2020 Ovulation, pregnancy, and lambing rates during nonbreeding season with or without exogenous gonadotropin stimulation Ovulation, pregnancy, and lambing rates during nonbreeding season with or without exogenous gonadotropin stimulation Hayder Habeeb,a,b Timothy Hazzard,a Fred Stormshak,a Michelle Kutzlera aDepartment of Animal and Rangeland Sciences, Oregon State University, Corvallis, OR bDepartment of Animal Resources, Al-Qasim Green University, Babylon, Iraq Abstract Our objective was to evaluate reproductive effects of varying gonadotropin dosages on anestrous ewes pretreated with progesterone and then exposed to a novel ram. Hypothesis was that a lower dosage of P.G. 600® (equine chorionic gonadotropin 80 IU/ml and human chorionic gonadotropin 40 IU/ml) induces estrus in anestrous ewes. Twenty-four anestrous ewes were treated with intravaginal progesterone-releasing devices for 9 days and given prostaglandin F2α 2 days prior to device withdrawal. On the day of progesterone withdrawal (day 0), ewes were given 5 ml of P.G. 600® (T1; n = 8), 1.5 ml of P.G. 600® (T2; n = 8) or 5 ml saline (control group, C; n = 8). Three rams were rotated every 4 hours through each group of ewes for 4 days. Venous blood samples were collected on day 0 prior to treatment (0 hour) and at 2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 144, 168, and 336 hours. Serum estradiol-17β and progesterone concentrations were determined by chemiluminescence. Ovulation and pregnancy rates were determined using transrectal ultrasonography on days 9 - 11 and on days 21 and 28. Serum progesterone concentrations increased (p < 0.00001) in T1 compared to T2 and C groups. Serum estradiol concentrations, ovulation, pregnancy, and fecundity rates, and weaning weights were not significantly different among groups. We concluded that gonadotropin treatment neither enhanced nor diminished reproductive productivity. Keywords: CIDR, corpora lutea, estrus induction, anestrus, progesterone Introduction Sheep are seasonally polyestrus, cycling during shortened daylight.1 Breeding management practices that adhere to the ewe’s natural breeding season limit ewe productivity (average of 1 lamb crop/ewe/year). To increase ewe’s productivity to 3 lamb crops for every 2 years or 5 lamb crops for every 3 years, ewes must be bred outside of the natural breeding season.2 As hours of daylight increase, decreased secretory pattern of melatonin results in dopamine increase in A15 dopamine neurons in the retrochiasmatic area in the hypothalamus3 that gradually inhibits kisspeptin from the arcuate nucleus, resulting in inhibition of gonadotropin releasing hormone (GnRH) secretion.4 Reduced secretion of GnRH results in lower luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion until ovarian follicular activity becomes static. With static follicular activity, estradiol-17β concentrations remain low, activating the estrogen responsive inhibitory neural system in the brain. Increasing brain sensitivity to estrogen makes estradiol the primary inhibitor for GnRH and LH pulse frequency.5,6 Introduction of a novel ram to anestrous ewes increases LH secretion7,8 that induces ovulation in approximately half of anestrous ewes without any other treatment.9,10 The mechanism of this phenomenon is not clear, but it is believed to be mediated through pheromones from the ram’s sudoriferous gland. Pheromones act through ewe’s olfactory bulb on the ventromedial nucleus and the preoptic area of the hypothalamus, leading to increased kisspeptin release11 that increases GnRH and LH pulse frequency.12 In ruminants, prior exposure to progesterone is necessary for both expression of estrous behavior and normal luteal lifespan.13 Therefore, using progesterone priming prior to novel ram exposure and breeding should result in a fertile estrus with ovulation and normal luteal function. This strategy has resulted in pregnancy in approximately 50% of ewes.14 Gonadotrophin treatment increases percentage of ewes ovulating following progesterone pretreatment and ram exposure. Multiple injections of porcine FSH increase rates of ovulation15 and pregnancy in ewes bred outside the breeding season.16 However, gonadotropin treatment decreases embryo Clinical Theriogenology • Volume 12 Number 1 • March 202023 viability, based on embryo collection studies.17 Similar to multiple injections of FSH, a single injection of equine chorionic gonadotropin (eCG) increases ovulation rate,18,19 but appears to decrease embryo viability.20 It is noteworthy that anestrous ewes treated with progesterone ovulate when they are also treated with gonadotropins or exposed to rams, but not after progesterone treatment alone.21 P.G. 600® (Intervet/Merck Animal Health, Madison, NJ) is approved for estrus induction in swine. It contains a combination of equine chorionic gonadotropin (eCG; 80 IU/ml) and human chorionic gonadotropin (hCG; 40 IU/ml). In United States, P.G. 600® is commonly used off-label to induce estrus and ovulation in anestrous ewes.22-25 Ewes are commonly given 5 ml P.G. 600®, containing 400 IU eCG and 200 IU hCG.22,24 However, this amount of gonadotropins to ewes during breeding season has overstimulated ovaries and reduced fertilization rates, as well as increased estradiol-17β concentrations.26 Therefore, we hypothesized that a lower dosage of P.G. 600® induces estrus in anestrous ewes without overstimulating ovaries. Objective of this study was to evaluate reproductive effects of varying gonadotropin dosages in anestrous ewes pretreated with progesterone and then exposed to a novel ram. Materials and methods Twenty-four multiparous Polypay ewes, 2 - 6 years old, were used for this experiment. The fecundity rate for these ewes prior to this experiment was 1.7 ± 0.4. Animal use was approved by the Oregon State University Institutional Animal Care and Use Committee (ACUP #4865). Estrus induction regimen used for this experiment was the same protocol that was used in the previous experiment conducted during breeding season.26 Briefly, during late spring (May in the Northern Hemisphere), an intravaginal progesterone-releasing device (0.3 g progesterone; Eazi-Breed™ CIDR, Zoetis, Kalamazoo, MI) was inserted and removed after 9 days. Cloprostenol (125 μg; Estrumate®, Intervet/Merck Animal Health, Madison, NJ) was injected intramuscularly 2 days prior to progesterone withdrawal. At progesterone withdrawal (day 0), ewes were treated with P.G. 600® (Intervet/Merck Animal Health, Madison, NJ) or saline. Treatment group 1 (T1) received 5 ml (400 IU eCG and 200 IU hCG) intramuscularly (n = 8) and treatment group 2 (T2) received 1.5 ml (120 IU eCG and 60 IU hCG) intramuscularly (n = 8). Control group (C) received an injection of 5 ml saline, equivalent volume of the higher dose of P.G. 600® (n = 8). Rams used were novel to ewes prior to their first exposure. Three rams were used and each remained for 4 hours in a given group of ewes over 4 days. Rams were rotated between ewe groups to eliminate any effect of ram preference on pregnancy rates. However, it is understood that this experimental design of rotating rams every 4 hours is not a practical management tool for sheep producers. Prior to treatment and immediately following progesterone withdrawal, jugular venous blood samples were collected (day 0). Additionally, blood samples were collected at 2, 4, 6, 8, 12, 24 (day 1), 48 (day 2), 72 (day 3), 96 (day 4), 120 (day 5), 144 (day 6), 168 (day 7), and 336 (day 14) hours after treatment. Blood samples were centrifuged at 1,620 × g after overnight storage at 4ºC. Sera were separated and stored at -20ºC until analyzed for progesterone and estradiol-17β. Serum progesterone and estradiol-17β concentrations were determined using chemiluminescence (IMMULITE®1000, Siemens Healthcare Diagnostics, Tarrytown, NY) in a single assay for each steroid. Progesterone and estradiol-17β intra-assay coefficient of variation was 4.6 and 10.9%, respectively. Assay detection limits for progesterone and estradiol-17β were 0.2 ng/ml and 20 pg/ml, respectively. Transrectal ultrasonography (7.5-MHz linear array, MINDRAY model #50L60EAV, Shenzhen, China) was used on days 9 - 11 after treatment, to determine number of ovulations per ewe by counting corpus luteum (CL) present on each ovary, as described.27 Mean ± standard deviation (SD) ovulation rate was calculated for each group. In addition, height and width of each CL was recorded and the mean ± SD corpora lutea diameter were calculated for each group. Transrectal ultrasonography was used on days 21 and 28 after treatment injection to image both uterine horns to determine pregnancy status. Pregnancy rate was calculated for each group. Following lambing, number of lambs per ewe (fecundity rate) and birth weights were recorded and mean ± SD fecundity rate and birth weight was calculated for each group. In Clinical Theriogenology • Volume 12 Number 1 • March 2020 24 addition, total litter weight was calculated for each ewe and then the mean ± SD