Available online at www.sciencedirect.com Theriogenology 71 (2009) 1358–1366 www.theriojournal.com Comparison of the effects of eFSH and deslorelin treatment regimes on ovarian stimulation and embryo production of donor mares in early vernal transition T. Raz a,*, S. Carley b, C. Card a a Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan S7N 5B4, Canada b Lakeland Veterinary Services Ltd., North Battleford, Saskatchewan S9A 3L8, Canada Received 24 May 2008; received in revised form 22 September 2008; accepted 28 September 2008 Abstract The objective was to compare the effects of eFSH and deslorelin treatment regimes on ovarian stimulation and embryo production of donor mares in early spring transition. Starting January 30th, mares kept under ambient light were examined by transrectal ultrasonography. When a follicle 25 mm was detected, mares were assigned to one of two treatment groups, using a sequential alternating treatment design. In the eFSH group, mares (n = 18) were treated twice daily with eFSH (12.5 mg im) until they achieved a follicle 35 mm; hCG was given 36 h later. In the deslorelin group, mares (n = 18) were treated twice daily with deslorelin (63 mg im) until a follicle 35 mm was detected, and then they were given hCG. Estrous mares were inseminated with fresh semen. Eight days after ovulation, embryo recovery attempts were performed. In each group, 14/18 (78%) mares ovulated following the eFSH or deslorelin treatment regimes. The mean (95% CI) interval from treatment initiation to ovulation was 8.2 d (7.3, 8.9) and 7.2 d (6.2, 8.1) in the eFSH and deslorelin groups, respectively. In the eFSH group, the number of ovulations was significantly higher (mean Æ S.E.M.; 3.4 Æ 0.4 vs. 1.1 Æ 0.1 ovulations), and more embryos were recovered (2.6 Æ 0.5 vs. 0.4 Æ 0.2 embryos/recovery attempt). We concluded that eFSH and deslorelin treatment regimes were equally effective in inducing ovulation in early transitional mares, within a predictable time of treatment; however, the eFSH regime increased the number of ovulations and embryos recovered per mare. # 2009 Elsevier Inc. All rights reserved. Keywords: Transitional mare; Embryo transfer; Superovulation; eFSH; Deslorelin 1. Introduction mares [1,2]. Following winter solstice, anestrous mares gradually obtain ovulatory competence during a pro- Mares are seasonal, polyestrus, long-day breeders. longed phase called vernal transition. This transition During the winter, the function of the mare’s hypotha- phase is characterized by a series of stages or events lamic–pituitary–ovarian axis changes; GnRH secretion is characterized by increased GnRH and gonadotropin minimal, and follicular waves cease in the majority of secretion, resurgence of follicular development, estrous behavior and, finally ovulation [2,3]. During the early transitional phase, the number of follicles with a diameter * Corresponding author. Tel.: +1 306 966 7169; 20 mm in the mare’s ovaries increases, and ovaries fax: +1 306 966 7159. usually contain several developing and atretic follicles E-mail addresses: [email protected], [email protected] (T. Raz). [4,5]. Duringthelatetransitionphase,mostmaresdevelop 0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.09.048 T. Raz et al. / Theriogenology 71 (2009) 1358–1366 1359 1–3 anovulatory follicular waves, each characterized by a Saskatchewan in Canada, which is located at 528070 large dominant follicle (35 mm); follicles continue to latitude in the Northern Hemisphere. Thirty-six emerge and regress until one is ultimately recruited to be mares, Quarter Horse/Percheron cross type, ages 3– the ovulatory follicle [4,5]. Increasing daylight length 10 y, with a body condition score of at least 5 out of 9, plays the major role in the resurgence of ovulatory were used for this study. They had no signs of competence, and the mean month that mares experience systemic disease or lameness, and had good perineal the first ovulation of the breeding season depends on the conformation. Mares were kept under ambient light in geographical latitude at which they live, genetics, nutri- outdoor paddocks with sheds, and were maintained on tion, climate, and other environmental factors [2,3,5–7]. alfalfa and grass hay, with access to water and trace- Successful regimes to stimulate ovarian cyclicity in mineralized salt, in accordance with the University of mares to overcome winter anestrus and/or the prolonged Saskatchewan’s Institutional Animal Care and Use transitional phase is of interest to the horse breeding Committee. industry, as economic pressures exist to produce foals Transrectal palpation and ultrasonographic exam- early in the year. This has become even more pronounced inations of the reproductive tracts were performed in the last three decades, since the use of embryo transfer starting on January 30th, 2006. Uterine and cervical and other technologies to improve embryo production tone were palpated and