PROCEDURAL IMPROVEMENTS FOR IN-VITRO PRODUCTION OF VIABLE UTERINE STAGE CAPRINE EMBRYOS

Efforts to improve proportions of caprine immature oocytes developing into viable uterine-stage embryos in vitro involved study of 1924 oocy tes in experiments designed to examine influences of fertilization med ia, sperm incubation temperatures, sperm treatment procedures, differe nt protein supplementations, and different insemination intervals. Ooc yte-cumulus complexes (OCCs) were matured during 27 h in TCM-199 suppl emented with 20% FBS, 100 mu g LH ml(-1), 0.5 mu g FSH ml(-1), and 1 m u g Estradiol-17-beta ml(-1) at 38.5 degrees C in a humidified 5% CO2, 5% O-2, and 90% N-2 atmosphere. Freshly collected sperm were washed a nd incubated at either 22 degrees C or 38.5 degrees C for 5 h and then treated with either 0.1 mu M calcium ionophore A23187 for 1 min, or w ith 7.35 mM calcium lactate in the presence of oocytes during the inse mination interval, or with 100 mu g heparin +2 mM caffeine ml(-1) for 15 min, The interval for insemination was experimentally varied, i.e. 14 or 24 h. Results showed that: (a) when used as a fertilization medi um mDM supported more blastocyst development than TALP (10.5% vs. 0%, P < 0.05); (b) incubation temperatures of 22 degrees C or 38.5 degrees C prepared goat spermatozoa equally for capacitation in mDM containin g 20% FBS; (c) when oocytes were inseminated with sperm incubated in m DM with 20% FBS and capacitated with calcium lactate more embryos reac hed the blastocyst stage (P < 0.05) than after incubation in the same conditions but after sperm capacitation with heparin, and A23187 (31.8 % vs. 24.2% and 10.2%, respectively; (d) a 24 h insemination interval was not superior to 14 h when sperm were incubated with either 20% FBS or 6 mg BSA ml(-1) and capacitated with calcium lactate (P > 0.05). T hree morulae resulting from the best conditions in this work (FBS, cal cium lactate, 14 h insemination) were transferred into the uterine hor n ipsilateral to the corpus luteum of a recipient and two normal femal e kids were born after normal gestation. This is the first report in w hich it has been possible to consistently rake caprine development to the blastocyst stage in vitro, and to obtain offspring following uteri ne transfer. Methodology reported here should facilitate implementatio n of new reproductive and genetic strategies in goat breeding.