2011: Effect of GnRH administration at day 5 or day 7 after AI on progesterone concentrations, corpus luteum volume and pregnancy in lactating dairy cows   Effect of GnRH administration at day 5 or day 7 after AI on progesterone concentrations, corpus luteum volume and pregnancy in lactating dairy cows Ramanathan K. Kasimanickam,a Mathew R. Mink,b Raymond L. Nebelb* aDepartment of Veterinary Clinical Sciences, Washington State University, Pullman, WA; bDepartment of Dairy Sciences, Virginia Tech, Blacksburg, VA Abstract The objective of the study was to determine the effect of gonadotropin releasing hormone (GnRH) administration on day 5 or day 7 after artificial insemination (AI) on progesterone concentration, total corpus luteum (CL) volume and pregnancy/AI (P/AI) in lactating dairy cows. Multiparous dairy cows (n=1054) from two dairy farms, one in Virginia and one in North Carolina, were enrolled in this study. All cows were synchronized with Presynch-CO-Synch protocol for first service. Following AI (day 0), cows were randomly allocated to one of the following three treatment groups: cows in the GnRH- 5 group (n=335) received 100 µg of GnRH im five days after insemination; cows in the GnRH-7 group (n=390) received 100 µg of GnRH im seven days after insemination and cows in the control group (n=329) received no treatment. Blood samples were collected from a subset of cows (n=52) at day -38, day -24, day -10, day 0 (day of AI) and day 14 for progesterone concentration determination. The progesterone concentrations at day -38, -28, and -10 determined ovarian cyclicity of the cows and the progesterone concentration at day14 (9 or 7 days following GnRH treatment) were used to detect differences in progesterone among treatment groups. The ovaries of the subset of cows were screened using ultrasonography for the formation of accessory CL and total volume of CLs (primary + accessory CLs) was recorded. Cows were examined for pregnancy by transrectal ultrasonography at 30 days and again at 60 days after insemination. The P/AI for GnRH-5, GnRH-7 and control groups were 31.6% (106/335), 33.8% (132/390) and 31.9% (105/329), respectively (p > 0.1). The total CL volume differed among the treatment groups (p < 0.05) but day 14 progesterone concentration did not differ among the treatment groups (p > 0.1). The progesterone concentration in the control group (n=17) was 5.9 ± 0.3 ng/ mL and total CL volume was 8.3 ± 0.6 cm3. The progesterone concentration in the GnRH-5 group (n=15) was 6.3 ± 0.4 ng/ mL and total CL volume was 12.6 ± 0.7 cm3. The progesterone concentration in the GnRH-7 group (n=20) was 6.6 ± 0.4 ng/ mL and total CL volume was 12.0 ± 0.8 cm3. In conclusion, GnRH administration at day 5 or day 7 following insemination neither increased progesterone concentrations nor improved P/AI compared to controls. Keywords: GnRH, progesterone, pregnancy rate, timed artificial insemination, dairy cattle, fertility Introduction Establishment and maintenance of pregnancy are complex processes that require precise communication between conceptus and dam. The role of progesterone is critical in these processes and insufficient luteal activity has been associated with infertility in cattle. Conception failure is coincident with less than normal concentrations of progesterone as early as six days after AI.1 Blood progesterone generally reaches greater concentrations earlier in pregnant than in non-pregnant cows suggesting that strategies to increase progesterone concentration earlier following insemination may be beneficial in reducing embryo loss. Embryo development is related to concentrations of progesterone and the ability of the conceptus to secrete interferon-τ.2 Mann et al tested the effect of timing of progesterone supplementation on embryo development and interferon-τ production in cows and concluded that both early (5 to 9 days) and later (12 to 16 days) progesterone supplementation resulted in significant increases in plasma progesterone. In addition they have found early progesterone supplementation resulted in a fourfold increase in trophoblast