litter weight calculated for each group. At weaning, weight was record and mean ± SD weaning weight was calculated for each group. A repeated measure analysis of covariance (ANCOVA) was used to analyze estradiol-17β and progesterone concentrations over time and among treatment groups. A one-way analysis of variance (ANOVA) was used to analyze data on ovulation rate, corpora lutea diameter, fecundity rate, birth weight, litter weight, and weaning weight among treatment groups. A Chi-square test was used to compare pregnancy rates among treatment groups. Significance was defined as p < 0.05. Results There was no effect of treatment on serum estradiol-17β concentrations (Figure 1). However, serum progesterone concentration was greater in T1 compared to T2 and C ewes at 168 and 360 hours (p < 0.00001; Figure 2). Ovulation rate was not significantly different among groups (Table). However, CL diameters were significantly greater in T1 (16.1 ± 3.6 mm) compared to T2 (14.2 ± 3.5 mm), but not compared to C (14.9 ± 3.4 mm). Fecundity rate, birth weight, litter weight, and weaning weight did not significantly differ among treatments (Table). Pregnancy rate was numerically lower in T1 (50%) and T2 (62.5%) compared to C (87.5%) (p = 0.1056). Figure 1. Ewes treated with 5 ml P.G. 600® (Group T1; n = 8), 1.5 ml P.G. 600® (Group T2; n = 8) or 5 ml saline (Group C; n = 8), were sampled hourly from the treatment injection time (0 hour) and at (2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 144, 168, and 336 hours) post treatment. There were no effects of treatment on estradiol-17β concentrations over time. Clinical Theriogenology • Volume 12 Number 1 • March 202025 Figure 2. Ewes treated with 5 ml P.G. 600® (Group T1; n = 8), 1.5 ml P.G. 600® (Group T2; n = 8) or 5 ml saline (Group C; n = 8), were sampled hourly from the treatment injection time (0 hour) and at (2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 144, 168, and 336 hours) post treatment. Serum progesterone concentrations were higher in T1 compared to T2 and C ewes at 168 and 360 hours (*p < 0.00001). Table. Results of P.G. 600® administration on reproductive characteristics of ewes during the non-breeding season. Ewes were treated with 5 ml P.G. 600® (Group T1; n = 8), 1.5 ml P.G. 600® (Group T2; n = 8) or 5 ml saline (Group C; n = 8). No significant differences among groups in all parameters. Discussion Ovulation rate in anestrous ewes in our study did not differ between treatment groups receiving progesterone and gonadotropins or progesterone alone, consistent with a previous study.28 That ovulation rate in anestrous ewes was not affected by gonadotropin dose was similar to an earlier report.15 It is important to note that in both studies,15,28 multiple injections of FSH were used instead of P.G. 600® and FSH was administered until 24 or 36 hours prior to progesterone removal instead of concurrent with progesterone removal (as in present study). Item Treatment Groups Group T1 T2 C Ovulation rate (mean ± SD) 2.23 ± 0.47 2.0 ± 0.26 2.0 ± 0.24 Pregnancy rate 50 % 62.5 % 87.5 % Fecundity rate (mean ± SD) 2.0 ± 0 2.25 ± 1.25 1.85 ± 0.37 Birth weight, kg (mean ± SD) 4.0 ± 0.53 3.85 ± 1.02 3.96 ± 0.94 Litter weight, kg (mean ± SD) 7.49 ± 1.02 8.66 ± 3.92 7.36 ± 1.56 Weaning weight, kg (mean ± SD) 20.45 ± 3.1 19.92 ± 5.04 21.2 ± 5.45 * * Clinical Theriogenology • Volume 12 Number 1 • March 2020 26 Pregnancy rate was not significantly different among groups (control = 87.5%, 1.5 ml P.G. 600® = 62.5%, and 5 ml P.G. 600® = 50%). Similar findings were reported earlier.22,29 Anestrous ewes treated with melengestrol acetate (MGA) and 5 ml of P.G. 600® had pregnancy rates not different from MGA treatment alone. Furthermore, treatment of anestrous ewes with intravaginal progesterone followed by 3 ml P.G. 600® resulted in pregnancy rates that were not different from treatment with intravaginal progesterone alone.25 However, a higher dose of P.G. 600® (> 3.49 ml) decreased lambing rate,30 a finding that served as the basis for our hypothesis. P.G. 600® (400 IU of eCG and 200 IU of hCG) increased serum progesterone concentrations (p < 0.00001) compared to controls (Figure 2), similar to an earlier report31 wherein ewes were treated with 300 IU eCG after progesterone withdrawal. However, the finding that higher progesterone concentration during early gestation increased embryo survival32 was not supported by our findings. Increases in serum progesterone concentration were likely due to increased CL