M.C. Wiltbank

Synchronization of ovulation in dairy cows using PGF2α and GnRH

This paper reports a new method for synchronizing the time of ovulation in cattle using GnRH and PGF2α. In Experiments 1 and 2, lactating dairy cows (n=20) ranging from 36 to 280 d postpartum and dairy heifers (n=24) 14 to 16 mo old were treated with an intramuscular injection of 100 μg GnRH at a random stage of the estrous cycle. Seven d later the cattle received PGF2α to regress corpora lutea (CL). Lactating cows and heifers received a second injection of 100 μg GnRH 48 and 24 h later, respectively. Lactating cows were artificially inseminated 24 h after the second GnRH injection. Ovarian morphology was monitored daily by trans-rectal ultrasonography from 5 d prior to treatment until ovulation. In Experiment 3, the flexibility in the timing of hormonal injections with this synchronization protocol was evaluated by randomly assigning 66 lactating dairy cows to 3 different treatment groups. Lactating cows received the injection of PGF2α 48 (Group 1), 24 (Group 2), and 0 h (Group 3) prior to the second injection of GnRH, which was administered at the same time in each group to ensure the second injection of GnRH was given when follicles were at a similar stage of growth. In Experiments 1 and 2, the first injection of GnRH caused ovulation and formation of a new or accessory CL in 1820 cows and 1324 heifers. In addition, this injection of GnRH initiated or was coincident with initiation of a new follicular wave in 2020 lactating cows and 1824 heifers. Corpora lutea regressed after PGF2α in 2020 cows and in 1824 heifers. All cows and 1824 heifers ovulated a newly formed dominant follicle between 24 and 32 h after the second injection of GnRH. Ten of 20 cows conceived to the timed artificial insemination. In Experiment 3, the conception rate in Groups 1 and 2 were greater than in Group 3, (55 and 46 % vs 11%, respectively). In summary, this protocol could have a major impact on managing reproduction in lactating dairy cows, because it allows for AI to occur at a known time of ovulation and eliminates the need for detection of estrus.

Synchronization rate, size of the ovulatory follicle, and pregnancy rate after synchronization of ovulation beginning on different days of the estrous cycle in lactating dairy cows

Recently a protocol was developed that precisely synchronizes the time of ovulation in lactating dairy cows (Ovsynch; GnRH-7d-PGF2 alpha-2d-GnRH). We evaluated whether initiation of Ovsynch on different days of the estrous cycle altered the effectiveness of this protocol. The percentage of cows (n = 156) ovulating to the first GnRH was 64% and varied (P < 0.01) by stage of estrous cycle. Treatment with PGF2 alpha was effective, with 93% of cows having low progesterone at second GnRH. The overall percentage of cows that ovulated after second GnRH (synchronization rate) was 87% and varied by response to first GnRH (92% if ovulation to first GnRH vs 79% if no ovulation; P < 0.05). There were 6% of cows that ovulated before the second injection of GnRH and 7% with no detectable ovulation by 48 h after second GnRH. Maximal diameter of the ovulatory follicle varied by stage of estrous cycle, with cows in which Ovsynch was initiated at midcycle having the smallest follicles. In addition, milk production and serum progesterone concentration on the day of PGF2 alpha affected (P < 0.05) size of the ovulatory follicle. Using these results we analyzed pregnancy rate at Days 28 and 98 after AI for cows (n = 404) in which Ovsynch was initiated on known days of the estrous cycle. Pregnancy rate was lower for cows expected to ovulate larger follicles than those expected to ovulate smaller follicles (P < 0.05; 32 vs 42%). Thus, although overall synchronization rate with Ovsynch was above 85%, there were clear differences in response according to day of protocol initiation. Cows in which Ovsynch was initiated near midcycle had smaller ovulatory follicles and greater pregnancy rates.

