Experiments to improve in vitro fertilization techniques for in vivo-matured porcine oocytes

Three experiments were designed for the in vitro fertilization of in vivo matured oocytes following different sperm treatments: In experiment I, spermatozoa (sperm rich fraction) were capacitated in isolated and ligated uterine horns of estrous gilts (Method A) for at least 3.5 hours and 1x10(6) sperm/ml were exposed to mature oocytes. Of 1586 oocytes 509 (32.1%) developed to the two to eight cell stage. Cleavage stage embryos (n=187) were transferred into nine recipients; 81 embryos (43.3%) were recovered after 72 hours and 21 (26%) had developed to the morula or blastocyst stage. The average number of nuclei (whole mount staining) was 108.4 and indicated normal developmental capacity. Another 127 embryos were transferred into three recipients, two became pregnant and one gilt of them delivered two offspring after 116 days of gestation. In experiment II, two different procedures for the capacitation of spermatozoa using modified TCM 199 medium were compared with that of Method A. Semen was either adjusted to a concentration of 2x10(8) sperm/ml with modified TCM 199E (Method B), or after centrifugation the sperm pellet was diluted in a 1:1 ratio with the same extender (Method C). Motility, and hyperactivity were significantly different (P 0.05) among treatments and were best after uterine incubation (Method A). The percentage of head-to-head aggregation increased significantly (P 0.05) after incubation in the uterine horn, and is suspected to represent a spontaneous acrosome reaction. A total of 492 oocytes were fertilized with semen capacitated by the three methods however, maximal fertilization (34.7%; P 0.05) was obtained with Method B. In experiment III, Method B was repeated and in order to minimize the rate of polyspermy, a total of 298 oocytes were exposed to different sperm concentrations (8x10(3); 4x10(3); 2x10(3) per oocyte). The reduction of spermatozoa exposed to oocytes improved the fertilization rate significantly (P 0.05) from 37.2 to 54.5%.