268PREGNANCIES FROM FROZEN IVF CATTLE EMBRYOS USING
SEX-SORTED AND UNSORTED SPERM
Previously we demonstrated that sex-sorted sperm could produce IVF embryos from juvenile and adult cattle at rates similar to those for unsorted sperm (Fry et al., 2003 Theriogenology 52, 198). In this study we investigated the pregnancy rates of recipient cattle following the transfer of frozen/thawed IVF embryos generated from young heifers using sex-sorted and unsorted sperm. COCs collected from FSH-stimulated Senepol or Beefex heifers by TVR were matured, fertilized with either sex-sorted or unsorted Senepol sperm and cultured for 6 days under our standard laboratory conditions (Fry et al., 2003 Theriogenology 59, 446, Earl et al., 1997 Theriogenology 47, 255). Embryos reaching the blastocyst or expanded blastocyst stage of development were frozen by the CL-V method of vitrification. Briefly, embryos were equilibrated for 5–10min in HEPES-199 media containing 20% FCS (HM), placed in HM containing 10% EG, 10% DMSO for approximately 2 minutes and then in HM containing 20% EG, 20% DMSO for between 20–60sec (Vatja et al., 1997 Cryoletters 18, 191). Vitrification was achieved by collecting between 5–10 IVF embryos in a 3-μL droplet and securing this droplet to a coded CL-V holder. The droplet was vitrified using the CL-V kit (Lindemans et al., 2004 Theriogenology in press) and then sealed in a precooled ‘‘straw’’ for storage in liquid nitrogen. To thaw, the ‘‘straw’’ with specimen was removed from storage;; the specimen droplet was withdrawn from the ‘‘straw’’ and placed directly into HM containg 0.2M sucrose (SM). After approximately 5–10min each embryo was assessed, loaded into a tomcat catheter in SM and transferred surgically into a recipeint cow within 10–15min of thaw. Of 129 Brahman and Brahman cross cows receiving 2 injections of 125μg cloprostenol 11 days apart, 60 exhibited oestrus 2–4 days after the second injection and 53 were deemed suitable for embryo transfer. Pregnancy was determined by ultrasound on Day 40. No difference in pregnancy rate was found between treatment groups (P>0.05; Table 1). The low submission rate (60/129) and pregnancy rate for the in vivo control group indicate that the fertility of the recipient cows may have been compromised by the drought conditions predominating in Central Queensland. Notwithstanding, the CL-V method for the vitrification of IVF embryos produced by either sex-sorted or unsorted sperm gave similar and very promising pregnancy results of around 40%. This provides new opportunities for the rapid banking of large numbers of sexed IVF embryos generated from elite cattle by TVR for user friendly embryo transfer programs. Table 1 Pregnancies from IVF embryos derived from sex-sorted and unsorted sperm and frozen by the CL-V method of vitrification, or from in vivo embryos frozen in glycerol
