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J. Anim Sci. 2008. 86:83-93. doi:10.2527/jas.2007-0277
© 2008 American Society of Animal Science
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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION

Gonadotropin-releasing hormone-induced ovulation and luteinizing hormone release in beef heifers: Effect of day of the cycle

J. A. Atkins, D. C. Busch, J. F. Bader, D. H. Keisler, D. J. Patterson, M. C. Lucy and M. F. Smith1

Division of Animal Sciences, University of Missouri, Columbia 65211


   Abstract
Top
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The COSynch protocol has been used to synchronize ovulation and facilitate fixed-time AI in beef cattle. Establishment and maintenance of pregnancy was negatively affected, in previous studies, by GnRH-induced ovulation of small dominant follicles (≤11 mm). The reason for the presence of small follicles at the second GnRH (GnRH 2) is not clear. The objectives of this study were 1) to determine the effect of ovulatory response at the first GnRH (GnRH 1) on diameter and variation in diameter of the largest follicle at GnRH 2, and 2) to determine the effect of day of the cycle (stage of a follicular wave) on GnRH-induced luteinizing hormone (LH) release, and the resulting ovulatory response after GnRH 1 and 2. Two experiments used pubertal beef heifers synchronized to be on different days of the estrous cycle (d 2, 5, 10, 15, and 18 after estrus) in which a dominant follicle would or would not respond to GnRH 1. Ovulatory response to GnRH 1 did not affect size or variation in diameter of the largest follicle at GnRH 2 in Exp. 1 or 2. In Exp. 1, ovulatory response after GnRH 1 (0/14a, 12/13b, 4/13ac, 9/13bc, and 2/10a in the d 2, 5, 10, 15, and 18 groups; a–cP < 0.05) and GnRH 2 (13/14a, 12/13a, 12/13a, 2/13b, and 2/10b in the d 2, 5, 10, 15, and 18 groups, respectively; a,bP < 0.05) was affected by day of the cycle. In Exp. 2, day of the cycle also affected the proportion of heifers ovulating after GnRH 1 (0/7a, 8/8b, 0/6a 5/8ab, and 5/8ab of the d 2, 5, 10, 15, and 18 heifers, respectively; a–cP < 0.05) and GnRH 2 (3/7ab, 8/8b, 5/6b, 1/8a, and 2/8a of the d 2, 5, 10, 15, and 18 heifers, respectively; a,bP < 0.05). In both experiments, heifers receiving GnRH 1 on d 15 and 18 had a greater (P < 0.05) occurrence of luteolysis before PGF2{alpha} injection and expression of estrus than heifers treated on d 2, 5, and 10. The GnRH-induced LH surge was of greatest magnitude in heifers receiving GnRH 1 on d 18 of the cycle followed by d 5, 15, 10, and 2 (9,054b, 5,774bc, 4,672c, 2,548c, and 915d arbitrary units; respectively; a–dP < 0.05). In summary, ovulatory response to GnRH 1 did not affect size of the dominant follicle at GnRH 2. Day of the cycle when GnRH 1 was delivered affected dominant follicle size at GnRH 2. Treatment with GnRH 1 in the earlier part of the estrous cycle (on or before d 10) increased the proportion of dominant follicles that were large enough to respond to GnRH 2 (≥10 mm) and increased ovulatory response after GnRH 2.

Key Words: beef heifer • follicle size • gonadotropin-releasing hormone • luteinizing hormone • ovulation


   INTRODUCTION
Top
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
A fixed-timed AI (TAI) protocol for cattle includes an injection of GnRH on d –9 (GnRH 1), plus PGF2{alpha} on d –2, and an injection of GnRH on d 0 (GnRH 2; day of insemination). The TAI protocols have consistently resulted in lower pregnancy rates than protocols involving estrous detection in heifers and cows (Pursley et al., 1997Go; Johnson and Day, 2004Go; Larson et al., 2006Go). Lower pregnancy rates might be due to presence of small physiologically immature dominant follicles at GnRH-induced ovulation and insemination. The GnRH-induced ovulation of small dominant follicles decreased pregnancy rates in beef heifers (Perry et al., 2007Go) and postpartum cows (Perry et al., 2005Go) after TAI compared with GnRH-induced ovulation of large follicles. We hypothesized that failure of the previous dominant follicle to ovulate in response to GnRH 1 and thereby synchronize a follicular wave results in the presence of a small dominant follicle at insemination (GnRH 2). Failure to ovulate to GnRH may be due to absence of a dominant follicle, presence of a follicle no longer able to ovulate (atretic), inadequate gonadotropin secretion in response to GnRH 1, or a combination of these. The preceding hypotheses were tested by administering GnRH 1 to heifers on days of the estrous cycle that were chosen to represent specific stages of follicular waves when dominant follicles would or would not ovulate in response to GnRH 1, and where the GnRH-induced luteinizing hormone (LH) release is known to differ.

