INTRACYTOPLASMIC SPERM INJECTION (ICSI) VERSUS CONVENTIONAL IVF ON ABATTOIR-DERIVED AND IN VITRO-MATURED EQUINE OOCYTES

Conventional IVF as well as several assisted microfertilization techni ques have shown limited success in the horse. After recent positive re sults achieved with intracytoplasmic injection of a single spermatozoo n (ICSI) in human IVF, we chose to try the method in the horse. We com pared conventional IVF to ICSI by fertilization rates of oocytes with compact and expanded cumuli and by developmental potential of the resu lting embryos. Cumulus-oocyte complexes (COCs) were obtained by aspira ting the follicular fluid from the ovaries of slaughtered mares. Compl exes showing complete cumulus investment, either compact or expanded, were randomly assigned to IVF or ICSI trials and separately cultured f or IVM. Frozen-thawed stallion spermatozoa were prepared for IVF with a swim-up procedure conducted in Talp-Hepes with heparin or for ICSI i n Earle's balanced salt solution (EBSS) supplemented with human serum albumin (HSA). Oocytes for IVF were partially decumulated by pipetting , whereas those for ICSI were totally denuded with 80 Ul/ml hyaluronid ase. Oocytes were fixed, stained and examined for signs of fertilizati on the day after IVF or ICSI. The percentage of normally fertilized oo cytes showing 2 pronuclei or cleavage was significantly higher with IC SI than IVF (29.8%, 17/57 vs 8.7%, 9/103; P<0.01). Significantly highe r fertilization rates were observed in oocytes retrieved with an expan ded cumulus when submitted to ICSI procedure as compared with IVF (52. 2%, 12/23 vs 17.1%, 6/35; P<0.01), whereas in oocytes recovered with a compact cumulus, fertilization rates were low (14.7%, 5/34 with ICSI and 4.4%, 3/68 with IVF; NS). Embryonal development did not occur afte r culture following IVF, as indicated by absence of cleavage in any of the 93 inseminated oocytes, Following ICSI, 7 of 55 injected oocytes cleaved, 5 of which had shown expanded cumuli; of the 5, 2 were at the 16-cell stage and one each at the 8-, 3- and 2-cell stage, respective ly. The other 2 fertilized oocytes, originating from compact cumuli, r eached 4- and 8- cell stages, respectively. These results indicate tha t ICSI can be applied successfully to in vitro matured equine oocytes to increase the fertilization rates. In addition, it seems that in vit ro cytoplasmic maturation of oocytes issuing from a compact cumulus ma y not be complete enough to lead to a successful fertilization and tha t ICSI may be a tool to evaluate ooplasmic maturation. (C) 1997 by Els evier Science Inc.