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1 Bovine Functional Genomics Laboratory, Animal and Natural Resources Institute, USDA-ARS, Beltsville, MD 20705
2 Department of Animal and Avian Sciences, University of Maryland, College Park 20742
Corresponding author: A. V. Capuco; e-mail: acapuco{at}anri.barc.usda.gov.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Key Words: heifer and mammary growth • lactation • somatotropin • protein feeding
Abbreviation key: PCA = perchloric acid, RUPbST = RUP diet plus bST administration.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Because the majority of skeletal growth occurs during the prepubertal period (Heinrichs and Hargrove, 1987), this period provides the greatest opportunity for enhancing skeletal growth. Although increased rates of skeletal and BW growth in prepubertal dairy heifers can be achieved by increasing the energy density of diets, increasing rates of BW gain to more than 1 kg/d reduces mammary parenchymal growth and increases mammary fat deposition (Sejrsen et al., 1982; Capuco et al., 1995), both of which may be factors associated with less milk production during the first lactation. It has been suggested that rapid rates of growth may be achieved without detrimental effects on subsequent milk production if rapid growth occurred without excessive fattening (Capuco et al., 1995; Silva et al., 2002). Additional dietary protein may prove efficacious in enabling high rates of body and skeletal gain without excessive fattening (Van Amburgh et al., 1991). Management systems that increase skeletal growth rate might be used to accelerate body growth without increasing fattening, thus preventing detrimental effects of accelerated growth on mammary development and potential effects on lactation (Kertz et al., 1987; Radcliff et al., 1997; VandeHaar, 1997; Lammers and Heinrichs, 2000).
Somatotropin, particularly when combined with increased intestinal protein (Houseknecht et al., 1992; Bruckental et al., 1997), enhanced N retention in Holstein steers, suggesting that lean tissue and skeletal growth may be improved in response to bST and additional dietary rumen-undegradable protein. Previous studies with bST showed positive effects on prepubertal growth of mammary secretory tissue (Tucker, 1987) and increased skeletal growth (Grings et al., 1990; Sejrsen, 1994; Radcliff et al., 1997, 2000). Collectively, these experiments suggest that bST in combination with added protein, provided as RUP, may be a practical means to optimize skeletal growth rates during the prepubertal period without the negative impact on mammary development.
The objectives of this study were to determine the effects of administering bST and additional dietary RUP on prepubertal growth of the mammary gland and subsequent milk production. Effects of RUP supplementation and bST administration on body composition, skeletal growth rates, and organ and tissue growth rates are the topics of companion reports (Moallem et al., 2004a; 2004b).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Calves were raised in individual calf hutches or pens until weaning. All calves were fed colostrum for 3 d after birth and thereafter were raised on 4.5 L/d of a commercial milk replacer, ad libitum water, and starter mix until weaning at 60 d of age. After weaning, heifers were fed starter mix and water ad libitum until 90 d of age before being transitioned to a TMR fed from 3 to 10 mo. At 3 mo of age, 8 heifers were killed to determine pretreatment body composition. The remaining 64 heifers were assigned randomly to each of 4 treatments and group-fed by treatment until slaughter or onset of puberty. Twenty-four heifers, 6 per treatment (3 each at 5 and 10 mo of age), were killed to determine effects of treatment on body composition. These ages were selected to represent the midpoint of prepubertal development (5 mo) and the peripubertal period (10 mo). Treatments consisted of recombinant bST with (RUPbST) or without 2% added dietary RUP, applied in a 2 x 2 factorial design. Sustained release recombinant bST (Posilac; Monsanto Co., St. Louis, MO), equivalent to 0.1 mg/kg of BW per d, was injected subcutaneously every 14 d in bST-treated heifers. The control diet was formulated according to 1989 NRC requirements to meet nutrient requirements including energy and protein needs for a 200-kg, large-breed heifer with a live-weight growth rate of 800 g/d (NRC, 1989).
Experimental diets were formulated to be equal in energy and RDP content, but differing in RUP content. It is important to emphasize that because the diets were not isonitrogenous, the RUP-supplemented diet contained additional crude protein supplied as RUP. The added RUP diets contained 16.9% CP, 9.0% RDP, and 7.9% RUP (DM basis), compared with 14.9% CP, 9.0% RDP, and 5.9% RUP in the control diet. Diets were fed as a TMR for ad libitum intake. Ingredient and chemical composition of diets are shown in Table 1
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From puberty until time of breeding, heifers were fed a diet that was 23% corn silage, 75.5% alfalfa haylage, plus a 1.5% vitamin, trace-mineral supplement. The diet met or exceeded NRC (1989) nutrient requirements for 450-kg heifers with BW gain of 0.8 kg/d. From puberty until pregnancy, heifers were fed a common diet that consisted of 12.2% corn silage, 77.0% alfalfa silage, and a 1.8% vitamin, trace-mineral supplement. After confirmation of pregnancy, heifers were commingled with other animals in the herd and fed diets that met or exceeded NRC requirements for growth and pregnancy, but varied in ingredient content depending upon availability of forages and other feed ingredients.
Additional details regarding heifer rearing, as well as body growth and composition data, are provided in companion reports (Moallem et al., 2004a; 2004b).
Udder Sampling and Mammary Compositional Analysis
Heifers were transported to the USDA abattoir (Beltsville Agriculture Research Center, MD), where they were slaughtered by exsanguination after stunning with a captive bolt gun. The udder was removed, trimmed of skin and teats, and separated into right and left halves. Each udder half was weighed. The right udder half was trimmed of fat based upon color of tissue and the mass of parenchyma and fat determined. Parenchyma was ground, and aliquots were frozen and stored at –20°C until compositional analyses (DNA, RNA, protein, and lipid) were performed. In addition, samples of mammary parenchyma were obtained from the mid parenchymal region within the left rear quarter and processed for quantification of cells expressing Ki-67 nuclear proliferation antigen as subsequently described.
Nucleic acids were quantified as previously described (Capuco et al., 2001). Briefly, mammary tissue was homogenized (1:15 wt/vol) in DNA assay buffer (50 mM Na2PO4, 2 M NaCl, 2 mM Na2EDTA) using a Tekmar homogenizer (Tekmar, Cincinnati, OH). DNA was quantified using Hoechst 33258 dye binding (Labarca and Paigen, 1980) against a standard curve prepared using calf thymus DNA. Fluorescence was read using a Bio-Tek FL600 plate reader with a 360/460 nm filter set (Bio-Tek Instruments, Inc., Winooski, VT). Sample RNA was determined by ultraviolet absorbance. For this purpose, an aliquot of the above mammary homogenate was diluted with an equal volume of phosphate buffer, and perchloric acid (PCA) was added to a final concentration of 0.3 N. After incubation on ice and centrifugation, the pellet was resuspended and washed with 0.2 N PCA. The washed pellet was resuspended in 0.3 N PCA and hydrolyzed at 37°C for 60 min. Then, the concentration of PCA in the hydrolysate was increased to 0.6 N, the tube was incubated on ice, and then centrifuged. The precipitate was washed 3 times with ice-cold 0.2 N PCA. The hydrolysate and subsequent washes were combined. A portion of the collected supernatants was diluted and absorbance measured at 260 and 232 nm with a Beckman DU 650 (Beckman Instruments, Inc., Fullerton, CA).
The quantity of mammary parenchymal lipid was determined gravimetrically by chloroform-methanol extraction (Folch et al., 1957) and quantity of parenchymal protein by using the Pierce BCA protein assay (Rockford, IL) on tissue homogenates and bovine serum albumin standards.
Immunohistochemistry
Mammary tissue samples for immunohistochemistry were fixed overnight in 10% neutral buffered formalin at 4°C and then stored in 70% ethanol until further processing. Tissues were then dehydrated through ethanol, cleared in xylene, and embedded in paraffin according to standard techniques (Luna, 1968). Tissues were sectioned at 5 µm onto silanated slides.
The nuclear proliferation antigen, Ki-67, was detected immunohistochemically as described previously (Capuco et al., 2001). Briefly, slides were dewaxed in xylene and hydrated in a graded series of ethanol to PBS (pH 7.4). Tissue sections were quenched with 3% H2O2 in PBS and then washed in PBS. Microwave antigen retrieval in 10 mM citrate buffer (pH 6.0) was then used. Slides were washed in PBS, blocked with 5% non-immune goat serum in PBS, and incubated overnight at 4°C with Ki-67 primary antibody (MIB-1 monoclonal antibody, Zymed Laboratories, San Francisco, CA). Cells labeled with primary antibody were stained using the Histostain SP kit (Zymed Laboratories). Slides were incubated for 30 min at room temperature with biotinylated secondary antibody, washed in PBS, and incubated with the streptavidin-peroxidase-conjugate for 10 min at room temperature. After washing in PBS, sections were incubated with diaminobenzidine, counterstained with hematoxylin or Azure II, and mounted with Permaslip (Alban Scientific Inc., St. Louis, MO).
For each tissue section, 10 randomly selected microscopic fields were photographed with a Spot digital camera (Diagnostic Instruments Inc., Sterling Heights, MI) on a Zeiss Axioskop microscope (Carl Zeiss Inc., Thornwood, NY) using a microscopic magnification of 600x. Epithelial cells within each digital micrograph were counted and scored. At least 1000 epithelial cells were scored per heifer and the percentage of epithelial cells expressing Ki-67 nuclear proliferation antigen was determined.
