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Response of Dairy Cows Treated with Bovine Somatotropin to a Luteolytic Dose of Prostaglandin F2α1
Response of Dairy Cows Treated with Bovine Somatotropin to a Luteolytic Dose of Prostaglandin F2a1 CRYSTAL J. KIRBY, STACEY J. WILSON, and MATTHEW C. LUCY2 Department of Animal Science, University of Missouri, Columbia 65211
ABSTRACT The objective of this study was to evaluate the timing of follicular waves in cows treated with bovine somatotropin (bST) by measuring ovarian responses to a luteolytic dose of PGF2a on d 12 of the estrous cycle. Thirty lactating cows (26 Holstein and 4 Guernsey) were assigned to receive bST (500 mg; n = 18) or saline (control; 1.5 ml; n = 12) every 14 d for three injection cycles. On d 12 of a synchronized estrous cycle, cows were injected with PGF2a to induce luteolysis. Following PGF2a, 9 cows ovulated from the dominant follicle during the first follicular wave ( 4 cows treated with bST and 5 control cows), and 14 cows ovulated from the dominant follicle during the second follicular wave ( 8 cows treated with bST and 6 control cows). Of the cows that ovulated during the second follicular wave, cows treated with bST had more class 3 follicles ( ≥10 mm) than did control cows. Concentrations of estradiol rose earlier after PGF2a injection in cows treated with bST than in control cows. This rise in estradiol was parallel to the development of dominant follicles. Serum concentrations of FSH were decreased in cows treated with bST. During the first and second estruses, equivalent numbers of cows treated with bST and control cows ovulated, but fewer cows treated with bST expressed estrus. These results are consistent with the hypothesis that cows treated with bST have reduced FSH, a faster turnover of dominant follicles, and differences in the timing of follicular waves. Treatment of cows with bST also increased the incidence of undetected estrus. ( Key words: bovine somatotropin, ovary, cow, luteolysis) Abbreviation key: CL = corpus luteum, 1FW cows = cows that ovulated from the dominant follicle during the first follicular wave, 2FW cows = cows that
Received August 18, 1995. Accepted March 15, 1996. 1Contribution from the Missouri Agricultural Experiment Station, Journal Series Number 12383. 2Corresponding author: Matthew C. Lucy, Department of Animal Science Research Center, University of Missouri, Columbia 65211. 1997 J Dairy Sci 80:286–294
ovulated from the dominant follicle during the second follicular wave. INTRODUCTION Administration of bST to dairy cows increases milk production by approximately 10 to 20% (23). Because of its galactopoietic effect, bST was developed as a recombinant hormone to increase the milk production of lactating dairy cows. One form of recombinant bST (sometribove; Monsanto Co., St. Louis, MO) was approved for sale in the US by the FDA in November 1993. Since that time, approximately 10% of dairy cows in the US have been treated with bST (12). This percentage is expected to increase as producers become more familiar with recombinant bST. Several studies (3, 5, 8, 19, 21, 30, 32) have examined the long-term effects of bST on reproduction in cows, and most have detected modest declines in reproductive performance of cows treated with bST. These declines include increased days open and a decrease in herd pregnancy rate. Many of these reproductive losses are associated with a temporary negative energy balance experienced by cows treated with bST. This physiological change appears to delay rebreeding. The combined effects of bST and IGF-I have measurable effects on ovarian function of cows. Treatment with exogenous bST increases the number of ovarian follicles within distinct classes of follicular diameter [3 to 5 mm in heifers and 6 to 9 mm in lactating cows (6, 11)]. There also appear to be effects on the corpus luteum ( CL) , including increased CL weight ( 1 8 ) and increased concentrations of progesterone in blood of cows treated with bST (9, 16, 30). In addition to changes in follicular numbers and CL size, our laboratory has detected a change in the timing of follicular waves in cows treated with bST (14). This change involves an advancement in the timing of the second follicular wave with earlier recruitment of second wave follicles and earlier development of the dominant follicle during the second follicular wave. This study was designed to test further changes in follicular dynamics of cows treated with bST. Lactating dairy cows treated with saline (control) or bST were given a luteolytic dose of PGF2a on d 12 of the estrous cycle. This treatment coincided with a period of dominance for the first follicular wave or recruit-
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ment of the second follicular wave (associated with first wave atresia of the dominant follicle). If bST changes the timing of the first or second follicular wave, then follicular response to PGF2a on d 12 should be different. Therefore, changes in patterns of follicular growth in cows treated with bST were evaluated based on responses of ovarian follicles to luteolysis on d 12. MATERIALS AND METHODS Cows and Treatments Twenty-six Holstein and 4 Guernsey cows were used. Cows were housed in a concrete floor, free-stall barn with fence-row feeders equipped with locking head gates. The study was conducted between November and December 1994 as a single replicate. Twice daily, all cows were fed a TMR consisting of corn silage, alfalfa haylage, alfalfa hay, hominy, ground corn, whole cottonseed, and corn and soybean premix formulated to meet or exceed the NRC ( 2 0 ) nutritional requirements (1.67 Mcal/kg of NEL, 17.8% CP, 35.2% ADF, and 5.8% ether extract). Cows were milked twice daily, and milk production was measured using electronic flow meters. Water was available for ad libitum intake. The University of Missouri Animal Care and Use Committee approved all experimental procedures. Eighteen cows (16 Holstein and 2 Guernsey) were randomly assigned to receive treatment with bST (500 mg of bST; Posilac; Monsanto Co.), and 12 cows (10 Holstein and 2 Guernsey) received sterile saline (control; Abbott Laboratories, North Chicago, IL). Injections were administered subcutaneously into the tailhead region using 16-gauge, 16-mm (5/ 8-inch) needles. Posilac, a sustained-release form of recombinant bST, was given once every 14 d (single injection cycle). Days postpartum were similar for bST and control cows and averaged 65 ± 5 d at first injection. Estrous cycles were synchronized so that estrus occurred 7 d after the start of the second injection cycle. Two norgestomet implants (SyncroMate-B; Sanofi Animal Health, Overland Park, KS) were implanted for 9 d, and two injections (25 and 12.5 mg) of PGF2a (Lutalyse; The Upjohn Co., Kalamazoo, MI) were administered on d 7 and 8 of norgestomet implant. Estrus (expected 2 d after implant removal) was detected by 30-min visual observations in the morning (0600 h), in the afternoon (1400 h), and at night (2200 h). Cows were not inseminated at the synchronized estrus. All cows not seen in estrus were examined by ultrasound for signs of ovulation on the 4th d after implant removal. A cow was diagnosed as having ovulated if a developing CL was detected by ultrasound. Cows that did not ex-
press estrus or that did not ovulate (based on ultrasound examination) were removed from study ( n = 6; 1 control cow and 5 cows treated with bST). Cows remaining in the study were injected with 25 mg of PGF2a on d 12 after norgestomet-synchronized estrus. This PGF2a injection occurred 4 d after the third injection cycle of saline or bST. Ultrasound and Data Collection Beginning on d 10 of the norgestomet-synchronized estrous cycle and continuing until PGF2a-induced estrus, ovaries were examined daily by transrectal ultrasonography. Ultrasound examinations were performed with an ultrasound scanner (Aloka 500V; Corometrics Medical Systems Inc., Wallingford, CT) with a 7.5-MHz linear transducer. Number and diameter of all follicles ≥3 mm in diameter were recorded. Follicles were grouped into three diameter classes for analysis: class 1 ( 3 to 5 mm), class 2 ( 6 to 9 mm), and class 3 ( ≥10 mm). The development of the ovarian follicle was followed by mapping the location of large follicles on the ovary. Based on the growth of large follicles, the ovarian follicle was classified as either the first or second wave dominant follicle. The maximum cross-sectional area of the CL and the area of the fluid-filled cavity within the CL were recorded. Daily ultrasound examinations ended on the day of estrus following PGF2a administration. Blood samples were collected by coccygeal venipuncture once daily on d 10 and 11 of the synchronized estrous cycle. Following PGF2a injection on d 12, blood was collected every 6 h for 24 h. Blood was collected twice daily (12-h interval) on d 13 and 14 and once daily from d 15 until estrus. Ten milliliters of blood were collected into Vacutainer tubes (Becton Dickinson and Co., Rutherford, NJ), stored overnight at 4°C, and centrifuged (3000 × g for 15 min) for collection of serum. Serum was stored at –20°C until analyses. Body weight was recorded after the morning milking and feeding on wk 1, 4, and 6 of the experiment. Body condition scoring ( 7 ) was performed at the beginning (wk 1 ) and at the end of the study (wk 6). Radioimmunoassays Concentrations of serum progesterone were determined in all samples using an extraction assay that had been previously validated ( 4 ) . Intraassay and interassay coefficients of variation for progesterone were 5.5 and 12.6%, respectively. Serum estradiol ( 1 4 ) and FSH ( 2 2 ) were determined in daily samples. Intraassay and interassay coefficients of variation for estradiol were 6.0 and 5.3%, respectively. Journal of Dairy Science Vol. 80, No. 2, 1997
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Serum FSH was analyzed in a single assay; the intraassay coefficient of variation was 10.2%. Serum bST ( 2 6 ) was analyzed in a single assay for samples that were collected prior to treatment and 0 and 48 h after PGF2a. The intraassay coefficient of variation was 15.3%.
interval to estrus after PGF2a and the diameter of the preovulatory follicle were tested using the model Yilm = m + Ai + Wl + AWil + eilm. The percentage of cows responding to synchronization was tested using a chisquare test. Data are presented as least squares means and standard errors of the least squares means. RESULTS
Statistical Analyses Data were analyzed by least squares analysis of variance ( 2 7 ) as a split-plot design. The statistical model used in the analysis of milk production and serum bST was Yijk = m + Ai + B ( A ) j + Ck + ACik + eijk, where Yijk = dependent observation, m = overall mean, Ai = effect of treatment i, B ( A ) j = effect of cow j nested within treatment i, Ck = effect of day k, ACik = treatment by day interaction, and eijk = residual effect. The main effect of treatment was tested using cow nested within treatment as the error term. Remaining terms were tested with the residual effect. A second model was used to test the effects of follicle diameter, number of follicles, CL area, progesterone, and FSH. This model included the effects of follicular wave: Yijkl = m + Ai + Wl + AWil + B(AW) j + Ck + ACik + WCkl + ACWikl + eijkl, where Wl = effect of follicular wave l (first vs. second), AWil = treatment by follicular wave interaction, WCkl = follicular wave by day interaction, and ACWikl = treatment by follicular wave by day interaction. The effects of treatment, follicular wave, and treatment by follicular wave interaction were tested; cow nested within treatment by follicular wave interaction was the error term. The
Estrous Expression After Synchronization of Estrus At the norgestomet-synchronized estrus, 92% (11 of 12) of the control cows were observed to be in estrus (standing to be mounted by a herdmate), and 39% ( 7 of 18) of the cows treated with bST were observed to be in estrus (Table 1; P < 0.005). All cows observed to be in estrus were detected during a 24-h period beginning 36 h after implant removal. Those cows that failed to display estrus were examined by ultrasound on the 4th d after implant removal. Based on these examinations, 1 control cow was diagnosed as cystic (absence of a CL; follicle >25 mm) and was removed from the study. Of the 11 cows treated with bST that did not express estrus, 1 failed to have CL regression in response to the PGF2a injection, and 4 had several class 3 follicles with no CL. These 5 cows were removed from the study. The remaining 6 treated cows had developing CL and were in the recruitment phase of the first follicular wave, indicating that they had ovulated. These 6 cows remained on the study.
TABLE 1. Estrous expression in lactating cows treated with 500 mg of sustained-release bST or saline (control). First estrus1
Second estrus2
Total3
Item
bST
Control
bST
Control
bST
Control
Observations, no.
18 (no.) 7 6 0 4 1 0 13 3
12 (no.) 11 0 1 0 0 0 11 2
13 (no.) 6 4 1 0 0 2 10 1
11 (no.) 10 0 1 0 1 0 9 2
31 (no.) 13 10 1 4 1 2 23 4
23 (no.) 21 0 1 0 1 0 20 4
Standing estrus4 Undetected ovulation4 Cystic ovary Abnormal ovary No CL5 regression Incomplete CL regression Total to ovulate Double ovulations
(%) 39 33 0 22 6 0 72 23
(%) 92 0 8 0 0 0 92 18
(%) 46 29 8 0 0 15 77 10
(%) 91 0 9 0 9 0 82 22
(%) 42 32 3 13 3 6 74 17
(%) 92 0 4 0 4 0 87 20
1Estrus was synchronized with two norgestomet implants for 9 d and two injections (25 and 12.5 mg) of PGF on d 7 and 8, 24 h 2a apart. 2Luteolysis was induced with 25 mg of PGF 2a on d 12 of the synchronized estrous cycle. 3Combination of first and second estruses. 4P < 0.01 (total estruses). 5Corpus luteum.
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Development of Follicular Waves and Ovulation After PGF2a Prior to injection of PGF2a on d 12 of the estrous cycle, there was no effect of treatment on double ovulations (Table 1). Following the injection of PGF2a, 9 cows ovulated from the first wave dominant follicle ( 4 cows treated with bST and 5 control cows) and 14 cows ovulated from a second wave dominant follicle ( 8 cows treated with bST and 6 control cows). The number of cows treated with bST and control cows ovulating from first or second wave follicles was similar (Table 2). Interval to estrus was shorter ( P < 0.05) for cows that ovulated from the dominant follicle during the first follicular wave ( 1FW cows; 3.3 ± 0.7 d ) than for cows that ovulated during the second follicular wave ( 2FW cows; 5.2 ± 0.5 d). The effect of treatment was not significant, and no treatment by wave interaction was detected. Diameter of the preovulatory follicle on the day of estrus was not affected by wave or treatment. In addition, neither treatment nor wave affected ovulation rate following PGF2a injection. Number and Diameters of Follicles and Concentrations of FSH in Serum Numbers of class 1, 2, and 3 follicles for 1FW cows and for 2FW cows in each treatment are presented in Table 2. Numbers of class 1 follicles decreased (day
effect; P < 0.0001; Figure 1A) following the injection of PGF2a and averaged 10.1 ± 0.7 follicles per cow on d 12 (day of PGF2a injection) and 4.5 ± 0.8 follicles per cow on d 15 ( 3 d after PGF2a injection). There was no effect of treatment or follicular wave on numbers of class 1 follicles. The numbers of class 2 follicles increased after PGF2a injection and then decreased (day effect; P < 0.001; Figure 1B). There was a main effect of follicular wave ( P < 0.02) for class 2 follicles because 1FW cows had fewer class 2 follicles than did 2FW cows (3.3 ± 0.7 vs. 5.5 ± 0.5 follicles per cow for 1FW and 2FW cows, respectively). There was no effect of treatment on numbers of class 2 follicles. Numbers of class 3 follicles increased (day effect; P < 0.001) from d 2 before (1.2 ± 0.1 follicles per cow) to d 4 after (2.3 ± 0.2 follicles per cow) PGF2a injection. Compared with results for 1FW cows, the number of class 3 follicles in 2FW cows tended to increase sooner after PGF2a injection (follicular wave by day interaction, P < 0.09; Figure 1C). A follicular wave by treatment interaction ( P < 0.01) was detected for numbers of class 3 follicles. The 2FW cows treated with bST had a greater number of class 3 follicles than did the 2FW cows treated with saline (2.2 ± 0.1 vs. 1.6 ± 0.1 follicles per cow for cows treated with bST and control cows, respectively; P < 0.002). At the same time, 1FW cows treated with bST had fewer class 3 follicles than did 1FW cows treated with saline (1.3 ± 0.3 vs. 1.9 ± 0.3 follicles per cow for
TABLE 2. Characteristics of follicular development and ovulation from d 10 until estrus after an injection of PGF2a on d 12 during a synchronized estrous cycle in lactating cows1 treated with 500 mg of sustained-release bST or saline (control). 1FW Cows
2FW Cows
Item
bST
Control
bST
Control
Cows, no.
4
5
8
6
Follicles, no. Class 1 ( 3 to 5 mm) Class 2 ( 6 to 9 mm) 2 Class 3 ( ≥10 mm) 3 Largest follicle, mm Second largest follicle, mm4 Dominant follicle, mm First follicular wave Second follicular wave Interval to estrus, d5 Diameter of ovulatory follicle, mm
X
SEM
X
SEM
X
SEM
X
SEM
5.9 2.9 1.3 17.4 8.1
1.3 1.0 0.3 1.2 1.1
7.3 3.7 1.9 16.6 10.3
1.5 1.1 0.3 1.3 1.2
7.0 5.1 2.2 15.7 11.1
0.8 0.6 0.2 0.7 0.7
8.1 6.0 1.6 16.3 9.6
1.0 0.7 0.2 0.9 0.8
17.4 . . . 3.0 18.7
1.3 . . . 1.1 1.2
16.6 . . . 3.5 18.5
1.4 . . . 1.0 1.1
15.0 11.3 4.0 18.0
0.8 1.0 0.7 0.8
16.0 8.8 6.3 18.2
0.9 1.2 0.8 1.0
11FW Cows = Cows that ovulated from the dominant follicle during the first follicular wave; 2FW cows = cows that ovulated from the dominant follicle during the second follicular wave. 2Follicular wave ( P < 0.02). 3Follicular wave by treatment interaction ( P < 0.01). 4Follicular wave by treatment interaction ( P < 0.08). 5Follicular wave ( P < 0.05).
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Figure 2. Least squares means for concentration of FSH in serum. Cows received 500 mg of sustained-release bST ( π; SEM = 0.05) or saline (control; ÿ; SEM = 0.05) every 14 d for three injection cycles. Prostaglandin F2a (25 mg) was injected on d 12 of a synchronized estrous cycle in the third injection cycle.