diameters in the 5 ml P.G. 600® treatment group.33-35 Similar to earlier findings,31 fecundity rate, birth weight, litter weight, and weaning weight of lambs did not significantly differ among treatment groups (Table 1). In conclusion, during nonbreeding season, administration of P.G. 600® did not affect ovulation rate, fecundity rate, lambing rate, birth, or weaning weights but did increase circulating progesterone concentrations (and CL diameter at 5 ml dosage). Furthermore, there was a trend for the higher dose of P.G. 600® to be detrimental to pregnancy rate in the Polypay breed. Acknowledgment Authors thank the Oregon Sheep Commission for funding this research and the Ministry of Higher Education and Scientific Research in Iraq for graduate student financial assistance. Conflict of interest Authors disclose that there was no actual or potential conflict of interest in conducting this research and with their ability to objectively present/review the research or data. References 1. Posbergh CJ, Murphy RJ, Thonney ML: Further testing of melatonin receptor 1a for out-of-season reproduction in the cornell flock and allelic frequencies compared with romney sheep. J Anim Sci 2017;9:1939-1944. 2. Keisler DH: Sheep breeding strategies. In: Youngquist RS, Threlfall WR, editors. Current therapy in large animal theriogenology. St. Louis, Missouri: Saunders; 2007. p. 659. 3. Thiéry JC, Gayrard S, Le Corre S, et al: Dopaminergic control of LH secretion by the A15 nucleus in anoestrous ewes. J Reprod Fertil Suppl 1995;49:285-296. 4. Weems P, Jeremy S, Iain J, et al: Effects of season and estradiol on KNDy Neuron peptides, colocalization with D2 dopamine receptors, and dopaminergic inputs in the ewe. Endocrinology 2017;158:831-841. 5. Barrell GK, Moenter SM, Caraty A, et al: Seasonal changes of gonadotropin-releasing hormone secretion in the ewe. Biol Reprod 1992;46:1130-1135. 6. Goodman RL, Bittman EL, Foster DL, et al: Alterations in the control of luteinizing hormone pulse frequency underlie the seasonal variation in estradiol negative feedback in the ewe. Biol Reprod 1982;27:580-589. 7. Martin GB, Scaramuzzi RJ, Lindsay DR: Effect of the introduction of rams during the anoestrous season on the pulsatile secretion of LH in ovariectomized ewes. J Reprod Fertil 1983;67:47-55. 8. Martin GB, Cognie Y, Schirar A, et al: Diurnal variation in the response of anoestrous ewes to the ram effect. J Reprod Fertil 1985;75:275-284. 9. Oldham CM, Martin GB, Knight TW: Stimulation of seasonally anovular merino ewes by rams. I. Time from introduction of the rams to the preovulatory LH surge and ovulation. Anim Reprod Sci 1979;1:283-290. 10. 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Knights M, Baptistea QS, Dixona AB: Effects of dosage of FSH, vehicle and time of treatment on ovulation rate and prolificacy in ewes during the anestrous season. Small Rumin Res 2003;50:1-9. 16. Knights M, Maze TD, Bridges PJ, et al: Short-term treatment with a Controlled Internal Drug Releasing (CIDR) device and FSH to induce fertile estrus and increase prolificacy in anestrous ewes. Theriogenology 2001;55:1181-1191. 17. Torrès S, Cognié Y, Colas G: Transfer of superovulated sheep embryos obtained with different FSH-P. Theriogenology 1987;27:407-419. 18. Cognie Y: Current technologies for synchronisation and artificial insemination of sheep. In: Oldham CM, Martin GB, Purvis IW, editors, Reproductive physiology of Merino sheep concept and consequences. Perth: School of Agriculture, University of Western Australia;1990. p. 211. 19. Barrett DMW, Bartlewski PM, Batista-Arteaga M, et al: Ultrasound and endocrine evaluation of the ovarian response to a single dose of 500 IU of eCG following a 12-day treatment with progestogen-releasing intravaginal sponges in the breeding and nonbreeding seasons in ewes. Theriogenology 2004;61:311-327. 20. Ryan JP, Hunton JR, Maxwell WMC: Increased production of sheep embryos following superovulation of merino ewes with a combination of pregnant mare serum gonadotrophin and follicle stimulating hormone. Reprod Fertil Dev1991;3:551-560. 21. Umberger SH, Jabbar G, Lewis GS: Seasonally anovulatory ewes fail to respond to progestogen treatment in the absence of gonadotropin stimulation. Theriogenology 1994;42:1329-1336. 22. Safranski TJ, Lamberson WR, Keisler DH: Use of melengestrol acetate and gonadotropins to induce fertile estrus in seasonally anestrous ewes. J Anim Sci 1992;70:2935-2942. 23. 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