- Email: [email protected]
Ovulation rate after GnRH or PGF2α administration in early postpartum dairy cows
Theriogenology 60 (2003) 341–348
Ovulation rate after GnRH or PGF2a administration in early postpartum dairy cows$ Ahmet Gu¨men1, Brad Seguin* Department of Clinical and Population Sciences, College of Veterinary Medicine, University of Minnesota, 435H Anim Sci/Vet Med, 1988 Fitch Avenue, St. Paul, MN 55108, USA Received 20 November 2001; accepted 8 November 2002
Abstract The objectives of this study evaluating induction of ovulation in early postpartum dairy cows were to: compare two methods of GnRH (100 mcg) administration (i.m. route and s.c. implant), and determine if prostaglandin F2a (PGF) causes release of LH or ovulation similar to that reported for GnRH. In trial #1, serum LH peaked at 2 h after i.m. administration of GnRH and was declining at 4 h. The s.c. GnRH implant also caused an elevation in serum LH at 2 and 4 h after treatment, with LH declining at 6 h. Serum LH was unchanged in control cows. Experimental treatment caused ovulation in 4 of 14 GnRH i.m. treated cows, 4 of 12 GnRH implanted cows and 0 of 13 control cows. Parity had no effect on LH response but did affect resulting ovulation rate as multiparous cows were more likely to ovulate than were primiparous cows in response to either GnRH treatment. All cows that ovulated had a follicle larger than 12 mm at the time of treatment. In trial #2, serum LH increased as before after i.m. administration of GnRH, however, serum LH was unchanged in cows treated with PGF or saline. Gonadotropin releasing hormone caused more cows to ovulate than did PGF or saline treatments, and GnRH shortened the interval from treatment to the onset of CL function over the PGF treatment; 13:9 2:6, 28:2 4:1 and 22:3 4:1 days for GnRH, PGF and saline, respectively. In summary, there was no difference in the ability of s.c. implantation and i.m. administration of GnRH to cause ovulation. Prostaglandin F2a did not cause release of LH or ovulation. In 22 early postpartum dairy cows treated with 100 mcg GnRH i.m. in these two trials, nearly all cows (95%) responded with a release of LH but only 45% (10/22) responded with an ovulation and subsequent formation of a CL. # 2003 Elsevier Science Inc. All rights reserved. Keywords: GnRH; PGF2a; Postpartum; Ovulation; Dairy cows
$ Presented in part as Abstract #220 at the 2000 ASAS/ADSA Midwestern Sectional Meetings in Des Moines, IA. * Corresponding author. Tel.: þ1-612-624-4741; fax: þ1-612-624-3233. E-mail address: [email protected] (B. Seguin). 1 Present address: Department of Dairy Science, University of Wisconsin-Madison, Madison, WI 53706, USA.
0093-691X/03/$ – see front matter # 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0093-691X(02)01372-9
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1. Introduction Delayed onset of estrous cyclicity after calving is a major hindrance to the desired rebreeding performance of dairy herds attempting to reach their goals for reproductive performance and production efficiency [1,2]. For example, in one survey of 448 postpartum periods on six well-managed dairy herds by milk progesterone profiling, 20% had delayed cyclicity due to no ovarian activity through Day 50 postpartum and only 54% showed normal resumption of estrous cyclicity by Day 50 [2]. Britt et al. [3] reported that a s.c. implant of gonadotropin releasing hormone (GnRH) on Day 14 postpartum caused, through its ability to release luteinizing hormone (LH), ovulation in 100% of lactating Holstein cows. In some studies, GnRH treatment at 14 days postpartum has improved first service conception rate for breeding beginning at 50 days postpartum in dairy cows [4]. However, several reports have shown no effect on rebreeding performance after early postpartum administration of GnRH [5,6] and a few have even found a negative effect [7,8]. Prostaglandin F2a and its analogues (PGF) have been used to manipulate the bovine estrous cycle through its luteolytic activity [9]. However, early postpartum administration of PGF products has shown beneficial effects in some trials [10–13] but others have shown no effect [14–17] on herd reproductive performance. Interestingly, in two reports progesterone concentration, i.e. presence or absence of a functional corpus luteum (CL), at the time of this PGF treatment did not correlate with positive effects on reproductive performance [11,12]. This suggests that some mechanism other than luteolysis may be involved, perhaps stimulation of follicular growth or ovulation by PGF. Therefore, the objectives of these trials in early postpartum dairy cows were to compare delivery of GnRH by i.m. administration versus s.c. implantation on LH response and ovulation, and determine if PGF causes release of LH or ovulation similar to that reported for exogenous GnRH.