Milk Yield and Composition
Cows were milked twice daily throughout lactation and milk yield was electronically recorded at each milking for the entire lactation. Milk samples were collected monthly, alternating between a.m. and p.m. sampling milkings and analyzed for fat and protein using an infrared analyzer (Bentley Instruments, St. Paul, MN) by Lancaster DHIA (Manheim, PA).
Statistical Analyses
Mammary growth data were analyzed by 2-way AN-OVA. The Ki-67 labeling index was arcsine transformed prior to ANOVA. Bonferroni’s multiple comparison test was used for post ANOVA comparisons (Prism, version 3; GraphPad Software, Inc., San Diego, CA). Milk production and component data were analyzed using the mixed models procedure in SAS (SAS Inst., Inc., Cary, NC). The statistical model included effects of bST, RUP, and RUP x bST effects. Replicate within treatment was used as the random term to test treatment effects.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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). Although time to puberty did not differ among treatments, RUP and bST supplementation increased BW (P < 0.05) at puberty (Table 3), and increased or tended (P < 0.1) to increase frame size as assessed by several measures (Table 2; Moallem et al., 2004a). From 3 until 10 mo of age, mammary parenchymal mass (right udder-half) increased from 16 to 364 g and parenchymal DNA increased from 115 to 1022 mg (data not shown). Throughout this time, parenchymal RNA, lipid, and protein were unaffected by RUP or bST treatment, as was mammary extraparenchymal fat. The mammary epithelial cell proliferation index (Ki-67 labeling index), assessed by expression of nuclear cell proliferation antigen, did not differ among control, bST, and RUP treatments (Figure 1). Thus, prepubertal growth of mammary glands appeared to be equivalent across treatments.
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Age at first calving did not differ between treatments (Table 3
) and averaged 27 mo of age. First-lactation milk yields did not differ among treatments (Table 3), averaging 9641 kg for 305-d lactation. Mean 305-d mature equivalent milk yield was 11,925 kg. Milk composition was not affected by treatment and there was no effect on 305-d fat and protein yields (P > 0.05). Milk fat and protein averaged 3.81 and 3.01%, respectively, among all cows throughout lactation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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). Body weight and skeletal growth rates were increased at an early age by RUP addition to the diet, with smaller responses at later stages of the prepubertal period (Moallem et al., 2004a). Conversely, effects of bST on rates of BW and skeletal growth were small early, but increased as heifers matured. Effects were additive and only the RUPbST group maintained increased growth throughout the entire prepubertal period. These data suggest that the 1989 NRC recommendation for dietary protein underestimates the requirement for early post-weaning heifers. Others made similar conclusions regarding the 1989 NRC recommendations because when all nutrients were increased by 15% various body growth traits were enhanced (Bortone et al., 1994). In the current study, protein was limiting during the early postweaning period of development, whereas endogenous somatotropin may have been limiting at 250 to 300 d of age, typically the time of or just before puberty. The combination of RUP and bST increased rates of BW gain and wither height growth by 0.17 kg and 0.024 cm per d, respectively, during the treatment period (90 d until puberty). This growth represented a 19% increase in BW gain and a 17% increase in wither height gain compared with controls (Moallem et al., 2004a). Enhanced body and skeletal growth by RUP and bST administration before puberty did not impair mammary growth. Mammary gland mass, composition, cell numbers, and epithelial proliferation index were assessed during the mid prepubertal (5 mo) and late or peripubertal (10 mo) periods. Mammary gland mass, DNA, RNA, and protein content were unaffected by treatment, indicating equivalent mammary growth in all treatments. Extraparenchymal fat and parenchymal lipid content did not differ among treatments, indicating that mammary fat deposition, including parenchymal adipocyte content, was not influenced significantly by increased growth rate. Lack of effect on mammary tissue accretion is further supported by Ki-67 immunohistochemistry. Treatment did not affect the Ki-67 labeling index of mammary epithelial cells, indicating that the epithelial growth fraction was not altered and that rates of epithelial cell proliferation during the pre-pubertal period were similar across treatments. These mammary findings are in contrast to reduced mammary growth in heifers reared to achieve high rates of gain by utilizing high-energy diets (Little and Kay, 1977; Sejrsen, 1978; Sejrsen et al., 1982). The nature of hormonal mediation of decreased mammary growth is unclear (Capuco et al., 2003), but has been hypothesized to be a consequence of reduced activity of somatotropin or its mediators on the mammary gland (Sejrsen et al., 1983). Consistent with that hypothesis, IGF-I concentrations were elevated by dietary RUP and by bST administration (Moallem et al., 2004a).
