B and IGF-1 did not differ (P ≥ 0.15) between treatments on d 42 but was greater (P ≤

0.02) for BST vs. SAL heifers on d 263 (Table 3-5). Overall liver mRNA expression of GHR-1A and IGFBP-3 did not differ (P ≥ 0.12) between treatments (Table 3-5).

Heifers assigned to BST had BW at puberty and percentage of mature BW on d 263 did not differ (P ≥ 0.16) compared to SAL heifers, but tended (P = 0.10) to achieve puberty 26 d earlier than SAL heifers (Table 3-6). Effects of treatment × year were not detected (P ≥ 0.17) for pregnancy percentage, pregnancy loss, calving date, and calf BW at birth, except for overall calving percentage (P = 0.03; Table 3-6). Heifers assigned to BST tended (P ≤ 0.10) to have greater overall pregnancy percentage and less pregnancy loss compared to SAL heifers (Table 3-

6). Overall calving percentage was greater (P ≤ 0.05) for BST vs. SAL heifers in yr 1 and 2, but did not differ (P = 0.68) between treatments in yr 3 (Table 3-6). Calving date and calf BW at birth did not differ (P ≥ 0.34) between treatments.

Effects of treatment × year and treatment × year × day of the study (or week of the calving season) were not detected (P ≥ 0.28) for puberty attainment (Figure 3-2) or calving distribution (Figure 3-3). Effect of treatment × day of the study was detected (P = 0.03) for puberty attainment. The percentage of pubertal heifers was greater (P ≤ 0.04) for BST vs. SAL heifers on d 244, 263, 284, and 296 and tended (P = 0.08) to be greater for BST vs. SAL heifers

on d 273 (Figure 3-2). Effect of treatment × week was not detected (P = 0.78) for calving distribution (Figure 3-3).

Discussion

Beef heifers early-weaned at 70 d of age and limit-fed a high concentrate diet for 90 d after weaning had similar BW and ADG during breeding season, but hastened puberty attainment compared to heifers that were weaned at 270 d of age and provided similar postweaning management (Moriel et al., 2014). The exact nutrition-mediated mechanisms involved in this early activation of the reproductive axis in beef heifers are unknown. However, circulating IGF-I impacts gonadotropin activity required for puberty achievement in beef heifers (Butler and

Smith, 1989; Schillo et al., 1992; Spicer and Echternkamp, 1995). Thus, metabolic imprinting may be explored by identifying strategies that can increase heifer ADG and plasma IGF-1 during the developmental phase leading to optimized future reproductive performance.

In the present study, heifers administered preweaning injections of bovine ST had an 8.6 ng/mL increase in mean plasma IGF-1 concentrations, a 7.5% increase in ADG from d 0 to 42, and tended to be heavier on d 42 compared to heifers administered saline solution. Other studies demonstrated that postweaning bovine ST injections increased plasma IGF-1 concentrations

(Cooke et al., 2013), but did not increase postweaning ADG of Angus × Holstein heifers administered 500 mg of sometribove zinc every 14 d from 6 to 10 mo of age (Carstens et al.,

1997) and Angus × Hereford heifers injected with 250 mg of sometribove zinc every 14 d from 6 to 13 mo of age (Cooke et al., 2013). The increase in BW gain and circulating IGF-1 concentrations following bovine ST injections varied from 0% to 45% compared to control treatments (Dalke et al., 1992; Houseknecht et al., 1992), and several factors, such as plane of nutrition, age, and animal size, may explain this large variation (Rausch et al., 2002). Body weight gain and circulating IGF-1 response to bovine ST are positively influenced by cattle age

and nutritional status (Rausch et al., 2002; Radcliff et al., 2004). Cattle somatotropic axis is functional at birth (Granz et al., 1997), and the response to ST begins as early as 1 d of age

(Govoni et al., 2004), gradually increasing as age increases (Velayudhan et al., 2007). Likewise, plasma IGF-1 concentrations following bovine ST injection were greater for Holstein heifers gaining 1.2 vs. 0.8 kg/d (Radcliff et al., 2004).

Multiple mechanisms may be involved in the BW gain of cattle following bovine ST injections, including the repartitioning of nutrients toward muscle rather than adipose tissue deposition (Breier, 1999), enhanced long-bone growth (Buskirk et al., 1996), improved nitrogen retention (Eisemann et al., 1986), and increased circulating IGF-1-induced synthesis of muscle

(Jiang and Ge, 2014) and noncarcass tissues (Early et al., 1990). Multiple 14 d apart administrations of bovine ST during the postweaning phase reduced subcutaneous fat thickness by 9.2% without impacting LM depth, marbling scores, and BW gain (Cooke et al., 2013).

Although body composition was not evaluated in the present study, it is unlikely that three 14 d apart injections of bovine ST substantially affected body composition and nutrient requirements of heifers, leading to similar overall pre- and post-weaning growth performance. Hip height throughout the postweaning phase did not differ between treatments, indicating that bone growth did not differ. Our results perhaps indicate that the increment on bovine ST-induced ADG from d

0 to 42 may be the result of increased feed intake and gut fill, as reported by Enright et al.

(1990). Heifer BW on d 0 and 42 were recorded after shrink, but it is possible that gut fill was not completely eliminated after shrink. The less ADG from d 42 to 127 for BST vs. SAL heifers, and lack of differences on overall preweaning ADG from d 0 to 127, supports this rationale of potential gut fill effects. In addition, muscle protein deposition from d 0 to 42 perhaps was not sufficient to dramatically impact heifer BW at weaning. Nevertheless, preweaning bovine ST

injections in the present study successfully increased plasma IGF-1 concentrations and ADG of heifers during the developmental phase of the reproductive axis in beef heifers (Day and

Anderson, 1998).

The binding of GH to GHR-1A stimulates hepatic synthesis of IGF-1 (Smith et al., 2002) and is highly correlated with liver mRNA expression of GHR-1A and IGF-1 (Lucy et al., 2001).

Transcription of the growth hormone receptor gene (GHR) is initiated from multiple transcription start sites, generating GHR-1A, GHR-1B, and GHR-1C mRNA that differ in the 5′- untranslated region, but still encode the same amino acid sequence (Jiang and Lucy, 2001). The

GHR-1A mRNA is only expressed in the liver (Lucy et al., 1998), whereas GHR-1B and GHR-

1C mRNA are expressed in a wide array of tissues, including liver, skeletal muscle, adipose tissue, and mammary gland (Jiang et al., 1999; Jiang and Lucy, 2001). Hepatic synthesis of IGF-

1 is regulated primarily at the transcriptional level (Thissen et al., 1994) and is the major source of circulating IGF-1 (Yakar et al., 1999), which is also responsible for stimulating the hepatic expression of IGFBP-3 mRNA (Thissen et al., 1994). Thus, an increased hepatic expression of

GHR-1A mRNA enhances the capacity for GH binding (Lapierre et al., 1992) and the hepatic synthesis of IGF-1 (Radcliff et al., 2004). Nutrient intake and BW gain positively affect the abundance of GHR-1A, IGF-1, and IGFBP-3 in the liver (Thissen et al., 1994; Smith et al., 2002;

Radcliff et al., 2004). Holstein heifers administered daily injections of bovine ST (25 µg/kg of

BW from 120 to 247 d of age) had greater liver mRNA expression of IGF-1, but similar liver mRNA expression of GHR-1A and IGFBP-3 (Radcliff et al., 2004).

In the present study, preweaning injections of bovine ST did not impact liver mRNA expression of GHR-1A and IGFBP-3 throughout the study, and GHR-1B and IGF-1 mRNA on d

42. Following bovine ST administration to lactating and nonlactating dairy cattle, plasma IGF-1

concentrations increase after 3 d, peak at approximately 7 to 8 d, and gradually return to baseline concentrations starting at 12 d post-injection (Bilby et al., 1999, 2004). Hence, it is possible that the timing of liver sample collection was not optimal to detect the peak expression of liver mRNA of IGFBP-3, IGF-1, GHR-1B, and GHR-1A. Detection of greater mean plasma IGF-1 concentrations from d 0 and 42, but similar liver mRNA expression of IGF-1 on d 42 between

BST vs. saline heifers support this rationale. Nevertheless, the primary goal for the collection of liver mRNA expression data was to evaluate any potential carryover effects of preweaning injections of bovine ST on postweaning liver gene expression. Preweaning injections of bovine

ST increased liver mRNA expression of GHR-1B and IGF-1 approximately 221 d after the last injection of bovine ST, despite the similar postweaning nutritional management and ADG between treatments, which may be an evidence that preweaning injections of bovine ST caused metabolic imprinting effects. Similarly, Moriel et al. (2014) reported that beef heifers early- weaned at 70 d of age and limit-fed a high concentrate diet for 90 d after weaning had similar

BW and ADG during breeding season compared to heifers normally weaned at 270 d of age, but had increased liver IGF-1 mRNA expression 70 d after all heifer groups were allocated to the same post-weaning nutritional management (Moriel et al., 2014). Further studies are required to identify the metabolic imprinting mechanisms influencing the postweaning gene expression and reproduction of BST-injected heifers.

Despite the greater liver mRNA expression of IGF-1 at the start of the breeding season, postweaning plasma IGF-1 concentrations were greater for BST heifers on d 234, but not at the start and 33 d after the start of the breeding season. This response indicates that the greater liver mRNA expression of IGF-1 on d 263 did not translate into greater systemic concentrations of

IGF-1 on that same day. The greater plasma IGF-1 concentrations of BST heifers on d 234,

however, may indicate that liver mRNA expression of IGF-1 was likely greater for BST vs. SAL heifers before the start of the breeding season and that the magnitude of differences on liver mRNA expression of IGF-1 was declining during the postweaning phase leading to similar plasma IGF-1 concentrations on d 263 and 296. Therefore, one could speculate that the potential metabolic imprinting effects of preweaning bovine ST injections on liver metabolism of IGF-1 may not have persisted after d 263.

The impact of bovine ST injections on puberty attainment of beef heifers has been variable. Injections of bovine ST (250 mg every 14 d from 120 to 232 d of age) did not impact attainment of puberty of Angus × Simmental crossbred heifers (Buskirk et al., 1996).

Postweaning bovine ST injections (250 mg of bovine ST every 14 d from 6 to 13 mo of age) increased the percentage of Angus × Hereford heifers attaining puberty at the start of breeding season (Cooke et al., 2013), but had no impact on attainment of puberty of Angus heifers administered bovine ST (350 mg every 14 d from 7 to 14.5 mo of age) compared to control- treated heifers (Hall et al., 1994). In the present study, pre-weaning injections of bovine ST tended to decrease age at puberty by 26 d and hastened the percentage of pubertal heifers immediately at and during the first 33 d after the initiation of the breeding season compared to saline injections, despite their similar nutritional management, ADG, and BW during breeding season. The tendency to advance puberty supports our hypothesis and is likely a result of the increased plasma IGF-1 concentrations and ADG during the developmental phase of the reproductive axis in beef heifers (Day and Anderson, 1998). The exact mechanism for such responses on puberty attainment of these heifers cannot be determined in the present study, and the discussion of all potential mechanism leading to the enhanced puberty attainment is beyond the scope of this article.

Heifers should attain puberty before the initiation of the breeding season because the percentage of pregnant heifers was 21% greater in heifers mated on third vs. first postpubertal estrous cycle (Byerley et al., 1987; Perry et al., 1991), and the timing of conception in the first breeding season impacts lifetime productivity (Lesmeister et al., 1973). Heifers classified as cyclic at the initiation of breeding season had greater overall pregnancy and calving percentages to the first breeding season (Moriel et al., 2017). Beef heifers that conceived early during their initial breeding season and calved within the first 21 d period of the calving season had greater overall pregnancy percentage and calf weaning weights for the first six parturitions (Cushman et al., 2013). They also remained in the herd longer compared to females that calved during the second and third 21 d period of calving season (Cushman et al., 2013). In agreement with our hypothesis and the rationale described above, preweaning injections of bovine ST increased the overall pregnancy percentages in yr 1, 2, and 3, and calving percentages in yr 1 and 2. Calving distribution, however, did not differ between treatments, which was unexpected. Thus, the greater overall reproductive efficiency of BST heifers is likely because of the greater percentage of pubertal heifers at the initiation and during the first 30 d of the breeding season, highlighting the importance of age at puberty attainment for B. indicus-influenced heifers. Taken together, the results of the current study indicate that major limiting factor for reproductive success of B. indicus-influenced heifers is the delayed attainment of puberty because of poor environment- induced growth performance of heifers. It also suggests that successful pregnancy and calving percentages can occur even under situations of poor growth performance, if heifers are administered preweaning injections of bovine ST and become pubertal before the initiation of breeding season.

It is important to note that heifers were slightly lighter than expected at the initiation of the breeding season (% of mature BW), and for that reason, only 20 to 40 % of all heifers were considered pubertal at the start of the breeding season. This is likely a result of the impacts of environmental conditions reducing postweaning growth performance of heifers, as reported previously in cohorts at the same location (Moriel et al., 2014). In addition, the fact that only 20 to 40 % of heifers were pubertal at the initiation of the breeding season indicates that bull breeding power was not a limiting factor for the reproductive performance of heifers.

Calving percentages in yr 3 did not differ between BST and SAL heifers. This response was surprising considering that this was the only variable measured in the present study that demonstrated an effect of treatment × year. Further evaluation revealed that heifers in yr 3, regardless of treatment, had the lightest mean BW during the postweaning phase compared to heifers in yr 1 and 2, which could be attributed to differences in environmental conditions as postweaning nutritional management of heifers was similar across year. Likewise, overall puberty attainment was less for yr 3 vs. 1 and 2 (30% vs. 42% and 52%, respectively), and overall pregnancy percentage was less in yr 2 and 3 vs. 1 (60% and 63% vs. 90%, respectively).

Hence, the similar postweaning nutritional management and ADG of heifers among year (P =

0.19), and lack of treatment and treatment × year interactions for postweaning BW and ADG, indicates that the similar calving percentage between treatments in yr 3 may be attributed to heifers being the lightest, which limited the overall puberty attainment and pregnancy percentage, and prevented similar treatment effects observed for calving percentages in yr 1 and

Conclusion

In conclusion, preweaning injections of bovine ST (250 mg every 14 d between 135 and

163 d of age) increased puberty attainment of beef heifers at the initiation of their first breeding

season, overall pregnancy percentage in all 3 yr, and calving percentage in 2 of 3 yr. In addition, preweaning injections of bovine ST led to long-term effects on plasma IGF-1 concentrations and liver mRNA expression of genes associated with energy metabolism and known for positively influencing reproduction in beef cattle. These latter responses may be indicators of metabolic imprinting, but further measures are warranted to elucidate the actual metabolic imprinting mechanisms that may be occurring.

Table 3-1. Average nutritional composition of concentrate offered during the post-weaning phase (d 127 to 346) to beef heifers that received a pre-weaning s.c. injection of saline solution (SAL; 5 mL; 0.9% NaCl) or 250 mg of sometribove zinc (BST; Posilac Elanco, Greenfield, IN) on d 0, 14, and 28 (n = 15 heifers/treatment annually; 3 yr). Item Post-weaning concentrate 1 Ingredient 2, kg DM daily Molasses 1.0 Crude glycerin 1.0 Dried distillers grains 0.59 Soybean meal 0.30 Ca carbonate 0.009 Phosphoric acid 0.009

TDN 3, % of DM 81.3 CP, % of DM 15.4 NEm 4, Mcal/kg of DM 2.05 NEg 4, Mcal/kg of DM 1.39 Ca, % of DM 0.55 P, % of DM 0.40

1 Concentrate samples were collected monthly from weaning (d 127) until the end of the study (d 346) for wet chemistry analysis of all nutrients. 2 Ingredients were hand-mixed immediately before feeding. Concentrate was provided 3 times weekly (Monday, Wednesday, and Friday) at 0800 h in amounts to achieve a target daily DM intake of 2.9 kg/heifer from day 127 to 346. 3 Calculated as described by Weiss et al. (1992). 4 Calculated using the equations proposed by the NRC (2000).

Table 3-2. Primer sequences and accession number for all gene transcripts analyzed by quantitative real-time PCR1. Target gene Primer sequence Accession no. Cyclophilin Forward 5'-GGTACTGGTGGCAAGTCCAT-3' NM_178320.2 Reverse 5'-GCCATCCAACCACTCAGTCT-3' IGF-1 Forward 5'-CTCCTCGCATCTCTTCTATCT-3' NM_001077828 Reverse 5'-ACTCATCCACGATTCCTGTCT-3' IGFBP-3 Forward 5'-AATGGCAGTGAGTCGGAAGA-3' NM_174556.1 Reverse 5'-AAGTTCTGGGTGTCTGTGCT-3' GHR-1A Forward 5'-CCAGCCTCTGTTTCAGGAGTGT-3' AY748827 Reverse 5'-TGCCACTGCCAAGGTCAAC-3' GHR-1B Forward 5'-AGCCTGGAGGAACCATACGA-3' - Reverse 5'-TAGCCCCATCTGTCCAGTGA-3' RSP9 Forward 5'-CCTCGACCAAGAGCTGAAG-3' DT860044 Reverse 5'-CCTCCAGACCTCACGTTTGTTC-3'

1Primer sequence for IGF-1, IGFBP-3, and GHR-1A genes were obtained from Coyne et al. (2011), whereas the primer sequence for Cyclophilin and RSP9 genes were obtained from Cooke et al. (2008) and Janovick-Guretzky et al. (2007), respectively. Primer sequence for GHR-1B gene was designed based on the bovine gene sequences deposited in the National Center for Biotechnology Information and using the Primer Express v. 3.0.1 software (Applied Biosystems, Foster City, CA).

Table 3-3. Pre- and post-weaning growth performance of beef heifers that received a s.c. injection of saline solution (SAL; 5 mL; 0.9% NaCl) or 250 mg of sometribove zinc (BST; Posilac, Elanco, Greenfield, IN) on d 0, 14, and 28 (n = 15 heifers/treatment annually; 3 yr)1. Treatment P-value Treatment SAL BST SEM Treatment yr Item × yr Pre-weaning (d 0 to 127) ADG, kg/d d 0 to 14 1.26 1.35 0.06 0.46 0.18 <0.01 d 14 to 28 0.99 1.09 0.04 0.78 0.03 <0.01 d 28 to 42 1.00 0.97 0.04 0.27 0.40 <0.01 d 0 to 42 1.07 1.15 0.03 0.56 0.03 0.07 d 42 to 127 0.80 0.74 0.02 0.48 0.04 <0.01 d 0 to 127 0.89 0.88 0.02 0.66 0.50 <0.01

Post-weaning (d 127 to 346) ADG, kg/d d 127 to 262 0.12 0.13 0.03 0.36 0.71 0.02 d 127 to 346 0.30 0.28 0.02 0.14 0.61 0.20 Hip height, cm d 179 115 116 0.60 0.66 0.66 <0.01 d 346 123 123 0.60 0.48 0.87 0.11 Hip height change, cm 7.8 7.4 0.37 0.79 0.41 <0.01 1 Individual heifer shrunk BW were assessed on d 0, 14, 28, and 42, after 6 h of feed and water withdrawal, and then every 28 d from d 127 to 346, after 16 h of feed and water withdrawal. Heifers and their dams were managed as a single group without access to concentrate supplementation during the pre-weaning phase (d 0 to 127). After weaning (d 127), heifers were sorted by treatment, allocated into 1 of 8 bahiagrass pastures (1 pasture/treatment), and offered the same concentrate supplementation strategy until d 346.