length (p < 0.01) and a six-fold increase in uterine concentration of interferon-τ (p < 0.05).3 Hence more exposure of an embryo to progesterone may increase its chances of secreting interferon-τ and thus survive. Exogenous progesterone can stimulate embryo development, so by manipulating post-insemination concentrations of progesterone, conception rates may ultimately improve.4 Several strategies have been used to increase progesterone concentration after breeding.5 Administration of GnRH and human chorionic gonadotropin (hCG) during the early luteal phase induces                                                              * Current address: Select Sires Inc., Plain City, OH Clinical Theriogenology • Volume 3 Number 4 • December 2011455   ovulation of the dominant follicle from the first follicular wave and results in formation of an accessory CL.6 Injection of GnRH or its agonists induced accessory CL when administered on days 5 to 12 of the estrous cycle.7,8 Differences in response to GnRH administration from days 5 to 12 of the estrous cycle exist and may be due to the difference in the size of the follicle present at that time. The follicle size on day 5 of the estrous cycle is smaller in cows with a longer inter-wave interval (two follicular waves) and renders them non-responsive to GnRH when administered at day 5. However these follicles may responsive to GnRH on day 7. The objective of the study was to determine the effect of GnRH administration on day 5 or day 7 after AI on progesterone concentration, total CL volume and P/AI in lactating dairy cows. Materials and methods Multiparous dairy cows (n=1054) from two dairy farms (one in Virginia and one in North Carolina) were enrolled in this study. All cows were synchronized with Presynch-CO-Synch protocol for first service (Figure). Briefly, cows received two injections of 25 mg prostaglandin F2α (PGF) im (Lutalyse®, Pfizer Animal Health, New York, NY) at 32 ± 3 days postpartum and 46 ± 3 days postpartum (Presynch); 100 μg GnRH im (Cystorelin®, Merial Ltd. Duluth, GA) at 60 ± 3 days postpartum; 25 mg PGF at 67 ± 3 days postpartum; and 100 μg GnRH + timed AI (TAI) at 70 ± 3 days postpartum (CO- Synch; day 0). Body condition scores (1 to 5 scale; 1-emaciated; 5-obese) of all cows were recorded at the time of AI. Following AI, cows were blocked by body condition score and parity, and were randomly allocated to one of the following three treatment groups: cows in the GnRH-5 group (n=335) received 100 µg of GnRH im on day 5 after insemination; cows in the GnRH-7 group (n=390) received 100 µg of GnRH im on day 7 after insemination and cows in the control group (n=329) received no treatment. Blood samples were collected from a subset of cows (n=52) at 32, 46, 60, 70, and 84 days in milk (DIM) for progesterone concentration determination and the ovaries were screened using ultrasonography. Total volume of CLs (primary and accessory CL) was recorded. The CL volume was calculated using the formula 4/3πr3. The progesterone concentration at 60 DIM (at first GnRH the initiation of the Ovsynch protocol) determined ovarian cyclicity of the cows. Progesterone concentrations <1 ng/mL indicated a non-cycling cow. The progesterone concentration at 84 DIM (14 days after insemination) was used to measure differences among treatment groups in progesterone concentrations following GnRH administration (7 or 9 days after GnRH injection). Cows were examined for pregnancy by transrectal ultrasonography (Sonosite 180 plus, Sonosite Inc., Bothell, WA) at 30 days and again at 60 days after insemination. Figure. PGF2 PGF2 GnRH PGF2 GnRH + TAI 14 d 14 d 7 d 72 h 5/7 d 32 ± 3 46 ± 3 60 ± 3 67 ± 3 70 ± 3 75/77 (Day -38) (Day -24) (Day -10) (Day -3) (Day 0) (Day 5/7) Progesterone assay Blood samples were collected via coccygeal venipuncture into blood collection tubes (Vacutainer®, Becton Dickinson, Franklin Lakes, NJ), placed immediately on ice. The samples were brought to the laboratory and centrifuged at 3,000 x g for 15 minutes. Harvested serum was stored at - 20°C until further processing. Progesterone