Reproductive Outcomes for Dairy Heifers Treated with Combinations of Prostaglandin F2α, Norgestomet, and Gonadotropin-Releasing Hormone1

Reproductive Outcomes for Dairy Heifers Treated with Combinations of Prostaglandin F2α, Norgestomet, and Gonadotropin-Releasing Hormone1 J. S. Stevenson,* J. F. Smith,* and D. E. Hawkins† *Department of Animal Sciences and Industry Kansas State University, Manhattan 66506-0201 †Department of Animal and Range Science New Mexico State University, Las Cruces 88003-0003

ABSTRACT We conducted three experiments to test various protocols for synchronizing estrus, ovulation, or both before insemination of heifers. In experiment 1, 23 controls received two PGF2α injections; 23 heifers were treated like the controls plus a norgestomet implant for 8 d, with the second PGF2α injection 24 h before implant removal; and 23 heifers were treated like the previous group plus 100 µg of GnRH 54 h after the second PGF2α injection. Although norgestomet and GnRH altered some estrual characteristics, conception rates in experiment 1 (n = 69) and experiment 2 (278 heifers receiving the same treatments as those in experiment 1) generally were not different among treatments. Reproductive outcomes were not improved by adding norgestomet and GnRH to a standard PGF2α protocol. In experiment 3, control heifers received PGF2α and were inseminated after detected estrus or at 72 to 80 h after a second injection of PGF2α given 14 d after the first injection. Select Synch heifers, treated with GnRH either 6 or 7 d before PGF2α, were inseminated after detected estrus, whereas Ovsynch heifers were treated like Select Synch heifers but also received a second GnRH injection approximately 36 h after PGF2α and were inseminated 18 h later. Estrus detection and pregnancy rates after Ovsynch were less than those of controls, whereas conception and pregnancy rates did not differ between control and Select Synch heifers. Therefore, the Select Synch protocol was equivalent to a standard PGF2α protocol, whereas Ovsynch was inferior to both of those protocols. (Key words: estrus synchronization, heifers, hormones, timed artificial insemination)

Received November 3, 1999. Accepted March 14, 2000. Corresponding author: J. S. Stevenson; e-mail: jstevens@oznet. ksu.edu. 1 Contribution Number 00-38-J from the Kansas Agricultural Experiment Station, Manhattan. 2000 J Dairy Sci 83:2008–2015

Abbreviation key: E2 = estradiol-17β, NORG = norgestomet, Ovsynch = injections of GnRH 7 d before and 36 h after PGF2α with AI at 18 h after the second GnRH injection, P4 = progesterone, RIA = radioimmunoassy, Select Synch = injection of GnRH 7 d before PGF2α and AI after detected estrus. INTRODUCTION A major factor in the total cost of raising dairy replacement heifers is the age at first calving (8). Average age changed little from 1960 to 1980, but more recent data suggest that age at first calving is declining (7). The literature provides numerous examples of how controlling the estrous cycle is an effective way to accommodate the use of AI (3, 13) in dairy replacements to impregnate heifers conveniently and reduce age at first calving. Despite the advent of several estrus-synchronization methods, some dairy producers do not use these methods or AI (24) for various reasons. Time required to detect estrus is often a major limitation to the use of AI in heifers. Programs that successfully incorporate fixed-time inseminations with limited labor could eliminate some of the disadvantages seen by those who do not use AI. Since the advent of transrectal ultrasonography and its use for monitoring development of individual follicles, our understanding of folliculogenesis has increased (5, 20). To develop various estrus-synchronization schemes that will accommodate greater synchrony for a fixed-time insemination, synchronizing follicular maturation with luteal regression is critical. Improved synchrony of estrus associated with coordinated follicular maturation and luteal regression from administration of GnRH 6 or 7 d before PGF2α (Select Synch) has been demonstrated in dairy heifers (27) and lactating cows (4, 26). Further tightening of synchrony between AI and ovulation occurs when a progestin such as norgestomet or an intravaginal progesterone (P4)containing device is combined with PGF2α (6). Use of the Ovsynch protocol in lactating dairy cows successfully produces pregnancy rates equal to those