2. Materials and methods 2.1. Trial #1 Thirty-nine Holstein dairy cows (19 primiparous and 20 multiparous) were randomly assigned at calving among 3 groups (Group 1, GnRH by i.m. administration; Group 2, GnRH by s.c. implant; Group 3, untreated controls) for one-time treatment at 14 days postpartum. All cows were milked twice daily in a parlor, housed in a tie-stall barn, and fed a total mixed ration to meet maintenance, growth, reproduction, and milk production requirements. Intramuscular administration of 100 mcg of GnRH (Cystorelin, Merial Inc., Iselin, NJ) (Group 1, n ¼ 14) was given by deep i.m. injection (1.5 in. 18 gauge needle) in the right semimembranous/semitendinosus muscles. Subcutaneous implant of 100 mcg of GnRH (Group 2, n ¼ 12) was made in an upper caudal area of the left side of the neck under local anesthesia similar to the procedure of Britt et al. [3]. The GnRH implants were prepared by pouring the GnRH dose into a no. 12 gelatin capsule, which was reassembled,
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quick-frozen in liquid nitrogen, and then lyophilized (Labconco, Kansas City, MO). Untreated cows (Group 3, n ¼ 13) served as controls. Blood samples were collected from tail vessels just before treatment and at 2, 4 and 6 h after treatment into vacutainer tubes (Monoject, Sherwood Medical, St. Louis, MO) to obtain blood serum for LH assay. Blood samples to obtain plasma for progesterone assay were also collected just before treatment and then on Days 7 and 14 after treatment in potassium oxalate (14 mg) and sodium fluoride (17.5 mg) vacutainer tubes (Monoject, Sherwood Medical, St. Louis, MO). Blood samples for plasma were centrifuged within 1 h after collection and for serum at 12–18 h after collection. Serum and plasma samples were stored at 5 8C until assayed. Serum LH was determined by double antibody radioimmunoassay as described by Niswender et al. [18]. The LH assay incorporated NIH-bLH B10 for iodination and references standards, and NIDDK-anti-oLH-1 as the primary antiserum. Hormone sensitivity, calculated two standard deviations below the mean counts per minute at maximum binding, was 0.6515 ng/ml. Coefficients of variation intra- and inter-assays for LH were 2.95 and 4.02%, respectively. Plasma progesterone was determined by radioimmunoassay (COAT-A-COUNT progesterone test (Diagnostic Products Corp., Los Angeles, CA)). Plasma progesterone hormone sensitivity was 0.0585 ng/ml. Coefficients of variation for intra- and inter-assays for progesterone were 4.26 and 4.49%, respectively. On treatment day, ovarian structures were examined by palpation per rectum and ultrasonography using an ultrasound machine equipped with a 7.5 MHz rectal transducer (Medison 500 linear-array real time B-mode ultrasound scanner, Universal Medical Systems Inc., Bedford Hills, NY). Ovulation as a result of treatment was determined by ultrasonography on Day 3 and by changes in plasma progesterone concentration subsequent to treatment. Palpation per rectum was done thereafter every 6 days immediately after blood sample collection to monitor ovarian (especially CL) status. 2.2. Trial #2 Twenty-five multiparous Holstein dairy cows were randomly assigned at calving among three groups (Group 1, GnRH; Group 2, PGF; Group 3, saline) for a one-time treatment 14–19 days postpartum. Group 1 cows (n ¼ 8) were treated with 100 mcg of GnRH to serve as a positive control, Group 2 cows (n ¼ 10) were given 25 mg of PGF (Lutalyse, Pharmacia & Upjohn Co., Kalamazoo, MI) to serve as the test group, and Group 3 cows (n ¼ 7) were given saline to serve as negative controls. All treatments were given i.m. as described above. Blood samples were collected just before treatment and at 1, 2, 4, 12 and 24 h after treatment as before for serum LH assays. Blood samples were also collected just before treatment and then every 3 days for 24 days for plasma progesterone assays as before. Progesterone and LH data were evaluated using ANOVA with repeated measurement within animal over time. Ovulation rates were compared by the chi-square test. Interval data from treatment to first rise in progesterone were evaluated using ANOVA with SAS [19].
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3. Results 3.1. Trial #1 There was no difference in LH among the three treatment groups at the time of treatment (0 h). The single i.m. administration of GnRH caused serum LH to rise (Fig. 1). This LH response occurred in all but one of the GnRH i.m. treated cows. Luteinizing hormone data for this nonresponding cow was excluded in the following calculations of group averages. Mean serum LH peaked at 16:9 2:4 ng/ml in the sample taken 2 h after GnRH treatment and the LH response was declining at 4 h (6:8 0:96 ng/ml). The s.c. GnRH implant also elevated mean serum LH at 2 and 4 h samples (9:7 1:53 and 8:4 1:2 ng/ml) and the LH response was declining at 6 h (4:9 0:35 ng/ml). There was no difference in the overall LH response between the i.m. treatment and s.c. implant groups. Serum LH profiles in both GnRH treated groups differed (P < 0:01) from the LH profile of the untreated control cows in which there was no change in serum LH. Parity had no effect on LH response in this trial. Ovulation rate was 4 of 14 (29%) in GnRH i.m.-treated cows, 4 of 12 (33%) in GnRH s.c.-implanted cows and 0 of 13 (0%) in control cows. More GnRH treated cows (GnRH i.m.-treated plus GnRH s.c.-implanted groups) ovulated (P < 0:025) than did untreated control cows, but there was no difference in ovulation rate between GnRH i.m. treated and GnRH implanted cows. Parity affected ovulation response (P < 0:01) as GnRH caused ovulation in 7 of 13 multiparous versus 1 of 13 primiparous cows. A follicle larger than 12 mm in diameter was found in all cows that responded to GnRH with ovulation but many non-ovulating cows (9/12 primiparous, 5/6 multiparous) also had a follicle of this size. No ovulations occurred from GnRH treatment where the largest follicle was less than 10 mm in diameter. Plasma progesterone increased from undetectable at Day 0 to more than 1 ng/ ml on Day 7 and to more than 2 ng/ml on Day 14, respectively in 4 of 14 and 4 of 14 GnRH i.m. treated cows, in 4 of 12 and 4 of 12 GnRH implanted cows, and in 0 of 13 and 0 of 13 untreated cows (Fig. 2). There was a significant treatment effect among the three groups
Fig. 1. Serum LH concentration (mean S:E:M:) following treatments on day 14 postpartum in dairy cows in trial #1. An asterisk () indicates difference (P < 0:01) from control cows.
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Fig. 2. Plasma progesterone concentration (mean S:E:M:) following treatments on Day 14 postpartum in dairy cows in trial #1. An asterisk () indicates difference (P < 0:05) between GnRH-treated versus control cows.
(P < 0:001), with controls differing from the two groups of GnRH-treated cows, but there was no difference between the GnRH i.m.-treated and the GnRH s.c.-implanted groups. 3.2. Trial #2 Single i.m. treatment with GnRH caused a rapid increase in serum LH which peaked at the 2 h sample and was declining at 4 h. Serum LH increased to more than 10 ng/ml in all (8/8) GnRH cows, but in none of the 10 PGF and 7 saline treated cows (Fig. 3). Six of the 8 GnRH, 2 of the 10 PGF and 1 of the 7 saline treated cows ovulated by 3 days after treatment. The ovulation rate in GnRH treated cows was higher (P < 0:01) than that for PGF and saline treated cows, but there was no difference between PGF and saline treated
Fig. 3. Serum LH concentration (mean S:E:M:) by time after i.m. administration of GnRH, prostaglandin F2a (PGF), or saline on Day 14 postpartum in dairy cows in trial #2. An asterisk () indicates difference (P < 0:001) between GnRH-treated versus PGF and saline-treated cows.
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Fig. 4. Plasma progesterone concentration (mean S:E:M:) within 24 days after i.m. administration of GnRH, prostaglandin F2a (PGF), and saline on Day 14 postpartum in dairy cows in trial #2.
cows. Plasma progesterone increased to more than 2 ng/ml in 6 of 8 GnRH, 2 of 10 PGF, and 1 of 7 saline treated cows by 12 days after treatment (Fig. 4). Average days from treatment to first progesterone concentration above 2 ng/ml were13:9 2:6, 28:2 4:1 and 22:3 4:1 days for GnRH, PGF and saline treated groups, respectively. This interval was shorter (P < 0:05) for GnRH cows than for PGF treated cows, but there was no difference between GnRH and saline treated cows, or between saline and PGF treated cows. Treatment induced ovulation was affected by follicle size. Within the GnRH group, all ovulating cows had a follicle larger than 10 mm in diameter when treated. Combining result across the two trials, GnRH (100 mcg i.m.) administration at or near 14 days postpartum caused LH release in 21 of these 22 cows (95%), but stimulated ovulation in only 10 of 22 cows (45%). Similarly for the combined data from the i.m. GnRH treated cows and the s.c. implanted GnRH treated cows, 33 of 34 cows (97%) had an LH response but only 14 of 34 cows (41%) ovulated.
4. Discussion As expected from the previous report by Britt et al. [3], our administration of a s.c. implant of GnRH resulted in a rapid rise in peripheral serum LH and we observed a similar response in nearly all cows treated with the same dose of GnRH given by i.m. administration. However, in our trials, only 41% of GnRH treated cows ovulated and subsequently formed a functional CL, whereas Britt et al. [3] reported an ovulation rate of 100%. There was a strong indication that ovulation in response to GnRH treatment was influenced by parity and by follicle size as first lactation cows and cows without a follicle greater than 10–12 mm in diameter were much less likely to ovulate. This result agrees with Sartori et al. [20], who showed that follicles smaller than 10 mm in diameter did not ovulate even with a very high dose (40 ng/ml) of LH. Others have studied the effects of GnRH administered intramuscularly on ovulation in early postpartum cows [21–24]; their ovulation rate
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responses were more like our results than the 100% ovulation rate reported by Britt et al. [3]. Although some studies have reported beneficial effects of early postpartum use of PGF products on subsequent rebreeding performance [10–13], we could not demonstrate any indication that PGF caused release of LH or stimulation of ovulation in early postpartum cows. Thus, the mechanism by which early postpartum use of PGF appears to improve subsequent rebreeding performance of cows with no progesterone, i.e. no CL function at the time of treatment, [11–13] remains unexplained. In dairy cows, negative energy balance is directly related to the postpartum interval to first ovulation, and follicle size was adversely affected by negative energy balance in early postpartum dairy cows [25]. Therefore, it appears likely that the ability of GnRH to induce ovulation and CL function in postpartum dairy cows will be greatest in individual cows and in herds where severity and duration of negative energy balance problems are successfully minimized. Unfortunately, it is the cows and herds with the greatest problems with negative energy balance and postpartum ovarian inactivity that would benefit most from a potential treatment like GnRH to cause ovulation and initiate estrous cycle activity. Overall, these results show that contrary to much current thinking veterinarians and reproductive physiologists should anticipate significant limitations in the ability of GnRH to cause ovulation and CL development in postpartum dairy cows. Our results indicate GnRH use to be successful for this purpose approximately 45% of the time and that results are likely to be disappointing in first lactation cows. No evidence for efficacy of PGF treatment in causing LH release and/or ovulation in early postpartum dairy cows was detected.
Acknowledgements Ahmet Gu¨ men was supported by a fellowship from the Ministry of National Education of Turkey. We greatly appreciate the assistance of S.M. Pancarci and J. Cassady. References [1] Ferguson JD. Diet, production and reproduction in dairy cows. Anim Feed Sci Tech 1996;59:173–84. [2] Opsomer G, Coryn M, Deluyker H, de Kruif A. An analysis of ovarian dysfunction in high yielding dairy cows after calving based on progesterone profiles. Reprod Dom Anim 1998;33:193–204. [3] Britt JH, Kittok R, Harrison DS. Ovulation, estrus and endocrine response after GnRH in early postpartnum cows. J Anim Sci 1974;39:915–9. [4] Nash JG, Ball L, Olson JD. Effects on reproductive performance of administration of GnRH to early postpartum dairy cows. J Anim Sci 1980;50:1017–21. [5] Britt JH, Harrison DS, Morrow DA. Frequency of ovarian follicular cysts, reasons for culling and fertility in Holstein–Friesian cows given gonadotropin-releasing hormone at 2 weeks after parturition. Am J Vet Res 1977;38:749–51. [6] Kesler DJ, Garverick HA, Youngquist RS, Elmore RG, Bierschwal CJ. Effect of days postpartum and endogenous reproductive hormones on GnRH-induced LH release in dairy cows. J Anim Sci 1977;46: 797–803. [7] Etherington WG, Bosu WTK, Martin SW, Cote JF, Doig PA, Leslie KE. Reproductive performance in dairy cows following postpartum treatment with gonadotropin releasing hormone and/or prostaglandin: a field trial. Can J Comp Med 1984;48:245–50.
