The University of Georgia College of Veterinary Medicine

Georgia Veterinary Scholar Program

Georgia Veterinary Scholar	Faculty Mentor

Meggan Ballowe University of Georgia Class of 2003	Dr. Hugh Dookwah

Role of sperm antigens in inducing in vitro maturation in canine oocytes

In vitro maturation of canine oocytes is the major limiting factor to the use of assisted reproduction techniques to treat subfertility and infertility in dogs. In vitro fertilization, intracytoplasmic sperm injection, and embryo transfer have yet to be accomplished in the dog for this reason. In contrast to other mammals, the bitch ovulates an immature oocyte at the germinal vesicle stage of maturation. In vivo, the canine oocyte matures 48-72 hours post-ovulation during which time it is exposed to hormonal changes and, if the bitch is mated, the presence of sperm within the oviduct. To date, attempts to trigger consistently high rates of in vitro oocyte maturation have failed in the dog. In this study, we investigated the role of soluble sperm factors in inducing the maturation of in vitro cultured oocytes. We compared the following sperm fractionation methods: 1)homogenization, 2) homogenization and sonication, and 3) homogenization and sonication following hypo-osmotic sperm swelling using a 75:25 ratio of 150 mosmol fructose/citrate solutions. We applied these methods to both capacitated and non-capacitated sperm. The soluble sperm fractions were then assayed using SDS-PAGE electrophoresis and quantified using Coomassie protein assay. Hypo-osmotic sperm swelling followed by homogenization and sonication yielded the largest amount of protein. We are currently investigating the effects of adding five sperm equivalents of soluble sperm fractions to oocytes under several culture conditions. These conditions include In Vitro Maturation media (IVM), 0.1% recombinant Follicle Stimulating Hormone (rFSH) and oviductal co-culture in varying combinations. We incubate in vitro cultured oocytes in 5% CO2 in air at 39oC for 72-96 hours. Preliminary data suggests increased maturation to metaphase or early anaphase stages (MI/AI) of development in cultured ova treated with hypo-osmotically swollen sperm fractions. Further study will concentrate on this sperm fraction in IVM on oviductal co-culture with and without 0.1% rFSH.