The objectives of the experiment were as follows: 1) to determine the effect of ovulatory response to GnRH 1 on dominant follicle diameter and variation in follicular diameter at GnRH 2, and 2) to determine the effect of day of the cycle (i.e., stage of a follicular wave) on GnRH-induced LH secretion and the ovulatory response of the corresponding dominant follicle.


   MATERIALS AND METHODS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
The experimental procedures were approved by the University of Missouri—Columbia Animal Care and Use Committee.

Experiment 1
Pubertal 20-mo-old beef heifers (n = 63) of mixed breeds were assigned by age, weight, and breed (Angus, Red Angus, Charolais, and Angus or Red Angus x Simmental cross) to 1 of 5 treatment groups based on day of the estrous cycle [d 2, 5, 10, 15, and 18 after estrus (d 0); group d 2, 5, 10, 15, and 18; n = 14, 13, 13, 13, and 10, respectively] at the beginning of the COSynch protocol [Figure 1Go; GnRH (GnRH 1; d –9; 100 µg as 2 mL of Cystorelin i.m.; Merial, Athens, GA) followed 7 d later with PGF2{alpha} (d –2; 25 mg as 5 mL of Lutalyse i.m., Pfizer Animal Health, New York, NY) and 48 h later GnRH and TAI (GnRH 2; d 0; 100 µg as 2 mL of Cystorelin i.m.); Geary et al., 1998Go]. Treatment groups were chosen to represent different stages of follicular waves when dominant follicles would or would not ovulate after GnRH 1 (Figure 1Go).


Figure 1
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  		Figure 1. Experimental design and treatment schedule for cycling beef heifers assigned to receive the COSynch protocol beginning on d 2, 5, 10, 15, or 18 after estrus (groups d 2, 5, 10, 15, and 18, respectively). Treatment groups were chosen so heifers would be at specific stages of dominant follicle development at GnRH 1. The COSynch protocol included a GnRH injection (GnRH 1; d –9), an injection of PGF2{alpha} (d –2) 7 d later, and a second injection of GnRH (GnRH 2; d 0) 48 h after PGF2{alpha} treatment. Ovulatory response to GnRH 1 and 2 was verified using transrectal ultrasonography 2 d after treatment. Heifers in which a large follicle had disappeared and luteal tissue had formed were considered to have ovulated following GnRH.

 
Presynchronization.
Heifers were presynchronized by using a controlled internal drug release (CIDR) insert for 7 d and an injection of PGF2{alpha} on the day before CIDR removal. Heifers within each treatment group displayed estrus on the same day ± 12 h. Estrus was monitored every 6 h by visual observation during the presynchronization period, and heifers were declared in estrus if they stood to be mounted. In addition, estrus was monitored twice daily throughout the treatment period until 2 d after GnRH 2.

Transrectal Ultrasonography.
Ovarian structures were monitored using an Aloka 500V ultrasound with a 7.5 mHz transducer (Aloka, Wallingford, CT). Follicles ≥5 mm and the presence of corpora lutea (CL) were recorded. Follicular diameter was measured at the widest point and at a right angle to the first measurement. Follicular diameter was calculated as the average of the 2 measurements. Transrectal ultrasonography was performed on d –9 (GnRH 1) and d 0 (GnRH 2 and TAI) to determine the diameter of the dominant follicle. Ovulation after GnRH 1 or 2 was determined on d –7 or 2, respectively, based on the disappearance of a dominant follicle and in some cases formation of luteal tissue. Ultrasound on d –9, –7, –5, –3, –2, and 0 was performed to characterize follicular waves and growth of dominant follicles during the treatment period. In addition, ovaries were scanned every other day beginning 8 (d 18 group), 6 (d 15 group), or 2 d (d 10 and 5 group) before GnRH 1 to characterize the dominant follicle before GnRH 1 [i.e., follicle wave number and stage of growth (increasing, plateau, or regressing)]. Heifers in the d 2 group were in estrus 2 d before GnRH 1, so no additional ultrasound was performed before GnRH 1.

Blood Collection and RIA.
Blood samples were collected via jugular venipuncture into 10-mL Vacutainer tubes (Fisher Scientific, Pittsburgh, PA) on d –9, –7, – 5, –3, –2, 0, and every other day until d 28 after AI (Figure 1Go). Blood was allowed to clot at room temperature for 1 h, and then clot at 4°C for 24 h, and was centrifuged at 2,000 x g for 25 min. Serum was collected and stored at –20°C until RIA. Serum concentrations of progesterone (P4) were measured using a Coat-a-count RIA kit (Diagnostic Products Corporation, Los Angeles, CA; Kirby et al., 1997Go). Intra- and interassay CV and assay sensitivity were 2.0%, 5.2%, and 0.1 ng/mL, respectively. Serum concentrations of estradiol-17β (E2) were measured using RIA (Kirby et al., 1997Go) in samples collected on d –9, –7, –5, –3, –2, 0, and 2. Intra- and interassay CV and assay sensitivity were 8.4%, 9.2%, and 0.25 pg/mL, respectively.

