Improved Methods for the Production of Embryos in Seasonally Anestrous Ewes

In a recent study from our laboratory, a combination of melatonin implants and controlled internal drug release devices increased the number of developing follicles in follicle stimulating hormone-treated anestrous ewes; however, CIDR treatment decreased the rate of in vitro fertilization (IVF). In the current study, effects of alternative CIDR treatments on follicular development and rate of IVF in anestrous ewes was evaluated. On d -60 from oocyte collection (d 0) all ewes (1CIDR and 2CIDR treatment groups; n=6/group) received a melatonin implant. In addition, CIDR's were implanted on d -22 and removed on d -17. On d -10 2CIDR ewes received a second CIDR for 8 d (removed on d -2). All ewes received FSH injections twice daily on d -2 and d -1. On d 0, ovaries were removed and oocytes were aspirated from follicles, matured in vitro for 17-24 h and then subjected to IVF on d 1 of culture. 1CIDR and 2CIDR ewes had a similar (P > 0.10) number of follicles > 1 mm in diameter (26.7 ± 8.6 and 24.3 ± 13.3, respectively) and rate of oocyte recovery (91.3 ± 3.5 and 99.3 ± 1.6%, respectively). On d 3 of culture, the rate of oocyte maturation (sum of fertilized and matured unfertilized oocytes [determined by DAPI staining]) was similar (P > 0.10) for 1CIDR and 2CIDR ewes (89.7 ± 7.4 and 87.6 ± 6.3%, respectively). However, oocytes collected from 2CIDR ewes had lower (P < 0.01) rates of IVF than oocytes collected from 1CIDR ewes (30.2 vs. 58.0%, respectively). Thus, IVF rates were adversely affected by an additional 8 d CIDR treatment. These data indicate that the interval between CIDR treatment and oocyte collection affects IVF rates in melatonin and FSH-treated ewes during anestrous. Therefore, progestogen treatment protocols used in ovine IVF programs should be carefully designed to minimize adverse effects on fertilization rates. Introduction It has been suggested that low fertilization rates during the non-breeding season may be caused by an altered endocrine status when compared with ewes during the normal breeding season (Stenbak et al., 2001). Numerous studies have focused on developing hormonal treatments to improve follicular development and induce a fertile estrus in ewes during the nonbreeding season (Robinson et al., 1991 and 1993; Gordon, 1997; Carlson, 2000; Knights et al., 2000, 2001). The main focus of previous studies has been to improve pregnancy rates and maximize reproductive performance in vivo. However in a recent in vitro fertilization (IVF) study from our laboratory (Luther et al., 2002), a combination of melatonin implants and CIDR devices increased the number of developing follicles in FSH-treated anestrous ewes. In addition, treatment with CIDR devices decreased the rates of IVF. Limited data are available concerning the effects of exogenous hormones, such as progesterone, on oocyte quality for in vitro fertilization (IVF) during the non-breeding season in adult ewes. Progesterone-based therapies have been widely used for inducing a fertile estrus in seasonally anestrous ewes (Robbinson et al., 1991; Jabbar et al., 1994; Knights et al., 2000, 2001). Knights et al. (2001) demonstrated that a 5-day treatment with progesterone, in combination with FSH, stimulated a fertile estrus just as effectively as a 12-day progesterone treatment; and that prolificacy was comparable to that observed during the normal breeding season. In FSH-treated pre-pubertal ewe lambs, Armstrong et al. (1994) found an increase in the number of follicles, and oocyte recovery and maturation rates when progestogen pre-treatment was withdrawn 2 days before oocyte collection as apposed to a continuous progestogen pretreatment. The aim of the current study was to evaluate the effects of exogenous progesterone treatment on follicular development and oocyte quality in melatonin and FSH-treated seasonally anestrous ewes. Oocyte quality was measured by the rates of maturation and in vitro fertilization. Material and Methods Animals and Experimental Design Crossbred Rambioullet/Targhee Western U.S. range ewes were used during the nonbreeding season from March to May. The treatment protocols were as follows: