.19... . . . 2 I u . . i. .11.... t .1... In" I w- my .m4........w.= “vain-u. .... mat... 3' .1» .M. n.7,... . .. LIBRARY Mlchlgan State University rues u RETURN BOX to remain a}:- ehockoin mm your moord. TO AVOID FINES Mum on or baton date duo. DATE DUE DATE DUE DATE DUE MSU I. An Affirmative ActIaVEqual OpportunIIy InctItqun W ABSTRACT EFFECTS OF PREPUBERTAL DIET AND INJECTION OF bST ON AGE AT PUBERTY, BODY GROWTH, CARCASS COMPOSITION, AND MAMMARY DEVELOPMENT OF DAIRY HEIFERS By Roy Patrick Radcliff Reducing the age at first calving would increase a farm’s profit margin. One way to reduce age at first calving is to reduce the age at puberty and breeding. Previous experiments have demonstrated that a high energy diet will decrease age at puberty and breeding, but such diets are detrimental to mammary development and future milk production. The objective of this thesis was to determine the effects of feeding dairy heifers either a high-protein, high- energy diet or a standard diet, both with and without injection of bovine somatotropin on body growth, carcass composition, age at puberty, and composition and metabolic activity of the mammary gland. The high diet increased growth rate by increasing growth of soft tissue. Bovine somatotropin increased the growth of muscle and bone, while decreasing adipose deposition. Diet had no effect on mammary cell numbers or metabolic activity, whereas bST increased both variables. Thus, a high-protein, high-energy diet combined with bST potentially may decrease age at first calving without reducing future milk yield. I dedicate this thesis to my closest family, who have given me more love and support than anyone could ever ask for. Jack and Joan Radcliff, my parents; Ed and Mary Norman, my uncle and aunt; Jackie and Linda Gorbey, my uncle and aunt. iii ACKNOWLEDGEMENTS I would like to thank a few people for their help and guidance during my first two years of graduate training at Michigan State University. First, I would like to thank the members of my committee, Drs. Michael VandeHaar, Andrew Skidmore and Thomas Herdt for their patience and guidance during these past two years. I would like to express my deepest gratitude to my major professor, Dr. Allen Tucker, for allowing me the Opportunity to work with him, learn from him, and for his wisdom and endless patience. I would also like to thank Mr. Larry Chapin for his help with duties at the barn and in the laboratory as well as with statistical analysis. I thank Dr. Roy Fogwell for his consultations on various subjects, friendship, and stories of home. I thank Dr. Paula Gaynor for all of her thought provoking conversation and for the time she spent helping me find a place to live. I thank Mr. Paul Dyk for assigning body condition scores to my heifers. I thank the crews at the dairy barn and the meats laboratory for everything they did. I would like to thank my fellow graduate students in the Animal Science Department for their support and assistance, especially Mr. Mario Binelli and Ms. Christine Simmons from the Animal Reproduction Laboratory. iv I would like to express my gratitude to Jason Dickman. His friendship, humor, and all of his help at the barn will never be forgotten. I would like to thank Dr. Ed Stanisiewski from The Upjohn Company for economic support of the project and donation of bST. I would like to thank Michigan State University Experiment Station for funding this project through the Status And Potential of Michigan Agriculture Program (SAPMA). TABLE OF CONTENTS LIST OF TABLES ............................................ viii LIST OF FIGURES ............................................ ix INTRODUCTION ............................................. 1 REVIEW OF LITERATURE ................................ ' . . . . 4 Mammary development from birth to conception ................. 4 Effect of diet on body growth ................................ 5 Effects of bST on body growth ............................... 8 Effects of prepubertal diet on mammary development ............. 9 Effects of prepubertal administration of bST on mammary development ...................................... 1 1 MATERIALS AND METHODS ................................. 15 Management of animals ................................... 15 Body measurements ...................................... 18 Blood collection and analysis ............................... 19 Tissue collection ........................................ 20 Carcass composition analysis ............................... 21 Mammary tissue analysis .................................. 21 Statistical analysis .................................... t. . . 22 RESULTS .................................................. 24 General ............................................... 24 Body growth .......... - ................................. 24 Carcass composition ...................................... 26 Mammary deveIOpment ................................... 28 Serum profiles of somatotropin and NEFA ..................... 30 DISCUSSION ............................................... 34 SUMMARY AND CONCLUSIONS .............................. 41 APPENDIX A ............................................... 43 APPENDIX B ............................................... 45 BIBLIOGRAPHY ............................................ 48 vii Table 1. Table 2. Table 3. Table 4. LIST OF TABLES Composition of standard and high diets ..................... 16 Least square means of individual treatments and significance of pooled main effects (diet and bST) and interaction of main effects on body growth ....................................... 25 Least square means of individual treatments and significance of pooled main effects (diet and bST) and interaction of main effects on carcass composition .................................. 