Statistical Analysis.
One-way ANOVA with day as the independent variable was used to test differences among treatment groups in average follicular diameter and serum concentrations of E2 at GnRH 1 and 2 with SAS (SAS Inst. Inc., Cary, NC). Differences in ovulatory response among treatment groups at GnRH 1 and 2 were tested using GenMod. Differences in average follicular diameter and variances in follicular diameter at GnRH 1 and 2 between heifers that did or did not ovulate after GnRH 1 were analyzed with the 2-sample t-test. Changes in serum concentrations of P4 over time (d 2 to 12 after AI) were analyzed for heifers ovulating large or small follicles after GnRH 2 by ANOVA for repeated measures (PROC MIXED; Littell et al., 1998Go).

Experiment 2
Pubertal 15-mo-old beef heifers (n = 43) of mixed breeds (Angus, Red Angus, Charolais, and Angus or Red Angus x Simmental cross) were assigned to 1 of 6 treatment groups as described in Exp. 1 (d 2, 5, 10, 15, and 18; n = 7, 8, 6, 8, and 8, respectively) at the start of the COSynch protocol (see Exp. 1). Heifers (n = 6) were also assigned to a control group that received PGF2{alpha} on d 6 of the estrous cycle and GnRH 1 48 h later. The control group was designed to assess the maximum amount of LH released in response to GnRH 1. Heifers were presynchronized to a specific day of the cycle at GnRH 1 and at PGF2{alpha} (controls) as described in Exp. 1. The HeatWatch Estrous Detection System (DDx Inc., Denver, CO) was used to monitor estrus during the presynchronization and synchronization periods until heifers ovulated or came into estrus after GnRH 2. Estrus was defined as 3 mounts lasting longer than 2 s per mount within a 4-h period.

Transrectal ultrasonography was used to measure the diameter of the dominant follicle, presence of a CL, and to verify ovulation of a dominant follicle after GnRH 1 and 2, as described in Exp. 1.

Blood Collection and RIA.
Blood samples were collected on d –9, –7, –2, and 0, as described in Exp. 1. In addition, at GnRH 1 (d –9), 10 mL samples were collected every 30 min before GnRH 1 (–30), immediately before GnRH 1 (0), and every 30 min until 240 min after GnRH 1 to characterize the GnRH-induced release of LH. Serum concentrations of P4 and E2 were measured by RIA, as described in Exp. 1, in samples collected on d –9, –7, –2, and 0. Intraassay CV and assay sensitivity were 3.1% and 0.1 ng/mL, respectively, for the P4 assay. Intra- and interassay CV and assay sensitivity were 7.3%, 5.6%, and 0.25 pg/mL, respectively, for the E2 assay. Circulating concentrations of LH were measured by RIA from serial samples collected around GnRH 1. Serum concentrations of LH were assayed in triplicate 25-µL aliquots using a liquid-phase double-antibody RIA validated for use in bovine serum, using the general procedures described by Hampton et al. (2003)Go. The primary antibody, however, consisted of an antibovine LH monoclonal antibody (518B7; Matteri et al., 1987Go) diluted to a final tube concentration of 1:1,000,000. Standard concentrations of bovine LH and increasing volumes of a bovine serum pool (25 µL to 300 µL) were added to the assay tubes in quadruplicate, and the total volume was balanced to 300 µL per tube with buffer. The intraassay CV was 6.3% (n = 4), with a minimum detectable concentration of 60 pg/tube (0.06 ng/mL).

Statistical Analysis.
Differences in follicle diameter, variance in follicle diameter, and ovulatory response were analyzed as described in Exp. 1. Peak LH concentrations and total area under the curve for LH release were analyzed with ANOVA for each of the treatments including the control group. Because of heterogeneity of variance among groups, E2 and LH concentrations were log transformed before analysis, but nontransformed means are reported. Log transformation of E2 at GnRH 2 did not correct for the heterogeneity of variance. Consequently, heifers were ranked from 1 to 37 (1 being the lowest value and 37 being the greatest) and the ranking was tested for differences (Conover and Inman, 1981Go).


   RESULTS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Experiment 1
Ovulatory Response to GnRH 1 and 2.
Diameter of the largest follicle at GnRH 1 ranged from 4 to 14.5 mm. Mean diameter of the largest follicle at GnRH 1 was greatest in the d 10 group followed by d 18, 15, 5, and 2 groups (Table 1Go). The proportion of heifers ovulating in response to GnRH 1 was 42%. Ovulatory response to GnRH 1 was affected by day of the cycle (Table 1Go).


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  		Table 1. Mean diameter (mm) of the largest follicle (±SEM) at GnRH 1 and GnRH 2, number (%) of heifers ovulating to GnRH 1 and 2, undergoing luteolysis before prostaglandin F2{alpha} (PGF2{alpha} ), in estrus from GnRH 1 to PGF2{alpha} , and pregnant after fixed-time artificial insemination (TAI) in experiment 1 and 2
 
Diameter of the largest follicle at GnRH 2 ranged from 4 to 17 mm. Mean diameter of the largest follicle at GnRH 2 was greater (P < 0.05) in the d 2, 5, and 10 groups compared with the d 15 group but similar to the d 18 group (Table 1Go). All heifers in the d 2, 5, and 10 groups had a class III follicle (≥9 mm; Moreira et al., 2000Go) at GnRH 2 (d 0); however, heifers in the d 15 (2/13) and 18 (5/10) groups had fewer class III follicles at GnRH 2 (P < 0.05; Figure 2Go). Total ovulatory response to GnRH 2 was 65% (41/63) and was affected by day of the cycle at GnRH 1. More heifers in the d 2, 5, and 10 groups ovulated at GnRH 2 compared with heifers in the d 15 or 18 groups (P < 0.05; Table 1Go).


Figure 2
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  		Figure 2. Mean number of class III follicles (≥9 mm) for each treatment (day of the cycle at the beginning of the COSynch protocol) throughout Exp. 1 [d –9 (GnRH 1), d –2 (PGF2{alpha} ), and d 0 (GnRH 2)]. Heifers in the d 2, 5, and 10 groups had at least one class III follicle at d 0. Heifers in the d 15 and 18 groups had fewer class III follicles at GnRH 2 (P < 0.05).

 
Follicular Dynamics.
Heifers in the d 2, 5, and 10 groups had synchronized growth of a class III follicle before the injection of GnRH 2 (Figure 2Go). In the d 15 group, there was a decrease in the number of class III follicles after GnRH 1; however, several heifers in the d 15 group did not have a class III follicle at GnRH 2 because they were in estrus and ovulated before the PGF2{alpha} injection. Heifers in the d 18 group had a poor ovulatory response to GnRH 1. Several heifers showed estrus from d –5 through d 0, which resulted in a decline in the number of heifers with class III follicles at GnRH 2 (Figure 2Go).

Onset of Luteolysis and Estrus.
Heifers in the d 15 and 18 groups had a greater incidence of luteolysis before PGF2{alpha} injection than heifers in the d 2, 5, and 10 groups (P < 0.01; Table 1Go). More heifers in the d 15 and 18 groups were in estrus between GnRH 1 and PGF2{alpha} injection compared with the d 2, 5, and 10 groups (P < 0.01; Table 1Go). Of the 7 heifers in the d 18 group that were detected in estrus before PGF2{alpha} , 2 were in estrus on d –5, 1 was in estrus on d –4, 3 were in estrus on d –3, and 1 was in estrus on d –2 (PGF2{alpha} ). In the d 15 group, 1 heifer was detected in estrus on d –3 and 5 other heifers were in estrus on d –2. Of the 6 d-15 heifers that displayed estrus on or before the day of PGF2{alpha} , 4 heifers ovulated and formed accessory luteal tissue after GnRH 1. The proportion of heifers detected in estrus around the time of GnRH 2 (±1 d of GnRH 2) for the d 2, 5, and 10 groups was 8/14, 4/13, and 6/10, respectively.

Serum Concentrations of Estradiol and Progester-one.
Changes in serum concentrations of E2 were similar to changes in class III follicles (Figure 3Go). Heifers in the d 2, 5, and 10 groups had an increase in serum concentrations of E2 before GnRH 2 (Figure 3Go). Heifers in the d 15 group had concentrations of E2 that peaked on d –3 and –2 and decreased thereafter. Heifers in the d 18 group had an increase in serum concentrations of E2 up to d –5 at which time 2 heifers showed estrus, followed by a steady decline until GnRH 2.


Figure 3
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  		Figure 3. Mean serum (± SEM) concentrations of estradiol during the COSynch protocol (GnRH 1 = d –9; PGF2{alpha} = d –2; and GnRH 2 and TAI = d 0) for treatment group d 2, 5, 10, 15, and 18. The COSynch protocol included a GnRH injection (GnRH 1; d –9), an injection of PGF2{alpha} (d –2) 7 d later, and a second injection of GnRH (GnRH 2; d 0) 48 h after PGF2{alpha} treatment.