concentration was determined using a Coat-A-Count kit (DPC Diagnostic Products Inc., Los Angeles, CA) solid-phase 125I radioimmunoassay. The intra-assay and inter-assay coefficients of variation were 6.34% and 7.29%, respectively. The assay sensitivity was 0.03 ng/mL. Control (n=17); GnRH-5 (n=15); GnRH-7 (n=20); Clinical Theriogenology • Volume 3 Number 4 • December 2011 456   Statistical analyses Data were analyzed by PROC LOGISTICS procedure of SAS (SAS version 9.12, SAS Institute Inc., Cary, NC) to determine the difference in P/AI among treatment groups. The variables included in the model were parity, season, farm, treatment x parity, treatment x season, and treatment x farm interactions. GLM procedure was used to determine the difference in progesterone and total CL volume among the treatment groups. Progesterone and total CL volume were tested for normality using Kolmogorov-Smirnov test and observed to be normally distributed. P  0.05 was considered as significant. Results The P/AI for GnRH-5, GnRH-7 and control groups were 31.6% (106/335), 33.8% (132/390) and 31.9% (105/329), respectively (Table; p > 0.1). The P/AI was not different for parity, season, farms, treatment x parity, treatment x season, or treatment x farm interactions (p > 0.1). The mean (±SD) CL volume differed among the treatment groups (Table; p < 0.05) but mean (±SD) progesterone concentration did not differ among the treatment groups (p > 0.1). The progesterone concentration for the control group (n=17) was 5.9 ± 0.3 ng/ mL and mean (±SD) total CL volume was 8.3±0.6 cm3. The progesterone concentration for the GnRH-5 group (n=15) was 6.3 ± 0.4 ng/ mL and total CL volume was 12.6±0.7 cm3. In the GnRH-5 group six cows failed to develop accessory CL. In the GnRH-5 group, the progesterone concentration and total CL volume for the cows that did not develop accessory CL were 4.7 ± 0.3 ng/ mL and 9.2±0.5 cm3, respectively, and for the cows that did develop accessory CL were 7.4 ± 0.3 ng/ mL and 14.8±0.7 cm3. The progesterone concentration for the GnRH-7 group (n=20) was 6.6 ± 0.4 ng/ mL and total CL volume was 12.0±0.8 cm3. In the GnRH-7 group ten animals failed to develop accessory CL. In the GnRH-7 group, the progesterone concentration and total CL volume for the cows that did not develop accessory CL were 6.6 ± 0.5 ηg/ mL and 7.7±0.8 cm3, respectively, and for the cows that did develop accessory CL were 6.5 ± 0.4 ηg/ mL and 16.2±0.9 cm3. The P/AI for the control, GnRH-5 and GnRH-7 groups at 30 days after insemination in the subset of cows was 76.5% (13/17), 80.0 % (12/15) and 65.0% (13/20), respectively. The P/AI in the subset of cows for the control, GnRH-5 and GnRH-7 groups at 60 days after insemination was 70.6% (12/17), 73.3 % (11/15) and 50.0% (10/20), respectively. One cow with accessory CL in GnRH-5 group and three cows with no accessory CL in the GnRH-7 group lost their pregnancies between 30 and 60 days of gestation. Of the 52 cows, ovarian cyclicity affected the P/AI [cycling: 54.5% (43/79); non-cycling 15.6% (5/32); p < 0.02]. There was no effect of treatment or treatment x ovarian cyclicity on P/AI (p > 0.1). Discussion Treatment with GnRH on day 5 or day 7 following insemination neither increased progesterone concentrations nor improved P/AI compared to controls. Corpus luteum formation and development is highly responsive to LH, and small luteal cells synthesize more progesterone when stimulated by LH.9 The administration of GnRH or LH during the early luteal phase induces ovulation of the first-wave dominant follicle and formation of a functional accessory CL. Schmitt et al observed that most of the increase of progesterone production after hCG injection was due to the formation of accessory CL.10 Furthermore, cows treated with hCG on day 5 after estrus had a greater increase in plasma progesterone from day 6 to 13 and greater circulating progesterone on day 13 of the estrous cycle. However, Sterry et al observed that GnRH did not affect circulating progesterone concentrations from 5 to 19 days after TAI.11 In this study, 40% and 50% of cows failed to