separately scored from 1 to 4 has grown and become more attractive. Many horse (1 – soft, 2 – moderately soft, 3 – moderately toned, 4 – owners would like to recover embryos from their toned). A B-mode ultrasound scanner (GE Ausonics, performance mare early in the year, before the show Universal Ultrasound, Bedford Hills, NY, USA) season; others would like to have their mare conceive equipped with a 5.0 MHz linear-array transducer was early in the year, after she has already produced a few used to monitor ovarian activity and to subjectively embryos. Various therapeutic strategies to advance the score endometrial edema from 0 (no edema, homo- first ovulation of the year have been investigated. In geneous grey) to 4 (marked edema with a distinct black addition to artificial photoperiod [8], hormonal strategies and white pattern) [24]. At the beginning of the study, employed include the use of GnRH and GnRH analogs the reproductive status of all mares was defined as [1,9–13], progesterone and progestins [14,15],hCG[16], winter anestrus due to the absence of luteal tissue and prostaglandins [17], prolactin [18], dopamine antagonists follicles >15 mm in diameter on repeated examina- [19], equine pituitary extract [20,21], and more recently, tions. Mares were examined every 2 or 3 d until a equine FSH (eFSH) [22,23]. However, information on follicle 18 mm in diameter was detected and daily efficacious and practical hormonal treatment regimes for thereafter until 3 d post ovulation(s), and on Day 8 after early transitional mares used for embryo transfer, with a ovulation (day of embryo recovery attempt). predictable interval to ovulation, embryo production, and continuation of cyclicity, is still limited. 2.2. Experimental design and treatment groups The objective of this study was to determine and compare the efficacy of deslorelin (GnRH agonist) and When a follicle 25 mm in diameter was detected, eFSH treatment regimes in donor mares in early spring mares were assigned to one of two treatment groups, transition. We investigated the effects of two treatment using a sequential alternating treatment design. Thus, protocols on folliculogenesis, interval to first ovulation, the first mare that had a follicle 25 mm in diameter ovulation rate, ovulation synchrony, embryo recovery was randomly assigned to a treatment group. Thereafter, rate, and continuation of cyclicity. We hypothesized that mares were assigned to a treatment group by alternate both treatment regimes would stimulate follicular sequence in order to balance the date of treatment growth and ovulation in early transitional mares, within initiation and reduce the effect of mare variability only a few days of treatment; however, we anticipated between groups. that the eFSH treatment would result in higher number In the eFSH group, mares (n = 18) were treated with of ovulations and higher embryo recovery rate. eFSH (12.5 mg im; eFSH1, Bioniche Animal Health Canada Inc., Belleville, ON, Canada) twice daily until a 2. Materials and methods follicle 35 mm in diameter was detected, and approximately 36 h later hCG (2000 IU im; Chorulon1, 2.1. Animals and reproductive tract examinations Intervet Canada Ltd., Whitby, ON, Canada) was administered. In the deslorelin group, mares (n = 18) This study was performed between January and were treated with deslorelin (63 mg, im; BET Pharm, June 2006, in the research facility of the University of Lexington, KY, USA) twice daily until a follicle 1360 T. Raz et al. / Theriogenology 71 (2009) 1358–1366 35 mm in diameter was detected, at which time hCG 2.5. Measurement of serum estradiol-17b and was administered. progesterone concentrations In both treatment groups, mares were artificially inseminated 24 h after a follicle 35 mm in diameter During the first cycle, jugular blood samples were was detected, and again every 48 h until ovulation (Day collected into sterile plain vacutainer tubes at the time of 0 = day of the first ovulation) with fresh semen treatment assignment, just prior to hCG administration collected from a stallion of proven fertility. A minimum (hCG-Day), on the day of ovulation (Day 0), and 8 d post dose of 5 Â 108 progressively motile spermatozoa ovulation (Day 8) just prior to the embryo recovery extended 1:1 with Kenney extender (EZ-Mixin, Animal attempt. For the second cycle, jugular blood samples Reproduction Systems, Chino, CA, USA) was used to were collected on hCG-Day, Day 0, and Day 8. The blood inseminate the mares. samples were centrifuged, and the sera were separated A treatment failure was defined as a mare that did not and stored frozen (À20 8C) until hormone assays were develop a follicle 35 mm in diameter within 12 d of performed. Serum concentrations of progesterone (P4) eFSH/deslorelin treatment, or if a mare developed a and estradiol-17b (E2) were determined using RIA follicle 35 mm but failed to ovulate within 72 h after validated for use in horses.