 
Average serum concentrations of E2 at GnRH 2 were greater in the d 2, 5, and 10 groups compared with the d 15 group (P < 0.01). In addition, serum concentrations of E2 in the d 2 and 10 groups were greater than the d 18 (P < 0.01) group.

Circulating concentrations of P4 (d 2 to 12; d 0 = GnRH 2 and AI) were greater (P = 0.05) among heifers that ovulated large (>11.5 mm) compared with small (≤11 mm) dominant follicles (Figure 4Go). Among heifers that responded to GnRH 2, there was no effect of expression of estrus or pregnancy on circulating concentrations of P4 from d 2 to 12 after insemination.


Figure 4
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  		Figure 4. Mean (±SEM) serum concentrations of progesterone from d 2 to 12 after insemination (GnRH 2 = d 0) in heifers ovulating small (≤11 mm) or large (>11.5 mm) follicles. Heifers that ovulated large follicles had greater (P < 0.05) serum concentrations of progesterone than heifers that ovulated small follicles. The CoSynch protocol included a GnRH injection (GnRH 1; d –9), an injection of PGF2{alpha} (d –2) 7 d later, and a second injection of GnRH (GnRH 2; d 0) 48 h after PGF2{alpha} treatment.

 
Effect of Ovulatory Response to GnRH 1 on Subsequent Dominant Follicle Diameter and Ovulatory Response at GnRH 2.
Ovulatory response to GnRH 2 was not dependent on ovulatory response after GnRH 1. Only 63% (17/27) of the heifers that ovulated after GnRH 1 also ovulated after GnRH 2. There was no effect of ovulatory response at GnRH 1 on the following variables: 1) dominant follicle diameter at GnRH 2; 2) variation in dominant follicle diameter at GnRH 2; 3) percentage ovulating after GnRH 2; or 4) percentage of heifers undergoing luteolysis before PGF2{alpha} (Table 2Go). When the analysis only included heifers in groups that had a dominant follicle large enough to respond to GnRH 1 (i.e., not the d 2 group), there was still no difference in the above measurements (data not shown).


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  		Table 2. Effect of ovulatory response at GnRH 1 in the COSynch protocol1 on mean follicular diameter (mm) at GnRH 1 and 2, variance in diameter of the largest follicle at GnRH 1 and 2, number (%) ovulating at GnRH 2, undergoing luteolysis before PGF2{alpha} and pregnant in Exp. 1 and 2
 
Experiment 2
Ovulatory Response to GnRH 1 and 2.
Diameter of the largest follicle at GnRH 1 ranged from 4 to 16.5 mm. Mean diameter of the largest follicle was smaller in the d 2 group compared with the d 5, 10, 15, and 18 groups (P < 0.01; Table 1Go). Excluding the control group, the total percentage of heifers ovulating after GnRH 1 and GnRH 2 was 49 and 52%, respectively, and was affected by day of the cycle (P < 0.05; Table 1Go). Each of the heifers in the control group had large follicles (14.1 mm ± 0.6) that ovulated after GnRH 1 (6/6). None of the heifers in the d 2 group, all of the d 5 heifers, none of the d 10 heifers, and 5/8 of the heifers in the d 15 and 18 groups ovulated after GnRH 1 (Table 1Go).

Diameter of the largest follicle at GnRH 2 ranged from 4 to 15 mm. Mean diameter of the largest follicle at GnRH 2 was greater (P ≤ 0.09) in the d 2, 5, and 10 groups compared with the d 15 and 18 groups (Table 1Go). The ovulatory response at GnRH 2 followed a similar pattern as Exp. 1 with the exception of heifers in the d 2 group. The d 5 and 10 groups had a large proportion of heifers ovulating followed by the d 2, 18, and 15 groups.

GnRH-induced LH Surge.
Peak circulating concentrations of LH occurred 90 min after the GnRH 1 injection and were greatest in the control group followed by heifers in the d 18, 5, 15, 10, and 2 groups (113a, 84b, 60b, 36c, 24c, and 10d ng/mL, respectively; a–dP < 0.05; Figure 5Go). Total LH released (arbitrary units) during the 4 h after GnRH 1 was greatest in the control group (13,642a) followed by heifers in the d 18 (9,054b), 5 (5,774bc), 15 (4,672c), 10 (2,548c), and 2 (915d; respectively; a–dP < 0.05) groups (data not included).


Figure 5
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  		Figure 5. Mean serum concentrations of luteinizing hormone (LH) in heifers after GnRH 1 on d 2, 5, 10, 15, and 18 after estrus or control heifers (PGF2{alpha} -induced luteolysis on d 6 of the estrous cycle, 2 d preceding GnRH). The COSynch protocol included a GnRH injection (GnRH 1; d –9), an injection of PGF2{alpha} (d –2) 7 d later, and a second injection of GnRH (GnRH 2; d 0) 48 h after PGF2{alpha} treatment.