Figure 1. Least squares means for the number of follicles in different follicular diameter classes: class 1 (A; 3 to 5 mm; SEM = 0.7), class 2 (B; 6 to 9 mm; SEM = 0.4), and class 3 (C; ≥10 mm; SEM = 0.1). Cows were treated with 500 mg of sustained-release bST or saline (control) every 14 d for three injection cycles. Prostaglandin F2a (25 mg) was injected on d 12 of a synchronized estrous cycle in the third injection cycle. Cows within each treatment were grouped according to whether they ovulated from the dominant follicle during the first follicular wave ( ÿ) or the second follicular wave ( o) . Journal of Dairy Science Vol. 80, No. 2, 1997
cows treated with bST and control cows, respectively). Concentrations of FSH in serum were similar in 1FW and 2FW cows (0.63 ± 0.04 and 0.61 ± 0.03 ng/ ml, respectively), but concentrations were decreased in serum of treated cows ( P < 0.02; Figure 2 ) compared with concentrations in control cows. Mean serum FSH was 0.56 ± 0.03 and 0.68 ± 0.03 ng/ml for treated and control cows, respectively. No treatment by follicular wave or treatment by day interaction was detected ( P > 0.10). During the experimental period, the diameter of the largest follicle (not necessarily a dominant follicle) on the ovary was similar for cows treated with bST and control cows (Table 2). There was a tendency ( P < 0.08) for a follicular wave by treatment interaction for the second largest follicle because 1FW treated cows had smaller second largest follicles than did 1FW control cows, and 2FW treated cows had a larger second largest follicle than 2FW control cows. The first-wave dominant follicle increased in diameter for 1FW cows (Figure 3A) and decreased in diameter for 2FW cows (Figure 3B; follicular wave by day interaction, P < 0.001). The growth of the dominant follicle during the first follicular wave was not different for cows treated with bST and control cows that ovulated during the first follicular wave (Figure 3A; Table 2). For 2FW cows, the dominant follicle of the first follicular wave decreased in diameter as the preovulatory follicle increased in diameter from d 2 before (5.3 ± 0.4 mm) to 4 d after
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(14.7 ± 0.4 mm; Figure 3B) PGF2a injection. The dominant follicle of the second wave tended ( P < 0.13) to be larger in cows treated with bST; an equivalent diameter of the second wave dominant follicle occurred approximately 2 d later in control cows than in cows treated with bST. Size of the CL The size of the CL (luteal tissue plus fluid-filled center cavity) was similar between cows treated with
bST and control cows (4.3 ± 0.1 cm2) . The 1FW cows had larger CL at the time of PGF2a injection (5.9 ± 0.2 vs. 5.1 ± 0.2 cm2 for 1FW and 2FW cows, respectively) that declined to a similar size by d 3 after PGF2a injection (2.1 ± 0.3 vs. 2.3 ± 0.2 cm2 for 1FW and 2FW cows, respectively; follicular wave by day interaction, P < 0.10). When the size of the fluid-filled center was subtracted from CL area, no differences were detected in follicular wave or treatment. Mean size of the luteal tissue area (CL minus hollow cavity) was 3.5 ± 0.1 cm2. Estradiol, Progesterone, and CL Decline Serum estradiol increased ( P < 0.001) from PGF2a injection until estrus (Figure 4A). Estradiol increased earlier in 1FW cows than in 2FW cows (follicular wave by day interaction, P < 0.03). Serum estradiol was similar for cows treated with bST and control cows that ovulated the first wave dominant follicle. However, for 2FW cows, estradiol increased earlier in cows treated with bST than in control cows (treatment by day interaction, P < 0.05). Before the injection of PGF2a, serum progesterone was greater ( P < 0.06) in 2FW cows (6.0 ± 0.3 ng/ml) than in 1FW cows (4.1 ± 0.4 ng/ml; Figure 4B). After the injection of PGF2a, progesterone rapidly decreased in 24 h to 0.6 ± 0.3 ng/ml for 2FW cows and 0.3 ± 0.3 ng/ ml for 1FW cows. There was no effect of treatment on the decline in progesterone after PGF2a injection. Expression of Estrus and Pregnancy After PGF2a on d 12
Figure 3. Least squares means for the diameter of the dominant follicle for cows that ovulated during the first follicular wave (1FW cows) (A; SEM = 0.4; π = cows treated with bST; ÿ = control cows) and for cows that ovulated during the second follicular wave (2FW cows) (B; SEM = 0.5 for first wave dominant follicle, π = cows treated with bST; ÿ = control cows; and SEM = 0.4 for second wave dominant follicle, ∫ = cows treated with bST; and o = control cows). Cows received 500 mg of sustained-release bST or saline (control) every 14 d for three injection cycles. Prostaglandin F2a (25 mg) was injected on d 12 of a synchronized estrous cycle in the third injection cycle.
Following injection of PGF2a on d 12, more control cows (10 of 11; 91%; P < 0.05) were observed to be in estrus than cows treated with bST ( 6 of 13; 46%; Table 1). One control cow was not detected to be in estrus because the CL failed to regress in response to the administration of PGF2a. For this cow, CL size did not decrease after PGF2a, and serum progesterone concentration did not decline. One control cow became cystic (no ovulation; follicle >25 mm) but expressed estrus. Of the cows treated with bST that were not observed in estrus, 1 cow became cystic (without estrous expression), and 2 had incomplete CL regression. In these 2 cows, the CL decreased in size, and serum progesterone decreased, but this small CL and low concentration of progesterone was sustained for >10 d. The remaining 4 (29%) cows treated with bST had unobserved ovulations. The responses to estrous synchronization for the first and second estruses are summarized in Table 1. More control cows were observed in estrus than were cows treated with bST (92% vs. 42%). Ten cows Journal of Dairy Science Vol. 80, No. 2, 1997
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treated with bST had undetected ovulations but no control cows did. Similar numbers of cows treated with bST and control cows became cystic or experienced failure of induced luteolysis after PGF2a administration. Following PGF2a-induced luteolysis, 18 cows were inseminated ( 8 cows treated with bST and 10 control cows). These included 6 1FW cows and 12 2FW cows. Of the cows inseminated, conception rates were 3 of 8 (38%) for cows treated with bST, 4 of 10 (40%) for control cows, 2 of 6 (33%) for 1FW cows, and 5 of 12 (42%) for 2FW cows.
Serum bST, Milk Production, BW, and Body Condition Score Prior to treatment, concentrations of bST in serum were similar for treated and control cows (5.1 ± 2.7 and 4.8 ± 2.9 ng/ml, respectively). Following treatment, concentrations of bST in serum were greater ( P < 0.001) in cows treated with bST and averaged ( 0 and 48 h after PGF2a administration) 29.7 ± 2.7 and 25.0 ± 2.7 ng/ml for treated cows and 3.4 ± 2.9 and 4.1 ± 2.9 ng/ml for control cows. Differences in milk production for treated and control cows did not achieve statistical significance (34.2 ± 0.2 vs. 31.3 ± 0.2 kg/d for treated and control cows, respectively), but there was a numerical increase in milk production of 8.5% for cows treated with bST. Body weight increased during the study ( P < 0.001) but was not different between cows treated with bST and control cows on wk 1 (548.0 ± 2.8 kg), wk 4 (557.5 ± 3.2 kg), and wk 6 (567.3 ± 3.3 kg). Body condition score (fivepoint scale, where 1 = thin to 5 = obese) was similar for cows treated with bST (1.7 ± 0.2) and for control cows (2.0 ± 0.2). DISCUSSION
Figure 4. Least squares means for serum concentrations of estradiol (A; SEM = 0.2) and progesterone (B; SEM = 0.2) for cows treated with 500 mg of sustained-release bST or saline (control) every 14 d for three injection cycles. Prostaglandin F2a (25 mg) was injected on d 12 ( d 0 on graph) of a synchronized estrous cycle in the third injection cycle. Cows were further separated based on ovulation of the dominant follicle during the first (1FW cows) or second (2FW cows) follicular wave. Legend: π = 1FW cows treated with bST, ÿ = 1FW cows (control), ∫ = 2FW cows treated with bST, and o = 2FW cows (control). Journal of Dairy Science Vol. 80, No. 2, 1997
Previously, Kirby et al. ( 1 4 ) reported that bST treatment advanced the development of the second follicular wave in cows by 48 h. Therefore, this study was designed to test further the changes in follicular dynamics in cows treated with bST. Prostaglandin F2a was injected on d 12 in an attempt to synchronize the ovulation of the second wave dominant follicle. Unfortunately, about 40% of cows were 1FW cows. However, for 2FW cows, bST apparently advanced the development of the second follicular wave. This conclusion is based on several observations. First, numbers of class 3 follicles were greater and increased earlier in cows treated with bST. Although differences in the development of the preovulatory follicle were not significant ( P = 0.13), the preovulatory follicle appeared to have developed earlier in cows treated with bST (Figure 3B). This change was also observed when serum concentrations of estradiol were examined for 2FW cows. Concentrations of estradiol rose earlier in cows treated with bST than in control cows. These differences suggest a change in the timing of the second follicular wave of about 2 d for cows treated with bST, which agrees with previous observations (14). The reason that follicular dynamics are changed in cows treated with bST is not completely understood. The development of the first wave dominant follicle is synchronized by the LH surge at the beginning of the
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estrous cycle (10, 24, 28, 31). Therefore, 1FW cows were expected to have similar development of this follicle (Figure 3A). The development of the second wave follicle is less synchronous because atresia of the first wave dominant follicle controls the timing of the development of the second wave dominant follicle (28). For an unknown reason, bST causes earlier atresia of the first wave dominant follicle. A possible explanation for this phenomenon is that bST (or IGFI released in response to bST) may accelerate the normal process of granulosa cell growth and differentiation. Dominant follicles have a finite lifespan (particularly in the presence of luteal progesterone and low LH pulse frequency); therefore, if bST accelerates the process, atresia may occur during the estrous cycle. A second possibility is that bST changes concentrations of gonadotropins within the blood. Previous reports (5, 30) have suggested a decrease in basal LH in cows treated with bST. In the present study, serum FSH decreased (Figure 2 ) as it did in a previous study by Kirby et al. (14). Mice expressing a bST transgene also have reduced concentrations of gonadotropins in blood ( 1 ) . Decreases in LH support have been associated with atresia of the first wave dominant follicle in cows (29). Whether LH is actually changed midcycle in cows treated with bST is unknown, however. One unexpected finding from this study was the inhibitory effect of bST on the expression of estrus (Table 1 ) that was observed after two different synchronization protocols. In either case, the total number of ovulations was not reduced in cows treated with bST. Instead, bST treatment caused an increase in the percentage of undetected ovulations. These undetected ovulations represented approximately 30% of the ovulations that occurred in cows treated with bST. Earlier reports by Morbeck et al. (19), Lefebvre and Block (15), and Waterman et al. ( 3 2 ) indicated that cows treated with bST did not express estrus as intensely as did control cows. Lefebvre and Block ( 1 5 ) tested the effects of bST on estrous expression in steroid-primed, ovariectomized heifers and demonstrated reduced estrous expression when heifers were treated with bST. Lefebvre and Block ( 1 5 ) concluded that bST altered behavioral centers within the brain that control estrus expression. Transgenic mice that express a bST transgene or normal mice administered bST also show decreased intensity of mating behavior ( 2 ) . In the present study, the timing of estradiol was changed by bST, but absolute concentrations of progesterone and estradiol were not different between treatments. This finding would suggest that differences in steroid concentrations were not a
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causative factor in the lack of estrus expression in cows treated with bST. Other direct effects on behavioral centers within the brain are more likely. Cows that lose BW or are in poor condition may have increased bST in blood ( 1 3 ) and decreased reproductive performance (25, 33). Therefore, inhibitory effects of bST on expression of estrus may be a naturally occurring, behavioral mechanism to control mating behavior in nutritionally challenged cows. Ovulation of first or second wave follicles after PGF2a injection was associated with differences in numbers of follicles and CL function. The 2FW cows were recruiting follicles at the time of PGF2a administration; therefore, a greater number of class 2 follicles (Figure 1B) was detected in 2FW cows. The increased number of class 2 follicles was followed by an increase in class 3 follicles in 2FW cows (Figure 1C). Class 3 follicles increased 2 d after PGF2a injection in 2FW cows, which corresponded to the time when class 2 follicles were decreasing. These data agree with a model for follicular growth in cows (17). At the time of PGF2a injection, the largest follicles were increasing in diameter for 1FW cows and decreasing in diameter for 2FW cows. For 1FW cows, the first wave dominant follicle grew, suppressed the growth of other follicles, and subsequently ovulated. For the 2FW cows, the largest follicle was not the dominant follicle because it failed to ovulate after PGF2a administration. For the 2FW cows, the dominant follicle was the second largest follicle that grew after PGF2a injection. In agreement with this model, estradiol rose earlier in 1FW cows, because the largest follicle was already mature and secreting estradiol. In 2FW cows, the second largest follicle was the growing dominant follicle, which required several days to reach mature size. Therefore, serum estradiol was slower to rise (Figure 4A). The 2FW cows had higher concentrations of progesterone than did cows ovulating from the first wave follicles. This difference may suggest that rate of development of the CL (measured by progesterone secretion) can affect the turnover and atresia of follicles. Theoretically, higher concentrations of progesterone that occurred earlier during the estrous cycle might have suppressed pulsatile secretion of LH, possibly leading to faster atresia of first wave dominant follicles in cows with greater progesterone (29). This earlier atresia would lead to an earlier recruitment of the second follicular wave in cows with rapidly developing CL. In summary, treatment of lactating cows with bST caused a change in the timing of follicular waves. This advancement in the wave was detected as an Journal of Dairy Science Vol. 80, No. 2, 1997
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earlier development of second wave dominant follicles following injection of PGF2a on d 12 of the estrous cycle. In addition to its effects on follicular dynamics, bST led to a decreased percentage of cows detected to be in standing estrus. This decrease was associated with an increase in the incidence of undetected ovulation. REFERENCES 1 Bartke, A., M. Cesim, K. Tang, R. W. Steger, V. Chandrashekar, and D. Turyn. 1994. Neuroendocrine and productive consequences of overexpression of growth hormone in transgenic mice. Proc. Soc. Exp. Biol. Med. 206:345. 2 Cecim, M., J. Kerr, and A. Bartke. 1995. Effects of bovine growth hormone (bGH) transgene expression or bGH treatment on reproductive functions in female mice. Biol. Reprod. 52:1144. 3 Cole, W. J., P. J. Eppard, B. G. Boysen, K. S. Madsen, R. H. Sorbet, M. A. Miller, R. L. Hintz, T. C. White, W. E. Ribelin, B. G. Hammond, R. J. Collier, and G. M. Lanza. 1992. Response of dairy cows to high doses of a sustained-release bovine somatotropin administered during two lactations. 2. Health and reproduction. J. Dairy Sci. 75:111. 4 Copelin, J. P., M. F. Smith, D. H. Keisler, and H. A. Garverick. 1989. Effect of active immunization of pre-partum and postpartum cows against prostaglandin F-2a on lifespan and progesterone secretion of short-lived corpora lutea. J. Reprod. Fertil. 87:199. 5 Dalton, J. C., and D. P. Marcinkowski. 1994. Effect of sometribove administration on LH concentrations in dairy cattle. Theriogenology 41:437. 6 De La Sota, R. L., M. C. Lucy, C. R. Staples, and W. W. Thatcher. 1993. Effects of recombinant bovine somatotropin (sometribove) on ovarian function in lactating and nonlactating dairy cows. J. Dairy Sci. 76:1002. 7 Edmonson, A. J., I. J. Lean, L. D. Weaver, T. Farver, and G. Webster. 1989. A body condition scoring chart for Holstein dairy cows. J. Dairy Sci. 72:68. 8 Esteban, E., P. H. Kass, L. D. Weaver, J. D. Rowe, C. A. Holmberg, C. E. Franti, and H. F. Troutt. 1994. Interval from calving to conception in high producing dairy cows treated with recombinant bovine somatotropin. J. Dairy Sci. 77:2549. 9 Gallo, G. F., and E. Block. 1991. Effects of recombinant bovine somatotropin on hypophyseal and ovarian functions of lactating dairy cows. Can. J. Anim. Sci. 71:343. 10 Ginther, O. J., J. P. Kastelic, and L. Knopf. 1989. Intraovarian relationships among dominant and subordinate follicles and the corpus luteum in heifers. Theriogenology 35:787. 11 Gong, J. G., T. Branley, and R. Webb. 1991. The effect of recombinant bovine somatotropin on ovarian function in heifers: follicular populations and peripheral hormones. Biol. Reprod. 45:941. 12 Kessler, D. A., and S. F. Sundlof. 1994. BST update. FDA Vet. 10:2. 13 Kirby, C. J., J. D. Armstrong, B. G. Huff, R. L. Stanko, R. W. Harvey, E. P. Heimer, and R. M. Campbell. 1993. Changes in serum somatotropin, somatotropin mRNA, and serum and follicular insulin-like growth factor-I in response to feed restriction in cows actively immunized against growth hormonereleasing factor. J. Anim. Sci. 71:3033.