27 Least square means of individual treatments and significance of pooled main effects (diet and bST) and interaction of main effects on mammary development .............................. 29 viii Figure 1. Figure 2. Figure 3. Figure 4. LIST OF FIGURES Concentrations of serum somatotropin in heifers fed high control diet (0; n= 10), high diet and injected with bST (O; n=10), standardcontrol diet ([1; n=9), standard diet and injected with bST (I; n=9) for 6 h on the day before slaughter. Each point represents the average of a treatment group. Arrow represents time of injection ................... 31 Concentrations of serum nonesterified fatty acids of heifers fed high control diet (0; n= 10), high diet and injected with bST (0; n= 10), standard control diet (El; n=9), or standard diet and injected with bST (I; n=9) from d 0 to slaughter. Each point represents the average of a treatment group. P=puberty, P-l4 = 14 days before onset of puberty, P+30 = 30 days after onset of puberty .............................. 33 Dry matter intake heifers fed high control diet (0; n=10), high diet and injected with bST (0; n= 10), standard control diet (El; n=9), standard diet and injected with bST (I; n=9). Each point represents the average weekly DMI of a treatment group .............................................. 46 Weekly body weights of heifers fed high control diet (0; n=10), high diet and injected with bST (0; n= 10), standard control diet (El; n=9), standard diet and injected with bST (I; n=9). Each point represents the average weekly weight of a treatment. .' ..... 47 INTRODUCTION Rearing replacement dairy heifers accounts for approximately 20% of total production costs on a dairy farm (Annexstad, 1986). During the time between birth and first calving expenses are generated in the form of feed, housing, and labor, while contributing nothing to the income of the farm._ Any way for the farmer to reduce the expense of rearing replacement heifers without impairing productivity after calving would increase overall profitability of the farm. Current recommendations suggest that 24 mo is an optimal age at first calving for dairy heifers. Calving at a later age increases the total cost of rearing replacement heifers by increasing the amount of time they are supported by the farm without generating income. However, calving at an earlier age could increase costs by increasing the incidence of dystocia and metabolic disorders at calving if the heifer has not attained the proper skeletal size and weight by the time of calving. While growing prepubertal heifers at a higher rate of gain with high energy diets could solve the size problem, it produces another problem: namely, it decreases milk production ( Swanson, 1960; Gardner et al., 1977; Sejrsen, 1978, and Little and Kay, 1979). During the period between 3 mo of age and puberty the mammary 2 gland grows allometrically, in other words, more rapidly than the rest of the body (Sinha and Tucker, 1969). The decrease in milk production associated with feeding high energy diets during this allometric growth phase has been attributed to a decrease in growth of mammary parenchyma and a concurrent increase in deposition of mammary adipose tissue. Van Amburgh and Galton (1994) fed dairy heifers a diet high in energy and balanced with elevated protein. As in other studies with diets formulated for high rates of gain, age at first calving was reduced. However, subsequent milk production was not reduced. The practical recommendation to dairy farmers to grow replacement heifers at 800 g/d during the prepubertal period comes from calculating the growth required to ensure adequate size and body weight for breeding by 15 mo of age. This growth rate allows the heifer to attain an adequate size for breeding by 15 mo of age, while at the same time allowing for greater parenchymal development without excess adipose deposition in the mammary gland (Sejrsen, 1994). Administration of bovine somatotropin (bST) increases body growth rate while decreasing carcass fat (McShane et al., 1989; Hufstedler et al., 1991; Moseley et al., 1992 and Vestergaard et al., 1993). In addition, injection of bST to dairy heifers during the prepubertal period increases mammary parenchymal tissue while decreasing mammary adipose tissue (Sejrsen et al., 1986 and Glasser et al., 1991). I hypothesized that a high-protein, high-energy diet combined with administration of bST would increase body growth rate and 3 mammary development, and thereby allow heifers to calve earlier than 24 mo of age without decreasing milk production. Specific objectives of this thesis were to determine effects of bST on body growth rates, carcass composition, age at puberty, and growth and metabolic activity of mammary parenchyma in heifers fed to gain either .8 kg/d or 1.2 kg/d. Information about body growth as well as mammary growth and metabolic activity could then be used to choose those treatments with the greatest potential to maximize body and mammary growth, thereby shortening the interval between birth and calving without reducing subsequent milk production. REVIEW OF LITERATURE Mammary development from birth to conception At the time of birth, the mammary gland of a heifer is an immature duct system consisting of primary and secondary sprouts and end buds (parenchyma), which are derived from ectoderm. This duct system is surrounded by a combination of mature smooth muscle, adipose, connective, blood, and lymph tissue (stroma), which are derived from mesoderm (Tucker, 1969). During the first 2 to 3 mo after birth, the mammary gland grows at a rate similar to the rest of the body (isometric growth). At 2 to 3 mo of age, the mammary gland begins to grow as much as three times faster than the rest of the body (allometric growth). This allometric growth continues through several estrous cycles after onset of puberty, at which time it returns to an isometric growth rate (Sinha and Tucker, 1969). Prepubertal growth of parenchymal tissue is characterized by extension of the ducts into the stroma in the form of a solid cord of cells, followed by canalization (lumen formation). Along with growth of parenchyma, there is concurrent growth of stroma (Reece, 1958). Extension of the ducts is the result of rapid proliferation of the end buds. At the tip of the end bud, a layer of undifferentiated cuboidal epithelial cells (cap cells) engage in intense mitotic 5 activity (Williams and Daniel, 1983). The progeny of these cells, not the parents, differentiate into epithelial cells, forming ducts and myoepithelial cells (Smith and Medina, 1988). After puberty, the gland consists of an extended duct system but alveoli have not yet formed. Alveoli are small almost spherical structures located at the end of ducts, and are made up of a single layer of epithelial cells. Milk synthesis and secretion occur in the alveoli. Alveolar development usually occurs after conception during a second allometric growth phase (Tucker, 1969). Effect of diet on body growth Growth can be defined as an increase in size or mass. Growth of an animal can be characterized by two processes, hypertrophy and hyperplasia. Different body tissues grow and mature at different rates throughout the life of an animal. Although the rate of growth can be controlled, the sequence of tissue maturation remains the same regardless of the growth rate of an animal (Batt, 1980). Tissues mature in the following order: neural tissue, bone, muscle, and finally adipose tissue. Because neural tissue is relatively mature at birth, in this review I will focus only on postnatal growth of bone, muscle, and adipose tissue, and how altering dietary nutrient content affects their growth. Normal bone growth throughout the life of cattle can be described as an increase in growth rate beginning soon after birth. However, the growth rate slows dramatically by 8 to 10 mo of age in cattle (Berg and Butterfield, 1968). However, new bone is constantly being laid down and reabsorbed by the 6 body throughout an animal’s life. Mature bone size is maintained by an equilibrium between new growth and reabsorption. As bone growth slows, muscle growth rate increases. If muscle growth is graphed as a function of time after birth, it is a sigmoidal curve: i.e., at first muscle growth is slow, then it increases dramatically before slowing again by 15 mo of age in Holstein steers (Berg and Butterfield, 1968). Net muscle growth is the difference between total protein synthesis and degradation by the body. During rapid muscle growth, total protein synthesis is 6 to 10 times greater than protein accretion (Eisemann et al., 1989b and Bergen and Merkel, 1991). Postnatal muscle growth is a process of increasing myofiber cross-sectional area and length, but fiber numbers do not change (Burleigh, 1974 and Goldspink, 1974). As a muscle fiber grows, nuclei are added from mitosis of satellite cells which reside between the basement membrane and the plasma membrane of the muscle fiber. Although the functions of lipid in an animal are numerous, .the major role of adipose tissue is long-term storage of energy (Leat and Cox, 1980). As the rate of muscle growth decreases, the rate of adipose accretion increases. As net growth of the body increases after 100 to 200 d of age in cattle both hypertrophy and hyperplasia occur in the carcass fat depots, whereas hypertrophy alone occurs in the perirenal depots of steers (Robelin, 1981 and Truscott, Wood, and Denny, 1983). After net growth of bone and muscle stop, adipose accretion may continue by hypertrophy of quiescent preadipocytes, as well as mature 7 adipocytes (Vernon, 1986) as long as nutrient availability permits. Accretion of adipose tissue is the difference between lipogenesis and lipolysis. When rates of lipogenesis and lipolysis become equal, lipid accretion stops. To live and grow, animals must obtain nutrients from outside the body. A certain amount of these nutrients are required just to provide energy for body functions to proceed normally. This is the amount required for maintenance. Once the quota for maintenance is met, excess nutrients are used for growth and production, or excreted. If the level of nutrient intake is not adequate for maintenance, the body then calls on its own reserves to meet these needs and growth is reduced. When level of nutrition is adequate, all growing tissues are served according to their needs. As an animal ages these needs change. For example, early in life bone will require more nutrients for growth. However, as the animal ages, bone requires less and muscle requires more nutrients. As muscle matures, more nutrients are available for storage in the form of adipose tissue. Nutrient intake and age of the animal affect how nutrients are partitioned to bone and muscle growth, adipose tissue accretion, or excreated (Koch et al., 1979). By increasing nutrient density of a diet, growth rates of body tissues are increased. When nutrition is limiting for a growing animal, the earlier developing tissues take priority for the supply of available nutrients. Thus, nerve and bone will grow normally while growth of muscle and adipose tissue are hindered. If nutrients are severely limited, growth of bone will slow and that of muscle and adipose will stop 8 (Hammond, 1960). Pomeroy (1941) demonstrated that if pigs weighing 136 kg were fed a low level of nutrition, bone growth continued normally but muscle growth and adipose accretion stopped. If nutrients were further restricted, muscle and adipose tissue even atrOphied. Similar results have been reported in lambs. For example, Palsson and Verges (1952) reported that severe restriction of nutrients early in life would decrease bone growth. Lambs fed adequate nutrition early in life followed by a period of restricted nutrition had the same amount of bone, but less muscle growth and adipose tissue accretion than nonrestricted lambs. These data indicate that to impair bone growth, dietary nutrients have to be limiting during the time bone growth rate is highest; i.e., early in life. Effects of bST on body growth In 1959, Brumby first showed the effects of growth hormone (bST) on growth of cattle. Since then, many researchers have investigated many different doses and many different periods of administration. During thistime bST has been shown to increase average daily gain (ADG) in lambs (Pell and Bates, 1987), crossbred heifers and steers (Hufstedler et al.,1991 and Moseley et al.1992), beef heifers (McShane et al. 1989 and Vestergaard et al. 1993) and dairy heifers (Sejrsen et al., 1986; Sandles et al., 1987a and Gringes et al., 1990). This increase in ADG can be attributed to an increase in bone and muscle growth. Increased bone growth is indicated by an increase in withers or hip height (Brumby, 1959 and Gringes et al. 1990), and pelvic area (McShane et al., 1989 9 and Gringes et al. 1990). Injection of bST increases muscle growth, as indicated by protein content in the 9-10-11 rib section (Peters, 1986; Hufstedler et al., 1991; Moseley et al., 1992 and Schwarz et al., 1993); DNA content of semimembranosus and tricep muscles (Malton et al., 1990); and rate of protein synthesis (Pell and Bates, 1987 and Eisemann et al., 1989A), and lean tissue mass (Vestegaard et al., 1993). The increase in growth of bone and muscle is accompanied by a concurrent decrease in adipose accretion (McShane et al., 1989; Hufstedler et al., 1991; Moseley et al., 1992; Schwarz et al., 1993 and Vestergaard et al., 1993). Thus exogenous bST will not only increase the growth rate of cattle but will also improve carcass composition by increasing muscle and reducing adipose accretion. The increase in growth from bST may prove to be economically beneficial to the dairy farm by decreasing the time required to bring a heifer into milk production and insuring that the heifer has attained an adequate body weight and skeletal size for ease of calving, and to support milk production after calving. Effects of prepubertal diet on mammary development There have been mixed reports of the effect of diet on mammary development. Early reports suggest that rapid body growth in heifers decreases subsequent milk production. This decrease in milk production associated with accelerated rates of gain was attributed to incomplete parenchymal development and excess fat deposition in the mammary gland (Swanson, 1960). Since then, there have been many reports that increased body growth rates are associated 10 with impaired mammary development (Gardener et al., 1977; Little and Kay, 1979; Sejrsen et al., 1982; Harrison et al., 1983; Petitclerc et al., 1984 and Stelwagen and Grieve, 1990). Sejrsen et al. (1982) concluded from comparison of animals fed ad libitum and restricted diets before and after puberty, that dietary impairment of mammary development occurred during the prepubertal allometric growth phase. This conclusion is supported by a report from Harrison et al. (1983), in which heifers reared at higher rates of gain for the first year of life had less mammary secretory tissue at 12 mo of age as well as after two lactations, and Lacasse et al. (1993) who reported that plane of nutrition after 1 year of life had no effect on first lactation milk yield. Sejrsen (1978 and 1994) attributed impairment of mammary development almost entirely to level of energy intake, and recommends feeding dairy heifers to gain no more than .6 to .7 kg/d before puberty. However, reports cited by Sejrsen to support this conclusion used feed restriction to reduce the level of energy intake. Impairment of prepubertal mammary development in dairy heifers fed ad libitum compared with heifers that are restricted-fed may be due to differences in endogenous hormone secretions. Sejrsen et. al. (1983) reported that restricted-fed dairy heifers had higher growth hormone concentrations than heifers fed ad libitum. These restricted heifers also had more parenchymal tissue and less adipose tissue in the mammary glands. Another explanation for increased mammary development in restricted-fed dairy heifers is that puberty is delayed and these heifers have more time for allometric growth of mammary parenchymal 11 tissue to occur. However, there have also been reports of accelerated body growth without impairment of mammary development (Peri et al., 1993 and Van Amburgh and Galton, 1994). In both of these reports the accelerated groups of heifers were fed a diet that was elevated in both protein and energy. Furthermore, Peri et al. (1993) reported no difference in serum growth hormone concentrations. Perhaps the difference in mammary development in earlier experiments as compared with these recent studies is that the former had. a dietary imbalance of protein and energy that in some way is inhibitory to mammary growth. Effects of prepubertal administration of bST on mammary development Somatotropin as well as other pituitary hormones are necessary for mammary development (Tucker, 1969). Effects of administration of exogenous somatotropin on mammary development have been investigated by several people since the