Effects of Melatonin and Controlled Internal Drug Release (CIDR) Device on Follicular Development and Oocyte Quality in the Anestrous Ewes Treated with Follicle Stimulating Hormone

Administration of exogenous melatonin (MEL) and progesterone (P4) in conjunction with follicle stimulating hormone (FSH) affects the number of developing follicles and oocyte quality in the anestrus ewe. Crossbred Rambioullet x Targhee Western range ewes (n=25) were randomly assigned to four treatment groups in a 2 x 2 factorial design [+/-MEL and +/CIDR device; MEL/CIDR, CIDR, MEL, and Control (no treatment), respectively]. MEL/CIDR and MEL ewes (n=14) received an 18 mg Melovine® (melatonin) implant for 42 days before oocyte collection. MEL/CIDR and CIDR ewes (n=11) were vaginally implanted with CIDR (Type G) devices (intravaginal pessaries containing P4) for five days before oocyte collection. Two days before oocyte collection all ewes received FSH injections twice daily. At slaughter, ovaries were removed, all visible follicles were counted, and oocytes were collected, matured and fertilized in vitro. The average number of follicles was greater (P<0.08) for MEL/CIDR ewes than Control ewes (37.3+5.5 and 22.6+5.5, respectively), but not different from MEL (31.3±5.5) and CIDR (25.8±5.5) ewes. Percentage of oocytes recovered from follicles was similar (P>0.10) for all treatments (overall 89.9±7.1%). Additionally, the rates of maturation of oocytes were similar (P>0.10) across treatments (overall 78.6±11.0%). Oocytes collected from CIDR treated ewes (CIDR and MEL/CIDR treatment groups), had lower (P<0.02) fertilization rates than ewes not treated with CIDR (MEL and Control; 10.3±2.0 and 10.1±2.0 vs. 18.5±2.0 and 20.0±2.0%, respectively). These data indicate that melatonin and P4 increases the number of developing follicles, and although P4 assists in the recruitment of more follicles, it decreases fertilization rates. INTRODUCTION The ewe is seasonally polyestrous and will stop cycling during late winter in response to the increasing photoperiod (Robinson et al., 1993; Bittman, 1984). Low fertilization rates during seasonal anestrous may be caused by an altered endocrine status when compared to the normal breeding season in the ewe (Stenbak et al., 2001). Numerous studies have focused on developing hormonal treatments to improve follicular development and induce estrus in ewes during seasonal anestrus (Robinson et al., 1991 and 1993; Gordon, 1997; Carlson, 2000; Knights et al., 2000, 2001). The main focus of these previous studies was to improve pregnancy rates and maximize reproductive performance in vivo. However, limited data are available concerning the effects of exogenous hormones, such as melatonin and progesterone, on oocyte quality for in vitro fertilization (IVF) during seasonal anestrus. Many studies have been conducted during seasonal anestrus to evaluate the effectiveness of different melatonin treatments on reproductive performance in vivo. Melatonin treatment has been shown to be an effective method to induce estrous cycles, increase ovulation rates, and increase lambing rates during seasonal anestrous (Waller, 1988; Haresign, 1990 and 1992; Robionson 1991 and 1993; Bister, 1999; Carlson, 2000). It has also been demonstrated that melatonin affects oocyte development and support fertilization and early embryonic development following IVF in rats and mice (Fernandez, 1995; Ishizuka, 2000). Another common method of inducing fertile estrus in the seasonal anestrous ewe is through the use of progesterone-based therapies (Robbinson et al., 1991; Jabbar et al., 1994; Knights et al., 2000, 2001). An improvement in fertility of ewes synchronized with higher doses of progesterone is due to an increase in sperm transport (Hawk, 1971), synchrony in the onset of estrus in relationship to the luteinizing hormone surge (Van Cleeff, 1998), and/or patterns of follicular development (Johnson, 1996). Knights et al. (2001) demonstrated that a 5-day treatment with progesterone, in combination with follicle stimulating hormone (FSH), stimulated a fertile estrus as effectively as a 12-day progesterone treatment with FSH. In addition, this resulted in prolificacy comparable to that observed during the normal breeding season. However, data concerning the effects of melatonin and/or progestagen treatment on quality