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Energy balance and body condition influence luteal function in holstein heifers

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Energy balance and body condition influence luteal function in holstein heifers
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  DOMESTIC ANIMAL ENDOCRINOLOGY Vol. 7 2):135-148, 1990 ENERGY BALANCE AND BODY CONDITION INFLUENCE LUTEAL FUNCTION IN HOLSTEIN HEIFERS 1 A. Villa-Godoy T.L. Hughes R.S. Emery W.J. Enright A.D. Ealy S.A. Zinn and R.L. Fogwell 2 Department of Animal Science, Michigan State University, East Lansing, 48824-1225 Received January 9 1989 ABSTRACT A factorial experiment was conducted to determine influence of energy balance (EB) and body condition (BC) on luteal function in heifers. Heifers with moderate (MBC) or fat (FBC) BC were fed individually to sustain positive EB (PEB) or to cause negative EB (NEB). Intake of feed was measured daily and body weight weekly. Progesterone was quantified daily in serum for 3.5 estrous cycles. On days 9, 10, or 11 after fourth estrus, blood was sampled every 15 rain for 12 hr to quantify luteinizing hormone (LH), growth hormone (GH), insulin and non-esterified fatty acids (NEFA). The next day, luteal cells were incubated and proportions of small to large cells were determined. After fourth estrus, area of progesterone profiles in serum for 10 days postestrus was reduced in all heifers relative to MBC.PEB heifers. But, luteal weight from FBC-PEB and MBC-NEB heifers was less than MBC-PEB heifers and FBC-NEB heifers were intermediate. Secretion of progesterone in vitro was increased by LId for PEB but not NEB heifers. MBC-NEB heifers had increased ratios of small to large luteal cells. Independent of BC, NEB decreased concentrations of insulin and increased GH and NEFA. Secretion of progesterune was not associated with LH, GH or insulin, but was correlated negatively with NEFA. We conclude that reduced concentrations of progesterone in serum of FBC- PEB and MBC-NEB heifers is due to impaired luteal development. But, reduced con- centrations of progesterone in serum of NEB heifers is due also to reduced basal (MBC) and LH-induced (MBC and FBC) secretion of progesterone by luteal cells. Body condition at onset of NEB may determine when effects of NEB on progesterone are detected. INTRODUCTION At least 80 of dairy cows experienced negative energy balance (NEB) during early lactation (1, 2). Variation in EB was determined largely by calories ingested and not by yield of milk (3, 4). Since cows (5) and ewes (6) with fat body condition (FBC) ingest less feed than animals with moderate BC, FBC may increase magnitude of NEB. Reduced concentrations of progesterone in serum have been associated with embryonic death (7) and low rate of con- ception (8). A consequence of NEB is that concentrations of progesterone in milk are reduced (4). In lactating cows, effects of EB and number of postpartum estrous cycles on progesterone are confounded (4) so heifers have been used previously to avoid this bias. But, in most studies heifers were fed in groups so energy status of individuals is variable and not defined clearly (9, 10). We hypothesized that BC will modify response of corpora lutea to NEB. Thus, our primary objective was to determine independent and associative effects of EB and BC on secretion of progesterone by corpora lutea in heifers. Luteinizing hormone (I J-I) is the major bovine luteotropin (11). For a variety of possible reasons, adverse effects of NEB on corpora lutea have not been Copyright 1990 by DOMENDO, NC. 135 0739-7240/90/ 3.00  136 VILLA GODOY ET AL. explained consistently by effects of NEB on LH 9, 10). Thus, a second objective was to determine if adverse effects of NEB on secretion of progesterone are associated with altered secretion or response to LH. Concentrations of insulin, growth hormone GH) and non-esterified fatty acids NEFA) in serum are affected by NEB 12, 13, 14). But it has not been determined whether these hormones or catabolites are modulated by BC or are correlated with luteal function. To examine potential explanations