Table 3-4. Pre- and post-weaning plasma IGF-1 concentrations of beef heifers that received a s.c. injection of saline solution (SAL; 5 mL; 0.9% NaCl) or 250 mg of sometribove zinc (BST; Posilac, Elanco, Greenfield, IN) on d 0, 14, and 28 (n = 15 heifers/treatment annually; 3 yr).1 Treatment P-value 2 Treatment Item SAL BST SEM P 3 Treatment × Day Pre-weaning plasma IGF-1, ng/mL Overall (d 0 to 42) 4 94.8 103.4 3.16 - 0.83 0.05

Post-weaning plasma IGF-1, ng/mL d 234 166.9 197.2 8.00 <0.01 0.04 0.19 d 263 181.9 184.5 8.00 0.82 d 296 181.0 181.0 8.00 0.98 1Blood samples were collected from jugular vein from all heifers on d 0, 14, 28, 42, 127, and then every 9 to 10 d from d 179 to 346. Blood samples for the assessment of plasma IGF-1 concentrations were selected to represent the period of pre-weaning injections (d 0, 14, 28, and 42), day of weaning (d 127), and then 28 d before (d 235), immediately at (d 263), and 33 d after (d 296) the start of the breeding season. 2 Effects of day, but not treatment × yr and treatment × yr × day of the study (P ≥ 0.14), were detected for pre- and post-weaning plasma IGF-1 concentrations (P < 0.0001). 3 P-value for the comparison of treatment within day. 4Average plasma IGF-1 concentrations of blood samples collected on d 14, 28, and 42, after covariate-adjusted for plasma IGF-1 concentrations obtained on d 0 (P < 0.0001).

Table 3-5. Liver mRNA expression (fold increase1; yr 3 only) of beef heifers that received a s.c. injection of saline solution (SAL; 5 mL; 0.9% NaCl) or 250 mg of sometribove zinc (BST; Posilac, Elanco, Greenfield, IN) on d 0, 14, and 28 (n = 15 heifers/treatment).2 Treatment P-value Treatment Gene 3 SAL bST SEM P 4 Day Treatment × Day ------Fold increase ------GHR-1A 5 1.77 1.84 0.18 - 0.15 <0.01 0.79 IGFBP-3 5 1.58 1.96 0.15 - 0.44 <0.01 0.12 GHR-1B d 42 3.59 4.36 0.41 0.22 0.02 <0.01 0.76 d 263 0.72 1.70 0.41 0.02 IGF-1 d 42 1.50 1.87 0.17 0.15 <0.01 0.04 0.22 d 263 0.93 1.85 0.17 <0.01 1 Responses were quantified based on the threshold cycle (CT) and were normalized to average CT of Cyclophilin and RSP9 (ΔCT) examined in the same sample and assessed at the same time as the targets. Within each target gene, results are expressed as relative fold change (2–ΔΔCT) using the average ΔCT of all samples as reference, as described by Ocón-Grove et al. (2008). 2 Heifers and their dams were managed as a single group without access to concentrate supplementation during the pre-weaning phase (d 0 to 127). After weaning (d 127), heifers were sorted by treatment, allocated into bahiagrass pastures (1 pasture/treatment), and offered the same concentrate supplementation strategy until d 346. 3 Liver mRNA expression of GHR-1A, GHR-1B, and IGF-1, but not IGFBP-3 (P = 0.77), were covariate-adjusted to respective mRNA expression obtained on d 0 (P ≤ 0.06). 4 P-value for the comparison of treatment within day. 5 Average liver mRNA expression of GHR-1A and IGFBP-3 obtained on d 0, 42, and 263.

Table 3-6. Reproductive performance of beef heifers that received a s.c. injection of saline solution (SAL; 5 mL; 0.9% NaCl) or 250 mg of sometribove zinc (BST; Posilac, Elanco, Greenfield, IN) on d 0, 14, and 28 (n = 15 heifers/treatment annually; 3 yr).1 Treatment P-value Treatment Item SAL BST SEM P 2 Treatment yr × yr Age at puberty 3, d 414 388 12.9 - 0.45 0.10 0.48 Body weight at puberty 4, kg 291 285 5.70 - 0.46 0.34 0.37 Mature BW d 263 5, % 54.7 56.2 1.22 - 0.69 0.16 <0.01 Overall pregnancy % 6 68.9 82.2 6.11 - 0.17 0.10 0.01 Overall calving % yr 1 73.3 93.3 6.48 0.05 0.03 0.02 0.08 yr 2 33.3 86.7 6.48 <0.01 yr 3 66.7 60.0 6.48 0.68 Pregnancy loss 6, % 11.1 2.2 3.66 - 0.28 0.08 0.20 Calving date 6, Julian d 277 284 5.80 - 0.25 0.34 0.38 Calf birth BW 6, kg 26.1 25.0 1.00 - 0.57 0.41 <0.01 1 After weaning (d 127), heifers were sorted by treatment, allocated into 1 of 8 bahiagrass pastures (1 pasture/treatment), and offered the same concentrate supplementation strategy until d 346. Heifers were exposed to yearling Angus bulls from d 263 to 346 (1 bull/group). Every 9 to 10 d, heifers were rotated among the same 8 bahiagrass pastures from d 127 to 346 and bulls rotated among heifer treatment groups from d 263 to 346. 2 P-value for the comparison of treatment within day. 3 Heifers were considered pubertal after the first increase in plasma progesterone concentrations that exceeded 1.0 ng/mL (Perry et al., 1991). 4 Body weight at puberty = initial BW of the respective 28-d interval + (ADG of the respective 28- d interval  number of days between the day at puberty attainment and initial BW collection). 5 Assuming a cow herd mature body weight of 499 kg (Moriel et al., 2017). 6 Pregnancy rates were determined via rectal palpation at approximately 45 d after the end of the breeding season. Pregnancy loss calculated as percentage of heifers that were categorized as pregnant at approximately 45 d after the end of breeding season, but did not deliver a live calf. Heifers were observed twice daily for calving. Calving date were determined using Julian date, and calf birth BW obtained within 12 h of birth.

SAL BST 270 260 250 240 230 220 210 200

190

weaning heifer body weight,body weaningkg heifer -

Pre 180 170 160 14 28 42 127 Day of the study

Figure 3-1. Pre- (A) and post-weaning (B) body weight of beef heifers that received a s.c. injection of saline solution (SAL; 5 mL; 0.9% NaCl) or 250 mg of sometribove zinc (BST; Posilac, Elanco) on d 0, 14, and 28 (n = 15 heifers/treatment annually; 3 yr). Body weight on d 0 did not differ among treatments (P ≥ 0.91), but was included as covariate in the BW analyses (P < 0.0001). Effects of treatment × day of the study were detected for pre-weaning BW (P = 0.01; SEM = 1.28), but not for post-weaning BW of heifers (P = 0.50; SEM = 4.75). †P = 0.09.

SAL BST 330

320

310

300

290

280

270 weaning heifer body kg weight, weaning heifer

- 260 Post 250

240 179 207 235 263 296 317 346 Day of the study

Figure 3-1. Continued.

SAL BST 100

20 Pubertal heifers, % of total total % heifers, ofheifers Pubertal 10

226 346 179 189 198 207 217 235 244 254 263 273 284 296 307 317 328 337 Day of the study

Figure 3-2. Percentage of pubertal beef heifers that received an s.c. injection of saline solution (SAL; 5 mL; 0.9% NaCl) or 250 mg of sometribove zinc (BST; Posilac, Elanco) on d 0, 14, and 28 (n = 15 heifers per treatment annually; 3 yr). Heifers were weaned on d 127. Heifers were considered pubertal after the first increase in serum progesterone concentrations that exceeded 1.0 ng/mL (Perry et al., 1991). Heifers were exposed to mature Angus bulls from d 263 to 346 (one bull per treatment). Effects of treatment × day of the study were detected (P = 0.03; SEM = 6.09) for puberty achievement from d 179 to 346. * P ≤ 0.05 and †P = 0.08.

SAL BST 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0

10.0 Calving distribution, % of heifers that calved that heifers of%distribution, Calving 0.0 1 2 3 4 5 6 7 8 9 10 11 12 Week of the calving season

Figure 3-3. Calving distribution (% of heifers that calved) of beef heifers that received an s.c. injection of saline solution (SAL; 5 mL; 0.9% NaCl) or 250 mg of sometribove zinc (BST; Posilac, Elanco) on d 0, 14, and 28 (n = 15 heifers per treatment annually; 3 yr). Heifers were weaned on d 127 and exposed to mature Angus bulls from d 263 to 346 (one bull per treatment). Every 9 to 10 d, heifers were rotated among the same bahiagrass pastures from d 127 to 346 and bulls rotated among heifer groups from d 263 to 346. Effects of year, treatment, treatment × year, treatment × week of calving season, and treatment × week of calving season × year were not detected (P ≥ 0.27) for calving distribution.