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after detected estrus (16). However in one study (26), Ovsynch seemed to be less effective than a PGF2α-based system. Comparisons of Ovsynch or timed AI protocols to various estrus-synchronization systems for use in heifers are warranted. The objective of the first two experiments was to determine if a progestin would alter reproductive outcomes by allowing more precise control of the onset of estrus when incorporated into a two-injection PGF2α protocol with or without the addition of GnRH to induce ovulation before insemination. The objective of the third experiment was to determine the reproductive outcomes of dairy heifers treated with GnRH before PGF2αinduced luteolysis and estrus followed by inseminations after detected estrus (GnRH + PGF2α; Select Synch) or at one fixed time after a second injection of GnRH (Ovsynch). MATERIALS AND METHODS Experiment 1 The first of two similarly designed experiments was conducted in six replications at the Kansas State University Dairy Teaching and Research Center during February, March, and August of 1996 and July, September, and October of 1998. Holstein heifers (n = 69) averaging 13.2 ± 0.1 (x ± SD) mo of age (range: 11.9 to 14.9 mo) were assigned randomly to each of three treatments (Figure 1): 1) 23 control heifers received two 25mg injections of PGF2α (Lutalyse, Pharmacia & Upjohn, Kalmazoo, MI) 14 d apart; 2) 23 heifers were treated like the controls plus they received one 6-mg ear implant containing norgestomet (NORG; Syncro-Mate-B, Mer-

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ial Limited, Iselin, NJ) for 8 d beginning 7 d before the second injection of PGF2α (removed 24 h after the second PGF2α); and 3) 23 heifers were treated like the NORG heifers plus they received 100 µg of GnRH (Cystorelin, Merial Limited, Iselin, NJ) 54 h after the second PGF2α injection (NORG + GnRH). A HeatWatch (DDX, Inc., Denver, CO) device was affixed to the rump of each heifer immediately before the first injection of PGF2α and maintained in place until after the first estrus following the second PGF2α injection. Heifers also were observed visually for signs of estrus at 12-h intervals beginning at the second injection of PGF2α. The following measurements were calculated from the information recorded by the HeatWatch software: interval from the injection of PGF2α to estrus, number and duration of individual standing events (minimum of 2 s was required) per period of estrus, and duration of estrus (interval between first and last standing events) to determine if estrual events were altered by treatments. One technician inseminated all heifers with semen from one sire at 8 to 16 h after first detected estrus (first standing event), or, in the absence of detected estrus, semen was placed in the uterine body 72 h after the second injection of PGF2α of all remaining heifers. Pregnancy was diagnosed by return to estrus after insemination or by palpation of the uterus and its contents between 40 and 54 d after insemination. Blood collection and radioimmunoassays. Blood was collected via puncture of the tail vein from 10 heifers in the NORG and NORG + GnRH treatments and from 10 controls (first three replications only) just before the first PGF2α injection and daily thereafter until implant removal (+24 h) and again before injection of GnRH (+54 h; Figure 1). Progesterone (P4) (21) and estradiol-17β (E2; 14) were measured in these serum samples using specific radioimmunoassy (RIA). Intraand interassay coefficients of variation were 4.4 and 4.3% for P4 (sensitivity = 12 ± 2 pg/ml in three assays) and 10.5 and 10.5% for E2 (sensitivity = 0.2 ± 0.02 pg/ ml in three assays), respectively. Experiment 2

Figure 1. Experimental design of treatments for heifers in experiments 1 and 2. The solid bar indicates when norgestomet (NORG) ear implants were in situ after the second injection of PGF2α. Blood (B) samples were collected daily from d −14 to +54 h in experiment 1 and on d −14, −13, −7, −6, 0, and +54 h in experiment 2.

The second experiment was conducted at two dairy farms near Las Cruces, New Mexico. Breeding-age Holstein heifers were assigned randomly to the same three treatments described in experiment 1 (Figure 1). Three replications occurred at one dairy in February (n = 106), March (n = 60), and May (n = 62) of 1996, and the fourth replication was conducted at a second dairy in February, 1996 (n = 61). Heifers were tail marked with chalk and then inseminated after the second PGF2α injection when the tail chalk was observed to be rubbed Journal of Dairy Science Vol. 83, No. 9, 2000

