PS6-20. Cat Blastocyst Production Using Two Methods of Epididymal Sperm Cool-storage.

Abstract In the event of orchiectomy or the unexpected death of a valuable male, epididymal sperm can be utilized for in vitro embryo production in felines. However, decisions regarding tissue handling must be made soon after death. In most cases, the sperm is not used immediately for fertilization but is either cryopreserved or used after oocyte maturation, approximately 24hr. later. In vitro viability of feline epididymal sperm has been demonstrated after 24hr. of cool storage (5ºC). However, a direct comparison of embryo production using sperm stored in extender versus sperm maintained in situ for 24hr. has not been reported. The objective of this experiment was to compare embryonic development rates using two methods of epididymal sperm cool-storage: in extender or in situ. Domestic cat ovaries and testicles were obtained from sterilization surgeries. Compacted cumulus oocyte complexes (COCs) were recovered by slicing the ovaries (469 grade-1 COCs in 6 replicates), and cultured in IVM medium (Stroebech, bovine suite). Sperm from one epididymis from each tomcat were recovered by swim-up: epididymides were sliced and incubated in sperm-wash medium (SW) for 10 min at 38.5ºC; the supernatant was recovered and centrifuged at 400g x 5 min. The pellet was resuspended in Kenney’s extender and cooled to 5ºC overnight (Group 1). The other epididymis from each tomcat was left attached to the testicle and cooled to 5ºC overnight (Group 2). At 21hr. from the start of COCs maturation, the cool epididymides were processed as per group 1, but the pellet was resuspended in SW. During the centrifugation time for group 2, the cool-extended sperm from group 1 were centrifuged at 400g x 5 minutes and resuspended in SW. Both groups were centrifuged again, concentration was adjusted to 1 x106 motile sperm/mL, and COCs were fertilized in IVF medium. At 18hr. from the start of fertilization, cumulus cells were removed by vortexing and presumptive zygotes were placed in in vitro culture conditions (IVC). Cleavage and blastocyst rates were calculated at 96 and 168hr. post-fertilization, respectively, based on the number of presumptive zygotes transferred to IVC. Total sperm motility was analyzed by paired t-test. For embryonic development, each well was considered an experimental unit; development rates were analyzed as repeated measures, main effects were treatment, time, and interaction, blocked by replicate. There was no difference (p ≥ 0.05) in total sperm motility. There was no difference in embryo development rates between the groups; there was an effect of time, and no interaction. Cleavage rates were 49.2±4.3 and 48.8±5.7 (%, LSM±SEM), and blastocyst rates were 20.8±3.4 and 19.8±5.5 for groups 1 and 2, respectively. These results indicate that both cool-storage methods are equally acceptable for blastocyst production when the sperm is used for IVF the day after testicular recovery.