LIST OF REFERENCES

Allen, C. C., B. R. C. Alves, X. Li, L. O. Tedeschi, H. Zhou, J. C. Paschal, P. K. Riggs, U. M. Braga-Neto, D. H. Keisler, G. L. Williams, and M. Amstalden. 2012. Gene expression in the arcuate nucleus of heifers is affected by controlled intake of high- and low- concentrate diets. J. Anim. Sci. 90:2222–2232. doi:10.2527/jas.2011-4684.

AOAC. 2006. Official methods of analysis. 18th ed. AOAC Int., Arlington, VA.

Armstrong, J. D., and A. M. Benoit. 1996. Paracrine , Autocrine , and Endocrine Factors that Mediate the Influence of Nutrition on Reproduction in Cattle and Swine : An In Vivo , IGF-I Perspective Paracrine , Autocrine , and Endocrine Factors that Mediate the Influence of Nutrition on Reproductio. J. Anim. Sci. 74:18–35.

Arthington, J. D., and L. R. Corah. 1995. Liver biopsy procedures for determining the trace mineral status in beef cows. Part II. (Video, AI 9134). Extension TV, Dep. Commun. Coop. Ext. Serv., Kansas State Univ., Manhattan.

Badinga, L., R. J. Collier, W. W. Thatcher, C. J. Wilcox, H. H. Head, and F. W. Bazer. 1991. Ontogeny of hepatic bovine growth hormone receptors in cattle. J. Anim. Sci. 69:1925– 1934.

Bagley, C. P. 1993. Nutritional management of replacement beef heifers: a review. J. Anim. Sci. 71:3155–3163. doi:/1993.71113155x.

Bass, J. J., S. C. Hodgkinson, B. H. Breier, W. D. Carter, and P. D. Gluckman. 1992. Effects of bovine somatotrophin on insulin-like growth factor-I, insulin, growth and carcass composition of lambs. Livest. Prod. Sci. 31:321–334. doi:10.1016/0301-6226(92)90078- I. Available from: http://www.sciencedirect.com/science/article/pii/030162269290078I

Bauman, D. E. 1999. Bovine somatotropin and lactation: From basic science to commercial application. In: Domestic Animal Endocrinology. Vol. 17. p. 101–116.

Bauman, D. E., D. H. Beermann, R. D. Boyd, P. J. Buttery, R. B. Campbell, W. V. Chalupa, T. D. Etherton, K. Klasing, G. T. Schelling, and N. C. Steele. 1994. Metabolic Modifiers: Effects on the Nutrient Requirements of Food-Producing Animals. National Academy Press, Washington, D.C. 1994.

Bauman, D. E., and R. G. Vernon. 1993. Effects of exogenous bovine somatotropin on lactation. Annu. Rev. Nutr. 437–461.

Baxter, R. C. 2013. Insulin-like growth factor binding protein-3 ( IGFBP-3 ): Novel ligands mediate unexpected functions. J. Cell Commun. Signal. 3:179–189. doi:10.1007/s12079- 013-0203-9.

Berge, P. 1991. Long-term effects of feeding during calfhood on subsequent performance in beef cattle. Livest. Prod. Sci. 28:179–201.

Bilby, C. R. 2005. Effects of polyunsaturated fatty acids and bovine somatotropin on endocrine function, embryo development, and uterine-conceptus interactions in dairy cattle. University of Florida.

Bilby, C. R., J. F. Bader, B. E. Salfen, R. S. Youngquist, C. N. Murphy, H. A. Garverick, B. A. Crooker, and M. C. Lucy. 1999. Plasma GH, IGF-I, and conception rate in cattle treated with low doses of recombinant bovine GH. Theriogenology. 51:1285–1296. doi:10.1016/S0093-691X(99)00072-2.

Bilby, T. R., A. Guzeloglu, S. Kamimura, S. M. Pancarci, F. Michel, H. H. Head, and W. W. Thatcher. 2004. Pregnancy and bovine somatotropin in nonlactating dairy cows: I. Ovarian, conceptus, and insulin-like growth factor system responses. J. Dairy Sci. 87:3256–3267. doi:10.3168/jds.S0022-0302(04)73462-1. Available from: http://dx.doi.org/10.3168/jds.S0022-0302(04)73462-1

Binelli, M., W. K. Vanderkooi, L. T. Chapin, M. J. Vandehaar, J. D. Turner, W. M. Moseley, and H. A. Tucker. 1995. Comparison of Growth Hormone-Releasing Factor and Somatotropin: Body Growth and Lactation of Primiparous Cows. J. Dairy Sci. 78:2129– 2139. doi:https://doi.org/10.3168/jds.S0022-0302(95)76840-0. Available from: http://www.sciencedirect.com/science/article/pii/S0022030295768400

Breier, B. H. 1999. Regulation of protein and energy metabolism by the somatotropic axis. Domest. Anim. Endocrinol. 17:209–218. doi:10.1016/S0739-7240(99)00038-7. Available from: http://www.sciencedirect.com/science/article/pii/S0739724099000387

Buskirk, D. D., D. B. Faulkner, W. L. Hurley, D. J. Kesler, F. A. Ireland, T. G. Nash, J. C. Castree, and J. L. Vicini. 1996. Growth, reproductive performance, mammary development, and milk production of beef heifers as influenced by prepubertal dietary energy and administration of bovine somatotropin. J. Anim. Sci. 74:2649–2662.

Butler, W. R., and R. D. Smith. 1989. Interrelationships between energy balance on postpartum reproductive function in dairy cattle. J. Dairy Sci. 72:767–783.

Byerley, D. J., R. B. Staigmiller, J. G. Berardinelli, and R. E. Short. 1987. Pregnancy rates of beef heifers bred either on pubertal or third estrus. J. Anim. Sci. 65:645–650. doi:10.2527/jas1987.653645x. Available from: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citati on&list_uids=3667429

Campbell, R. G., R. J. Johnson, M. R. Taverner, and R. H. King. 1991. Interrelationships Between Exogenous Porcine Somatotropin (pST) Administration and Dietary Protein and Energy Intake on Protein Deposition Capacity and Energy Metabolism of Pigs. J. Anim. Sci. 69:1522–1531.

Cappellozza, B. I., R. F. Cooke, M. M. Reis, P. Moriel, D. H. Keisler, and D. W. Bohnert. 2014. Supplementation based on protein or energy ingredients to beef cattle consuming low- quality cool-season forages: II. Performance, reproductive, and metabolic responses of replacement heifers. J. Anim. Sci. 92:2725–2734. doi:10.2527/jas2013-7442.

Capper, J. L., E. Castaneda-Gutierrez, R. A. Cady, and D. E. Bauman. 2008. The environmental impact of recombinant bovine somatotropin (rbST) use in dairy production. Proc. Natl. Acad. Sci. 105:9668–9673. doi:10.1073/pnas.0802446105. Available from: http://www.pnas.org/cgi/doi/10.1073/pnas.0802446105

Carlacci, L., K. C. Chou, and G. M. Maggiora. 1991. A Heuristic Approach to Predicting the Tertiary Structure of Bovine Somatotropin. Biochemistry. 30:4389–4398. doi:10.1021/bi00232a004.

Carriquiry, M., W. J. Weber, and B. A. Crooker. 2008. Administration of Bovine Somatotropin in Early Lactation: A Meta-Analysis of Production Responses by Multiparous Holstein Cows. J. Dairy Sci. 91:2641–2652. doi:10.3168/jds.2007-0841. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0022030208711391

Carstens, G. E., D. E. Glaser, F. M. Byers, L. W. Greene, and D. K. Lunt. 2010. Effects of bovine somatotropin treatment and intermittent growth pattern on mammary gland development in heifers. 2378–2388.

Carstens, G. E., D. E. Glaser, F. M. Byers, L. W. Greene, and D. K. Lunt. 1997. Effects of bovine somatotropin treatment and intermittent growth pattern on mammary gland development in heifers. J. Anim. Sci. 75:2378–2388.

Carter-Su, C., J. Schwartz, and L. S. Argetsinger. 2016. Growth hormone signaling pathways. Growth Horm. IGF Res. 28:11–15. doi:10.1016/j.ghir.2015.09.002. Available from: http://linkinghub.elsevier.com/retrieve/pii/S1096637415300290

Caton, J. S., and B. W. Hess. 2010. Maternal plane of nutrition: Impacts on fetal outcomes and postnatal offspring responses. Page 104-122 in: Proc. 4th Grazing Livestock Nutrition Conference. B. W. Hess, T. DelCurto, J. G. P. Bowman and R. C. Waterman (eds.) West. Sect. Am. Soc. Anim. Sci., Champaign, IL.

Cole W. J., K. S. Madsen, R. L. Hintz, R. J. Collier. 1991. Effect of recombinantly-derived bovine somatotropin on reproductive performance of dairy cattle. Theriogenology: 36:573-595.

Cooke, R. F., J. D. Arthington, D. B. Araujo, G. C. Lamb, and A. D. Ealy. 2008. Effects of supplementation frequency on performance, reproductive, and metabolic responses of Brahman-crossbred females. J. Anim. Sci. 86:2296-2309.

Cooke, R. F., D. W. Bohnert, C. L. Francisco, R. S. Marques, C. J. Mueller, and D. H. Keisler. 2013. Effects of bovine somatotropin administration on growth, physiological, and reproductive responses of replacement beef heifers. J. Anim. Sci. 91:2894–2901. doi:10.2527/jas.2012-6082.

