131 Embryo production after intracytoplasmic sperm injection using stallion semen refrozen in different extenders

Intracytoplasmic sperm injection (ICSI) allows efficient use of low stores of stallion semen for equine embryo production. Post-thawing re-extension and refreezing yields multiple straws, thus improving semen availability and increasing the potential of obtaining offspring from a stallion. However, there is conflicting anecdotal information on the effectiveness of ICSI with refrozen stallion semen. The objective of this study was to evaluate the effect of the extender used for refreezing semen on equine embryo production after ICSI. Semen from three stallions was extended with INRA 96, cushioned-centrifuged, extended to 200 × 10⁶ sperm mL⁻¹ with a low egg yolk/glycerol/amide extender (BotuCrio®, Botupharma; Extender 1 [E1]) and frozen in 0.5-mL straws (F1). The F1 semen was then thawed, extended to 2 × 10⁶ sperm mL⁻¹ with each of three extenders, and refrozen in 0.5-mL straws (F2). In addition to E1, extenders used for refreezing (R) included E2, a high-egg yolk/glycerol extender (SBS CryoSystem Spectrum Red, Minitube), and E3, a low egg yolk/milk/glycerol extender (E-Z Freezin^TM, “MFR5”, Animal Reproduction Systems, Inc.). Equine oocytes obtained from slaughterhouse ovaries or by transvaginal aspiration were matured for 30 h. For ICSI, straws of semen in all four treatments were thawed and prepared by centrifugal washing twice in GMOPS medium supplemented with 10% FBS. Motile sperm with normal morphology were immobilized in hyaluronan solution and injected into oocytes using a piezo drill with fluorocarbon ballast. Cleavage rates were evaluated after 5 days of culture (ICSI = Day 0) and blastocyst development rates were evaluated after 7 to 10 days of culture. Nine ICSI replicates were performed (three per stallion) with all treatments included in each replicate. The order of use of the different treatments was randomized within each replicate. A total of 184 mature oocytes were injected (45–47 per semen treatment). Cleavage and blastocyst rates per injected oocyte were evaluated by Fisher’s exact test. The cleavage rates for F1, R-E1, R-E2, and R-E3 were 65%, 38%, 47%, and 50%, respectively. The blastocyst production rates were 26%, 11%, 16%, and 17%, respectively. The cleavage rate for R-E1 was lower (P < 0.05) than for F1, and the blastocyst development rate in R-E1 tended to be lower (P = 0.06) than for F1. In conclusion, refreezing semen extender affects embryo production after ICSI in horses, and selection of semen cryopreservation procedures is important to optimize results. Refrozen stallion semen can be effectively used for ICSI provided an extender such as MFR5 is used for refreezing. Although a limited number of extenders were evaluated, use of extenders without amides may be advisable when refreezing stallion semen for ICSI at this time.