for how NEB and/or BC affect progesterone, our third objective was to determine association of insulin, GH or NEFA with concentrations of progesterone in serum MATERIALS AND METHODS Design and General Procedures. Twenty nulligravid Holstein heifers were grouped by body weight and assigned to treatments within a 2x2 factorial experiment. Main effects were: 1) body condition BC); moderate MBC) or fat FBC), and 2) energy balance EB); positive PEB) or negative NEB). Heifers were fed total mixed rations TMR; Table 1; 15) and had free access to water and trace mineralized salt. The study consisted of three phases: conditioning, adaptation and experimental. The conditioning phase ~ 6 too) started with two groups of heifers of similar age ~ 10 mo) and body weight 277 + 5 kg) that were fed diets A or B once daily Table 1) and this phase ended when ranges of BC for the two groups MBC and FBC) did not overlap see Results). During a 20-day adaptation phase, heifers were restrained in gang-lock stanchions and meal-fed individually during two periods of 90 min daily 0500 to 0630 and 1700 to 1830 hr). During adaptation, all heifers were offered 1.6 kg of DM per 100 kg body weight of diet A Table 1). Same diet was used to reduce heterogeneity of ruminal fermentation present during conditioning phase 16, 17). But distinctions of BC achieved during conditioning phase MBC and FBC) were maintained during adaptation. Ovaries of heifers were examined rectally at least twice and presence of a corpus luteum designated a heifer as postpubertal. Estrus was synchronized with prostaglandin F2~ to homogenize stage of an estrous cycle at onset of experimental phase. During the experimental phase 3.5 estrous cycles) heifers were observed for signs of estrus for periods of 30 min at intervals of ~8 hr. Estrus was when a heifer stood to be mounted for -->2 sec and concurrently had low progesterone <1 ng/ml) in serum. During experimental phase heifers were fed individually two meals daily and two diets were offered. Diet A Table 1) was offered to MBC and FBC heifers to support PEB and daily body weight gain of ~ .8 kg. Remaining MBC and FBC heifers received diet C Table 1) and experienced NEB. Thus, four treatment combinations number of heifers) were established for the experimental phase: MBC-PEB 5), FBC-PEB 6), MBC-NEB 5) and FBC-NEB 4). Amounts of dietary dry matter DM) and crude protein CP) offered per 1 O0 kg body weight BW) were same for PEB and NEB heifers. Specifically all heifers were offered 1.62 kg DM/IO0 kg BW and 0.25 kg CP/IO0 kg BW. Thus energy was the only known dietary variable between PEB and NEB heifers. In the experimental phase, BW was expected to increase in PEB and to decrease in NEB heifers. Independent of BW change, dietary calories and kg DM per 100 kg BW remained constant within energy status. Thus, total feed offered daily to each heifer was adjusted weekly for changes in BW of that individual  ENERGY FATNESS AND CORPORA LUTEA 137 heifer. For example, as a NEB heifer lost BW, total feed offered daily per heifer was reduced so feed offered per 1 O0 kg BW was not changed over time. Body Weight, Condition Score and Energy Balance. Heifers were weighed at 1500 hr on two consecutive days each week. Average weekly change in body weight (BW) was used to estimate daily changes. In alternate weeks, body condition of heifers was scored (I to 4; 4 = fat) by four observers (I 8). Intake of feed was recorded daily and EB was estimated daily for individual heifers as the difference between energy intake (DM intake >( NEm, Table 1) and net energy required for maintenance (NE=, Mcal ---- .077 W.~5; 19). Daily intake of crude protein for NEB heifers (1 .I kg) and for PEB heifers (1.8 kg) satisfied requirements so all heifers were in positive nitrogen balance. Luteal Function. During the experimental phase jugular blood was sampled daily at 1500 hr and progesterone was quantified in serum (20). At 10, 11 or 12 days postestrus of the fourth estrous cycle, corpora lutea were collected via supravaginal incision (21). Corpora lutea were rinsed immediately with Hank's buffered saline containing HEPES (20 raM), bovine serum albumin (BSA) fraction V (. I ), penicillin G (sodium salt, .07 g/l) and streptomycin sulfate (. 