Costa, R. H., V. V. Kalinichenko, A. X. L. Holterman, and X. Wang. 2003. Transcription Factors in Liver Development, Differentiation, and Regeneration. Hepatology. 38:1331–1347. doi:10.1016/j.hep.2003.09.034.

Coyne, G. S., D. A. Kenny, and S. M. Waters. 2011. Effect of dietary n-3 polyunsaturated fatty acid supplementation on bovine uterine endometrial and hepatic gene expression of the insulin-like growth factor system. Theriogenology 75:500–512.

Cushman, R. A., L. K. Kill, R. N. Funston, E. M. Mousel, and G. A. Perry. 2013. Heifer calving date positively influences calf weaning weights through six parturitions. J. Anim. Sci. 91:4486–4491. doi:10.2527/jas2013-6465.

Daftary, S. S., and A. C. Gore. 2003. Developmental changes in hypothalamic insulin-like growth factor-1: Relationship to gonadotropin-releasing hormone neurons. Endocrinology. 144:2034–2045. doi:10.1210/en.2002-221025.

Daftary, S. S., and A. C. Gore. 2005. IGF-1 in the brain as a regulator of reproductive neuroendocrine function. Exp. Biol. Med. 230:292–306. doi:10.1177/153537020523000503.

Dalke, B. S., R. A. Roeder, T. R. Kasser, J. J. Veenhuizen, C. W. Hunt, D. D. Hinman, and G. T. Schelling. 1992. Dose–response effects of recombinant bovine somatotropin implants on feedlot performance in steers. J. Anim. Sci. 70:2130–2137.

Day, M. L., and L. H. Anderson. 1998. Current concepts on Control of Puberty. J. Anim. Sci. 76:1–15.

Day, M. L., K. Imakawa, P. L. Wolfe, R. J. Kittok, and J. E. Kinder. 1987. Endocrine mechanisms of puberty in heifers: estradiol negative feedback regulation of luteinizing hormone secretion. Biol. Reprod. 37:1054–1065. doi:10.1095/biolreprod31.2.332.

Day, M. L., and G. P. Nogueira. 2013. Management of age at puberty in beef heifers to optimize efficiency of beef production. Anim. Front. 3:6–11. doi:10.2527/af.2013-0027. Available from: http://www.animalfrontiers.org/cgi/doi/10.2527/af.2013-0027

Dodson, S. E., B. J. McLeod, W. Haresign, a R. Peters, and G. E. Lamming. 1988. Endocrine changes from birth to puberty in the heifer. J. Reprod. Fertil. 82:527–538. doi:10.1530/jrf.0.0820527.

Donkin, S. 2012. The Role of Liver Metabolism During Transition on Postpartum Health and Performance. 23rd Annu. Florida Rumin. Nutr. Symp. 97–107. Available from: http://dairy.ifas.ufl.edu/RNS/2012/8DonkinRNS2012.pdf

Dunshea, F. R., D. M. Harris, D. E. Bauman, R. D. Boyd, and A. W. Bell. 1992. Effects of porcine somatotropin on in vivo glucose kinetics and lipogenesis in growing pigs. J. Anim. Sci. 70:141–151. doi:10.1016/0309-1740(93)90040-O.

Early, R. J., B. W. McBride, and R. 0. Ball. 1990. Growth and metabolism in somatotropin- treated steers: II. Carcass and noncarcass tissue components and chemical composition. J. Anim. Sci. 68:4144-4152.

Edens, A., and F. Talamantes. 1998. Alternative Processing of Growth Hormone Receptor Transcripts. Endocr. Rev. 19:559–582.

Eisemann, J. H., H. F. Tyrrell, A. C. Hammond, P. J. Reynolds, D. E. Bauman, G. L. Haaland, J. P. McMurtry, and G. A. Varga. 1986. Effect of bovine growth hormone administration on metabolism of growing Hereford heifers: dietary digestibility, energy and nitrogen balance. J. Nutr. 116:157–163.

Enright, W. J., J. F. Quirke, P. D. Gluckman, B. H. Breier, L. G. Kennedy, I. C. Hart, J. F. Roche, A. Coert, and P. Allen. 1990. Effects of long-term administration of pituitary- derived bovine growth hormone and estradiol on growth in steers. J. Anim. Sci. 68:2345- 2356.

Etherton, T. D., and D. E. Bauman. 1998. Biology of somatotropin in growth and lactation of domestic animals. Physiol. Rev. 78:745–761.

Etherton, T. D., and I. Louveau. 1992. Manipulation of adiposity by somatotropin and B- adrenergic agonists: a comparison of their mechanisms of action. In: Proceedings of the Nutrition Society. p. 419–431.

Evans, A. C. O., G. P. Adams, and N. C. Rawlings. 1994. Follicular and hormonal development in prepubertal heifers from 2 to 36 weeks of age. J. Reprod. Fertil. 102:463–470. doi:10.1530/jrf.0.1020463.

FASS. 2010. Guide for the care of use of animals in research and teaching.

Fenech, M. 2010. Dietary reference values of individual micronutrients and nutriomes for DNA damage prevention. Am. J. Clin. Nutr. 91:1438–1454. doi:10.3945/ajcn.2010.28674D.1.

Ferrell, C. L. 1982. Effects of postweaning rate of gain on onset of puberty and productive performance of heifers of different breeds. J. Anim. Sci. 55:1272–1283. doi:10.2527/jas1982.5561272x.

Freetly, H. C., K. A. Vonnahme, A. K. McNeel, L. E. Camacho, O. L. Amundson, E. D. Forbes, C. A. Lents, and R. A. Cushman. 2014. The consequence of level of nutrition on heifer ovarian and mammary development. J. Anim. Sci. 92:5437–5443. doi:10.2527/jas2014- 8086.

Gallaher, R. N., C. O. Weldon, and J. G. Futral. 1975. An aluminum block digester for plant and soil analysis. Soil Sci. Soc. Am. J. 39:803–806.

Gasser, C. L. 2013. Joint Alpharma-beef Species symposium: Considerations on puberty in replacement beef heifers. J. Anim. Sci. 91:1336–1340. doi:10.2527/jas.2012-6008.

Gasser, C. L., E. J. Behlke, D. E. Grum, and M. L. Day. 2006a. Effect of timing of feeding a high-concentrate diet on growth and attainment of puberty in early-weaned heifers. J. Anim. Sci. 84:3118–3122. doi:10.2527/jas.2005-676.

Gasser, C. L., G. A. Bridges, M. L. Mussard, D. E. Grum, J. E. Kinder, and M. L. Day. 2006b. Induction of precocious puberty in heifers III: Hastened reduction of estradiol negative feedback on secretion of luteinizing hormone. J. Anim. Sci. 84:2050–2056. doi:10.2527/jas.2005-638.

Gasser, C. L., C. R. Burke, M. L. Mussard, E. J. Behlke, D. E. Grum, J. E. Kinder, and M. L. Day. 2006c. Induction of precocious puberty in heifers II: Advanced ovarian follicular development. J. Anim. Sci. 84:2042–2049. doi:10.2527/jas.2005-637.

Gasser, C. L., D. E. Grum, M. L. Mussard, F. L. Fluharty, J. E. Kinder, and M. L. Day. 2006d. Induction of precocious puberty in heifers I: Enhanced secretion of luteinizing hormone. J. Anim. Sci. 84:2035–2041. doi:10.2527/jas.2005-636.

Gluckman, P. D., B. H. Breier, and S. R. Davis. 1987. Physiology of the Somatotropic Axis with Particular Reference to the Ruminant. J. Dairy Sci. 70:442–466. doi:https://doi.org/10.3168/jds.S0022-0302(87)80028-0. Available from: http://www.sciencedirect.com/science/article/pii/S0022030287800280

Gong, J. G., G. Baxter, T. A. Bramley, and R. Webb. 1997. Enhancement of ovarian follicle development in heifers by treatment with recombinant bovine somatotrophin: a dose- response study. J Reprod Fertil. 110:91–97. doi:10.1530/jrf.0.1100091. Available from: http://www.reproduction-online.org/cgi/content/abstract/110/1/91

Gong, J. G., T. A. Bramley, and R. Webb. 1993. The effect of recombinant bovine somatotrophin on ovarian follicular growth and development in heifers. J. Endocrinol. 97:247–254.

Govoni, K. E., T. A. Hoagland, and S. A. Zinn. 2004. The ontogeny of the somatotropic axis in male and female Hereford calves from birth to one year of age and its response to administration of exogenous bovine somatotropin. J. Anim. Sci. 82:1646–1655.

Granz, S., F. Ellendorff, R. Grossmann, Y. Kato, E. Muhlbauer, and F. Elsaesser. 1997. Ontogeny of growth hormone and LH beta-, FSH beta- and alpha-subunit mRNA levels in the porcine fetal and neonatal anterior pituitary. J. Neuroendocrinol. 9:439–449.

Groenewegen, P. P., B. W. McBride, J. H. Burton, and T. H. Elsasser. 1990. Efect of Bovine Somatotropin on the Growth Rate, Hormone Profiles and Carcass Composition of Holstein Bull Calves. Domest. Anim. Endocrinol. 7:43–54.