Reduction in size of the ovulatory follicle reduces subsequent luteal size and pregnancy rate

We hypothesized that reducing the size of the ovulatory follicle using aspiration and GnRH would reduce the size of the resulting CL, reduce circulating progesterone concentrations, and alter conception rates. Lactating dairy cows (n=52) had synchronized ovulation and AI by treating with GnRH and PGF2alpha as follows: Day -9, GnRH (100 microg); Day -2, PGF2alpha (25 mg); Day 0, GnRH (100 microg); Day 1, AI. Treated cows (aspirated group; n=29) had all follicles > 4 mm in diameter aspirated on Days -5 or -6 in order to start a new follicular wave. Control cows (nonaspirated group: n=23) had no follicle aspiration. The size of follicles and CL were monitored by ultrasonography. The synchronized ovulation rate (ovulation rate to second GnRH injection: 42/52=80.8%) and double ovulation rate of synchronized cows (6/42=14.3%) did not differ (P > 0.05) between groups. Aspiration reduced the size of the ovulatory follicle (P < 0.0001; 11.5 +/- 0.2 vs 14.5 +/- 0.4 mm), and serum estradiol concentrations at second GnRH treatment (P < 0.0002; 2.5 +/- 0.4 vs 5.7 +/- 0.6 pg/mL). The volume of CL was less (P < 0.05) for aspirated than nonaspirated cows on Day 7 (2,862 +/- 228 vs 5,363 +/- 342 mm3) or Day 14 (4,652 +/- 283 vs 6,526 +/- 373 mm3). Similarly, serum progesterone concentrations were less on Day 7 (P < 0.05) and Day 14 (P < 0.10) for aspirated cows. Pregnancy rate per AI for synchronized cows was lower (P < 0.05) for aspirated (3/21=14.3%) than nonaspirated (10/21=47.6%) cows. In conclusion, ovulation of smaller follicles produced lowered fertility possibly because development of smaller CL decreased circulating progesterone concentrations.

Follicular deviation and acquisition of ovulatory capacity in bovine follicles.

Abstract Selection of dominant follicles in cattle is associated with a deviation in growth rate between the dominant and largest subordinate follicle of a wave (diameter deviation). To determine whether acquisition of ovulatory capacity is temporally associated with diameter deviation, cows were challenged with purified LH at known times after a GnRH-induced LH surge (experiment 1) or at known follicular diameters (experiments 2 and 3). A 4-mg dose of LH induced ovulation in all cows when the largest follicle was ≥12 mm (16 of 16), in 17% (1 of 6) when it was 11 mm, and no ovulation when it was ≤10 mm (0 of 19). To determine the effect of LH dose on ovulatory capacity, follicular dynamics were monitored every 12 h, and cows received either 4 or 24 mg of LH when the largest follicle first achieved 10 mm in diameter (experiment 2). The proportion of cows ovulating was greater (P < 0.05) for the 24-mg (9 of 13; 69.2%) compared with the 4-mg (1 of 13; 7.7%) LH dose. To determine the effect of a higher LH dose on follicles near diameter deviation, follicular dynamics were monitored every 8 h, and cows received 40 mg of LH when the largest follicle first achieved 7.0, 8.5, or 10.0 mm (experiment 3). No cows with a follicle of 7 mm (0 of 9) or 8.5 mm (0 of 9) ovulated, compared with 80% (8 of 10) of cows with 10-mm follicles. Thus, follicles acquired ovulatory capacity at about 10 mm, corresponding to about 1 day after the start of follicular deviation, but they required a greater LH dose to induce ovulation compared with larger follicles. We speculate that acquisition of ovulatory capacity may involve an increased expression of LH receptors on granulosa cells of the dominant follicle and that this change may also be important for further growth of the dominant follicle.