develop accessory CL when GnRH was administered on day 5 and day 7, respectively. It should be noted that progesterone determination was from a subset of 52 cows only. Interestingly, in the GnRH-5 group, the progesterone concentration was higher in cows that developed accessory CL compared to cows that did not develop accessory CL; however this difference was not observed in the GnRH-7 group. In both the GnRH-5 and the GnRH-7 group the total CL volume was higher in cows that developed accessory CL compared to cows that did not. These observations (increased total CL volume and similar progesterone concentrations in cows with accessory CL compared to cows with no accessory CL) indicate that individual variation in dairy cow metabolism might play a role in modulating circulating progesterone concentrations. Higher producing cows generally consume more dry matter, and feed intake can affect plasma progesterone concentrations of dairy cows.12,13 Clinical Theriogenology • Volume 3 Number 4 • December 2011457   Previous studies have found conflicting results on conception rates in cows receiving hCG.14-16 Sianangama and Rajamahendran and Santos et al observed a remarkable increase in conception rate in cows receiving hCG on day 7 and day 5 after AI, respectively.16,17 However, other authors report no differences in conception rate between control and hCG-treated cows.14,15 In the latter studies, the conception rate of control cows (conception rate > 59%) was markedly similar to those in this study.13,14 It is likely that such highly fertile groups of cows might not benefit from hCG treatment. In addition, a limited number of cows and different protocols and dosage regimens were utilized, which might have affected the ability to detect any difference between treatment groups. Sterry et al conducted experiments in which cows were assigned to either control (n = 223), intravaginal progesterone (n = 218), or GnRH 5 days after TAI (n = 227) treatments in one experiment and in another cows were assigned to control (n = 160), GnRH 5 days after TAI (n = 159), or GnRH 7 days after TAI (n = 163).11 They observed that the treatment did not affect P/AI in these experiments; however, when data were combined to compare control animals (n = 383) with those treated with GnRH 5 days after TAI (n = 386), P/AI tended to be greater in those treated with GnRH 5 days after TAI (49.1%) than in control cows (45.8%). They also have observed that the effect resulted from a GnRH treatment x cyclicity status interaction in which P/AI for non-cycling cows receiving GnRH 5 days after TAI was greater than for noncycling control cows (45.5 vs. 31.1%). No treatment x cyclicity interactions observed in the current study. Blood progesterone (timing and magnitude) generally reaches greater concentrations earlier in pregnant than in non-pregnant cows suggesting that strategies to increase progesterone concentration earlier following insemination may be beneficial in reducing embryo loss. Santos et al observed an increase in conception rate for cows receiving hCG on day 5 after AI.17 Ginther et al indicated that deviation of the dominant follicle takes place two to three days after a new cohort of follicles is recruited.15 The dominant follicle acquires LH receptors between two and four days after wave emergence when it reaches 8 to 10 mm in diameter.18,19 Therefore, on day 5 of the estrous cycle, dairy cows should have a dominant follicle that is responsive to LH and is capable of ovulating when ovulation is induced by hCG. It is reasonable to expect a similar response when GnRH is administered on day 5 or day 7. However, it is possible that the increased response to hCG administration on day 5 is due to its longer half-life compared to GnRH. In conclusion, GnRH administration on day 5 or day 7 following insemination neither increased progesterone concentrations nor improved P/AI. Table. Effect of treatment, parity, season and farms on the odds of pregnancy in lactating dairy cows Predictor Coefficient ± SE P Odds Ratio 95% Lower Upper Constant -0.6265 ± 0.3451 0.069 Treatment† 