Hall, J. B. 2013. Nutritional development and the target weight debate. Vet. Clin. North Am. - Food Anim. Pract. 29:537–554. doi:10.1016/j.cvfa.2013.07.015. Available from: http://dx.doi.org/10.1016/j.cvfa.2013.07.015

Hall, J. B., K. K. Schillo, B. P. Fitzgerald, and N. W. Bradley. 1994. Effects of recombinant bovine somatotropin and dietary energy intake on growth, secretion of luteinizing hormone, follicular development, and onset of puberty in beef heifers. J. Anim. Sci. 72:709-718.

Hess, B. W., S. L. Lake, E. J. Scholljegerdes, T. R. Weston, V. Nayigihugu, J. D. C. Molle, and G. E. Moss. 2005. Nutritional controls of beef cow reproduction. J. Anim. Sci. 83:E90– E106. doi:/2005.8313_supplE90x.

Houseknecht, K. L., D. E. Bauman, D. G. Fox, and D. F. Smith. 1992. Abomasal infusion of casein enhances nitrogen retention in somatotropin-treated steers. J. Nutr. 122:1717– 1725.

Janovick-Guretzky, N.A., Dann, H.M., Carlson, D.B., Murphy, M.R., Loor, J.J., Drackley, J.K., 2007. Housekeeping gene expression in bovine liver is affected by physiological state, feed intake, and dietary treatment. J. Dairy Sci. 90:2246–2252.

Jiang, H., and M. C. Lucy. 2001. Variants of the 5’-untranslated region of the bovine growth hormone receptor mRNA: Isolation, expression and effects on translational efficiency. Gene 265:45-53.

Jiang, H., C. S. Okamura, and M. C. Lucy. 1999. Isolation and characterization of a novel promoter for the bovine growth hormone receptor gene. J. Biol. Chem. 274:7893-7900.

Jiang, H., and X. Ge. 2014. Mechanism of growth hormone stimulation of skeletal muscle growth in cattle. J. Anim. Sci. 2014.92:21–29.

Jiang, H., Y. Wang, M. Wu, Z. Gu, S. J. Frank, and R. Torres-Diaz. 2007. Growth hormone stimulates hepatic expression of bovine growth hormone receptor messenger ribonucleic acid through signal transducer and activator of transcription 5 activation of a major growth hormone receptor gene promoter. Endocrinology. 148:3307–3315. doi:10.1210/en.2006-1738.

Jirtle, R. L., and M. K. Skinner. 2007. Environmental epigenomics and disease susceptibility. Nat. Rev. Genet. 8:253-262.

Jungermann, K., and N. Katz. 1989. Functional specialization of different hepatocyte populations. Physiol. Rev. 69:708–64. doi:10.1152/physrev.1989.69.3.708. Available from: http://physrev.physiology.org/content/69/3/708.abstract%5Cnhttp://www.ncbi.nlm.nih.go v/pubmed/2664826

Juul, A., P. Bang, N. T. Hertel, K. Main, P. Dalgaard, K. Jorgensen, J. Muller, K. Hall, and N. E. Skakkebaek. 1994. Serum Insulin-Like Growth Factor-1 in 1030 Healthy Children, Adolescents, and Adults: Relation to Age, Sex, Stage of Puberty, testicular Size, and Body Mass Index. J. Clin. Endocrinol. Metab. 78:744–752.

Juul, A., P. Dalgaard, W. F. Blum, P. Bang, K. Hall, K. F. Michaelsen, J. Muller, and N. E. Skakkebaek. 1995. Serum Levels of Insulin-Like Growth Factor (IGF)- Binding Protein- 3 (IGFBP-3) in Healthy Infants, Children, and Adolescents: The Relation to IGF-1, IGF- 2, IGFBP-1, IGFBP-2, Age, Sex, Body Mass Index, and Pubertal Maturation. J. Clin. Endocrinol. Metab. 80:2534–2542.

Kaung, H. L.1994. Growth dynamics of pancreatic islet cell populations during fetal and neonatal development of the rat. Dev Dyn. 200:163-175.

Kim, J. W. 2014. Modulation of the Somatotropic Axis in Periparturient Dairy Cows. Asian- Australasian J. Anim. Sci. 27:147–154. doi:http://dx.doi.org/10.5713/ajas.2013.13139.

Kobayashi, Y., M. J. VandeHaar, H. A. Tucker, B. K. Sharma, and M. C. Lucy. 1999. Expression of Growth Hormone Receptor 1A Messenger Ribonucleic Acid in Liver of Dairy Cows During Lactation and After Administration of Recombinant Bovine Somatotropin. J. Dairy Sci. 82:1910–1916. doi:10.3168/jds.S0022-0302(99)75426-3. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0022030299754263

Kojima, M., H. Hosoda, Y. Date, M. Nakazato, H. Matsuo, and K. Kangawa. 1999. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature. 402:656–660. doi:10.1038/45230.

Kurz, S. G., R. M. Dyer, Y. Hu, M. D. Wright, and M. L. Day. 1990. Regulation of luteinizing hormone secretion in prepubertal heifers fed an energy-deficient diet. Biol. Reprod. 43:450–456. doi:10.1095/biolreprod43.3.450.

Lapierre, H., C. K. Reynolds, T. H. Elsasser, P. Gaudreau, P. Brazeau, and H. F. Tyrrell. 1992. Effects of growth hormone-releasing factor and feed intake on energy metabolism in growing beef steers: Net hormone metabolism by portal-drained viscera and liver. J. Anim. Sci. 70:742–751.

Le Roith, D., C. Bondy, S. Yakar, J. L. Liu, and A. Butler. 2001. The somatomedin hypotheis. Endocr. Rev. 22:53–74. doi:10.1210/edrv.22.1.0419. Available from: https://academic.oup.com/edrv/article-lookup/doi/10.1210/edrv.22.1.0419

Lesmeister, J. L., P. J. Burfening, and R. L. Blackwell. 1973. Date of First Calving in Beef Cows and Subsequent Calf Production. J. Anim. Sci. 36:1–6. doi:10.2134/jas1973.3611.

Lobie, P. E., García-Aragón, J., Lincoln, D. T., Barnard, R., Wilcox, J. N., and Waters, M. J., 1993. Localization and ontogeny of growth hormone receptor gene expression in the central nervous system. Dev. Brain Res.74.275.

Lorenz K. Studies in animal and human behavior. Cambridge, MA: Harvard University Press, 1970.

Lucas, A. 1991. Programming by early nutrition in man. Wiley, Chichester (Ciba Foundation Symposium 156) p38-55.

Lucy, M. C. 2008. Functional Differences in the Growth Hormone and Insulin-like Growth Factor Axis in Cattle and Pigs: Implications for Post-partum Nutrition and Reproduction. Reprod. Domest. Anim. 43:31–39. doi:10.1111/j.1439-0531.2008.01140.x.

Lucy, M. C., C. K. Boyd, A. T. Koenigsfeld, and C. S. Okamura. 1998. Expression of somatotropin receptor messenger ribonucleic acid in bovine tissue. J. Dairy Sci. 81:1889– 1895. doi:10.3168/jds.S0022-0302(98)75760-1. Available from: http://dx.doi.org/10.3168/jds.S0022-0302(98)75760-1

Lucy, M. C., J. C. Byatt, T. L. Curran, D. F. Curran, and R. J. Collier. 1994. Placental lactogen and somatotropin: hormone binding to the corpus luteum and effects on the growth and functions of the ovary in heifers. Biol. Reprod. 50:1136–44. doi:10.1095/biolreprod50.5.1136.

Lucy, M. C., H. Jiang, and Y. Kobayashi. 2001. Changes in the Somatotrophic Axis Associated with the Initiation of Lactation. J. Dairy Sci. 84:E113–E119. doi:10.3168/jds.S0022- 0302(01)70205-6. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0022030201702056

Lui, J. C. 2017. Molecular and Cellular Endocrinology Regulation of body growth by microRNAs. Mol. Cell. Endocrinol. 456:2–8. doi:10.1016/j.mce.2016.10.024. Available from: http://dx.doi.org/10.1016/j.mce.2016.10.024

Mani, A. M., M. A. Fenwick, Z. Cheng, M. K. Sharma, D. Singh, and D. C. Wathes. 2010. IGF1 induces up-regulation of steroidogenic and apoptotic regulatory genes via activation of phosphatidylinositol-dependent kinase/AKT in bovine granulosa cells. Reproduction. 139:139–151. doi:10.1530/REP-09-0050.

Martin, J. L., K. A. Vonnahme, D. C. Adams, G. P. Lardy, and R. N. Funston. 2007. Effects of dam nutrition on growth and reproductive performance of heifer calves. J. Anim. Sci. 85:841–847. doi:10.2527/jas.2006-337.

Mazerbourg, S., C. A. Bondy, J. Zhou, and P. Monget. 2003. The insulin-like growth factor system: A key determinant role in the growth and selection of ovarian follicles? A comparative species study. Reprod. Domest. Anim. 38:247–258. doi:10.1046/j.1439- 0531.2003.00440.x.

Moallem, U., G. E. Dahl, E. K. Duffey, A. V Capuco, and R. A. Erdman. 2004. Bovine Somatotropin and Rumen-Undegradable Protein Effects on Skeletal Growth in Prepubertal Dairy Heifers*. J. Dairy Sci. 87:3881–3888. doi:https://doi.org/10.3168/jds.S0022-0302(04)73527-4. Available from: http://www.sciencedirect.com/science/article/pii/S0022030204735274

Moore, J. E., and G. O. Mott. 1974. Recovery of residual organic matter from “in vitro” digestion of forages. J. Dairy Sci. 57:1258–1259.