Physiological classification of anovulatory conditions in cattle

Evaluation of follicular growth patterns by ultrasound combined with measurement of circulating reproductive hormones has allowed designation of three functionally critical follicular sizes during the final stages of follicular growth: emergence (-4 mm), deviation (-9 mm), and ovulation (variable from 10 to 20 mm). Classification of anovulatory conditions on the basis of these three critical points is logical and provides for rational diagnosis and treatment of the underlying physiological condition. In extreme undernutrition, there is growth of follicles to emergence but not to deviation; however, the underlying pathophysiology is not defined because of relatively few scientific investigations of this condition. Anovulatory conditions with growth of follicles to deviation but not to ovulatory size have been extensively studied. Undernutrition and/or suckling can cause this anovulatory condition. It is characterized by a greater negative feedback effect of estradiol on GnRH/LH pulses than found in normally cycling cows. Another anovulatory condition that is common in high producing lactaing dairy cows is characterized by growth of follicles to larger than ovulatory size, such as is observed in cows with follicular cysts. This condition is characterized by an insensitivity of the hypothalamus to the positive feedback effects of estradiol. Thus, these last two common anovulatory conditions appear to be primarily due to changes in the responsiveness of the hypothalamus to estradiol. Treatments that increase circulating progesterone concentrations can help in the treatment of these two conditions by potentially altering GnRH/LH pulses and allowing the final stages of follicular growth or resetting the hypothalamic responsiveness to the positive feedback effects of estradiol.

Emergence and deviation of follicles during the development of follicular waves in cattle

The nature of emergence and deviation of follicles during follicular waves in cattle was studied in 3 experiments by re-examining data from previous projects. Wave emergence was defined as the day or examination (when more than 1 examination per day) the future dominant follicle was 4 mm (Day 0 or Examination 0). Deviation was defined as the beginning of the greatest difference in growth rates between the 2 largest follicles and between 2 consecutive examinations. The search for deviation in an individual wave was done retrospectively from the examination with the maximum diameter of the second largest follicle. In Experiment 1, follicles were assessed ultrasonically for 28 waves every 8 h. The number of examinations that encompassed the emergence of all growing 3-mm follicles was 10.0 +/-0.5 (mean +/-SEM; equivalent to 3.3 d) and extended from mean Examination -3.1 +/-0.3 to mean Examination 6.0 +/-0.6. A mean of 24 growing 3-mm follicles was found, and the maximal attained diameters were 4 mm (46%), 5 mm (25%), and >/=6 mm (29%). More (P<0.05) 3-mm follicles at Examinations -2 and -1 grew to >/=6 mm than to 4 or 5 mm, whereas more 3-mm follicles at Examinations 2 to 6 grew to only 4 mm. On average, the future dominant follicle appeared as a 3-mm follicle (Examination -2.1 +/-0.2) 6 and 10 h earlier (P<0.03) than for the largest (Examination -1.4 +/-0.3) and second-largest (Examination -0.8 +/-0.4) future subordinates, respectively. This result supported the hypothesis that the future dominant follicle has, on the average, an early developmental advantage. In Experiment 2 (n=33 waves), data were normalized to the day at the beginning of deviation (Day 2.8 +/-0.2) when the mean diameters of the dominant and largest subordinate follicle were 8.5 +/-0.2 mm and 7.2 +/-0.2 mm, respectively. This result suggests that the follicle selected to become dominant, as manifested by deviation, is the first follicle to develop to a decisive stage. In Experiment 3 (n=19 waves), FSH concentrations were lower (P<0.05) on the day at the beginning of deviation (8.5 +/-0.5 ng/ml) than on the day before (10.1 +/-0.8 ng/ml), with no continuing decrease after deviation. This temporal result suggests that the attainment of approximate basal levels of FSH is a component of the deviation mechanism.

ASSOCIATIONS BETWEEN EMERGENCE OF FOLLICULAR WAVES AND FLUCTUATIONS IN FSH CONCENTRATIONS DURING THE ESTROUS CYCLE IN EWES

Folliculogenesis was studied daily in 16 interovulatory intervals in 5 Polypay ewes from mid February through April using transrectal ultrasonic imaging. The 3-mm follicles attaining > or = 5 mm on Days--1 (ovulation=Day 0) to 11 showed significant peak numbers on Days 0, 5 and 10. The number of 3- and 4-mm follicles that did not reach > 4 mm was not significant, indicating that these follicles did not manifest a wave pattern. A follicular wave was defined as one or more follicles growing to > or = 5 mm; the day the follicles were 3 mm was the day of wave emergence, and the first wave after ovulation was Wave 1. Waves 1, 2 and 3 emerged on Days -1 to 2,4 to 7 and 8 to 10, respectively. Four interovulatory intervals in April were short (9 to 14 d), indicating the end of the ovulatory season. In the remaining 12 intervals, the ovulatory wave was Wave 3 in one interval, Wave 4 in 8 intervals, and Wave 5 or 6 in 3 intervals. The ovulatory wave followed the rhythmic pattern of Waves 1 to 3 by emerging on Days 11 to 14 in 50% of the intervals. In the remaining intervals, either the ovulatory wave was Wave 4 but did not emerge until Day 16 or other waves intervened between Wave 3 and the ovulatory wave. The longest intervals (22, 24 and 24 d) had >4 waves. Based on a cycle-detection program, peak values of FSH fluctuations were temporally associated with the emergence of waves as indicated by the following: 1) tendency (P < 0.08) for an increase in FSH concentrations between 3 and 2 days before emergence of a wave; 2) close agreement between mean number of waves per interval (mean +/- SEM, 4.1 +/- 0.3) and mean number of identified FSH fluctuations (4.5 +/- 0.3); 3) close agreement in length of interwave intervals (4.0 +/- 0.3) and interpeak (FSH) intervals (3.6 +/- 0.2); 4) positive correlation (r(2)=0.8) for number of the 2 events (follicular waves and FSH fluctuations) within intervals; and 5) a closer (P < 0.01) temporal relationship between the 2 events than would have been expected if the events were independent. The results support a relationship between transient increases in FSH concentrations and emergence of follicular waves throughout the interovulatory interval in Polypay ewes, with the 2 events occurring approximately every 4 d.

FOLLICULAR AND HORMONAL DYNAMICS DURING THE FIRST FOLLICULAR WAVE IN HEIFERS

A few days after the first follicular wave emerges as 4-mm follicles, follicular deviation occurs wherein 1 follicle of the wave continues to grow (dominant follicle) while the others regress. The objectives of this study were to characterize follicle growth and associated changes in systemic concentrations of gonadotropins and estradiol at 8-h intervals encompassing the time of follicle deviation. Blood samples from heifers (n = 11) were collected and the ovaries scanned by ultrasound every 8 h from 48 h before to 112 h after the maximal value for the preovulatory LH surge. The follicular wave emerged at 5.8 +/- 5.5 h (mean +/- SEM) after the LH surge, and at this time the future dominant follicle (4.2 +/- 0.8 mm) was larger (P < 0.001) than the future largest subordinate follicle (3.6 +/- 0.1 mm). There was no difference in growth rates between the 2 follicles from emergence to the beginning of the deviation (0.5 mm/8 h for each follicle), indicating that, on average, the future dominant follicle maintained a size advantage over the future subordinate follicle. Deviation occurred when the 2 largest follicles were 8.3 +/- 0.2 and 7.8 +/- 0.2 mm in diameter, at 61.0 +/- 3.7 h after wave emergence. Diameter deviation was manifested between 2 adjacent examinations at 8-h intervals. Mean concentrations of FSH decreased, while mean concentrations of LH increased 24 and 32 h before deviation, respectively, and remained constant (no significant differences) for several 8-h intervals encompassing deviation. In addition to the increase and decrease in circulating estradiol concentrations associated with the preovulatory LH surge, an increase (P < 0.05) occurred between the beginning of deviation and 32 h after deviation. The results supported the hypotheses that deviation occurs rapidly (within 8 h), that elevated systemic LH concentrations are present during deviation, and that deviation is not preceded by an increase in systemic estradiol.