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off (based on twice daily observation) while they were restrained in lockups at the feed bunk. The GnRH injection in the NORG + GnRH treatment was administered to ensure that ovulation would occur within 24 to 32 h (15) to accommodate insemination after less precise detection by observations. Inseminations were made as soon as tail chalk was observed to be rubbed off, and then tails of heifers were rechalked. When the tail chalk was observed to be rubbed off again, heifers were reinseminated (12 to 24 h after the first insemination), accounting for double inseminations in 27% of heifers at one dairy. Heifers also were inseminated based on visual signs of estrus with semen from six sires. After two or three unsuccessful inseminations, heifers were exposed to Holstein cleanup bulls. Pregnancy was diagnosed by return to estrus after the synchronized estrus and insemination or by calvings resulting from the synchronized insemination (duration of normal gestation = 279 ± 7 d). Blood collection and RIA. Blood was collected via tail vein puncture just before the first PGF2α injection (d −14), before implant insertion (d −7), before the second PGF2α injection (d 0), before implant removal (+24 h), and before the injection of GnRH (+54 h; Figure 1). Progesterone (21) was measured in these serum samples with a specific RIA as in experiment 1. Intra- and interassay coefficients of variation were 4.9 and 5.9% for P4 (sensitivity 10 ± 2 pg/ml in 10 assays), respectively. Those heifers with serum concentrations of progesterone <1 ng/ml on d −14, −7, and 0 were classified as noncyclic (prepubertal). Statistical analyses. Rates of estrus detection (number of heifers detected in estrus during 7 d after PGF2α divided by the number of heifers treated) or AI submission rate in experiment 2 based on tail chalk removal or visually detected estrus; conception (number of pregnant heifers after synchronized insemination divided by the number of heifers inseminated); pregnancy (number of pregnant heifers after synchronized insemination divided by the number of heifers treated); and calving (number of heifers that eventually calved as the result of insemination or natural service divided by the number of heifers treated) were calculated. Intervals from the second PGF2α injection to first standing event of estrus (experiment 1) or to first insemination (experiment 2) were calculated. All of the preceding variables were analyzed by procedure GLM in SAS (18) using a model consisting of treatment (both experiments), replication (experiment 1), or herd (experiment 2), and their interactions. In experiment 2, sires and technicians were confounded with herd so their effects were part of any differences in herds. The effects of puberty (estrus cyclicity before insemination in experiment 2) and its interaction with treatJournal of Dairy Science Vol. 83, No. 9, 2000

ment were tested in the previously described models. All other continuous variables of experiment 1 (number and duration of individual standing events during estrus and duration of estrus) were analyzed with treatment as the only source of variation. Other categorical variables were tested by chi-square using the CochranMantel-Haenszel statistic of SAS (18). To determine differences in estrus synchrony after PGF2α in experiment 1, we determined a measure of the variation associated with mean intervals to estrus by Levene’s test (10, 12). In experiment 1, concentrations of P4 and E2 from d −7 to + 2 were analyzed as a split-plot ANOVA with repeated measures (18). In both experiments, the error term for testing effects of treatment on concentrations of P4 and E2 (experiment 1 only) was heifer within treatment, whereas for testing treatment effects on P4 in experiment 2, the error term was heifer within treatment × herd. Experiment 3 A third experiment was conducted in 19 replications at the Kansas State University Dairy Teaching and Research Center between October 1993 and February 1998. Holstein heifers (n = 353), averaging 13.6 ± 0.9 mo (x ± SD) of age (range: 11 to 16.5 mo) and 402 ± 73 kg (x ± SD) of BW (range: 338 to 520 kg) were assigned randomly but unequally to three treatments: 1) one or two injections (14 d apart) of PGF2α (Lutalyse; PGF2α control; n = 131); 2) 100 µg of GnRH (Cystorelin or Factrel, Fort Dodge Laboratories, Fort Dodge, IA) 6 (n = 52) or 7 d (n = 57) before 25 mg of PGF2α (Lutalyse; Select Synch); or 3) Select Synch plus a second 100-µg injection of GnRH (Cystorelin) given 35.7 ± 7.3 h (x ± SD; range: 24 to 48 h) after PGF2α (Ovsynch; n = 113). Heifers were observed for estrus at least twice daily about 12 h apart, beginning at the time of the second or only PGF2α injection. Inseminations in the PGF2α controls were performed 8 to 16 h after detected estrus or once between 72 and 80 h after the second injection of PGF2α in the absence of detected estrus for all remaining heifers. Inseminations in the Select Synch treatment were performed 8 to 16 h after detected estrus for heifers detected in estrus up to 7 d after PGF2α. Inseminations of all heifers in the Ovsynch treatment were performed at 18.2 ± 3.3 (x ± SD) h (range: 15 to 25 h) after the second GnRH injection. Blood was collected from 46 control heifers and 44 heifers each in the Select Synch and Ovsynch treatments just before the second or only PGF2α injection to measure serum concentrations of P4 as in experiment 1 and 2. Intraassay coefficient of variation was 8.2% (sensitivity = 10 ± 2 pg/ml).