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14 Kirby, C. J., M. F. Smith, D. H. Keisler, and M. C. Lucy. 1997. Follicular function in lactating dairy cows treated with sustained-released bovine somatotropin. J. Dairy Sci. 80:275. 15 Lefebvre, D. M., and E. Block. 1992. Effect of recombinant bovine somatotropin on estradiol-induced estrous behavior in ovariectomized heifers. J. Dairy Sci. 75:1461. 16 Lucy, M. C., T. L. Curran, R. J. Collier, and W. J. Cole. 1994. Extended function of the corpus luteum and earlier development of the second follicular wave in heifers treated with bovine somatotropin. Theriogenology 41:561. 17 Lucy, M. C., J. D. Savio, L. Badinga, R. L. De La Sota, and W. W. Thatcher. 1992. Factors that affect ovarian follicular dynamics in cattle. J. Anim. Sci. 70:3615. 18 Lucy, M. C., W. W. Thatcher, R. J. Collier, F. A. Simmen, Y. Ko, J. D. Savio, and L. Badinga. 1995. Effects of somatotropin on the conceptus, uterus, and ovary during maternal recognition of pregnancy in cattle. Domest. Anim. Endocrinol. 12:73. 19 Morbeck, D. E., J. H. Britt, and B. T. McDaniel. 1991. Relationships among milk yield, metabolism, and reproductive performance of primiparous Holstein cows treated with somatotropin. J. Dairy Sci. 74:2153. 20 National Research Council. 1989. Nutrient Requirements of Dairy Cattle. 6th rev. ed. Natl. Acad. Sci., Washington, DC. 21 Oldenbroek, J. K., G. J. Garssen, L. J. Jonker, and J.I.D. Wilkinson. 1993. Effects of treatment of dairy cows with recombinant bovine somatotropin over three or four lactations. J. Dairy Sci. 76:453. 22 Page, R. D., D. H. Keisler, R. L. Butcher, R. A. Dailey, and E. K. Inskeep. 1987. Prepubertal and peripubertal changes in secretory patterns of LH and FSH in beef heifers. Anim. Reprod. Sci. 14:85. 23 Peel, C. J., and D. E. Bauman. 1987. Somatotropin and lactation. J. Dairy Sci. 70:474. 24 Rajamahendran, R., and C. Taylor. 1990. Characterization of ovarian activity in postpartum dairy cows using ultrasound imaging and progesterone profiles. Anim. Reprod. Sci. 22:171. 25 Randel, R. D. 1990. Nutrition and postpartum rebreeding in cattle. J. Anim. Sci. 68:853. 26 Safranski, T. J. 1994. Growth hormone transgenic livestock: a mouse model. Ph.D. Diss., Univ. Missouri, Columbia. 27 SAS User’s Guide: Statistics, Version 5 Edition. 1985. SAS Inst., Inc., Cary, NC. 28 Savio, J. D., L. Keenman, M. P. Boland, and J. F. Roche. 1988. Pattern of growth of dominant follicles during the estrous cycle of heifers. J. Reprod. Fertil. 83:663. 29 Savio, J. D., W. W. Thatcher, L. Badinga, R. L. De La Sota, and D. Wolfenson. 1993. Regulation of dominant follicle turnover during the oestrous cycle in cows. J. Reprod. Fertil. 97:197. 30 Schemm, S. R., D. R. Deaver, L. C. Griel, Jr., and L. D. Muller. 1990. Effects of recombinant bovine somatotropin on luteinizing hormone and ovarian function in lactating dairy cows. Biol. Reprod. 42:815. 31 Sirois, J., and J. E. Fortune. 1988. Ovarian follicular dynamics during the estrous cycle in heifers monitored by real-time ultrasonography. Biol. Reprod. 39:308. 32 Waterman, D. F., W. J. Silvia, R. W. Hemken, G. Heersche, Jr., T. S. Swenson, and R. G. Eggert. 1993. Effect of bovine somatotropin on reproductive function in lactating dairy cows. Theriogenology 40:1015. 33 Wettemann, R. P. 1980. Postpartum endocrine function of cattle, sheep and swine. J. Anim. Sci. 51(Suppl. 2):2.
ABSTRACT The objective of this study was to evaluate the timing of follicular waves in cows treated with bovine somatotropin (bST) by measuring ovarian responses to a luteolytic dose of PGF2a on d 12 of the estrous cycle. Thirty lactating cows (26 Holstein and 4 Guernsey) were assigned to receive bST (500 mg; n = 18) or saline (control; 1.5 ml; n = 12) every 14 d for three injection cycles. On d 12 of a synchronized estrous cycle, cows were injected with PGF2a to induce luteolysis. Following PGF2a, 9 cows ovulated from the dominant follicle during the first follicular wave ( 4 cows treated with bST and 5 control cows), and 14 cows ovulated from the dominant follicle during the second follicular wave ( 8 cows treated with bST and 6 control cows). Of the cows that ovulated during the second follicular wave, cows treated with bST had more class 3 follicles ( ≥10 mm) than did control cows. Concentrations of estradiol rose earlier after PGF2a injection in cows treated with bST than in control cows. This rise in estradiol was parallel to the development of dominant follicles. Serum concentrations of FSH were decreased in cows treated with bST. During the first and second estruses, equivalent numbers of cows treated with bST and control cows ovulated, but fewer cows treated with bST expressed estrus. These results are consistent with the hypothesis that cows treated with bST have reduced FSH, a faster turnover of dominant follicles, and differences in the timing of follicular waves. Treatment of cows with bST also increased the incidence of undetected estrus. ( Key words: bovine somatotropin, ovary, cow, luteolysis) Abbreviation key: CL = corpus luteum, 1FW cows = cows that ovulated from the dominant follicle during the first follicular wave, 2FW cows = cows that
Received August 18, 1995. Accepted March 15, 1996. 1Contribution from the Missouri Agricultural Experiment Station, Journal Series Number 12383. 2Corresponding author: Matthew C. Lucy, Department of Animal Science Research Center, University of Missouri, Columbia 65211. 1997 J Dairy Sci 80:286–294
ovulated from the dominant follicle during the second follicular wave. INTRODUCTION Administration of bST to dairy cows increases milk production by approximately 10 to 20% (23). Because of its galactopoietic effect, bST was developed as a recombinant hormone to increase the milk production of lactating dairy cows. One form of recombinant bST (sometribove; Monsanto Co., St. Louis, MO) was approved for sale in the US by the FDA in November 1993. Since that time, approximately 10% of dairy cows in the US have been treated with bST (12). This percentage is expected to increase as producers become more familiar with recombinant bST. Several studies (3, 5, 8, 19, 21, 30, 32) have examined the long-term effects of bST on reproduction in cows, and most have detected modest declines in reproductive performance of cows treated with bST. These declines include increased days open and a decrease in herd pregnancy rate. Many of these reproductive losses are associated with a temporary negative energy balance experienced by cows treated with bST. This physiological change appears to delay rebreeding. The combined effects of bST and IGF-I have measurable effects on ovarian function of cows. Treatment with exogenous bST increases the number of ovarian follicles within distinct classes of follicular diameter [3 to 5 mm in heifers and 6 to 9 mm in lactating cows (6, 11)]. There also appear to be effects on the corpus luteum ( CL) , including increased CL weight ( 1 8 ) and increased concentrations of progesterone in blood of cows treated with bST (9, 16, 30). In addition to changes in follicular numbers and CL size, our laboratory has detected a change in the timing of follicular waves in cows treated with bST (14). This change involves an advancement in the timing of the second follicular wave with earlier recruitment of second wave follicles and earlier development of the dominant follicle during the second follicular wave. This study was designed to test further changes in follicular dynamics of cows treated with bST. Lactating dairy cows treated with saline (control) or bST were given a luteolytic dose of PGF2a on d 12 of the estrous cycle. This treatment coincided with a period of dominance for the first follicular wave or recruit-
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ment of the second follicular wave (associated with first wave atresia of the dominant follicle). If bST changes the timing of the first or second follicular wave, then follicular response to PGF2a on d 12 should be different. Therefore, changes in patterns of follicular growth in cows treated with bST were evaluated based on responses of ovarian follicles to luteolysis on d 12. MATERIALS AND METHODS Cows and Treatments Twenty-six Holstein and 4 Guernsey cows were used. Cows were housed in a concrete floor, free-stall barn with fence-row feeders equipped with locking head gates. The study was conducted between November and December 1994 as a single replicate. Twice daily, all cows were fed a TMR consisting of corn silage, alfalfa haylage, alfalfa hay, hominy, ground corn, whole cottonseed, and corn and soybean premix formulated to meet or exceed the NRC ( 2 0 ) nutritional requirements (1.67 Mcal/kg of NEL, 17.8% CP, 35.2% ADF, and 5.8% ether extract). Cows were milked twice daily, and milk production was measured using electronic flow meters. Water was available for ad libitum intake. The University of Missouri Animal Care and Use Committee approved all experimental procedures. Eighteen cows (16 Holstein and 2 Guernsey) were randomly assigned to receive treatment with bST (500 mg of bST; Posilac; Monsanto Co.), and 12 cows (10 Holstein and 2 Guernsey) received sterile saline (control; Abbott Laboratories, North Chicago, IL). Injections were administered subcutaneously into the tailhead region using 16-gauge, 16-mm (5/ 8-inch) needles. Posilac, a sustained-release form of recombinant bST, was given once every 14 d (single injection cycle). Days postpartum were similar for bST and control cows and averaged 65 ± 5 d at first injection. Estrous cycles were synchronized so that estrus occurred 7 d after the start of the second injection cycle. Two norgestomet implants (SyncroMate-B; Sanofi Animal Health, Overland Park, KS) were implanted for 9 d, and two injections (25 and 12.5 mg) of PGF2a (Lutalyse; The Upjohn Co., Kalamazoo, MI) were administered on d 7 and 8 of norgestomet implant. Estrus (expected 2 d after implant removal) was detected by 30-min visual observations in the morning (0600 h), in the afternoon (1400 h), and at night (2200 h). Cows were not inseminated at the synchronized estrus. All cows not seen in estrus were examined by ultrasound for signs of ovulation on the 4th d after implant removal. A cow was diagnosed as having ovulated if a developing CL was detected by ultrasound. Cows that did not ex-
press estrus or that did not ovulate (based on ultrasound examination) were removed from study ( n = 6; 1 control cow and 5 cows treated with bST). Cows remaining in the study were injected with 25 mg of PGF2a on d 12 after norgestomet-synchronized estrus. This PGF2a injection occurred 4 d after the third injection cycle of saline or bST. Ultrasound and Data Collection Beginning on d 10 of the norgestomet-synchronized estrous cycle and continuing until PGF2a-induced estrus, ovaries were examined daily by transrectal ultrasonography. Ultrasound examinations were performed with an ultrasound scanner (Aloka 500V; Corometrics Medical Systems Inc., Wallingford, CT) with a 7.5-MHz linear transducer. Number and diameter of all follicles ≥3 mm in diameter were recorded. Follicles were grouped into three diameter classes for analysis: class 1 ( 3 to 5 mm), class 2 ( 6 to 9 mm), and class 3 ( ≥10 mm). The development of the ovarian follicle was followed by mapping the location of large follicles on the ovary. Based on the growth of large follicles, the ovarian follicle was classified as either the first or second wave dominant follicle. The maximum cross-sectional area of the CL and the area of the fluid-filled cavity within the CL were recorded. Daily ultrasound examinations ended on the day of estrus following PGF2a administration. Blood samples were collected by coccygeal venipuncture once daily on d 10 and 11 of the synchronized estrous cycle. Following PGF2a injection on d 12, blood was collected every 6 h for 24 h. Blood was collected twice daily (12-h interval) on d 13 and 14 and once daily from d 15 until estrus. Ten milliliters of blood were collected into Vacutainer tubes (Becton Dickinson and Co., Rutherford, NJ), stored overnight at 4°C, and centrifuged (3000 × g for 15 min) for collection of serum. Serum was stored at –20°C until analyses. Body weight was recorded after the morning milking and feeding on wk 1, 4, and 6 of the experiment. Body condition scoring ( 7 ) was performed at the beginning (wk 1 ) and at the end of the study (wk 6). Radioimmunoassays Concentrations of serum progesterone were determined in all samples using an extraction assay that had been previously validated ( 4 ) . Intraassay and interassay coefficients of variation for progesterone were 5.5 and 12.6%, respectively. Serum estradiol ( 1 4 ) and FSH ( 2 2 ) were determined in daily samples. Intraassay and interassay coefficients of variation for estradiol were 6.0 and 5.3%, respectively. Journal of Dairy Science Vol. 80, No. 2, 1997
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Serum FSH was analyzed in a single assay; the intraassay coefficient of variation was 10.2%. Serum bST ( 2 6 ) was analyzed in a single assay for samples that were collected prior to treatment and 0 and 48 h after PGF2a. The intraassay coefficient of variation was 15.3%.