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[8] Etherington WG, Martin SW, Dohoo IR, Bosu WTK. Interrelationships between postpartum events, hormonal therapy, reproductive abnormalities and reproductive performance in dairy cows: a path analysis. Can J Comp Med 1985;49:261–7. [9] Seguin BE, Tate DJ, Otterby DE. Use of cloprostenol in a reproductive management system for dairy cattle. J Am Vet Med Assoc 1983;183:533–7. [10] Etherington WG, Kelton DF, Adams JE. Reproductive performance of dairy cows following treatment with fenprostalene, dinoprost, or cloprostenol between 24 and 31 days postpartum: a field trial. Theriogenology 1994;42:739–52. [11] McClary DG, Putnam MR, Wright JC, Sartin JL. Effect of early postpartum treatment with prostaglandin F2a on subsequent fertility in the dairy cow. Theriogenology 1989;31:565–70. [12] Young IM, Anderson DB, Plenderleith RWJ. Increased conception rate in dairy cows after early post partum administration of prostaglandin F2a THAM. Vet Rec 1984;115:429–31. [13] Young IM, Anderson DB. Improved reproductive performance from dairy cows treated with dinoprost tromethamine soon after calving. Theriogenology 1986;26:199–208. [14] Amstrong JD, O’Gorman J, Roche JF. Effects of prostaglandin on the reproductive performance of dairy cows. Vet Rec 1989;125:597–600. [15] Gaitus FL, Urgel JC. Reproductive parameters in dairy cows with normal puerperium treated with prostaglandin F2a-tham on day 14 postpartum. Zuchthygiene 1989;24:201–6. [16] Glansvill SF, Dobson H. Effect of prostaglandin treatment on the fertility of problem cows. Vet Rec 1991;128:374–6. [17] Morton JM, Allen JD, Harris DJ, Miller GT. Failure of a single postpartum prostaglandin treatment to improve the reproductive performance of dairy cows. Aust Vet J 1992;69:158–60. [18] Niswender GD, Reichert LE, Midgley AR, Nalbandov AV. Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology 1969;84:1166–73. [19] SAS1 User’s Guide: Statistics. 1998. SAS Institute, Inc., Cary, NC. [20] Sartori R, Fricke PM, Ferreira JCP, Ginther OJ, Wiltbank MC. Follicular deviation and acquisition of ovulatory capacity in bovine follicles. Biol Reprod 2001;65:1403–9. [21] Kesler DJ, Garverick HA, Youngquist RS, Elmore RG, Bierschwal CJ. Ovarian and endocrine responses and reproductive performance following GnRH treatment in early postpartum dairy cows. Theriogenology 1978;9:363–9. [22] Fernandes LC, Thatcher WW, Wilcox CJ, Call EP. LH release in response to GnRH during the postpartum period of dairy cows. J Anim Sci 1978;46:443–8. [23] Labib F, El-Azab MA, Sharawy SM. Effect of administration of Gonadotropin-releasing hormone (Gn-RH) during early postpartum period on reproductive performance of dairy cows. Assiut Vet J 1988;20:156–61. [24] Zailed AA, Garverick HA, Bierschwal CJ, Elmore RG, Youngquist RS, Sharp AJ. Effect of ovarian activity and endogenous reproductive hormones on GnRH induced ovarian cycles in postpartum dairy cows. J Anim Sci 1980;50:508–13. [25] Butler WR, Smith RD. Interrelationships between energy balance and postpartum reproductive function in dairy cattle. J Dairy Sci 1989;72:767–83.
Ovulation rate after GnRH or PGF2a administration in early postpartum dairy cows$ Ahmet Gu¨men1, Brad Seguin* Department of Clinical and Population Sciences, College of Veterinary Medicine, University of Minnesota, 435H Anim Sci/Vet Med, 1988 Fitch Avenue, St. Paul, MN 55108, USA Received 20 November 2001; accepted 8 November 2002
Abstract The objectives of this study evaluating induction of ovulation in early postpartum dairy cows were to: compare two methods of GnRH (100 mcg) administration (i.m. route and s.c. implant), and determine if prostaglandin F2a (PGF) causes release of LH or ovulation similar to that reported for GnRH. In trial #1, serum LH peaked at 2 h after i.m. administration of GnRH and was declining at 4 h. The s.c. GnRH implant also caused an elevation in serum LH at 2 and 4 h after treatment, with LH declining at 6 h. Serum LH was unchanged in control cows. Experimental treatment caused ovulation in 4 of 14 GnRH i.m. treated cows, 4 of 12 GnRH implanted cows and 0 of 13 control cows. Parity had no effect on LH response but did affect resulting ovulation rate as multiparous cows were more likely to ovulate than were primiparous cows in response to either GnRH treatment. All cows that ovulated had a follicle larger than 12 mm at the time of treatment. In trial #2, serum LH increased as before after i.m. administration of GnRH, however, serum LH was unchanged in cows treated with PGF or saline. Gonadotropin releasing hormone caused more cows to ovulate than did PGF or saline treatments, and GnRH shortened the interval from treatment to the onset of CL function over the PGF treatment; 13:9 2:6, 28:2 4:1 and 22:3 4:1 days for GnRH, PGF and saline, respectively. In summary, there was no difference in the ability of s.c. implantation and i.m. administration of GnRH to cause ovulation. Prostaglandin F2a did not cause release of LH or ovulation. In 22 early postpartum dairy cows treated with 100 mcg GnRH i.m. in these two trials, nearly all cows (95%) responded with a release of LH but only 45% (10/22) responded with an ovulation and subsequent formation of a CL. # 2003 Elsevier Science Inc. All rights reserved. Keywords: GnRH; PGF2a; Postpartum; Ovulation; Dairy cows
$ Presented in part as Abstract #220 at the 2000 ASAS/ADSA Midwestern Sectional Meetings in Des Moines, IA. * Corresponding author. Tel.: þ1-612-624-4741; fax: þ1-612-624-3233. E-mail address: [email protected] (B. Seguin). 1 Present address: Department of Dairy Science, University of Wisconsin-Madison, Madison, WI 53706, USA.