PROGRAMMED BREEDING OF HEIFERS Table 1. Reproductive characteristics of Kansas Holstein dairy heifers (Experiment 1). Treatment Item Heifers, no. Estrus detection rate, % PGF2α to estrus,2 h Standing events,2 no. Duration of individual standing events, s Duration of estrus, h Conception rate, % At estrus Fixed time

2 × PGF2α 23

NORG1 23

NORG + GnRH 23

86.9 54.6 ± 3.5 20.6 ± 3.5 3.1 ± 0.2

78.9 64.2 ± 3.7b 14.0 ± 3.5 2.7 ± 0.2b

47.8a 59.0 ± 4.9 12.9 ± 4.0 2.7 ± 0.2b

15.6 ± 1.6 69.6 14/18 2/5

10.9 ± 1.6a 39.1 8/17 1/6

10.0 ± 1.9a 52.2 7/10 5/13

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those heifers that were inseminated by appointment at 72 h after the second PGF2α injection in the absence of detected estrus than for those inseminated 8 to 16 h after detected estrus (64 vs. 35%). Figure 2 illustrates daily serum concentrations of E2 and P4 in control and the combined NORG and NORG + GnRH heifers during the synchronization period. No differences were detected in concentrations of P4, whereas a tendency (P < 0.10) for a treatment × day interaction occurred for concentrations of E2. In summary, norgestomet had little effect on serum concentrations of E2 and P4.

Different (P < 0.05) from control. Different (P < 0.10) from control. 1 Norgestomet. 2 Replication effect (P < 0.05). a b

Statistical analyses. Rates of estrus detection, conception, and pregnancy were defined and calculated as in experiments 1 and 2. Procedures GLM and CATMOD were used to analyze all categorical data, and procedure GLM was also used to analyze noncategorical data (18). The proportion of heifers detected in estrus, interval to estrus after PGF2α, and concentrations of P4 were analyzed in a model, including treatment and replication. Models used to analyze conception and pregnancy rates included treatment, replication, and technicians and sires nested within replication. Mean differences were separated by a priori orthogonal contrasts in which each treatment was compared to the control. RESULTS Experiment 1 Table 1 summarizes the reproductive characteristics of heifers in each treatment. Compared with 2 × PGF2α controls, percentage of heifers detected in estrus was less (P < 0.05) for NORG + GnRH heifers, but intervals from PGF2α to estrus (first standing event) after the second PGF2α injection tended (P = 0.07) to be greater for NORG heifers. No difference in the precision of estrus synchrony was detected when variances of treatment intervals to estrus after PGF2α were tested. Although several characteristics of estrus in heifers treated with norgestomet seemed to be suppressed, duration of estrus was less (P < 0.05) by about 50% for both NORG treatments than for the control. Conception rates were not different among treatments, but there were insufficient observations to detect differences of the magnitudes observed. However, across treatments, conception was less (P < 0.01) for

Figure 2. Daily mean concentrations of estradiol-17β (E2) and progesterone (P4) in serum of dairy heifers (experiment 1). Control heifers (open circles; n = 10) received two injections of PGF2α 14 d apart (designated by arrows). The remaining heifers (solid circles; n = 10) were treated like the control heifers plus they received one 6mg ear implant of norgestomet (solid bar) from d −7 to +24 h after the second PGF2α injection. Journal of Dairy Science Vol. 83, No. 9, 2000

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Experiment 2 Table 2 summarizes the reproductive characteristics of all New Mexico heifers assigned to the same three treatments tested in experiment 1. Just 7.7% of the heifers had baseline concentrations (<1 ng/ml) of P4 on d −14, −7, and 0 of the synchronization schemes, suggesting that the incidence of delayed puberty was small and unequally distributed among treatments (Table 2). Among prepubertal heifers, the AI submission rate (100 vs. 50%) was greater (treatment × puberty interaction; P < 0.001) in the two treatments that included norgestomet. None of four control prepubertal heifers conceived, whereas three of seven NORGtreated and seven of nine NORG + GnRH-treated heifers conceived at the synchronized estrus. These positive stimulating effects of norgestomet in peripubertal heifers are consistent with reports in beef heifers (13, 25). Interval from the second injection of PGF2α to insemination was less (P < 0.05) in one herd (2.7 ± 0.1 d) than in the other (3.4 ± 0.2 d). Rates of conception (P = 0.10), pregnancy (P = 0.11), and calving (P = 0.08) tended to