Moreira, F., C. Orlandi, C. A. Risco, R. Mattos, F. Lopes, and W. W. Thatcher. 2001. Effects of Presynchronization and Bovine Somatotropin on Pregnancy Rates to a Timed Artificial Insemination Protocol in Lactating Dairy Cows. J. Dairy Sci. 84:1646–1659. doi:10.3168/jds.S0022-0302(01)74600-0. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0022030201746000

Moreira, F., L. Badinga, C. Burnley, and W. W. Thatcher. 2002. Bovine somatotropin increases embryonic development in superovulated cows and improves post-transfer pregnancy rates when given to lactating recipient cows. Theriogenology. 57:1371–87.

Moriel, P., S. E. Johnson, J. M. B. Vendramini, V. R. G. Mercadante, M. J. Hersom, and J. D. Arthington. 2014. Effects of calf weaning age and subsequent management system on growth and reproductive performance of beef heifers. J. Anim. Sci. 92:3096–3107. doi:10.2527/jas.2013-7389.

Moriel, P., P. A. Lancaster, G. C. Lamb, J. M. B. Vendramini, and J. D. Arthington. 2017. Effects of post-weaning plane of nutrition and estrus synchronization on reproductive performance of Bos indicus-influenced beef heifers. J. Anim. Sci. 95:242. doi:10.2527/asasann.2017.497. Available from: https://www.animalsciencepublications.org/publications/jas/abstracts/95/supplement4/24 2a

Moriel, P., R. F. Cooke, D. W. Bohnert, J. M. B. Vendramini, and J. D. Arthington. 2012. Effects of energy supplementation frequency and forage quality on performance, reproductive, and physiological responses of replacement beef heifers. J. Anim. Sci. 90:2371–2380.

Mulliniks, J. T., S. R. Edwards, J. D. Hobbs, Z. D. Mcfarlane, and E. R. Cope. 2017. Postweaning feed efficiency decreased in progeny from high milk producing beef cows. 235–239. doi:10.2527/asasws.2017.0007.

Nanke, K. E., J. G. Sebranek, K. J. Prusa, and L. F. Miller. 1993. Effects of Porcine Somatotropin (PST) Administration on the Fat/Lean Content and Processing Properties of Pork Belliest. Meat Sci. 35:341–353.

Neibergs, H. L., and K. A. Johnson. 2012. Nutrition and the genome. J. Anim. Sci. 90:2308– 2316. doi:10.2527/jas2011-4582.

NRC. 1996. Nutrient Requirements of Beef Cattle. Revised 7th ed. Natl. Acad. Press, Washington, DC.

NRC. 2000. Nutrient Requirements of Beef Cattle. Revised 7th ed. Natl. Acad. Press, Washington, DC.

Ocón-Grove, O. M., F. N. T. Cooke, I. M. Alvarez, S. E. Johnson, T. L. Ott, and A. D. Ealy. 2008. Ovine endometrial expression of fibroblast growth factor (FGF) 2 and conceptus expression of FGF receptors during early pregnancy. Domest. Anim. Endocrinol. 34:135– 145.

Oosthuizen, N., P. L. P. Fontes, D. D. Henry, F. M. Ciriaco, C. D. Sanford, and L. B. Canal. 2017. Administration of Recombinant Bovine Somatotropin Prior to Fixed-time Artificial Insemination and the Effects on Pregnancy Rates and Embryo Development in Beef Heifers.

Patel, M., and M. Srinivasan. 2002. Metabolic programming: Causes and consequences. J. Bio. Chem. 277:1629-1632.

Patel, M. S., and M. Srinivasan. 2011. Metabolic programming in the immediate postnatal life. Ann. Nutr. Metab. 58:18-28.

Perry, R. C., L. R. Corah, R. C. Cochran, J. R. Brethour, K. C. Olson, and J. J. Higgins. 1991. Effects of hay quality, breed, and ovarian development on onset of puberty and reproductive performance of beef heifers. J. Prod. Agric. 4:13-18.

Phillips, W. A., J. W. Holloway, and S. W. Coleman. 1991. Effect of pre- and postweaning management system on the performance on Brahman crossbred feeder calves. J. Anim. Sci. 69:3102–3111. doi:10.2527/1991.6983102x.

Radcliff, R. P., B. L. McCormack, B. A. Crooker, and M. C. Lucy. 2003. Growth hormone (GH) binding and expression of GH receptor 1A mRNA in hepatic tissue of periparturient dairy cows. J. Dairy Sci. 86:3920-3926.

Radcliff, R. P., M. J. VandeHaar, L. T. Chapin, T. E. Pilbeam, D. K. Beede, E. P. Stanisiewski, and H. A. Tucker. 2000. Effects of diet and injection of bovine somatotropin on prepubertal growth and first-lactation milk yields of Holstein cows. J. Dairy Sci. 83:23–9. doi:10.3168/jds.S0022-0302(00)74850-8. Available from: http://www.ncbi.nlm.nih.gov/pubmed/10659959

Radcliff, R. P., M. J. VandeHaar, Y. Kobayashi, B. K. Sharma, H. A. Tucker, and M. C. Lucy. 2004. Effect of dietary energy and somatotropin on components of the somatotropic axis in Holstein heifers. J. Dairy Sci. 87:1229–35. doi:10.3168/jds.S0022-0302(04)73273-7. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15290971

Radunz, A. E., F. L. Fluharty, A. E. Relling, T. L. Felix, L. M. Shoup, H. N. Zerby, and S. C. Loerch. 2012. Prepartum dietary energy source fed to beef cows : II . Effects on progeny postnatal growth , glucose tolerance , and carcass composition. J. Anim. Sci. 4962–4974. doi:10.2527/jas2012-5098.

Rausch, M. I., M. W. Tripp, K. E. Govoni, W. Zang, W. J. Weber, B. A. Crooker, T. A. Hoagland, and S. A. Zinn. 2002. The influence of level of feeding on growth and serum insulin-like growth factor I and insulin-like growth factor-binding proteins in growing beef cattle supplemented with somatotropin. J. Anim. Sci. 80:94–100.

Rawlings, N. C., A. C. O. Evans, A. Honaramooz, and P. M. Bartlewski. 2003. Antral follicle growth and endocrine changes in prepubertal cattle, sheep and goats. Anim. Reprod. Sci. 78:259–270. doi:10.1016/S0378-4320(03)00094-0.

Raymond, R., C. W. Bales, D. Ph, D. E. Bauman, D. Clemmons, R. Kleinman, D. Lanna, S. Nickerson, and K. Sejrsen. 2009. Recombinant Bovine Somatotropin ( rbST ): A Safety Assessment Recombinant Bovine. J. Dairy Sci.

Rhoads, R. P., J. W. Kim, M. E. Van Amburgh, R. A. Ehrhardt, S. J. Frank, and Y. R. Boisclair. 2007. Effect of nutrition on the GH responsiveness of liver and adipose tissue in dairy cows. J. Endocrinol. 195:49–58. doi:10.1677/JOE-07-0068.

Ribeiro, E. S., R. G. S. Bruno, A. M. Farias, J. A. Hernández-Rivera, G. C. Gomes, R. Surjus, L. F. V. Becker, A. Birt, T. L. Ott, J. R. Branen, R. G. Sasser, D. H. Keisler, W. W. Thatcher, T. R. Bilby, and J. E. P. Santos. 2014. Low Doses of Bovine Somatotropin Enhance Conceptus Development and Fertility in Lactating Dairy Cows. Biol. Reprod. 90:1–12. doi:10.1095/biolreprod.113.114694. Available from: https://academic.oup.com/biolreprod/article-lookup/doi/10.1095/biolreprod.113.114694

Rice, L. E. 1991. Nutrition and the Development of Replacement Heifers. Vet. Clin. North Am. Food Anim. Pract. 7:27–39. doi:https://doi.org/10.1016/S0749-0720(15)30808-2. Available from: http://www.sciencedirect.com/science/article/pii/S0749072015308082

Riggs, A. D., R. A. Martienssen, V. E. Russo. 1996. Introduction. In: V. E. Russo, R. A. Martienssen, A. D. Riggs, editors, Epigenetic mechanisms of gene regulation. Cold Spring Harbor Laboratory Press, Plainview, NY. p. 1-4.

Roberts, A. J., R. N. Funston, and G. E. Moss. 2001. Insulin-Like Growth Factor Binding Proteins in the Bovine Anterior Pituitary. Endocrine. 14:399–406.

Roberts, A. J., S. I. Paisley, T. W. Geary, E. E. Grings, R. C. Waterman, and M. D. MacNeil. 2007. Effects of restricted feeding of beef heifers during the postweaning period on growth, ef ciency, and ultrasound carcass characteristics. J. Anim. Sci. 85:2740-2745.

Roberts, A. J., 3rd Nugent, R. A., J. Klindt, and T. G. Jenkins. 1997. Circulating Insulin-Like Growth Factor I , Insulin-Like Growth Factor Binding Proteins , Growth Hormone , and Resumption of Estrus in Postpartum Cows Subjected to Dietary Energy Restriction. J. Anim. Sci. 1909–1917.

Santos, J. E. P., S. O. Juchem, R. L. A. Cerri, K. N. Galvão, R. C. Chebel, W. W. Thatcher, C. S. Dei, and C. R. Bilby. 2004. Effect of bST and Reproductive Management on Reproductive Performance of Holstein Dairy Cows. J. Dairy Sci. 87:868–881. doi:10.3168/jds.S0022-0302(04)73231-2. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0022030204732312

Schams, D., B. Berisha, M. Kosmann, R. Einspanier, and W. M. Amselgruber. 1999. Possible role of growth hormone, IGFs, and IGF-binding proteins in the regulation of ovarian function in large farm animals. Domest. Anim. Endocrinol. 17:279–285. doi:10.1016/S0739-7240(99)00044-2.

Schillo, K. K., D. J. Dierschke, and E. R. Hauser. 1982. Regulation of luteinizing hormone secretion in prepubertal heifers: increased threshold to negative feedback action of estradiol. J. Anim. Sci. 54:325–336.

Schillo, K. K., J. B. Hall, and S. M. Hileman. 1992. Effects of nutrition and season on the onset of puberty in the beef heifer. J. Anim. Sci. 70:3994–4005. doi:10.2527/1992.70123994x.

Schlegel, M. L., W. G. Bergen, A. L. Schroeder, M. J. VandeHaar, and S. R. Rust. 2006. Use of bovine somatotropin for increased skeletal and lean tissue growth of Holstein steers. J. Anim. Sci. 84:1176–1187.

Schneider, J. E. 2004. Energy balance and reproduction. Physiol. Behav. 81:289–317. doi:10.1016/j.physbeh.2004.02.007.

Schoppee, P. D., J. D. Armstrong, R. W. Harvey, M. D. Whitacre, A. Felix, and R. M. Campbell. 1996. Immunization against growth hormone releasing factor or chronic feed restriction initiated at 3.5 months of age reduces ovarian response to pulsatile administration of gonadotropin-releasing hormone at 6 months of age and delays onset of puberty in heifer. Biol. Reprod. 55:87–98. doi:10.1095/biolreprod55.1.87. Available from: http://www.ncbi.nlm.nih.gov/pubmed/8793063

Short, R. E., and R. A. Bellows. 1971. Relationships among weight gains, age at puberty and reproductive performance in heifers. J. Anim. Sci. 32:127-131.

Short, R. E., R. B. Staigmiller, R. A. Bellows, and R. C. Greer. 1994. Breeding heifers at one year of age: Biological and economical considerations. In: M. J. Fields and R. S. Sand(ed.) Factors Affecting Calf Crop. p. 55-68. CRC Press, Boca Raton, FL.

Silva, J. R. V, J. R. Figueiredo, and R. van den Hurk. 2009. Involvement of growth hormone (GH) and insulin-like growth factor (IGF) system in ovarian folliculogenesis. Theriogenology. 71:1193–1208. doi:10.1016/j.theriogenology.2008.12.015.

Slagboom, P. E., and J. Vijg. 1989. Genetic instability and aging: theories, facts, and future perspectives. Genome. 31:373–385. doi:10.1139/g89-057.

Smith, J. M., M. E. Van Amburgh, M. C. Díaz, M. C. Lucy, and D. E. Bauman. 2002. Effect of nutrient intake on the development of the somatotropic axis and its responsiveness to GH in Holstein bull calves. J. Anim. Sci. 80:1528–1537. doi:10.2527/2002.8061528x.

Spicer, L. J., and S. E. Echternkamp. 1995. The ovarian insulin and insulin-like growth factor system with an emphasis on domestic animals. Domest Anim Endocrinol. 12:223–245. doi:0739-7240(95)00021-6 [pii]. Available from: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citati on&list_uids=7587167

Tarazon-Herrera, M. a, J. T. Huber, J. E. P. Santos, and L. G. Nussio. 2000. Effects of bovine somatotropin on milk yield and composition in Holstein cows in advanced lactation fed low- or high-energy diets. J. Dairy Sci. 83:430–434. doi:10.3168/jds.S0022- 0302(00)74899-5. Available from: http://dx.doi.org/10.3168/jds.S0022-0302(00)74899-5

Thakur, M. K., T. Oka, and Y. Natori. 1993. Gene expression and aging. Mech. Ageing Dev. 66:283–298.

Thiagalingam, S., K.-H. Cheng, H. J. Lee, N. Mineva, A. Thiagalingam, and J. F. Ponte. 2003. Histone deacetylases: unique players in shaping the epigenetic histone code. Ann. N. Y. Acad. Sci. 983:84–100. doi:10.1111/j.1749-6632.2003.tb05964.x.

Thissen, J. P., J.M. Ketelslegers, and L. E. Underwood. 1994. Nutritional regulation of the insulin-like growth factors. Endocr. Rev. 15:80–101.

Turner, J. W. 1980. Genetic and biological aspects of Zebu adaptability. J. Anim. Sci. 50:1201– 1205. doi:10.2134/jas1980.5061201x.

Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods for dietary fiber, neutral detergent fiber, and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74:3583–3597.

Velayudhan, B. T., K. E. Govoni, T. A. Hoagland, and S. A. Zinn. 2007. Growth rate and changes of the somatotropic axis in beef cattle administered exogenous bovine somatotropin beginning at two hundred, two hundred fifty, and three hundred days of age. J. Anim. Sci. 85:2866. doi:10.2527/jas.2007-0281. Available from: https://www.animalsciencepublications.org/publications/jas/abstracts/85/11/0852866

Velazquez, M. A., L. J. Spicer, and D. C. Wathes. 2008. The role of endocrine insulin-like growth factor-I (IGF-I) in female bovine reproduction. Domest. Anim. Endocrinol. 35:325–342. doi:10.1016/j.domaniend.2008.07.002. van der Walt, J. G. 1994. Somatotropin Physiology - a Review. South African J. Anim. Sci.- Suid-Afrikaanse Tydskr. Vir Veekd. 24:1–9.

Waterland, R. A., and C. Garza. 1999. Potential mechanisms of metabolic imprinting that lead to chronic disease. Am. J. Clin. Nutr. 69:179–197. doi:10.3945/an.112.002683.

Wathes, D. C., M. Fenwick, Z. Cheng, N. Bourne, S. Llewellyn, D. G. Morris, D. Kenny, J. Murphy, and R. Fitzpatrick. 2007. Influence of negative energy balance on cyclicity and fertility in the high producing dairy cow. Theriogenology. 68:232–241. doi:10.1016/j.theriogenology.2007.04.006.

Weiss, W. P., H. R. Conrad, and N. R. St. Pierre. 1992. A theoretically-based model for predicting total digestible nutrient values of forages and concentrates. Anim. Feed Sci. Technol. 39:95-110.

Weller, M. M. D. C. A., R. L. Albino, M. I. Marcondes, W. Silva, K. M. Daniels, M. M. Campos, M. S. Duarte, M. L. Mescouto, F. F. Silva, and S. E. F. Guimarães. 2016. Effects of nutrient intake level on mammary parenchyma growth and gene expression in crossbred (Holstein × Gyr) prepubertal heifers. J. Dairy Sci. 99:9962–9973. doi:10.3168/jds.2016-11532. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0022030216307202

Wiltbank, J. N., C. W. Kasson, and J. E. Ingalls. 1969. Puberty in Crossbred and Straightbred Beef Heifers on two Levels of Feed. J. Anim. Sci. 29:602–605. doi:10.2527/jas1969.294602x. Available from: http://dx.doi.org/10.2527/jas1969.294602x

Wiseman, A. R., M D Redden, C M Spencer, A L McGee, R Reuter, D L Lalman; 23 Effects of Timing of Weaning on Calf Performance and Maintenance Energy Requirements in Primiparous Beef Cows., Journal of Animal Science, Volume 96, Issue suppl_1, 1 March 2018, Pages 12–13, https://doi.org/10.1093/jas/sky027.024

Yakar, S., J. Liu, B. Stannard, A. Butler, D. Accili, B. Sauer, and D. LeRoith. 1999. Normal growth and development in the absence of hepatic insulin-like growth factor. Proc. Natl. Acad. Sci. USA 96:7324–7329.

Yelich, J. V., R. P. Wetteman, T. T. Marston, and L. J. Spicer. 1996. Luteinizing hormone, growth hormone, insulin-like growth factor-I, insulin and metabolites before puberty in heifers fed to gain at two rates. Domest. Anim. Endocrinol. 13:325-338.

Yung, M. C., M. J. VandeHaar, R. L. Fogwell, and B. K. Sharma. 1996. Effect of Energy Balance and Somatotropin on Insulin-like Growth factor I in Serum and on Weight and Progesterone of Corpus Luteum in Heifers. J Anim Sci. 74:2239–2244.

Zhu, M. J., S. P. Ford, P. W. Nathanielsz, and M. Du. 2004. Effect of maternal nutrient restriction in sheep on the development of fetal skeletal muscle. Biol. Reprod. 71:1968- 1973.

BIOGRAPHICAL SKETCH

Matheus Betelli Piccolo was born in Jundiaí, São Paulo, Brazil, in June 1993. He is son of Teresa Cristina Betelli Piccolo and José Alberto Piccolo. Matheus obtained his B.S. in Animal

Sciences from São Paulo State University (UNESP, Botucatu, Brazil) in 2015. During his undergrad program, he was mentored by Dr. José Luiz Moares Vasconcelos, who gave him the opportunity to work closely to livestock producers and to engage in research development through Conapec Jr.

During his final semester in college, he completed his final internship at the Mountain

Research Station in Waynesville, NC. Under the guidance of Dr. Philipe Moriel, working in research trials related to beef cattle nutrition and immunity.

In the first semester of 2016, worked as the field technician, conducting field research with beef cattle with Elanco Animal Health in the state of Mato Grosso do Sul, Brazil. On June

2016 was accepted at the UF graduate school and began working on his M.S. degree. He intends to complete his program by summer 2018 working on metabolic imprinting on beef heifer development under the orientation of Dr. Philipe Moriel.