First-lactation milk yields did not differ among treatments. Due to delays in breeding, mean age at first calving was 27 mo. Because postpubertal growth rates were similar among treatments, however, differences in body size tended to persist after puberty (Moallem et al., 2004a). Although treatment means for growth traits were not statistically different at 644 d of age, the magnitude of differences among treatment means for BW and skeletal growth parameters were analogous to those observed at 341 d of age. Thus, although not directly assessed, both RUP and bST-treated heifers should have been of larger body frame size and weight at calving. Increased prepubertal growth rates did not decrease subsequent milk yields. Milk yields for RUP or bST-treated heifers were numerically 7 to 16% greater than controls.
It has been suggested that CP is a limiting factor for developing accelerated heifer growth (Van Amburgh et al., 1991; VandeHaar, 1997). Results from the current study indicate that limiting protein is particularly problematic during the early postweaning period. By supplying a diet of high protein and energy from 4 mo of age until the luteal phase of the fifth estrous cycle, Radcliff et al. (1997) increased growth rate to 1200 g/ d (controls at 800 g/d) without negatively impacting mammary development, and reduced age at puberty without hindering BW or skeletal size at puberty. Administration of bST to heifers on high-gain or control diets increased BW, skeletal size, and mammary growth (47%). In a subsequent experiment (Radcliff et al., 2000), heifers were reared on analogous diets for BW gains of 800 vs. 1200 g/d. A third group of heifers reared on the high-gain diet was injected daily with bST (25 µg/kg of BW). Heifers were bred after BW exceeded 363 kg and treatments (diet and bST) were continued until pregnancy was confirmed. Heifers in both high-gain groups were 90 d younger than control heifers at first breeding and parturition. Postpartum BW, BCS, and skeletal size did not differ among treatments. Milk production of heifers reared for high rate of gain was 14% less milk than for heifers reared at the standard rate of gain, even though the diet was formulated for high protein content. In contrast, prepubertal bST treatment prevented the decline in milk production observed in the high-gain group. In light of results from their first experiment (Radcliff et al., 1997), it was hypothesized that the high-gain group would not produce less milk than heifers in the low-gain group and that bST injection would increase milk production beyond that of heifers on the standard diet. The decline in milk production may have resulted from early breeding in the high-gain groups, but was prevented by bST administration. In the current study, RUP and bST increased skeletal and BW gains without significantly decreasing age at puberty or calving.
Lammers and Heinrichs (2000) evaluated the impact of increasing dietary protein from 11.8 to 15.6% in the diet of heifers from 7 to 12 mo of age. As in the present experiment, they observed increased skeletal growth rates with small increases in average daily gain. Effect on mammary growth was not directly evaluated and subsequent lactational performance has not been reported. However, increased teat length was interpreted as indicative of increased mammary growth with added dietary protein. In the present experiment, mammary growth was not significantly increased by RUP or bST treatments.
The relationship between heifer rearing and subsequent lactational performance is complex and the number of mammary epithelial cells at puberty seemingly does not always equate to milk production effects (Capuco et al., 2003). The current study clearly demonstrated that supplemental dietary RUP and bST administration increased heifer growth (Moallem et al., 2004a; 2004b). Such a management approach permits earlier breeding, or breeding at a constant age, but at greater BW and frame size. Mammary growth and lactational performance was similar in all treatments, suggesting that implementation of this rearing scheme might produce heifers with mammary development and milk production similar to those reared with lower rates of gain.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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FOOTNOTES |
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Present address: Department of Animal Sciences, University of Illinois, Urbana, 61801-4734.
Present address: Department of Dairy Cattle, Institute of Animal Science, Volcani Center, Bet-Dagan, 50250, Israel.
Received for publication October 27, 2003. Accepted for publication June 9, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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  G. I. Zanton and A. J. Heinrichs Review: Limit-Feeding with Altered Forage-to-Concentrate Levels in Dairy Heifer Diets Professional Animal Scientist, August 1, 2009; 25(4): 393 - 403. [Abstract] [PDF]
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 L. F. P. Silva, B. E. Etchebarne, M. S. Weber Nielsen, J. S. Liesman, M. Kiupel, and M. J. VandeHaar Intramammary Infusion of Leptin Decreases Proliferation of Mammary Epithelial Cells in Prepubertal Heifers J Dairy Sci, August 1, 2008; 91(8): 3034 - 3044. [Abstract] [Full Text] [PDF]
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M. J. Meyer, A. V. Capuco, D. A. Ross, L. M. Lintault, and M. E. Van Amburgh Developmental and nutritional regulation of the prepubertal heifer mammary gland: I. Parenchyma and fat pad mass and composition. J Dairy Sci, November 1, 2006; 89(11): 4289 - 4297. [Abstract] [Full Text] [PDF]
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