0093-691X/03/$ – see front matter # 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0093-691X(02)01372-9
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1. Introduction Delayed onset of estrous cyclicity after calving is a major hindrance to the desired rebreeding performance of dairy herds attempting to reach their goals for reproductive performance and production efficiency [1,2]. For example, in one survey of 448 postpartum periods on six well-managed dairy herds by milk progesterone profiling, 20% had delayed cyclicity due to no ovarian activity through Day 50 postpartum and only 54% showed normal resumption of estrous cyclicity by Day 50 [2]. Britt et al. [3] reported that a s.c. implant of gonadotropin releasing hormone (GnRH) on Day 14 postpartum caused, through its ability to release luteinizing hormone (LH), ovulation in 100% of lactating Holstein cows. In some studies, GnRH treatment at 14 days postpartum has improved first service conception rate for breeding beginning at 50 days postpartum in dairy cows [4]. However, several reports have shown no effect on rebreeding performance after early postpartum administration of GnRH [5,6] and a few have even found a negative effect [7,8]. Prostaglandin F2a and its analogues (PGF) have been used to manipulate the bovine estrous cycle through its luteolytic activity [9]. However, early postpartum administration of PGF products has shown beneficial effects in some trials [10–13] but others have shown no effect [14–17] on herd reproductive performance. Interestingly, in two reports progesterone concentration, i.e. presence or absence of a functional corpus luteum (CL), at the time of this PGF treatment did not correlate with positive effects on reproductive performance [11,12]. This suggests that some mechanism other than luteolysis may be involved, perhaps stimulation of follicular growth or ovulation by PGF. Therefore, the objectives of these trials in early postpartum dairy cows were to compare delivery of GnRH by i.m. administration versus s.c. implantation on LH response and ovulation, and determine if PGF causes release of LH or ovulation similar to that reported for exogenous GnRH.
2. Materials and methods 2.1. Trial #1 Thirty-nine Holstein dairy cows (19 primiparous and 20 multiparous) were randomly assigned at calving among 3 groups (Group 1, GnRH by i.m. administration; Group 2, GnRH by s.c. implant; Group 3, untreated controls) for one-time treatment at 14 days postpartum. All cows were milked twice daily in a parlor, housed in a tie-stall barn, and fed a total mixed ration to meet maintenance, growth, reproduction, and milk production requirements. Intramuscular administration of 100 mcg of GnRH (Cystorelin, Merial Inc., Iselin, NJ) (Group 1, n ¼ 14) was given by deep i.m. injection (1.5 in. 18 gauge needle) in the right semimembranous/semitendinosus muscles. Subcutaneous implant of 100 mcg of GnRH (Group 2, n ¼ 12) was made in an upper caudal area of the left side of the neck under local anesthesia similar to the procedure of Britt et al. [3]. The GnRH implants were prepared by pouring the GnRH dose into a no. 12 gelatin capsule, which was reassembled,
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quick-frozen in liquid nitrogen, and then lyophilized (Labconco, Kansas City, MO). Untreated cows (Group 3, n ¼ 13) served as controls. Blood samples were collected from tail vessels just before treatment and at 2, 4 and 6 h after treatment into vacutainer tubes (Monoject, Sherwood Medical, St. Louis, MO) to obtain blood serum for LH assay. Blood samples to obtain plasma for progesterone assay were also collected just before treatment and then on Days 7 and 14 after treatment in potassium oxalate (14 mg) and sodium fluoride (17.5 mg) vacutainer tubes (Monoject, Sherwood Medical, St. Louis, MO). Blood samples for plasma were centrifuged within 1 h after collection and for serum at 12–18 h after collection. Serum and plasma samples were stored at 5 8C until assayed. Serum LH was determined by double antibody radioimmunoassay as described by Niswender et al. [18]. The LH assay incorporated NIH-bLH B10 for iodination and references standards, and NIDDK-anti-oLH-1 as the primary antiserum. Hormone sensitivity, calculated two standard deviations below the mean counts per minute at maximum binding, was 0.6515 ng/ml. Coefficients of variation intra- and inter-assays for LH were 2.95 and 4.02%, respectively. Plasma progesterone was determined by radioimmunoassay (COAT-A-COUNT progesterone test (Diagnostic Products Corp., Los Angeles, CA)). Plasma progesterone hormone sensitivity was 0.0585 ng/ml. Coefficients of variation for intra- and inter-assays for progesterone were 4.26 and 4.49%, respectively. On treatment day, ovarian structures were examined by palpation per rectum and ultrasonography using an ultrasound machine equipped with a 7.5 MHz rectal transducer (Medison 500 linear-array real time B-mode ultrasound scanner, Universal Medical Systems Inc., Bedford Hills, NY). Ovulation as a result of treatment was determined by ultrasonography on Day 3 and by changes in plasma progesterone concentration subsequent to treatment. Palpation per rectum was done thereafter every 6 days immediately after blood sample collection to monitor ovarian (especially CL) status. 2.2. Trial #2 Twenty-five multiparous Holstein dairy cows were randomly assigned at calving among three groups (Group 1, GnRH; Group 2, PGF; Group 3, saline) for a one-time treatment 14–19 days postpartum. Group 1 cows (n ¼ 8) were treated with 100 mcg of GnRH to serve as a positive control, Group 2 cows (n ¼ 10) were given 25 mg of PGF (Lutalyse, Pharmacia & Upjohn Co., Kalamazoo, MI) to serve as the test group, and Group 3 cows (n ¼ 7) were given saline to serve as negative controls. All treatments were given i.m. as described above. Blood samples were collected just before treatment and at 1, 2, 4, 12 and 24 h after treatment as before for serum LH assays. Blood samples were also collected just before treatment and then every 3 days for 24 days for plasma progesterone assays as before. Progesterone and LH data were evaluated using ANOVA with repeated measurement within animal over time. Ovulation rates were compared by the chi-square test. Interval data from treatment to first rise in progesterone were evaluated using ANOVA with SAS [19].
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3. Results 3.1. Trial #1 There was no difference in LH among the three treatment groups at the time of treatment (0 h). The single i.m. administration of GnRH caused serum LH to rise (Fig. 1). This LH response occurred in all but one of the GnRH i.m. treated cows. Luteinizing hormone data for this nonresponding cow was excluded in the following calculations of group averages. Mean serum LH peaked at 16:9 2:4 ng/ml in the sample taken 2 h after GnRH treatment and the LH response was declining at 4 h (6:8 0:96 ng/ml). The s.c. GnRH implant also elevated mean serum LH at 2 and 4 h samples (9:7 1:53 and 8:4 1:2 ng/ml) and the LH response was declining at 6 h (4:9 0:35 ng/ml). There was no difference in the overall LH response between the i.m. treatment and s.c. implant groups. Serum LH profiles in both GnRH treated groups differed (P < 0:01) from the LH profile of the untreated control cows in which there was no change in serum LH. Parity had no effect on LH response in this trial. Ovulation rate was 4 of 14 (29%) in GnRH i.m.-treated cows, 4 of 12 (33%) in GnRH s.c.-implanted cows and 0 of 13 (0%) in control cows. More GnRH treated cows (GnRH i.m.-treated plus GnRH s.c.-implanted groups) ovulated (P < 0:025) than did untreated control cows, but there was no difference in ovulation rate between GnRH i.m. treated and GnRH implanted cows. Parity affected ovulation response (P < 0:01) as GnRH caused ovulation in 7 of 13 multiparous versus 1 of 13 primiparous cows. A follicle larger than 12 mm in diameter was found in all cows that responded to GnRH with ovulation but many non-ovulating cows (9/12 primiparous, 5/6 multiparous) also had a follicle of this size. No ovulations occurred from GnRH treatment where the largest follicle was less than 10 mm in diameter. Plasma progesterone increased from undetectable at Day 0 to more than 1 ng/ ml on Day 7 and to more than 2 ng/ml on Day 14, respectively in 4 of 14 and 4 of 14 GnRH i.m. treated cows, in 4 of 12 and 4 of 12 GnRH implanted cows, and in 0 of 13 and 0 of 13 untreated cows (Fig. 2). There was a significant treatment effect among the three groups
Fig. 1. Serum LH concentration (mean S:E:M:) following treatments on day 14 postpartum in dairy cows in trial #1. An asterisk () indicates difference (P < 0:01) from control cows.
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Fig. 2. Plasma progesterone concentration (mean S:E:M:) following treatments on Day 14 postpartum in dairy cows in trial #1. An asterisk () indicates difference (P < 0:05) between GnRH-treated versus control cows.