interval to estrus after PGF2a and the diameter of the preovulatory follicle were tested using the model Yilm = m + Ai + Wl + AWil + eilm. The percentage of cows responding to synchronization was tested using a chisquare test. Data are presented as least squares means and standard errors of the least squares means. RESULTS
Statistical Analyses Data were analyzed by least squares analysis of variance ( 2 7 ) as a split-plot design. The statistical model used in the analysis of milk production and serum bST was Yijk = m + Ai + B ( A ) j + Ck + ACik + eijk, where Yijk = dependent observation, m = overall mean, Ai = effect of treatment i, B ( A ) j = effect of cow j nested within treatment i, Ck = effect of day k, ACik = treatment by day interaction, and eijk = residual effect. The main effect of treatment was tested using cow nested within treatment as the error term. Remaining terms were tested with the residual effect. A second model was used to test the effects of follicle diameter, number of follicles, CL area, progesterone, and FSH. This model included the effects of follicular wave: Yijkl = m + Ai + Wl + AWil + B(AW) j + Ck + ACik + WCkl + ACWikl + eijkl, where Wl = effect of follicular wave l (first vs. second), AWil = treatment by follicular wave interaction, WCkl = follicular wave by day interaction, and ACWikl = treatment by follicular wave by day interaction. The effects of treatment, follicular wave, and treatment by follicular wave interaction were tested; cow nested within treatment by follicular wave interaction was the error term. The
Estrous Expression After Synchronization of Estrus At the norgestomet-synchronized estrus, 92% (11 of 12) of the control cows were observed to be in estrus (standing to be mounted by a herdmate), and 39% ( 7 of 18) of the cows treated with bST were observed to be in estrus (Table 1; P < 0.005). All cows observed to be in estrus were detected during a 24-h period beginning 36 h after implant removal. Those cows that failed to display estrus were examined by ultrasound on the 4th d after implant removal. Based on these examinations, 1 control cow was diagnosed as cystic (absence of a CL; follicle >25 mm) and was removed from the study. Of the 11 cows treated with bST that did not express estrus, 1 failed to have CL regression in response to the PGF2a injection, and 4 had several class 3 follicles with no CL. These 5 cows were removed from the study. The remaining 6 treated cows had developing CL and were in the recruitment phase of the first follicular wave, indicating that they had ovulated. These 6 cows remained on the study.
TABLE 1. Estrous expression in lactating cows treated with 500 mg of sustained-release bST or saline (control). First estrus1
Second estrus2
Total3
Item
bST
Control
bST
Control
bST
Control
Observations, no.
18 (no.) 7 6 0 4 1 0 13 3
12 (no.) 11 0 1 0 0 0 11 2
13 (no.) 6 4 1 0 0 2 10 1
11 (no.) 10 0 1 0 1 0 9 2
31 (no.) 13 10 1 4 1 2 23 4
23 (no.) 21 0 1 0 1 0 20 4
Standing estrus4 Undetected ovulation4 Cystic ovary Abnormal ovary No CL5 regression Incomplete CL regression Total to ovulate Double ovulations
(%) 39 33 0 22 6 0 72 23
(%) 92 0 8 0 0 0 92 18
(%) 46 29 8 0 0 15 77 10
(%) 91 0 9 0 9 0 82 22
(%) 42 32 3 13 3 6 74 17
(%) 92 0 4 0 4 0 87 20
1Estrus was synchronized with two norgestomet implants for 9 d and two injections (25 and 12.5 mg) of PGF on d 7 and 8, 24 h 2a apart. 2Luteolysis was induced with 25 mg of PGF 2a on d 12 of the synchronized estrous cycle. 3Combination of first and second estruses. 4P < 0.01 (total estruses). 5Corpus luteum.
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Development of Follicular Waves and Ovulation After PGF2a Prior to injection of PGF2a on d 12 of the estrous cycle, there was no effect of treatment on double ovulations (Table 1). Following the injection of PGF2a, 9 cows ovulated from the first wave dominant follicle ( 4 cows treated with bST and 5 control cows) and 14 cows ovulated from a second wave dominant follicle ( 8 cows treated with bST and 6 control cows). The number of cows treated with bST and control cows ovulating from first or second wave follicles was similar (Table 2). Interval to estrus was shorter ( P < 0.05) for cows that ovulated from the dominant follicle during the first follicular wave ( 1FW cows; 3.3 ± 0.7 d ) than for cows that ovulated during the second follicular wave ( 2FW cows; 5.2 ± 0.5 d). The effect of treatment was not significant, and no treatment by wave interaction was detected. Diameter of the preovulatory follicle on the day of estrus was not affected by wave or treatment. In addition, neither treatment nor wave affected ovulation rate following PGF2a injection. Number and Diameters of Follicles and Concentrations of FSH in Serum Numbers of class 1, 2, and 3 follicles for 1FW cows and for 2FW cows in each treatment are presented in Table 2. Numbers of class 1 follicles decreased (day
effect; P < 0.0001; Figure 1A) following the injection of PGF2a and averaged 10.1 ± 0.7 follicles per cow on d 12 (day of PGF2a injection) and 4.5 ± 0.8 follicles per cow on d 15 ( 3 d after PGF2a injection). There was no effect of treatment or follicular wave on numbers of class 1 follicles. The numbers of class 2 follicles increased after PGF2a injection and then decreased (day effect; P < 0.001; Figure 1B). There was a main effect of follicular wave ( P < 0.02) for class 2 follicles because 1FW cows had fewer class 2 follicles than did 2FW cows (3.3 ± 0.7 vs. 5.5 ± 0.5 follicles per cow for 1FW and 2FW cows, respectively). There was no effect of treatment on numbers of class 2 follicles. Numbers of class 3 follicles increased (day effect; P < 0.001) from d 2 before (1.2 ± 0.1 follicles per cow) to d 4 after (2.3 ± 0.2 follicles per cow) PGF2a injection. Compared with results for 1FW cows, the number of class 3 follicles in 2FW cows tended to increase sooner after PGF2a injection (follicular wave by day interaction, P < 0.09; Figure 1C). A follicular wave by treatment interaction ( P < 0.01) was detected for numbers of class 3 follicles. The 2FW cows treated with bST had a greater number of class 3 follicles than did the 2FW cows treated with saline (2.2 ± 0.1 vs. 1.6 ± 0.1 follicles per cow for cows treated with bST and control cows, respectively; P < 0.002). At the same time, 1FW cows treated with bST had fewer class 3 follicles than did 1FW cows treated with saline (1.3 ± 0.3 vs. 1.9 ± 0.3 follicles per cow for
TABLE 2. Characteristics of follicular development and ovulation from d 10 until estrus after an injection of PGF2a on d 12 during a synchronized estrous cycle in lactating cows1 treated with 500 mg of sustained-release bST or saline (control). 1FW Cows
2FW Cows
Item
bST
Control
bST
Control
Cows, no.