Table 2. Reproductive performance of New Mexico Holstein dairy heifers in response to treatments (Experiment 2). Treatment Item

2 × PGF2α

NORG1

NORG + GnRH

Heifers, no. Prepubertal Pubertal Heifers, no. Herd A Herd B AI submission rate,2 % Prepubertal Pubertal PGF2α to AI,3 d

101 4 97 101 78 23 96.0 50.0 97.9 2.9 ± 0.1

92 7 85 92 74 18 95.7 100.0 95.3 3.2 ± 0.1

85 9 76 85 67 18 100.0 100.0 100.0 3.1 ± 0.1

Conception rate,4 % Herd A Herd B

52.5 44.9 78.3

52.2 52.7 50.0

61.2 53.7 88.9

Pregnancy rate,5 % Herd A Herd B Calving rate,6 %

52.5 44.9 78.3 83.2

53.3 54.1 50.0 84.8

61.2 53.7 88.9 95.3

1

Norgestomet. Percentage of heifers submitted was based mostly on tail chalk removal monitored on twice daily basis. Treatment (P < 0.001), herd (P < 0.05), and treatment × pubertal status interaction (P < 0.001). 3 For heifers detected in estrus during 6 d after PGF2α. Effects of herd (P < 0.05) and treatment (P < 0.05): control vs. others (P < 0.05). 4 Effects of herd (P < 0.01), treatment (P = 0.10), and treatment × herd (P = 0.06). 5 Effects of herd (P < 0.01), treatment (P = 0.11), treatment × herd (P < 0.05). 6 Effect of treatment (P = 0.08): control vs. NORG + GnRH (P < 0.05). 2

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Figure 3. Average concentrations of serum progesterone (P4) in dairy heifers (experiment 2) on d −14, −13, −7, 0, and +1 from the second injection of PGF2α (d 0). Control heifers (open bars; n = 98) received two injections of PGF2α 14 d apart (d −14 and 0). The remaining heifers (combined NORG and NORG + GnRH treatments; solid bars; n = 177) were treated like the control heifers plus they received one 6-mg ear implant of norgestomet from d −7 to +1 after the second PGF2α injection. NORG = Norgestomet.

be altered by treatment. However, a treatment × herd interaction occurred for rates of conception (P = 0.06) and pregnancy (P < 0.05). For both measures, heifers receiving norgestomet had better fertility in herd A, whereas in herd B, the NORG treatment was more detrimental than the other treatments. Those heifers that were inseminated twice after tail chalk removal only tended (P = 0.12) to have greater conception rates than those inseminated once (71 vs. 60.2%). Calving rates were slightly greater (P < 0.05) in NORG + GnRH heifers than controls. Mean serum concentrations of P4 on d −14, −13, −7, 0, and +1 in the control and combined NORG-treated heifers are illustrated in Figure 3. Overall, serum P4 was not different between controls and NORG treatments on d 0 and +1 when treatment effects were possible. Similar rates of luteal function and luteolysis were detected between groups (control vs. combined treatments in which norgestomet was administered: NORG and NORG + GnRH) after PGF2α, and nearly all heifers had a decrease in concentrations of P4 after PGF2α (Table 3). Further relationships between serum P4 and conception rates are summarized in Table 3. Conception rates did not differ between control and combined norgestomet treatments, whether comparison was based on presence or absence of luteal activity on d 0 or successful luteolysis by 24 h after PGF2α. Across treatments, heifers with luteal activity before PGF2α had conception

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PROGRAMMED BREEDING OF HEIFERS Table 3. Conception rate (CR) in heifers treated with PGF2α or norgestomet (NORG) with and without GnRH as a function of luteal activity (concentration of progesterone; P4) before and after PGF2α (experiment 2). Treatments Item