(P < 0:001), with controls differing from the two groups of GnRH-treated cows, but there was no difference between the GnRH i.m.-treated and the GnRH s.c.-implanted groups. 3.2. Trial #2 Single i.m. treatment with GnRH caused a rapid increase in serum LH which peaked at the 2 h sample and was declining at 4 h. Serum LH increased to more than 10 ng/ml in all (8/8) GnRH cows, but in none of the 10 PGF and 7 saline treated cows (Fig. 3). Six of the 8 GnRH, 2 of the 10 PGF and 1 of the 7 saline treated cows ovulated by 3 days after treatment. The ovulation rate in GnRH treated cows was higher (P < 0:01) than that for PGF and saline treated cows, but there was no difference between PGF and saline treated
Fig. 3. Serum LH concentration (mean S:E:M:) by time after i.m. administration of GnRH, prostaglandin F2a (PGF), or saline on Day 14 postpartum in dairy cows in trial #2. An asterisk () indicates difference (P < 0:001) between GnRH-treated versus PGF and saline-treated cows.
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Fig. 4. Plasma progesterone concentration (mean S:E:M:) within 24 days after i.m. administration of GnRH, prostaglandin F2a (PGF), and saline on Day 14 postpartum in dairy cows in trial #2.
cows. Plasma progesterone increased to more than 2 ng/ml in 6 of 8 GnRH, 2 of 10 PGF, and 1 of 7 saline treated cows by 12 days after treatment (Fig. 4). Average days from treatment to first progesterone concentration above 2 ng/ml were13:9 2:6, 28:2 4:1 and 22:3 4:1 days for GnRH, PGF and saline treated groups, respectively. This interval was shorter (P < 0:05) for GnRH cows than for PGF treated cows, but there was no difference between GnRH and saline treated cows, or between saline and PGF treated cows. Treatment induced ovulation was affected by follicle size. Within the GnRH group, all ovulating cows had a follicle larger than 10 mm in diameter when treated. Combining result across the two trials, GnRH (100 mcg i.m.) administration at or near 14 days postpartum caused LH release in 21 of these 22 cows (95%), but stimulated ovulation in only 10 of 22 cows (45%). Similarly for the combined data from the i.m. GnRH treated cows and the s.c. implanted GnRH treated cows, 33 of 34 cows (97%) had an LH response but only 14 of 34 cows (41%) ovulated.
4. Discussion As expected from the previous report by Britt et al. [3], our administration of a s.c. implant of GnRH resulted in a rapid rise in peripheral serum LH and we observed a similar response in nearly all cows treated with the same dose of GnRH given by i.m. administration. However, in our trials, only 41% of GnRH treated cows ovulated and subsequently formed a functional CL, whereas Britt et al. [3] reported an ovulation rate of 100%. There was a strong indication that ovulation in response to GnRH treatment was influenced by parity and by follicle size as first lactation cows and cows without a follicle greater than 10–12 mm in diameter were much less likely to ovulate. This result agrees with Sartori et al. [20], who showed that follicles smaller than 10 mm in diameter did not ovulate even with a very high dose (40 ng/ml) of LH. Others have studied the effects of GnRH administered intramuscularly on ovulation in early postpartum cows [21–24]; their ovulation rate
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responses were more like our results than the 100% ovulation rate reported by Britt et al. [3]. Although some studies have reported beneficial effects of early postpartum use of PGF products on subsequent rebreeding performance [10–13], we could not demonstrate any indication that PGF caused release of LH or stimulation of ovulation in early postpartum cows. Thus, the mechanism by which early postpartum use of PGF appears to improve subsequent rebreeding performance of cows with no progesterone, i.e. no CL function at the time of treatment, [11–13] remains unexplained. In dairy cows, negative energy balance is directly related to the postpartum interval to first ovulation, and follicle size was adversely affected by negative energy balance in early postpartum dairy cows [25]. Therefore, it appears likely that the ability of GnRH to induce ovulation and CL function in postpartum dairy cows will be greatest in individual cows and in herds where severity and duration of negative energy balance problems are successfully minimized. Unfortunately, it is the cows and herds with the greatest problems with negative energy balance and postpartum ovarian inactivity that would benefit most from a potential treatment like GnRH to cause ovulation and initiate estrous cycle activity. Overall, these results show that contrary to much current thinking veterinarians and reproductive physiologists should anticipate significant limitations in the ability of GnRH to cause ovulation and CL development in postpartum dairy cows. Our results indicate GnRH use to be successful for this purpose approximately 45% of the time and that results are likely to be disappointing in first lactation cows. No evidence for efficacy of PGF treatment in causing LH release and/or ovulation in early postpartum dairy cows was detected.
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