4
5
8
6
Follicles, no. Class 1 ( 3 to 5 mm) Class 2 ( 6 to 9 mm) 2 Class 3 ( ≥10 mm) 3 Largest follicle, mm Second largest follicle, mm4 Dominant follicle, mm First follicular wave Second follicular wave Interval to estrus, d5 Diameter of ovulatory follicle, mm
X
SEM
X
SEM
X
SEM
X
SEM
5.9 2.9 1.3 17.4 8.1
1.3 1.0 0.3 1.2 1.1
7.3 3.7 1.9 16.6 10.3
1.5 1.1 0.3 1.3 1.2
7.0 5.1 2.2 15.7 11.1
0.8 0.6 0.2 0.7 0.7
8.1 6.0 1.6 16.3 9.6
1.0 0.7 0.2 0.9 0.8
17.4 . . . 3.0 18.7
1.3 . . . 1.1 1.2
16.6 . . . 3.5 18.5
1.4 . . . 1.0 1.1
15.0 11.3 4.0 18.0
0.8 1.0 0.7 0.8
16.0 8.8 6.3 18.2
0.9 1.2 0.8 1.0
11FW Cows = Cows that ovulated from the dominant follicle during the first follicular wave; 2FW cows = cows that ovulated from the dominant follicle during the second follicular wave. 2Follicular wave ( P < 0.02). 3Follicular wave by treatment interaction ( P < 0.01). 4Follicular wave by treatment interaction ( P < 0.08). 5Follicular wave ( P < 0.05).
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Figure 2. Least squares means for concentration of FSH in serum. Cows received 500 mg of sustained-release bST ( π; SEM = 0.05) or saline (control; ÿ; SEM = 0.05) every 14 d for three injection cycles. Prostaglandin F2a (25 mg) was injected on d 12 of a synchronized estrous cycle in the third injection cycle.
Figure 1. Least squares means for the number of follicles in different follicular diameter classes: class 1 (A; 3 to 5 mm; SEM = 0.7), class 2 (B; 6 to 9 mm; SEM = 0.4), and class 3 (C; ≥10 mm; SEM = 0.1). Cows were treated with 500 mg of sustained-release bST or saline (control) every 14 d for three injection cycles. Prostaglandin F2a (25 mg) was injected on d 12 of a synchronized estrous cycle in the third injection cycle. Cows within each treatment were grouped according to whether they ovulated from the dominant follicle during the first follicular wave ( ÿ) or the second follicular wave ( o) . Journal of Dairy Science Vol. 80, No. 2, 1997
cows treated with bST and control cows, respectively). Concentrations of FSH in serum were similar in 1FW and 2FW cows (0.63 ± 0.04 and 0.61 ± 0.03 ng/ ml, respectively), but concentrations were decreased in serum of treated cows ( P < 0.02; Figure 2 ) compared with concentrations in control cows. Mean serum FSH was 0.56 ± 0.03 and 0.68 ± 0.03 ng/ml for treated and control cows, respectively. No treatment by follicular wave or treatment by day interaction was detected ( P > 0.10). During the experimental period, the diameter of the largest follicle (not necessarily a dominant follicle) on the ovary was similar for cows treated with bST and control cows (Table 2). There was a tendency ( P < 0.08) for a follicular wave by treatment interaction for the second largest follicle because 1FW treated cows had smaller second largest follicles than did 1FW control cows, and 2FW treated cows had a larger second largest follicle than 2FW control cows. The first-wave dominant follicle increased in diameter for 1FW cows (Figure 3A) and decreased in diameter for 2FW cows (Figure 3B; follicular wave by day interaction, P < 0.001). The growth of the dominant follicle during the first follicular wave was not different for cows treated with bST and control cows that ovulated during the first follicular wave (Figure 3A; Table 2). For 2FW cows, the dominant follicle of the first follicular wave decreased in diameter as the preovulatory follicle increased in diameter from d 2 before (5.3 ± 0.4 mm) to 4 d after
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(14.7 ± 0.4 mm; Figure 3B) PGF2a injection. The dominant follicle of the second wave tended ( P < 0.13) to be larger in cows treated with bST; an equivalent diameter of the second wave dominant follicle occurred approximately 2 d later in control cows than in cows treated with bST. Size of the CL The size of the CL (luteal tissue plus fluid-filled center cavity) was similar between cows treated with
bST and control cows (4.3 ± 0.1 cm2) . The 1FW cows had larger CL at the time of PGF2a injection (5.9 ± 0.2 vs. 5.1 ± 0.2 cm2 for 1FW and 2FW cows, respectively) that declined to a similar size by d 3 after PGF2a injection (2.1 ± 0.3 vs. 2.3 ± 0.2 cm2 for 1FW and 2FW cows, respectively; follicular wave by day interaction, P < 0.10). When the size of the fluid-filled center was subtracted from CL area, no differences were detected in follicular wave or treatment. Mean size of the luteal tissue area (CL minus hollow cavity) was 3.5 ± 0.1 cm2. Estradiol, Progesterone, and CL Decline Serum estradiol increased ( P < 0.001) from PGF2a injection until estrus (Figure 4A). Estradiol increased earlier in 1FW cows than in 2FW cows (follicular wave by day interaction, P < 0.03). Serum estradiol was similar for cows treated with bST and control cows that ovulated the first wave dominant follicle. However, for 2FW cows, estradiol increased earlier in cows treated with bST than in control cows (treatment by day interaction, P < 0.05). Before the injection of PGF2a, serum progesterone was greater ( P < 0.06) in 2FW cows (6.0 ± 0.3 ng/ml) than in 1FW cows (4.1 ± 0.4 ng/ml; Figure 4B). After the injection of PGF2a, progesterone rapidly decreased in 24 h to 0.6 ± 0.3 ng/ml for 2FW cows and 0.3 ± 0.3 ng/ ml for 1FW cows. There was no effect of treatment on the decline in progesterone after PGF2a injection. Expression of Estrus and Pregnancy After PGF2a on d 12
Figure 3. Least squares means for the diameter of the dominant follicle for cows that ovulated during the first follicular wave (1FW cows) (A; SEM = 0.4; π = cows treated with bST; ÿ = control cows) and for cows that ovulated during the second follicular wave (2FW cows) (B; SEM = 0.5 for first wave dominant follicle, π = cows treated with bST; ÿ = control cows; and SEM = 0.4 for second wave dominant follicle, ∫ = cows treated with bST; and o = control cows). Cows received 500 mg of sustained-release bST or saline (control) every 14 d for three injection cycles. Prostaglandin F2a (25 mg) was injected on d 12 of a synchronized estrous cycle in the third injection cycle.
Following injection of PGF2a on d 12, more control cows (10 of 11; 91%; P < 0.05) were observed to be in estrus than cows treated with bST ( 6 of 13; 46%; Table 1). One control cow was not detected to be in estrus because the CL failed to regress in response to the administration of PGF2a. For this cow, CL size did not decrease after PGF2a, and serum progesterone concentration did not decline. One control cow became cystic (no ovulation; follicle >25 mm) but expressed estrus. Of the cows treated with bST that were not observed in estrus, 1 cow became cystic (without estrous expression), and 2 had incomplete CL regression. In these 2 cows, the CL decreased in size, and serum progesterone decreased, but this small CL and low concentration of progesterone was sustained for >10 d. The remaining 4 (29%) cows treated with bST had unobserved ovulations. The responses to estrous synchronization for the first and second estruses are summarized in Table 1. More control cows were observed in estrus than were cows treated with bST (92% vs. 42%). Ten cows Journal of Dairy Science Vol. 80, No. 2, 1997
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treated with bST had undetected ovulations but no control cows did. Similar numbers of cows treated with bST and control cows became cystic or experienced failure of induced luteolysis after PGF2a administration. Following PGF2a-induced luteolysis, 18 cows were inseminated ( 8 cows treated with bST and 10 control cows). These included 6 1FW cows and 12 2FW cows. Of the cows inseminated, conception rates were 3 of 8 (38%) for cows treated with bST, 4 of 10 (40%) for control cows, 2 of 6 (33%) for 1FW cows, and 5 of 12 (42%) for 2FW cows.