NORG and NORG + GnRH

Control

Luteal activity on d 01 CR if P4 >1 ng/ml CR if P4 <1 ng/ml Luteolysis by 24 h2 CR if P4 <1 ng/ml CR if P4 >1 ng/ml

No./no. %

No./no. (%)

88/101 48/88 5/13 60/88 33/60 15/28

142/177 82/142 18/35 100/142 60/100 22/42

(87.1) (54.5) (38.5) (68.2) (55.0) (53.6)

(80.2) (57.8) (51.4) (70.4) (60.0) (52.4)

1 Proportion of heifers in which concentrations of P4 were >1 ng/ml at the time of the second PGF2α injection (d 0). 2 Proportion of heifers in which concentrations of P4 were >1 ng/ml at the time of the second PGF2α injection that decreased to <1 ng/ml within 24 h.

rates of 57% compared with 48% in those with no luteal activity. For heifers in which luteolysis occurred, conception rates were 60% compared with 53% for heifers in which luteolysis did not occur. Experiment 3 Reproductive traits of Select Synch heifers treated with GnRH 6 or 7 d before PGF2α were similar (interval to estrus: 61 vs. 56 h; estrus detection rate: 79 vs. 88%; conception rate: 66 vs. 72%; and pregnancy rate: 52 vs. 63%). Therefore, these two groups of heifers were combined as one treatment and compared to the 2×PGF2α control and Ovsynch treatments. Overall reproductive traits of heifers for these comparisons are summarized in Table 4. Estrus-detection rates in the Ovsynch treatment were reduced (P < 0.01) as a result of the second GnRH injection compared with the control and Select Synch heifers. Because the sec-

Figure 4. Percentage of distribution of estrus after PGF2α (experiment 3) for control heifers treated with PGF2α (open bars; n = 92) and heifers treated with either Select Synch and Ovsynch combined (solid bars; n = 151). Asterisks indicate where means differed (P < 0.05).

ond GnRH injection induces the preovulatory LH surge and LH subsequently causes estrogen secretion by the preovulatory follicle to cease (26), further mounting and standing activity generally is limited. For those heifers detected in estrus, intervals to detected estrus (x ± SE) after PGF2α were less (P < 0.01) after the Ovsynch (56 ± 3 h) and Select Synch treatments (52 ± 5 h) compared with the control (65 ± 3 h). Serum concentrations of P4 just before the second or only injection of PGF2α were greater (P < 0.01) for controls than for heifers in the other treatments (5.2 ± 0.4 vs. 3.9 ± 0.3 ng/ml), suggesting that injection of GnRH at random stages of the estrous cycle 7 d before PGF2α resulted in reduced concentrations of P4 7 d later, consistent with our earlier report (26). Distribution of estrus after PGF2α between the controls and the combined GnRH treatments is illustrated in Figure 4. Compared with controls, proportionally

Table 4. Reproductive traits of heifers treated with PGF2α, Select Synch, or Ovsynch (experiment 3). Treatments1 Item

PGF2α control

Select Synch

Ovsynch

Estrus detection rate Conception rate Pregnancy rate Cumulative pregnancy rate

117/131 76/131 76/131 128/131

No./no. (%) 91/109 (83.5) 63/91 (69.2) 63/109 (57.8) 106/109 (97.2)

34/113 48/113 48/113 110/113

(89.3) (58.0) (58.0) (97.7)

(30.1)** (42.5) (42.5)* (97.3)

1 PGF2α Control = heifers given PGF2α were inseminated after estrus was detected or at 72 to 80 h after the second of two PGF2α injections given 14 d apart. Select Synch = heifers were given GnRH either 6 (n = 52) or 7 d (n = 57) before PGF2α and were inseminated after detected estrus. Ovsynch = heifers were given GnRH 7 d before PGF2α, followed by a second GnRH injection 35.7 h after PGF2α, with insemination occurring 18.2 h after GnRH. *Different (P < 0.05) from PGF2α control. **Different (P < 0.01) from PGF2α control.