of ovine oocytes are not available at present. The aim of this study was to evaluate the effects of exogenous melatonin and progesterone on follicular development and oocyte quality in FSH-treated ewes. Oocyte quality was measured by the rate of maturation, fertilization, and morula and blastocyst formation following in vitro fertilization procedures. MATERIALS AND METHODS Animals and Experimental Design Seasonally anestrous, crossbred Rambioullet/Targhee Western range ewes (n=25) were randomly assigned to four treatment groups (n=4-7/group) in a 2x2 factorial design [+/melatonin (MEL) and +/-CIDR device]. Ewes received an 18 mg Melovine® (melatonin; Sanofi Sante Nutrition Animal, La Ballastiere, France) implant for 42 days before oocyte collection and were vaginally implanted with CIDR devices for five days before slaughter (day of oocyte collection). Two days before slaughter all ewes received FSH injections as described by Stenbak et al. (2001). This study was conducted during the period from March to May Follicular Evaluation and Oocyte Collection Ovaries were removed at slaughter and placed in phosphate buffer solution (PBS) containing penicillin/streptomycin (Gibco, Gaithesburg, MD) at 39° C. The number of visible follicles on each ovary were counted. Oocytes were collected using a no. 15 scalpel and a Pasteur pipette in a petri dish containing oocyte collection media (Stenbak et al., 2001). Each follicle was cut with a scalpel and washed/flushed two or three times. Oocytes were then evaluated based on morphology and categorized as healthy or atretic according to Thompson et al. (1995). All oocytes were washed three times before being transferred into maturation medium containing epidermal growth factor (EGF; Choi et al., 2001; Grazul-Bilska et al., 2001; Stenbak et al., 2001) stabilized under mineral oil. In Vitro Maturation Oocytes were matured for 21-24 h at 39 C, 5% CO2, and 95% air, and then oocytes were evaluated again for health based on morphology (Thompson et al., 1995). Only healthy-looking oocytes were used for IVF. The cumulus cells were removed by using 0.1% hyaluronidase (Type I-S; Sigma) treatment (Stenbak et al., 2001). Following cumulus cell removal oocytes were transferred to stabilized fertilization medium, consisting of synthetic oviductal fluid (SOF; O’Brien et al., 1997; Tervit et al., 1992; Walker et al., 1996; Wang et al., 1998; Stenbak et al., 2001) and 2% heat inactivated sheep serum collected on day 0 of the estrous cycle. In Vitro Fertilization and Culture Frozen semen, pooled from 4 Hampshire rams, was thawed and viable sperm were separated using the swim-up technique in modified sperm washing medium (Irvine Scientific, Santa Ana, CA; Yovich, 1995; Stenbak et al., 2001). The oocytes were fertilized with 0.5-1.0 x 10 viable sperm/mL (up to 20 oocytes/500 L well). The oocytes were incubated with the sperm for 17 to 20 h at 39 C, 5% CO2, 5% O2, and 90 N2. Then zygotes were cultured in SOF medium without glucose (Catt et al., 1997, Wang, 1998; Stenbak et al., 2001). The dishes were evaluated approximately 48 h after adding sperm to determine the rate of fertilization based on the number of cleaved oocytes. Oocyte Staining to Determine Maturation Status Oocytes that failed to fertilize were fixed in methanol and then stained with 0.1 μg/ml of 4,6-diamino-2phenylindole (DAPI; Molecular Probes Inc., Eugene, OR, USA) in methanol for 15 minutes and mounted on slides (Jablonka-Shariff and Olson, 2000). The evaluation of nuclear status was done by epifluorescence microscopy (Gardner et al., 1997). Oocytes in the germinal vesicle stage, containing diplotene chromatin were considered to be immature. Mature oocytes demonstrated exclusion of the first polar body and therefore, were found to be in Metaphase II (Gaudet et al., 1997). Statistical Analysis Numbers of follicles and oocytes collected, and numbers and percentages of matured oocytes and cleaved zygotes were analyzed by using the general linear models procedure of the Statistical Analysis System (User’s Guide, 1985). When the F-test was significant, differences between specific means were evaluated using the least square differences test (Kirk, 1982). Rates of oocyte maturation and fertilization were analyzed by using the Chi-Squared procedure of the Statistical Analysis System (User’s Guide, 1985).