Serum bST, Milk Production, BW, and Body Condition Score Prior to treatment, concentrations of bST in serum were similar for treated and control cows (5.1 ± 2.7 and 4.8 ± 2.9 ng/ml, respectively). Following treatment, concentrations of bST in serum were greater ( P < 0.001) in cows treated with bST and averaged ( 0 and 48 h after PGF2a administration) 29.7 ± 2.7 and 25.0 ± 2.7 ng/ml for treated cows and 3.4 ± 2.9 and 4.1 ± 2.9 ng/ml for control cows. Differences in milk production for treated and control cows did not achieve statistical significance (34.2 ± 0.2 vs. 31.3 ± 0.2 kg/d for treated and control cows, respectively), but there was a numerical increase in milk production of 8.5% for cows treated with bST. Body weight increased during the study ( P < 0.001) but was not different between cows treated with bST and control cows on wk 1 (548.0 ± 2.8 kg), wk 4 (557.5 ± 3.2 kg), and wk 6 (567.3 ± 3.3 kg). Body condition score (fivepoint scale, where 1 = thin to 5 = obese) was similar for cows treated with bST (1.7 ± 0.2) and for control cows (2.0 ± 0.2). DISCUSSION
Figure 4. Least squares means for serum concentrations of estradiol (A; SEM = 0.2) and progesterone (B; SEM = 0.2) for cows treated with 500 mg of sustained-release bST or saline (control) every 14 d for three injection cycles. Prostaglandin F2a (25 mg) was injected on d 12 ( d 0 on graph) of a synchronized estrous cycle in the third injection cycle. Cows were further separated based on ovulation of the dominant follicle during the first (1FW cows) or second (2FW cows) follicular wave. Legend: π = 1FW cows treated with bST, ÿ = 1FW cows (control), ∫ = 2FW cows treated with bST, and o = 2FW cows (control). Journal of Dairy Science Vol. 80, No. 2, 1997
Previously, Kirby et al. ( 1 4 ) reported that bST treatment advanced the development of the second follicular wave in cows by 48 h. Therefore, this study was designed to test further the changes in follicular dynamics in cows treated with bST. Prostaglandin F2a was injected on d 12 in an attempt to synchronize the ovulation of the second wave dominant follicle. Unfortunately, about 40% of cows were 1FW cows. However, for 2FW cows, bST apparently advanced the development of the second follicular wave. This conclusion is based on several observations. First, numbers of class 3 follicles were greater and increased earlier in cows treated with bST. Although differences in the development of the preovulatory follicle were not significant ( P = 0.13), the preovulatory follicle appeared to have developed earlier in cows treated with bST (Figure 3B). This change was also observed when serum concentrations of estradiol were examined for 2FW cows. Concentrations of estradiol rose earlier in cows treated with bST than in control cows. These differences suggest a change in the timing of the second follicular wave of about 2 d for cows treated with bST, which agrees with previous observations (14). The reason that follicular dynamics are changed in cows treated with bST is not completely understood. The development of the first wave dominant follicle is synchronized by the LH surge at the beginning of the
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estrous cycle (10, 24, 28, 31). Therefore, 1FW cows were expected to have similar development of this follicle (Figure 3A). The development of the second wave follicle is less synchronous because atresia of the first wave dominant follicle controls the timing of the development of the second wave dominant follicle (28). For an unknown reason, bST causes earlier atresia of the first wave dominant follicle. A possible explanation for this phenomenon is that bST (or IGFI released in response to bST) may accelerate the normal process of granulosa cell growth and differentiation. Dominant follicles have a finite lifespan (particularly in the presence of luteal progesterone and low LH pulse frequency); therefore, if bST accelerates the process, atresia may occur during the estrous cycle. A second possibility is that bST changes concentrations of gonadotropins within the blood. Previous reports (5, 30) have suggested a decrease in basal LH in cows treated with bST. In the present study, serum FSH decreased (Figure 2 ) as it did in a previous study by Kirby et al. (14). Mice expressing a bST transgene also have reduced concentrations of gonadotropins in blood ( 1 ) . Decreases in LH support have been associated with atresia of the first wave dominant follicle in cows (29). Whether LH is actually changed midcycle in cows treated with bST is unknown, however. One unexpected finding from this study was the inhibitory effect of bST on the expression of estrus (Table 1 ) that was observed after two different synchronization protocols. In either case, the total number of ovulations was not reduced in cows treated with bST. Instead, bST treatment caused an increase in the percentage of undetected ovulations. These undetected ovulations represented approximately 30% of the ovulations that occurred in cows treated with bST. Earlier reports by Morbeck et al. (19), Lefebvre and Block (15), and Waterman et al. ( 3 2 ) indicated that cows treated with bST did not express estrus as intensely as did control cows. Lefebvre and Block ( 1 5 ) tested the effects of bST on estrous expression in steroid-primed, ovariectomized heifers and demonstrated reduced estrous expression when heifers were treated with bST. Lefebvre and Block ( 1 5 ) concluded that bST altered behavioral centers within the brain that control estrus expression. Transgenic mice that express a bST transgene or normal mice administered bST also show decreased intensity of mating behavior ( 2 ) . In the present study, the timing of estradiol was changed by bST, but absolute concentrations of progesterone and estradiol were not different between treatments. This finding would suggest that differences in steroid concentrations were not a
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causative factor in the lack of estrus expression in cows treated with bST. Other direct effects on behavioral centers within the brain are more likely. Cows that lose BW or are in poor condition may have increased bST in blood ( 1 3 ) and decreased reproductive performance (25, 33). Therefore, inhibitory effects of bST on expression of estrus may be a naturally occurring, behavioral mechanism to control mating behavior in nutritionally challenged cows. Ovulation of first or second wave follicles after PGF2a injection was associated with differences in numbers of follicles and CL function. The 2FW cows were recruiting follicles at the time of PGF2a administration; therefore, a greater number of class 2 follicles (Figure 1B) was detected in 2FW cows. The increased number of class 2 follicles was followed by an increase in class 3 follicles in 2FW cows (Figure 1C). Class 3 follicles increased 2 d after PGF2a injection in 2FW cows, which corresponded to the time when class 2 follicles were decreasing. These data agree with a model for follicular growth in cows (17). At the time of PGF2a injection, the largest follicles were increasing in diameter for 1FW cows and decreasing in diameter for 2FW cows. For 1FW cows, the first wave dominant follicle grew, suppressed the growth of other follicles, and subsequently ovulated. For the 2FW cows, the largest follicle was not the dominant follicle because it failed to ovulate after PGF2a administration. For the 2FW cows, the dominant follicle was the second largest follicle that grew after PGF2a injection. In agreement with this model, estradiol rose earlier in 1FW cows, because the largest follicle was already mature and secreting estradiol. In 2FW cows, the second largest follicle was the growing dominant follicle, which required several days to reach mature size. Therefore, serum estradiol was slower to rise (Figure 4A). The 2FW cows had higher concentrations of progesterone than did cows ovulating from the first wave follicles. This difference may suggest that rate of development of the CL (measured by progesterone secretion) can affect the turnover and atresia of follicles. Theoretically, higher concentrations of progesterone that occurred earlier during the estrous cycle might have suppressed pulsatile secretion of LH, possibly leading to faster atresia of first wave dominant follicles in cows with greater progesterone (29). This earlier atresia would lead to an earlier recruitment of the second follicular wave in cows with rapidly developing CL. In summary, treatment of lactating cows with bST caused a change in the timing of follicular waves. This advancement in the wave was detected as an Journal of Dairy Science Vol. 80, No. 2, 1997
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earlier development of second wave dominant follicles following injection of PGF2a on d 12 of the estrous cycle. In addition to its effects on follicular dynamics, bST led to a decreased percentage of cows detected to be in standing estrus. This decrease was associated with an increase in the incidence of undetected ovulation. REFERENCES 1 Bartke, A., M. Cesim, K. Tang, R. W. Steger, V. Chandrashekar, and D. Turyn. 1994. Neuroendocrine and productive consequences of overexpression of growth hormone in transgenic mice. Proc. Soc. Exp. Biol. Med. 206:345. 2 Cecim, M., J. Kerr, and A. Bartke. 1995. Effects of bovine growth hormone (bGH) transgene expression or bGH treatment on reproductive functions in female mice. Biol. Reprod. 52:1144. 3 Cole, W. J., P. J. Eppard, B. G. Boysen, K. S. Madsen, R. H. Sorbet, M. A. Miller, R. L. Hintz, T. C. White, W. E. Ribelin, B. G. Hammond, R. J. Collier, and G. M. Lanza. 1992. Response of dairy cows to high doses of a sustained-release bovine somatotropin administered during two lactations. 2. Health and reproduction. J. Dairy Sci. 75:111. 4 Copelin, J. P., M. F. Smith, D. H. Keisler, and H. A. Garverick. 1989. Effect of active immunization of pre-partum and postpartum cows against prostaglandin F-2a on lifespan and progesterone secretion of short-lived corpora lutea. J. Reprod. Fertil. 87:199. 5 Dalton, J. C., and D. P. Marcinkowski. 1994. Effect of sometribove administration on LH concentrations in dairy cattle. Theriogenology 41:437. 6 De La Sota, R. L., M. C. Lucy, C. R. Staples, and W. W. Thatcher. 1993. Effects of recombinant bovine somatotropin (sometribove) on ovarian function in lactating and nonlactating dairy cows. J. Dairy Sci. 76:1002. 7 Edmonson, A. J., I. J. Lean, L. D. Weaver, T. Farver, and G. Webster. 1989. A body condition scoring chart for Holstein dairy cows. J. Dairy Sci. 72:68. 8 Esteban, E., P. H. Kass, L. D. Weaver, J. D. Rowe, C. A. Holmberg, C. E. Franti, and H. F. Troutt. 1994. Interval from calving to conception in high producing dairy cows treated with recombinant bovine somatotropin. J. Dairy Sci. 77:2549. 9 Gallo, G. F., and E. Block. 1991. Effects of recombinant bovine somatotropin on hypophyseal and ovarian functions of lactating dairy cows. Can. J. Anim. Sci. 71:343. 10 Ginther, O. J., J. P. Kastelic, and L. Knopf. 1989. Intraovarian relationships among dominant and subordinate follicles and the corpus luteum in heifers. Theriogenology 35:787. 11 Gong, J. G., T. Branley, and R. Webb. 1991. The effect of recombinant bovine somatotropin on ovarian function in heifers: follicular populations and peripheral hormones. Biol. Reprod. 45:941. 12 Kessler, D. A., and S. F. Sundlof. 1994. BST update. FDA Vet. 10:2. 13 Kirby, C. J., J. D. Armstrong, B. G. Huff, R. L. Stanko, R. W. Harvey, E. P. Heimer, and R. M. Campbell. 1993. Changes in serum somatotropin, somatotropin mRNA, and serum and follicular insulin-like growth factor-I in response to feed restriction in cows actively immunized against growth hormonereleasing factor. J. Anim. Sci. 71:3033.
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