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more (P < 0.05) heifers treated with GnRH before PGF2α were in estrus less than 24 h after PGF2α. As a result, proportionally fewer (P < 0.05) heifers treated with GnRH before PGF2α were detected in estrus between 49 and 96 h. As a result of the earlier onset of estrus in heifers treated with GnRH before PGF2α, conception rates seemed to be lower after the Ovsynch heifers were timeinseminated but clearly not in the Select Synch heifers that were inseminated after detected estrus (Table 4). Among the control heifers, conception rates were 57.3% (67 of 117) for those inseminated after detected estrus, and 64.3% (9 of 14) for those inseminated once between 72 and 80 h after PGF2α. Pregnancy rates were reduced (P < 0.05) in the Ovsynch heifers compared with controls. Cumulative pregnancy rates before culling of nonpregnant heifers were not different among treatments. DISCUSSION Our hypothesis for experiments 1 and 2 was that norgestomet would suppress estrus for 24 h, while luteal regression was induced by PGF2α and until the norgestomet implant was removed. This seemed to occur because interval to estrus was or tended to be prolonged in one of two groups treated with norgestomet in experiment 1. Dominant follicles that form during two-, three-, or four-wave estrous cycles are capable of complete maturation and ovulation, if GnRH injections precede precocious luteal regression induced by injections of PGF2α (15). In the absence of a function corpus luteum (1, 22), a maturing follicle persists in the ovaries and fails to turn over in the presence of limited concentrations of progestins, which are produced via feeding of melengestrol acetate (9), exposure to a removable progestin implant (17), or intravaginal progesteronecontaining device (6). Furthermore, once the corpus luteum dies or its demise is induced by injections of PGF2α, the continued presence of progestins will prevent the onset of estrus (3). Doses of progestins typically administered in estrus-synchronization programs are not sufficient to prevent increased LH pulse frequency (9, 17) or the development of persistent follicles (9, 17, 22). Although these persistent follicles eventually ovulate after the removal of the exogenous progestin, fertility of the oocyte is compromised (11), leading to decreased fertilization rates and increased embryonic death within a few days (2). Persistent follicles should not have developed during norgestomet treatment in a high percentage of heifers because of a potential luteolytic response to the first injection of PGF2α (indicated in Figures 2 and 3). However, based on results observed in experiment 1 and those of herd B in experiment 2 (treatment × herd interactions; P < 0.05) suppression Journal of Dairy Science Vol. 83, No. 9, 2000

of conception rates suggests that persistent follicles in some heifers may have occurred. Conception and pregnancy rates among heifers in experiments 1 or 2 indicated that a standard two-injection PGF2α protocol is an acceptable method of synchronizing estrus in dairy heifers. It also was the least costly, assuming that PGF2α is $3 per dose, GnRH is $4 per dose, and norgestomet purchased as SyncroMate-B is $7 per dose. (Note: Since the completion of these studies, Syncro-Mate-B has been withdrawn from the market.) The cost of the control was $6 compared with $13 for the NORG treatment and $17 for the NORG + GnRH treatment. Although both treatments that included norgestomet provided significant improvement in the reproductive outcomes of a small number of prepubertal, later developing heifers (about 8% of total), their costs were two- to threefold greater than that of the PGF2α control. In experiment 3, the Ovsynch protocol produced results inferior to those obtained with Select Synch and a standard PGF2α protocol that allowed insemination at detected estrus after one or two injections plus timed inseminations at 72 to 80 h in heifers failing to be detected in estrus after both injections. The Ovsynch protocol produced pregnancy rates exceeding 40%, which were slightly better than those reported in our previous study (16) with heifers, but not as great as those observed after the Select Synch or standard PGF2α protocols. Pregnancy rates could be improved after the Ovsynch protocol if heifers were inseminated as Select Synch heifers (based on detected estrus) up about 40 h after PGF2α (Figure 4) and then only administer the second GnRH injection and inseminate the remaining heifers according to the Ovsynch protocol (19). Costs of the PGF2α control ($3 to $6) and Select Synch ($7) were quite similar, whereas Ovsynch ($10) was more expensive. Compared with the other two alternatives tested in experiment 3, unless labor costs or skills involved for detection of estrus are limiting, the greater cost associated with administering the Ovsynch protocol in heifers is not justified, when estrus detection rates are >80% and conception rates exceed 50%, as in our study. ACKNOWLEDGMENTS The authors thank the following New Mexico dairy producers for their assistance in conducting experiment 2: George Segura (Big Sky Dairy) and Joe Segura (Valley View Dairy). We express appreciation to Betty A. Hensley for technical assistance in the laboratory and to Richard K. Scoby for help at the Kansas State University Dairy Teaching and Research Center.

PROGRAMMED BREEDING OF HEIFERS

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