 
Heifers that ovulated in response to GnRH 1 had more total LH released (P < 0.03) than heifers that did not ovulate; however, this observation was confounded by treatment (day of the cycle). It was not possible to compare GnRH-induced LH release in heifers that did or did not ovulate within each day of the cycle because none of the heifers in the d 2 group, all of the heifers in the d 5 group, and none of the heifers in the d 10 group ovulated after GnRH 1. However, total LH secreted after GnRH 1 treatment was not different in heifers that did or did not ovulate in the d 15 and 18 groups (data not shown).

Onset of Luteolysis and Estrus.
Similar to Exp. 1, more heifers in the d 15 and 18 groups experienced luteolysis and displayed estrus on or before PGF2{alpha} than heifers in the d 2, 5, and 10 groups (P < 0.01; Table 1Go). Five of the heifers in the d 15 group were in estrus before PGF2{alpha} (2 on d –4, 1 on d –3, and 2 on d –2). The remaining 3 heifers in the d 15 group were in estrus the day before GnRH 2 (d –1). Three heifers in the d 18 group were in estrus before PGF2{alpha} (2 on d –6 and 1 on d –5). Of the remaining heifers (d 18 group), 1 was in estrus the day before GnRH 2 (d –1), 2 ovulated (disappearance of a large follicle via ultrasound) without showing estrus, and 2 were in estrus the day of GnRH 2.

Serum Concentrations of Estradiol.
Serum concentrations of E2 at GnRH 1 were positively associated with total LH released in response to GnRH (r = 0.81; P < 0.01) and were greatest in the control group followed by heifers in the d 18, 5, 15, 10, and 2 groups (9.4a, 4.3b, 3.7b, 1.5c, 1.4c, 0.9c pg/mL, respectively; a–cP < 0.05; Table 3Go). Mean serum concentrations of E2 at GnRH 2 were greatest in the d 5 group followed by d 10, 2, 18, and 15 groups (Table 3Go).


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  		Table 3. Mean serum concentrations of estradiol (±SEM) at GnRH 1 and GnRH 2 in the COSynch protocol1 in Exp. 1 and Exp. 2 for different treatment groups2
 
Effect of Ovulatory Response to GnRH 1 on Subsequent Dominant Follicle Diameter and Ovulatory Response at GnRH 2.
Similar to Exp. 1, there was no effect of ovulatory response to GnRH 1 on diameter of the largest follicle at GnRH 2, variation diameter of the largest follicle at GnRH 2, percentage ovulating after GnRH 2, or proportion of heifers undergoing luteolysis before PGF2{alpha} (Table 2Go). In addition, heifers with the smallest follicles at GnRH 2 were from the d 15 and 18 groups and had undergone luteolysis before PGF2{alpha}.


   DISCUSSION
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The GnRH-induced ovulation of small dominant follicles decreased pregnancy rates in heifers (Perry et al., 2007Go) and cows (Vasconcelos et al., 2001Go; Perry et al., 2005Go). However, cows that showed estrus and spontaneously ovulated small dominant follicles had pregnancy rates comparable with cows in which large dominant follicles were induced to ovulate with GnRH. Therefore, the physiological maturity of a follicle (i.e., ability to initiate estrus) and not its absolute diameter appears to be more important in pregnancy.

Dominant follicle diameter at GnRH 2 of a COSynch protocol ranged from 8 to 16 mm in heifers (Perry et al., 2007Go) and 9 to ≥17 mm in cows (Perry et al., 2002Go). The proportion of heifers and cows that had a small dominant follicle (≤11 mm) at GnRH 2 was 20.3% (Perry et al., 2007Go) and 26%, respectively (Perry et al., 2002Go). The presence of small dominant follicles at GnRH 2, therefore, is not uncommon. The reason for the presence of small dominant follicles at GnRH 2 is unclear, but may be because of failure of heifers and cows to initiate a follicular wave after GnRH 1. Failure of GnRH 1 to initiate a new follicular wave could lead to asynchronous follicular waves, small dominant follicles, and more variation in dominant follicle diameter at GnRH 2 compared with animals that have a synchronized follicular wave after GnRH 1. Only 66% of cows (Geary et al., 1998Go) and 50% of heifers (Pursley et al., 1995Go) at random stages of the cycle responded to GnRH 1. In the current study, the collective ovulatory response to GnRH 1 (Exp.1 and 2) was 45%. Heifers tend to have a lower ovulatory response to GnRH 1 than cows because of follicular waves of shorter duration and dominant follicles that have a lesser maximum diameter compared with cows (Pursley et al., 1995Go; Adams, 1999Go). The GnRH-induced LH release was reduced in heifers compared with cows (Lucy and Stevenson, 1986Go), which may contribute to decreased ovulatory response in heifers.