A new presynchronization system (Double-Ovsynch) increases fertility at first postpartum timed AI in lactating dairy cows

This study evaluated a novel presynchronization method, using Ovsynch prior to the Ovsynch-timed AI protocol (Double-Ovsynch) compared to Presynch-Ovsynch. Lactating Holstein (n=337) cows, were assigned to two treatment groups: (1) Presynch (n=180), two injections of PGF 14 d apart, followed by the Ovsynch-timed AI protocol 12 d later; (2) Double-Ovsynch (n=157), received GnRH, PGF 7 d later, and GnRH 3 d later, followed by the Ovsynch-timed AI protocol 7 d later. All cows received the same Ovsynch-timed AI protocol: GnRH (G1) at 68+/-3 DIM (mean+/-SEM), PGF 7 d later, GnRH (G2) 56h after PGF, and AI 16 to 20h later. Pregnancy was diagnosed 39-45 d after timed AI. Double-Ovsynch increased the pregnancies per AI (P/AI) compared to Presynch-Ovsynch (49.7% vs 41.7%, P=0.03). Surprisingly, Double-Ovsynch increased P/AI only in primiparous (65.2% vs 45.2%; P=0.02) and not multiparous (37.5% vs 39.3%) cows. In a subset of 87 cows, ovarian ultrasonography and progesterone (P4) measurements were performed at G1 and 7 d later. Double-Ovsynch decreased the percentage of cows with low P4 (<1ng/mL) at G1 (9.4% vs 33.3%) and increased the percentage of cows with high P4 (> or =3ng/mL) at PGF (78.1% vs 52.3%). Thus, presynchronization of cows with Double-Ovsynch increased fertility in primiparous cows compared to a standard Presynch protocol, perhaps due to induction of ovulation in non-cycling cows and improved synchronization of cycling cows. Future studies are needed, with a larger number of cows, to further test the hypothesis of higher fertility with Double-Ovsynch, and to elucidate the physiological mechanisms that underlie apparent changes in fertility with this protocol.

Follicular-Fluid Factors and Granulosa-Cell Gene Expression Associated with Follicle Deviation in Cattle

Abstract Intrafollicular changes in the largest follicle (F1) and second-largest (F2) follicle were examined in relation to follicle diameter deviation. Deviation is characterized by continued growth of the largest follicle and the cessation of growth of the smaller follicles. Granulosa cells and follicular fluid were obtained from slaughterhouse ovaries (n = 95 pairs, experiment 1), and follicular fluid was collected in vivo (n = 28 heifers, experiment 2). Several ranges in the diameter of F1 were used to represent the progressive growth of the follicle. The diameter range with the first significant increase in the difference between F1 and F2 was determined for each end point and was used as an indicator of the sequence of events associated with diameter deviation. An increased difference for diameter and for estradiol concentration occurred (P < 0.05) simultaneously at the 8.5- to 8.9-mm range in both experiments. In experiment 1, the increased difference between F1 and F2 in LH receptor (LHr) mRNA expression occurred (P < 0.05) at the 8.0- and 8.4-mm range. In F2 of experiment 2, there was a progressive decrease (P < 0.05) in free insulin-like growth factor (IGF)-1 and a progressive increase (P < 0.05) in IGF binding protein (BP)-2 across the follicle-diameter ranges (7.5-11.2 mm). No differences were detected between F1 and F2 for 3β-hydroxysteroid dehydrogenase mRNA expression in experiment 1 and testosterone, total inhibin, and dimeric inhibin-A concentrations in experiment 2. The results indicated that the acquisition of granulosa cell LHrs by F1, as indicated by increased LHr mRNA expression, occurred one diameter range before an increased difference between F1 and F2 for diameter or estradiol concentrations. On a temporal basis, it is concluded that LHr acquisition plays a role in the establishment of diameter deviation. In addition, the reduced growth of F2 may have involved the reduced bioavailability of IGF-1 in association with elevated IGFBPs.