0.1119 ± 0.2542 0.66 1.12 0.68 1.84 Parity‡ -0.1481 ± 0.1073 0.168 0.86 0.7 1.06 Season§ 0.0612 ± 0.0932 0.511 1.06 0.89 1.28 Farm** -0.1894 ± 0.1431 0.462 0.89 0.71 1.05 Treatment x Parity 0.0176 ± 0.7945 0.824 1.02 0.87 1.19 Treatment x Season -0.0440 ± 0.0679 0.517 0.96 0.84 1.09 Treatment x Farm 0.0248 ± 0.0574 0.852 0.98 0.81 1.10 †Treatment: GnRH administration on day 5 or day 7 or control; ‡Parity Categories: 1, 2 or 3; §Season: Fall, Winter, Spring or Summer; **2 farms References 1. Thatcher WW, Guzeglu A, Mattos R, et al: Uterine conception interactions and reproductive failure in cattle. Theriogenology 2001;56:1435-1450. 2. Mann GE, Lamming GE, Robinson RS, et al: The regulation of interferon-τ production and uterine hormone receptors during early pregnancy. J Reprod Fertil 1999;54(Suppl):317-328. Clinical Theriogenology • Volume 3 Number 4 • December 2011 458   3. Mann GE, Fray MD, Lamming GE: Effects of time of progesterone supplementation on embryo development and interferon-τ production in the cow. Vet J 2006;171:500-503. 4. Thatcher WW, Staples CR, Danet-Desnoyers G, et al: Embryo health and mortality in sheep and cattle. J Anim Sci 1994;72:16-30. 5. Binelli M, Thatcher WW, Mattos R, et al: Antiluteolytic strategies to improve fertility in cattle. Theriogenology 2001;56:1451-1463. 6. Schmitt EJP, Diaz T, Barros CM, et al: Differential response of the luteal phase and fertility in cattle following ovulation of the first-wave follicle with human chorionic gonadotropin or an agonist of gonadotropin-releasing hormone. J Anim Sci 1996;74:1074-1083. 7. Pursley JR, Mee MO, Wiltbank MC: Synchronization of ovulation in dariy cows using PGF2alpha and GnRH. Theriogenology 1995;44:915-923 8. Vasconcelos, JLM, Silcox RW, Rose GJM, et al: Synchronization rate, size of ovulatory follicles, and conception rate after syncronization of ovulation beginning on different days of estrous cycle in lactating dairy cows. Theriogenology 1999;52:1067-1078 9. Hoyer PB, Niswender GD: The regulation of steroidogenesis is different in the two types of ovine luteal cells. Can J Physiol Pharmacol 1985;63:240-248. 10. Schmitt EJP, Barros CM, Fields PA, et al: A cellular and endocrine characterization of the original and induced corpus luteum after administration of a gonadotropin-releasing hormone agonist or human chorionic gonadotropin on day five of the estrous cycle. J Anim Sci 1996;74:1915-1929. 11. Sterry RA, Welle ML, FrickePM: Treatment with gonadotropin-releasing hormone after first timed artificial insemination improves fertility in noncycling lactating dairy cows. J Dairy Sci 2006;89:4237-4245. 12. Vasconcelos JL, Bungert MKA, Tsai SJ, et al: Acute reduction in serum progesterone concentrations due to feed intake in pregnant lactating dairy cows [abstract]. J Dairy Sci 1998;81(Suppl 1):226. 13. Wiltbank M, Fricke P, Sangsritavong S, et al: Mechanisms that prevent and produce double ovulations in dairy cattle. J Dairy Sci 2000;83:2998-3007. 14. Eduvie LO, Seguin BE: Corpus luteum function and pregnancy rate in lactating dairy cows given human chorionic gonadotropin at middiestrus. Theriogenology 1982;17:415-422. 15. Helmer SD, Britt JH: Fertility of dairy cattle treated with human chorionic gonadotropin (hCG) to stimulate progesterone secretion. Theriogenology 1986;26:683-695. 16. Sianangama PC, Rajamahendran R: Characteristics of corpus luteum formed from the first wave dominant follicle following hCG in cattle. Theriogenology 1996;45:977-990. 17. Santos JEP, Thatcher WW, Pool L, et al: Effect of human chorionic gonadotropin on luteal function and reproductive performance of high-producing lactating Holstein dairy cows. J Anim Sci 2001;79:2881-2894. 18. Ginther OJ, Wiltbank MC, Fricke PM, et al: Selection of the dominant follicle in cattle. Biol Reprod 1996;55:1187- 1194. 19. Xu Z, Garverick HA, Smith GW, et al: Expression of follicle-stimulating hormone and luteinizing hormone receptors messenger ribonucleic acids in bovine follicles during the first follicular wave. Biol Reprod 1995;53:951-957. 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