1 g/I). Rinsed corpora lutea were placed in fresh buffer and transported on ice to our laboratory. After removal of adherent interstitial tissue, corpora lutea were weighed. To dissociate ceils, .5mm slices of luteal tissue were placed in Hank's buffered saline containing collagenase 6 (Worthington type IV, 2000 U/ gm tissue), deoxyribonuclease I T (.02 ) and BSA 7 (.1 ). Slices were incubated at 37 C in an atmosphere of 95 02 and 5 CO2 for 4 to 6 hr in a shaking water bath. Viability of luteal cells (22) was >90 for all corpora lutea. Dissociated viable cells (1.01 + .13 X 106) were incubated at 37 C in atmospheric air for 2 hr and were suspended in medium 199 s containing HEPES, BSA, penicillin G and streptomycin in concentrations described above and pH 7.35 was maintained by Hank's buffer. In addition, medium contained 0, .1, 1, 10, or 100 ng/ml of bovine luteinizing hormone (LH, NIH-LH-B8). Each dose of LH was replicated 5 times per corpus luteum. After incubation, cells and media were frozen together and stored at --20 C. To prepare samples for assay cells and media were thawed and frozen six times. Extracts were assayed for progesterone (20). Progesterone quantified in media plus cells represents synthesis and release and will be presented as secretion. Secretion of progesterone from luteal cells in vitro will be expressed as ng progesterone per 106 cells. Aliquots of dissociated luteal cells (>__ 1 X 106) were fixed in paraformal- dehyde (l )-phosphate buffer (0.1M, pH 7.4). Cells were washed three times with 0.1M phosphate buffer, smeared onto slides that were precoated with BSA and allowed to dry. Cells were stained with hematoxylin-eosin and only nu- cleated cells were inventoried. Stained cells were examined at 400X with a light microscope equipped with an ocular micrometer. Luteal cells were classified as small (10 to 15 I~) or large (>__22 gin) as described by Koos (23). One slide was prepared per corpus luteum and five microscopic fields were selected randomly per slide. Within each field 200 contiguous nucleated cells were classified as small or large. From 1000 cells per corpus luteum data were expressed as ratio of small to large cells (Sm:Lg). Sampling and Analysis for Hormones and MetaboHtes. On days 9, 10 or 11 postestrus of the fourth estrous cycle (24 hr before lutectomy) jugular blood was sampled every 15 rain during 12 hr (1 hr before to 11 hr after  138 VILLA GODOY ET AL. feeding) to quantify LH (24), GH (25) and insulin (Appendix) in serum. Blood was also sampled hourly before and after feeding to quantify NEFA in plasma (26). Measures of precision for all assays are in Table A-1. Statistical Analysis. All variables that were measured repeatedly, including BC, EB, BW, GH, LH, insulin and progesterone were examined by split-plot analysis of variance with estrous cycle or dose of LH as subplots (27). Differences among treatment combinations and interactions between EB and BC were determined by Dunnett s test (28). EB, BC and BW were averaged per estrous cycle and examined to determine that all four treatment combinations were established. Area of profiles for progesterone in serum included all values two standard deviations above baseline (.3 ng/ml) for 10 days postestrus of all estrous cycles within heifer. Secretory characteristics for LH were as defined by Hughes (29) and by Pulsar (30) for GH and insulin. Basal insulin was mean concentration in preprandial samples. Luteal development was described by luteal weight or slopes for progesterone profiles in serum during first 10 days postestrus of each cycle (31). As for area, srcin of profiles to evaluate slopes was when progesterone exceeded baseline by two standard deviations. Association of luteal function with GH, NEFA or insulin was examined by linear regression (28). Concentration of progesterone in serum at lutectomy was the dependent variable. Insulin, GH and NEFA were examined separately as individual independent variables. RESULTS At beginning of experimental phase (Figure 1) BC differed (P < .05) between FBC (3.0 - .1; range. 