The general pattern in ovulatory response to GnRH 1 among heifers from the d 2, 5, 10, and 15 groups was expected. The ovulatory response to GnRH 1 among heifers in the d 18 group was less than predicted. Ovulatory response of a dominant follicle in cattle during the growth phase, plateau phase, or atretic phase was 100, 33, and 0%, respectively (Silcox et al., 1993Go). In the current study, heifers in the d 2 group had recently ovulated and did not have a dominant follicle that could have ovulated or luteinized in response to GnRH 1. Heifers in the d 5 and 15 groups had dominant follicles that were growing, and the ovulatory response to GnRH 1 was greater. In the d 10 group, growth of the largest follicle ceased in most heifers and the ovulatory response to GnRH 1 was less. The d 18 group also had several heifers in which growth of the dominant follicle had decreased, and perhaps these heifers were having 3 follicular waves per cycle. Eight heifers in the d 18 group did not ovulate to GnRH 1, and 7 of these heifers initiated new follicular waves around the time of GnRH 1. Moreira et al. (2000)Go reported similar ovulatory responses to GnRH 1 in dairy heifers on d 2, 5, 10, and 15 after estrus, but all heifers on d 18 of the estrous cycle ovulated to GnRH 1. The discrepancy between the data reported by Moreira et al. (2000)Go and the current study may be the relative proportions of heifers with 2 or 3 follicular waves per cycle.

In addition to the physiological status of a dominant follicle, the magnitude of the GnRH-induced LH release may play a role in the ovulatory response to GnRH 1. Although bovine follicles attain ovulatory capacity around 10 mm, a larger dose of LH was required to induce ovulation of a 10-mm follicle compared with larger follicles (Sartori et al., 2001Go). The amount of LH required to cause ovulation of a follicle ≥13 mm was only 4 mg compared with 24 to 40 mg for a 10-mm follicle (Sartori et al., 2001Go).

Steroid environment is known to influence the magnitude and duration of the GnRH-induced LH release. Increased circulating concentrations of P4 inhibit, whereas elevated circulating concentrations of E2 (in the absence of P4) increase GnRH-induced gonadotropin secretion (Price and Webb, 1988Go). The GnRH-induced LH release was consistent with the known effects of P4 and E2 on d 2, 5, 10, 15, and 18 as well as the control group. Examination of the relationship between magnitude of the GnRH-induced LH release and ovulatory response in the d 2, 5, and 10 groups was not possible because every heifer either ovulated (d 5) or did not ovulate (d 2 and 10) in response to GnRH 1. In the d 15 and 18 groups (comprised of ovulatory and nonovulatory heifers), there was no association between magnitude of the GnRH-induced LH release and ovulatory response. The physiological status of the dominant follicle, therefore, was more important than the magnitude of the GnRH-induced LH surge on ovulatory response.

Ovulatory response to GnRH 2 was 61% (Exp. 1 and 2), and the effect of day of the cycle was similar for both experiments except for the d 2 group. The discrepancy in the d 2 group was likely because of a difference in age of the heifers between experiments. Heifers in Exp. 1 and 2 were approximately 20 and 15 mo of age, respectively. The younger heifers in Exp. 2 likely had shorter follicular waves compared with the older heifers in Exp. 1. At GnRH 2, a greater proportion of the younger heifers (Exp. 2) had initiated a new follicular wave, indicating that the previous dominant follicle was no longer dominant. In prepubertal heifers, duration of a follicle wave and maximum dominant follicle diameter increased with age (Adams, 1999Go). Shorter follicular waves in the younger heifers could explain the lower ovulatory response at GnRH 2 in Exp. 2 compared with Exp. 1.

The lower ovulatory response in the d 15 and 18 groups at GnRH 2 was because of the large proportion of heifers that experienced luteolysis and estrus before PGF2{alpha} . Interestingly, the d 15 group had a large proportion of heifers ovulate and form accessory CL (aCL) after GnRH 1, but the aCL were not able to inhibit many of these heifers from showing estrus. The reduced ability of the aCL to inhibit estrus could be because of inadequate P4 production (Keisler and Keisler, 1989Go), premature regression of the aCL, or both. In the d 15 group, it was not possible to distinguish between P4 from the aCL and the primary CL (formed after spontaneous ovulation). In the d 18 group, 2 heifers that ovulated in response to GnRH 1 were undergoing luteolysis at d –9, as evidenced by decreasing P4 concentrations. After aCL formation in the preceding group, circulating concentrations of P4 did not increase above 1 ng/mL through the remaining synchronization period. In ewes, GnRH-induced CL had reduced luteal phase P4 concentrations compared with control ewes (Keisler and Keisler, 1989Go). Similar results were reported in dairy cattle (Lucy and Stevenson, 1986Go). A preovulatory follicle may require exposure to a normal follicular phase (i.e., luteolysis followed by increasing estradiol production) so that the subsequent CL produces adequate amounts of P4 for the establishment and maintenance of pregnancy. Alternatively, the aCL may be responsive to endogenous PGF2{alpha} at an earlier time point than a primary CL. The aCL formed in the presence of increased circulating concentrations of P4 regressed after PGF2{alpha} administration as early as 2 d after formation (Howard and Britt, 1990Go). Further examination of the function of aCL formed during high and low circulating concentrations of P4 is warranted.

