Effect of sperm cryopreservation and treatment with calcium ionophore or heparin on in vitro fertilization of horse oocytes

Little information is available on methods of sperm capacitation for IVF in the horse. In this study, we summarized results of several independent tri als that compared acrosome reaction, hyperactivation and chromatin integrit y of fresh or cryopreserved stallion spermatozoa after treatment with hepar in or with calcium ionophore. We also examined the influence of spermatozoa storage (fresh vs. cryopreserved), capacitation treatment, oocyte maturati on time and cumulus morphology on the penetration rate and fertilization ra te. We recovered cumulus-oocyte-complexes (COCs) from ovaries by ultrasound guided follicle aspiration or by scraping of follicles from ovaries obtain ed at a slaughterhouse. Upon recovery, we evaluated the cumulus morphology, and the COCs were matured in vitro for 18 to 24 or 26 to 40 h, Fresh semen and cryopreserved semen were treated either with heparin (200 mug/mL) or c alcium ionophore (7.14 muM). Overall, 28.4% (99/349) of the oocytes were pe netrated, and 12.9% (45/349) were fertilized. Fresh spermatozoa treated wit h calcium ionophore showed a higher penetration rate than cryopreserved spe rmatozoa (36.0 vs. 0%). Fresh and heparin-treated spermatozoa showed a pene tration rate of 29.1%, and the same treatment for cryopreserved spermatozoa showed a penetration rate of 33.7%; none of these differences was signific ant (P>0.05). Fertilization rates after the calcium and heparin treatment f ollowed the same trend and also showed no significant differences. Prolonge d maturation period resulted in higher penetration (P<0.05) and fertilizati on rates in compact (26 to 40 h: 37.7 and 13.1% vs. 18 to 24 h: 13.1 and 2. 8%) and in tendency in expanded COCs (26 to 40 h: 40.0 and 30.3% vs. 18 to 24 h: 29.4 and 13.5%). In oocytes with only a few cumulus cells, the rates tended to be higher after the shorter incubation (IS to 24 h: 33.5 and 18.8 % vs. 26 to 40 h: 17.2 and 6.5%), We observed hyperactivation more frequent ly in fresh than in cryopreserved semen after different treatments (43.2, 3 9.1 and 35.4% for heparin, calcium ionophore and control vs. 15.7, 10.8 and 5.7%, respectively). We observed significant changes in the acrosome react ion of fresh spermatozoa after heparin treatment (62.6 vs. 48.2%, P<0.05), as well as in cryopreserved spermatozoa after calcium ionophore treatment ( 31.7 vs. 17.6%, P<0.05). The chromatin integrity was significantly reduced after heparin treatment of fresh spermatozoa, in comparison to control and calcium ionophore (81.0 vs. 87.3 and 86.6, P<0.02). We also observed a simi lar reduction of chromatin quality after heparin treatment in cryopreserved spermatozoa, but the difference was significant only between heparin and c alcium ionophore treatment [77.4 vs. 86.4 (P<0.02) and 84.9]. The results i n the this retrospective study show that capacitating fresh spermatozoa wit h calcium ionophore, or using heparin in cryopreserved spermatozoa, results in higher penetration and fertilization rates of in vitro matured horse oo cytes. A prolonged maturation time of 26 to 40 h is necessary for compact c umulus oocyte complexes to achieve the fertilization capacity. Further inve stigation is needed to show the developmental capacity of these fertilized oocytes. (C) 2001 by Elsevier Science Inc.