2.8 to 3.5) and MBC heifers (2.4 _+ .1; range 2.2 to 2.5). For PEB heifers, BC at end of conditioning phase did not vary significantly throughout the experimental phase (Figure 1). In contrast, BC of NEB heifers declined (P < .05) and was less (P < .05) than BC of corresponding control heifers by the second experimental estrous cycle (Figure 1). At onset of experimental phase, body weight of FBC heifers (455 + 7 kg) was greater (P < .05) than MBC heifers (429 + 6 kg). As designed for experimental phase, PEB heifers gained and NEB heifers lost body weight and condition (Figure 1). But, duration of NEB and accumulated energy loss (Figure 1) in FBC heifers (46 _+ .6d , -98 -+ 13 Mcal) were greater (P < .01) than for MBC heifers (38 + 3d, -57 -+ 4 Mcal). There were no main effects of EB or BC on luteal weight or ratio of small to large (Sm:Lg) luteal cells (Table 2) but, compared with MBC-PEB luteal weight was reduced (P < .05) by FBC- PEB and MBC-NEB but FBC-NEB was intermediate. In addition, proportion of Sm:Lg luteal cells in MBC-NEB heifers was greater (P < .05) than in all other heifers (Table 2). During the fourth estrous cycle, there were significant (P < .05) main effects of EB and BC on area under profiles of progesterone (Table 3). But, relative to MBC-PEB, area of progesterone profiles were reduced (P < .05) by FBC- PEB in first, second and tended (P < .07) to be reduced in third estrous cycle. Relative to MBC-PEB, NEB reduced area of progesterone profiles in second through fourth cycle of MBC heifers but only in the fourth cycle of FBC heifers (Table 3). During the first 10 days postestrus, rate that progesterone increased (Figure 2) in MBC-PEB heifers did not differ among the four estrous cycles. During  ENERGY FATNESS AND CORPORA LUTEA TABLE 1 COMPOSITION OF TOTAL MIXED RATIONS (TMR) 139 Diet ~ Item A B C Phases used Conditioning + + Adaptation + Experimental + + Ingredients, MfalfaI-Iaylage 24.9 4.4 22.4 Corn silage 53.4 16.9 48.9 High moisture 16.2 73.1 12.0 ear COlXl Protein supplement, 5.0 5.1 16.2 (44 ) Vitamin premix .5 .5 .5 Chemical analysis ~., Dry matter, 40.0 50.9 46.4 Energy, Mcal/kg 1.5 1.6 1.5 Crude protein, 13.6 13.3 17.7 Acid detergent 21.7 14.3 15.5 fiber, =Dry matter basis. ~Methods for chemical analysis in Pritchard and Staubus (15). ¢Concentration of minerals in total mixed rations were: Ca (.71 ), P (.3.9 ), K (1.1 ), Mg (.26 ), S (.21 ), Na (.13 ), Mn (57ppm), Fe (228 ppm), Cu (8 ppm), anti ~,.n (48 ppm). ~Net energy for maintenance (NE=) calculated from chemical analysis of TMR. all estrous cycles progesterone increased slower (P < .05) in FBC-PEB than in MBC-PEB heifers. But, NEB slowed rate that progesterone increased (P < .05) in MBC heifers during third and fourth cycle and in FBC heifers during fourth cycle. Thus, adverse effects of NEB and FBC on luteal development were evident whether examined by luteal weight (Table 2) or slope of progesterone profiles (Figure 2). EB interacted with BC to reduce (P < .01) basal (0 ng LH) secretion of progesterone in MBC-NEB relative to other treatment combinations (Figure 3). BC did not alter response of luteal cells to LH in vitro but NEB independently reduced (P< .01) LH-induced secretion of progesterone (Figure 3). Secretory profiles of LH, GH and insulin were examined at mid-diestrus of the fourth estrous cycle. There were no main effects or interactions of EB and BC on mean LH, (1.1 + 0.2 ng/ml), baseline LH (0.9 +- 0.2 ng/ml), amplitude of pulses (2.9 -+ 0.3 ng/ml) or frequency of pulses per 12 hr (2.0 + 0.3). Among treatment combinations, all differences in secretory characteristics of GH or insulin were due to EB and not to BC or interactions of EB and BC. T~LE A-1. F--VrlMAT~S OF PI~ lSlON FOR A.~AYS Number Coe•cient of variation, Hormone Assays Concenu'ation Inwassay Interassay Progesterone Low 21 0.2 ng/ml 9.6 12.7 High 21 6.7 ng/ml 4.2 6.7 Cells & medium 25 13.7 ng/ml 7.4 9.9 LH 1 10.5 ng/ml 8.5 GH 5.4 ng/ml 6.2 Insulin Fasted 1 66.7 I~U/ml 6.8 Fed 1 177.5 ItU/ml 4.5 NEFA 13 135.8 pLEg/1 2.0 16.4
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