Intracytoplasmic sperm injection in the horse: the ultimate blind date

Background: Intracytoplasmic sperm injection (ICSI) has become a useful technology to produce foals when availability of semen is limited or when in-vitro fertilization is desired, as is the need for subfertile mares. However, its application into clinical practice is challenging. The purpose of this review was to discuss some fundamental molecular aspects of oocyte maturation that should be considered when performing ICSI and to report factors of age and subfertility affecting the success of a commercial ICSI program. Review: The molecular synchrony of oocyte maturation included nuclear, epigenetic and cytoplasmic maturation. Oocyte developmental competence was found to be dependent on the ability to remain in meiotic arrest until the initiation of final maturation, requiring the adequate timing involved with follicular maturation prior to ovulation. Studies performed in cattle and humans have demonstrated that in-vivo oocyte maturation results in high pregnancy rates per oocyte fertilized. Therefore, determining precise maturation of the oocyte would be valuable for the timing of ICSI and subsequent embryo development. Reproductive aging in the mare was characterized by a decline in fertility. Using RT-PCR, quantitative and temporal differences were found in mRNA content of key regulatory maturation genes in granulosa and cumulus cells and in oocytes during in vivo maturation in young and old mares. These results suggested premature oocyte maturation in aged mares that potentially could result in subfertility. Consequently, the timing of oocyte retrival after gonadotropin administration should be carefully evaluated when performing ICSI. In a commercial program, equine patients were classified into normal mares (2.5 to 15 years), problem mares (15-23 years that had not been producing embryos or pregnancies) and old mares (>24 years). Old mares were assessed for endocrine, physical and nutritional imbalances. Follicular and oocyte maturation were induced with a dominant follicle >30 mm in diameter after a normal growth and blood flow and uterine edema with a combination of hCG and GnRH. Transvaginal oocyte retrieval was performed 20 hours after administration of gonadotropins. Oocytes were further cultured in vitro for 12 to 20 hours. Frozen semen was used for all sperm injections. Injected oocytes were further cultured in vitro for at least 24 hours. Embryos were then transferred surgically into oviducts of synchronized recipients. Oocyte recovery rate was 94% (523/557 cycles), cycles per month were 3.3, 2 and 1.3 for young, problem and old mares respectively. Cleavage rates were different (p<0.05) between young (82 %), problem (70 %) and old (52%) mares. Pregnancy rates at day 60 were also different (p<0.05) for young (68 %), problem (50 %) and old (23%) mares. Number of pregnancies obtained from a single straw of frozen semen ranged from 2 to 12. Reproductive senescence was observed in 10% of old mares. In addition, Cushing’s disease and elevated diestrus FSH were observed in 80% of the old mares. Foaling rates were evaluated in 55 pregnancies; 5% were lost in the last trimester and the remaining foals have shown no apparent abnormalities. Conclusion: More studies are needed to further elucidate the mechanisms of oocyte maturation and activation in the horse, as well as more objective methods to determine oocyte maturity and quality. A clinical ICSI program required an understanding of gamete physiology and detailed mare reproductive management. Our clinical data demonstrated that aging affects fertility profoundly in ways that may be difficult to address with current technology; nonetheless, ICSI has provided the equine industry an alternative to produce offspring from valuable mares and stallions that are subfertile.