observation of reduced somatotropin concentrations in serum of heifers reared at an accelerated growth rate (Sejrsen et al., 1983). Sejrsen et al. (1986) administered either pituitary-extracted somatotrOpin or vehicle for 15.6 wk beginning at 8 mo of age, to either Danish Friesian or Red Danish milkbreed monozygotic twins. Heifers treated with somatotropin had 18% more parenchyma and 26% less fat in the mammary gland than control heifers. Sandles and Peel (1987b) reported similar results. Administration of somatotropin, for 21 wk beginning at 3.5 mo of age, increased mammary DNA 20% and reduced the 12 amount of adipose tissue in the mammary gland 16% compared with controls. Injection of bST, for 15 wk beginning at 45 d of age, increased mammary development in lambs (McFadden et al., 1990). Glasser et al. (1993) reported that bST administered for 10 mo, beginning at 6 mo of age, increased parenchymal tissue 45% and decreased extra-parenchymal tissue 36% in Angus x Holstein heifers reared at either a constant growth rate of .8 kg/d or an intermittent growth rate. Intermittent growth was produced by two consecutive cycles. In each cycle, feed was restricted to produce .2 kg/d gain for 3 mo followed by a period of unrestricted gain. These results demonstrate the ability of exogenous somatotropin to enhance mammary development in cattle. However, the effect of somatotropin on mammary development is not independent of other hormones. For example, Purup et al. (1993) reported that bST failed to increase mammary development in ovariectomized heifers when compared with ovariectomized, vehicle-treated heifers, indicating that the increase in mammary development produced by somatotropin is also dependent on ovarian hormones such as estrogen or progesterone. ' Somatotropin receptor mRNA has been identified in both rat and bovine mammary glands by in situ hybridization (Glimm et al., 1990). Therefore, somatotropin may bind to mammary cells and act locally to stimulate mammary development. However, mouse mammary glands incubated in whole organ culture with either rat, ovine, or bovine somatotropin failed to develop until supraphysiological concentrations were used (Plaut et al., 1993). Collier et al. 13 (1993) also reported no effect of bST on proliferation of isolated bovine mammary epithelial cells. Furthermore, many efforts to demonstrate somatotropin binding to mammary epithelial cells have failed (Gertler et al., 1984; Akers, 1985; and Kazmer et al., 1986). The results of these studies suggest that somatotropin does not directly affect mammary growth. An alternative to direct stimulation of mammary growth by somatotropin could be indirect stimulation through growth factors. It has. been postulated that the effect of somatotropin on mammary development is mediated through insulin-like growth factor-I (IGF-I). Injecting dairy cattle with bST increases mammary development (Sejrsen, 1986; Sandles and Peel, 1987; McFadden et al., 1990, and Glasser et al., 1993), as well as serum IGF-I concentrations (Bauman and Vernon, 1993 and Dahl, 1993). Injection of bST also increases IGF-I mRNA and IGF-I receptor mRNA in the liver (VanderKooi, 1993). Incubating isolated bovine mammary epithelial cells in collagen gel with IGF-I alone or combined with epidermal growth factor increased cell proliferation (Collier et al., 1993). In contrast, incubation of mouse mammary glands from 34 to 37 d old BALB/c mice in whole gland culture with concentrations of IGF-I ranging from 10 ng/ml to 1 ug/ml did not stimulate mammary development (Plant et al., 1993). Determining the role of IGF-I in mammary development is even more complicated when data from in vivo experiments are considered. For example, restricting dietary intake not only increases mammary development and serum somatotropin concentration, (Sejrsen et al., 1982), but decreases 14 concentrations of IGF—I in serum (Breier, 1988). Thus, the roles of somatotropin and IGF-I are an enigma. A direct effect of somatotropin on mammary development, when increased by either dietary manipulation or injection of bST. is doubtful due to the fact that somatotropin receptors are absent from the mammary gland (Gertler et al., 1984; Akers, 1985, and Kazmer et al., 1986). The indirect role of somatotropin mediated through IGF—I on mammary development is questionable. Injection of bST increases mammary growth as well as concentrations of IGF-I in serum; however dietary restriction increases mammary development and serum somatotropin concentrations, but decreases concentrations of IGF-I in serum. Thus, results concerning the role of IGF-I on mammary development are not clear, and more research is needed to help clarify this question. Although somatotropin’s mechanism of action is still not known, somatotropin’s effect on, and importance to, mammary development are well documented. MATERIALS AND METHODS Management of animals Thirty-eight Holstein heifers, born between June 15 and July 15 were purchased and housed at the Michigan State University Dairy Teaching and Research Center located on College Road, East Lansing, Michigan. All animals were allowed 30 d to acclimate to new surroundings and monitored for illnesses resulting from shipping or commingling. Heifers were blocked by weight into groups of four. Within each block, heifers were randomly assigned to one of 4 treatments. Heifers were fed one of two diets from 4 mo of age until the early luteal phase of their fifth estrous cycle. Nine heifers were fed a total mixed ration that met the current recommended amounts of protein and energy formulated to produce (.8 kg gain per day (standard control diet; SC). Nine heifers were fed the standard control diet and injected daily with recombinant