Dominant follicle size at GnRH 2 was not affected by ovulatory response to GnRH 1. In Exp. 1 and 2, 46 and 36% (respectively) of the heifers had follicles ≤11 mm at GnRH 2, and almost half of these heifers ovulated after GnRH 1. In both Exp. 1 and 2, heifers with the smallest follicles (4 to 9 mm) at GnRH 2 were detected in estrus before GnRH 2 and were in the d 15 and 18 groups. However, the reason for the presence of follicles with diameters of 9.5 to 11 mm at GnRH 2 in heifers in the remaining groups was less clear. For example, 6 of the 14 heifers in the d 2 group (Exp. 1) had follicles in the 9.5 to 11 mm range even though they had the maximum time for dominant follicle growth before GnRH 2. These data indicate that these small follicles may have been growing at a slower rate than the larger follicles.

Ovulatory response to GnRH 1 did not affect ovulatory response to GnRH 2. Similarly, Lamb et al. (2006)Go reported the inclusion of GnRH 1 did not affect estrus response or pregnancy rate compared with heifers without GnRH 1 administered. In the current study, day of the cycle at GnRH 1 was more important than ovulatory response to GnRH 1 in predicting ovulatory response to GnRH 2. The combined percentage of heifers that ovulated in response to GnRH 2 in the d 2, 5, and 10 groups was 92.5 and 76% in Exp. 1 and 2, respectively, whereas the combined ovulatory response in the d 15 and 18 groups was 17 and 19% in Exp. 1 and 2, respectively. The lower ovulatory response in the d 15 and 18 groups was because a majority of the heifers underwent luteolysis and exhibited estrus before or at the time of PGF2{alpha} injection.

In postpartum cows, GnRH-induced ovulation of physiologically immature follicles decreased pregnancy rates and increased late embryonic/fetal mortality (Perry et al., 2005Go). The reason for the reduced ability to establish and maintain a pregnancy after ovulation of physiologically immature follicles is unknown but may involve reduced luteal function (Murdoch and van Kirk, 1998Go; Vasconcelos et al., 2001Go; Perry et al., 2002Go, 2005Go), inadequate uterine environment (Moore, 1985Go; Murdoch and van Kirk, 1998Go; Bridges et al., 2006Go), compromised oocyte quality (Arlotto et al., 1996Go; Brevini-Gandolfi and Gandolfi, 2001Go), or both. Cows induced to ovulate small follicles had a diminished rise in circulating P4 from d 2 to 16 (d 0 = GnRH) compared with cows that were induced to ovulate a large follicle or which ovulated spontaneously after estrus regardless of size (Perry et al., 2005Go). Similarly in Exp. 1, heifers that ovulated small follicles (≤11 mm) had a lesser rise in P4 compared with heifers that ovulated larger follicles. Secretion of E2 may be an important determinant of physiological maturity of a follicle. In Exp. 1, increasing concentrations of E2 were associated with increasing follicle size. Furthermore, extending the proestrus period from 1.5 to 2.5 d and increasing the preovulatory E2 concentrations resulted in greater subsequent luteal P4 and greater pregnancy rates regardless of size of the follicle that ovulated (Bridges et al., 2006Go). Follicular concentrations of E2 have been associated with greater P4 synthesis after gonadotropin stimulation (McNatty, 1979Go) and with induction of endometrial P4 receptors (Ing and Tornesi, 1997Go).

In summary, presence of small dominant follicles at GnRH 2 was not affected by ovulatory response to GnRH 1. The day of the cycle at GnRH 1 affected dominant follicle size and ovulatory response at GnRH 2. Heifers receiving GnRH 1 in the latter part of the cycle had a greater incidence of luteolysis before PGF2{alpha} and earlier onset of estrus regardless of presence of an aCL after GnRH 1. Therefore, presynchronizing heifers to be in the early stage of the estrous cycle (≤d 10) at GnRH 1 would increase the proportion of heifers having a follicle large enough to respond to GnRH 2 and increase the ovulatory response after GnRH 2.

1 Corresponding author: Smithmf{at}missouri.edu

Received for publication May 17, 2007. Accepted for publication August 27, 2007.


   LITERATURE CITED
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 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 


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