bovine somatotropin (bST; SB). Ten heifers were fed a total mixed ration containing elevated protein and energy formulated to produce 1.2 kg gain per day (high control diet; HC). Ten heifers were fed the high control diet and injected daily with bST (HB). Feed samples were collected every other week to assess protein and fiber content. Nutrient composition of diets is described in Table 1. Feed was offered ad libitum from 15 16 TABLE 1. Composition of Standard and High diets. Standard High Ingredients Grain, % 10%1 75%2 Haylage, % 90%3 25 %‘ Nutrient composition NDF, % of DM 49.6 19.4 NEm,‘ Meal/kg 1.17 1.83 NE”6 Meal/kg .57 1.20 CP, % of DM 16.3 19.4 Absorbable protein, % of DM 7 8.8 13.3 Rumen-undegraded protein ', % of CP 26.9 38.0 Absorbable protein/NE” 7.5 7.3 ‘ On a DM basis, contained 93.4% ground corn, .8% monocalcium phosphate, 1.5% white salt, .9% trace mineral-vitamin premix and 3. 9% DECCOX-le (Purina Mills; .5 % decoquinate) and was formulated so that diet provided 100% of protein, mineral, and vitamin requirements (NRC, 1989). 1 On a DM basis, contained 73.6% ground corn, 20.0% soybean meal, 3.7% animal protein supp., .9% limestone, 3.7% white salt, ..89% trace mineral-vitamin premix, and .5% DECCOX-le (Purina Mills; .5% decoquinate) and was formulated so that diet provided 100% mineral and vitamin requirements (NRC, 1989). 3 Haylage contained 1.05 Mcal NED/kg, .45 Mcal NEJkg, 17.3% CP, 25% rumen- undegraded protein, and 55.8% NDF. ‘ Haylage contained 1.28 Meal NED/kg, .65 Mcal NE/kg, 18.8% CP, 24% rumen- undegraded protein, and 47% NDF. ’ Net energy for maintenance. 17 Table 1 (cont’d) ° Net energy for gain. 7 as calculated by Spartan Dairy ration balancing program. ' Estimated from NRC experimental d 0 to slaughter. Fresh feed was offered everyday between 0845 and 0930 h. Orts for each pen were weighed daily and recorded to calculate average daily dry matter intake for each treatment group. At each daily feeding (0845 to 0930 h) all heifers were restrained in gang-lock stanchions and heifers that received bST were injected i.m. with bST (25 ug/kg BW, Somavubove, The Upjohn Co., Kalamazoo, MI) in the semitendinosus muscle using a 3-ml disposable syringe with a 23-gauge, 1.9-cm needle. This dosage was determined to be an optimal dose to improve carcass composition in crossbred steers (Moseley et al., 1992 ). Every 72 h, bST was reconstituted in sterile bottles with sterile water to a concentration of 14 mg/ml, diluted with sodium monobasic buffer (2 mg/ml, pH 11.2), and stored at 4'C. The concentration of bST was adjusted throughout the experiment so that all doses could be delivered in a volume of 1 to 2 ml for every heifer. Heifers were grouped by treatment and allowed free access to an outside paddock. They were exposed to ambient temperatures and 16 h of light (0600 to 2200 h), a photoperiod that stimulates mammary development (Petitclerc et al., 1985). Serum concentrations of progesterone were monitored as an 18 indicator of puberty as described in "Blood collection and analysis“. Estrus detection commenced twice daily for 30 min per pen after the first heifer attained puberty. Heifers were injected with Lutalyse' (The Upjohn Co., Kalamazoo, MI) during their third luteal phase, and again 11 d later. Nine (1 after their second injection of Lutalyse', heifers were transported in a trailer to the abattoir at the Michigan State University Meats Laboratory at 0630 h for slaughter. Body measurements All heifers were weighed before feeding on two consecutive days each week to monitor body weight gain. The average of the two weights was then assigned as the weekly weight. Weekly weights were used to calculate average daily gain (ADG). Diets were adjusted to maintain desired BW gains (Appendix A). Withers heights were measured every 2 wk while heifers were restrained in the gang lock stanchions. Commencing at 250 kg BW, body condition was scored (BCS) on a scale of 1 to 5 (Wildman et al., 1982) every 2 wk by three experienced examiners. The three scores for each heifer were then averaged and assigned to that heifer as her score. Twenty-four h after slaughter, pelvic area was calculated from two linear measurements of the left half of the carcass; one from the third coccygeal vertebrae to the pubis symphysis, and a second from the midline of the carcass to the pelvic wall. The second measurement was multiplied by 2 to represent the total width of the pelvic opening, and then multiplied by the first to produce the total pelvic area. 19 Blood collection and analysis All blood samples were stored at room temperature for approximately 6 h and then 4'C for approximately 15 h. Serum was harvested after centrifugation at 1550 x g for 25 min, and frozen at -20°C until assayed. Beginning when calves weighed 205 kg, blood samples were collected twice weekly via jugular venipuncture with Vacutainers’ (Becton Dickenson & Co., Rutheford, NJ). To monitor for the onset of puberty, this serum was assayed to quantify progesterone (P4) concentrations (Spicer et al., 1981). A heifer was considered pubertal when P4 concentrations were _>_ 1 ng/ ml in three consecutive serum samples. Two days before slaughter, each heifer was fitted with a sterile indwelling jugular catheter (18 gauge; Ico-Rally, Palo Alto, CA). Twenty four hours later, blood samples were collected at 20-min intervals for 6 h (0800 h to 1400 h). Catheter patency was maintained between samples by flushing the catheter with 3.5% sodium citrate in sterile water. Heifers in the SB and HB groups were injected with bST immediately after collection of the 0900 h sample. Serum concentrations of growth hormone were quantified according to Gaynor et al. (1995). Nonesterified fatty acids (NEFA; NEFA-C kit, Waco Chemicals USA, Dallas, TX; as modified by Johnson, et al., 1993) were quantified in a serum sample collected via venipuncture of each heifer at 0930 h on experimental d 10 and 58, z 14 d before puberty, z 30 d after puberty, and at the time of slaughter. Because animals were slaughtered according to date of puberty, the 20 amount of time between experimental d 58 and z 14 d before puberty ranged from 37 to 187 (1. Tissue collection All heifers were weighed, stunned by captive bolt and killed by exsanguination at the Michigan State University Meats Laboratory. The number of heifers killed each week depended on date of first ovulation, and ranged from 1 to 6 heifers. Heifers were slaughtered an average of 74 d after first ovulation. Immediately after exsanguination, heads were removed, sawed open along the coronal plane from immediately above the eyes to the top of the ears, to expose the brain tissue. Pituitary glands were subsequently removed and separated into anterior and posterior lobes. Anterior pituitaries were weighed and frozen by submersion in liquid nitrogen. Mammary glands were quickly removed and bisected into right and left halves. The left half was weighed, placed in a plastic bag, and frozen by submersion in a tub of dry ice and 95% ethanol. Frozen half udders were stored at -20°C until analyzed as described in section ”Mammary tissue analysis“. Internal organs were removed soon after the carcass was split open. The gall-bladder was removed from the liver and the liver was weighed. The rumen was emptied of it’s contents and visually examined for lesions. After the hide was removed, the carcass was then hoisted to the rail and split into halves along the spine. The carcass halves were then weighed. Perirenal fat was 21 removed from the left half beginning at the 4th lumbar vertebra and proceeding forward to the adrenal gland and then weighed. The carcasses were washed, wrapped with wet drapes, and hung in chambers at 2’C. Carcass composition analysis Twenty-four hours after slaughter, the left half of each carcass was cut between 7th and 8th, and the 12th and 13th ribs. The rib section including the 8th through 12th ribs was removed. The 9-10-11th rib section was then dissected according to the methods of Hankins and Howe (1946). The 9-10-11th rib section was then weighed and deboned. Next, bone and soft tissue were weighed. Soft tissue was ground, mixed and subsampled for analysis of protein, fat, and water content. Protein was determined in fresh samples by the macro- Kjeldahl procedure (AOAC, 1984). Fat was determined by Soxlet ether extraction of fresh samples. Water was determined by the difference in weight after drying in an oven at 110‘C for 24 h. Mammary tissue analysis The frozen left half of the udder was cut transversely with a band saw into 5- to IO-mm thick slices. All slices from the anterior and posterior ends of the gland that did not contain parenchymal tissue were discarded. Slices were placed on a cutting board and allowed to thaw slightly. Skin, teats, and 22 supramammary lymph nodes were dissected from the parenchyma with a scalpel and discarded. Fat located beyond the border of the parenchyma (in those slices that contained parenchyma) was removed and weighed. This fat was defined as extra-parenchymal fat. The remaining tissue will be referred to as mammary parenchymal tissue. The frozen mammary parenchymal tissue was weighed and then ground with dry ice into a fine powder with a blender. The powder was mixed and subsampled for subsequent analysis for DNA and RNA content (Tucker, 1964), dry matter, and fat by Soxlet ether extraction. Statistical analysis The data for total DNA, DNA adjusted for body weight, total parenchyma, total extra-parenchymal fat, extra-parenchymal fat adjusted for body weight, total intra—parenchymal fat, intra-parenchymal fat adjusted for body weight, percentage of carcass fat, percentage of carcass water, total carcass fat, total perirenal fat, and perirenal fat adjusted for body weight were transformed by natural logarithm to eliminate heterogeneous variance. For the variable Of body weight at puberty, one heifer tested positive as an outlier using a standardized residual test and was removed from the data set. All data were analyzed by ANOVA Least squares means of main effects, diet and bST, and for any diet x bST interactions were compared using an f test (Gill, 1978). Overall mean serum somatotropin concentrations were calculated from samples that were collected on the day before slaughter at 20-min intervals 22 supramammary lymph nodes were dissected from the parenchyma with a scalpel and discarded. Fat located beyond the border of the parenchyma (in those slices that contained parenchyma) was removed and weighed. This fat was defined as extra-parenchymal fat. The remaining tissue will be referred to as mammary parenchymal tissue. The frozen mammary parenchymal tissue was weighed and then ground with dry ice into a fine powder with a blender. The powder was mixed and subsampled for subsequent analysis for DNA and RNA content (Tucker, 1964), dry matter, and fat by Soxlet ether extraction. Statistical analysis The data for total DNA, DNA adjusted for body weight, total parenchyma, total extra-parenchymal fat, extra-parenchymal fat adjusted for body weight, total intra-parenchymal fat, intra-parenchymal fat adjusted for body weight, percentage of carcass fat, percentage of carcass water, total carcass fat, total perirenal fat, and perirenal fat adjusted for body weight were transformed by natural logarithm to eliminate heterogeneous variance. For the variable uf body weight at puberty, one heifer tested positive as an outlier using a standardized residual test and was removed from the data set. All data were analyzed by ANOVA. Least squares means of main effects, diet and bST, and for any diet x bST interactions were compared using an f test (Gill, 1978). Overall mean serum somatotropin concentrations were calculated from samples that were collected on the day before slaughter at 20—min intervals 23 from 0800 to 0900 h, and from 0900 to 1400 h , and for serum NEFA concentrations from d 10 to d 58, and from 14 d before puberty to slaughter. Overall mean concentrations of somatotropin were transformed by natural logarithm to eliminate heterogeneous variance. Overall mean NEFA and natural logarithm transformed somatotropin concentrations were analyzed by ANOVA. Main effects were compared using an f test. Values presented for mean concentrations of somatotropin are least square means of untransformed data. Student’s t test was used for independent comparisons between treatments (Gill, 1978). The criterion for statistical significance was P < .05; therefore, comparisons in which the P value was greater than .05 were considered not significant. RESULTS General Treatments commenced for an average of 219, 220, 262, 288 d for high diet, high diet + bST, standard diet, and standard diet + bST, respectively. There were no significant diet x bST interactions for any variable examined in this thesis. Because there were no significant diet x bST interactions, the appropriate data were pooled and results presented as main effects of diet and bST. Neither diet nor bST affected anterior pituitary weight. There were no visible lesions in the rumen epithelium of any of the heifers. None of the livers from these heifers were condemned due to abscesses or fat. Body growth Initial body weight was not different among treatment groups (Table 2). Compared with the standard diet, the high diet increased body weight and BCS at slaughter. Compared with noninjection, injection of bST increased body weight at slaughter but did not affect BCS at slaughter. Feeding the high diet to heifers increased ADG over that of heifers fed the standard diet. Similarly, injection of bST increased ADG over that of noninjected heifers. Compared with the standard diet, the high diet decreased age at 24 doze—285 Ema x 85 . .o u = 53, 333.8 mm 838 we 5:... 33:3 339— A .o— n e 53, toga—:28 mm 83:. we Btu Eaves: “do—eon a 92 + 3% Essa... u mm .36 .858 285; u Om Ha... + 3:. as u E as .288 as: u Oz. 25 on. :5. R. 5 SN - SN 5 RN 2N nee 8; 220.. E. 8. 9.. a a: E a a: m: Es €82. a Ema; SEE E. 8. mm. : an RN : on.” 8... EV €83 3 says EN. 2. so. 2 5 m: a 3N 8N 81:83:. a? a. go. so. as. a. a. m8. o3 NS A29: 09. a. on. so. a. 3 ON _. mm mm usage... a mom N. 3. so. a a: ER 2 5. am as: .2522... a 2%? a. «m. 8. m we 5 m we on as: Ewes RES 8 a 2 3 a ..5 .5; 55 .23 .mm .Om 3% .mm D: $92800 .5sz aces co acute EOE Lo .8203an 28 ohms Ba .33 acute ES: “co—eon Lo 8522:? can 9:253: 3:232: we ESE 88:3 .83 .N mam—4a. 26 puberty, but the high diet did not affect body weight or withers height at puberty, or pelvic area at slaughter (Table 2). In contrast, injection of bST did not affect age at puberty but increased body weight and withers height at puberty and pelvic area at slaughter. Carcass composition Compared with the standard diet, the high diet increased carcass weight as well as dressing percentage (Table 3). Compared with noninjection, injection of bST increased carcass weight but did not affect dressing percentage. Compared with the standard diet, the high diet increased total liver weight as well as liver weight adjusted for differences in body weight. Compared with noninjection, injection of bST also increased total liver weight and liver weight when adjusted for differences in body weight. Compared with the standard diet, the high diet decreased percentage of carcass protein and water, but increased percentage of carcass fat (Table 3). In contrast, injection of bST increased percentage of carcass protein and water, but injection of bST decreased percentage of fat. 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S 3 35 mg 8:9: Ems; 55 em. :5. so. 3. ca 3. R. E 3 EV Ema; as: S. a... so. 3” 3... 8m 3 can can Seneca ”seen E. 8. so. 2 me E 5 mm a as: is». £28 a a S S a .8. Ema 35 .23 .mm .8 .23 .mm .0: 322280 doEmanoo $8.30 no 308% :38 we 830885 was can new “33 acute 5.2: 383 .«o 088$:me ES 3:253: 3:239: no 88.: 83:9. ammo..— .n mam—<9 28 Table 3 (cont’d) ‘ n = 9 per treatment. 5 Diet x bST interaction. perirenal fat adjusted for differences in body weight. Mammary development Compared with the standard diet, the high diet did not affect total mammary DNA content or DNA content adjusted for differences in body weight, total mammary RNA content or mammary RNA content adjusted for differences in body weight, RNA/DNA ratio or total weight of dissectable parenchyma (Table 4). In contrast, injection of bST increased total mammary DNA content, as well as mammary DNA content adjusted for differences in body weight, total mammary RNA content, as well as mammary RNA content adjusted for differences in body weight, the RNA/DNA ratio, and total weight of dissectable parenchyma. Neither diet nor injection of bST affected the concentration of DNA in the mammary gland. However both the high diet and injection of bST: increased the concentration of RNA in the mammary gland. Compared with the standard diet, the high diet increased the total amount of extra-parenchymal fat as well as extra-parenchymal fat adjusted for differences in body weight. The high diet not affect the total amount of intra-parenchymal fat, but decreased the amount of intra-parenchymal fat when adjusted for differences in body weight. 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