Effects of heparin and sperm concentration on cleavage and blastocyst development rates of bovine oocytes inseminated with flow cytometrically-sorted sperm.
The objective was to determine the optimal concentration of heparin for sperm capacitation, as well as the optimal sperm concentration for in vitro fertilization using flow cytometrically-sorted sperm from individual bulls. A total of 5327 bovine oocytes and sperm from four bulls were examined. Oocytes from slaughterhouse ovaries were matured in TCM199 for 22-24 h. Flow cytometrically-sorted sperm as well as unsorted control sperm from the same bulls were cryopreserved. For sperm from each of the four bulls, oocytes were inseminated in a three-by-three factorial design plus one control group (three heparin concentrations: 0, 2, and 10 microg/ml and three sperm concentrations: 0.5 x 10(6), 1.5 x 10(6), and 4.5 x 10(6) ml(-1); 10 microg/ml of heparin and 1.5 x 10(6) ml(-1) of sperm were used for the unsorted control). Presumptive zygotes were cultured in chemically defined media, CDM-1 and CDM-2 for 52-54 h and 96 h, respectively. Samples of about 10 oocytes from each of the 10 treatment groups per replicate were fixed at 18-20 h after insemination to determine sperm pronuclei formation and polyspermy. Increased polyspermy resulted as heparin and sperm concentrations increased (P < 0.05). A higher rate of polyspermy was found in oocytes inseminated with unsorted control sperm compared with sorted sperm (P < 0.05). Sperm of one of four bulls tested required no heparin and lower concentration (0.5 x 10(6) ml(-1)) to obtain optimal cleavage and blastocyst rates while optimal parameters for another bull were higher heparin (10 microg/ml) and sperm concentrations (4.5 x 10(6) ml(-1)). Optimal parameters for the other two were intermediate levels of heparin and sperm. Sperm appeared to be partially capacitated during the flow cytometric-sorting process used for sex pre-determination. When heparin and sperm concentrations were optimized for individual bulls, blastocyst production per oocyte was similar for sorted and unsorted sperm for three of the four bulls studied.
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XY, Inc., 2301 Research Boulevard, Suite 110, Fort Collins, CO 80526, USA.
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D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D002417:Cattle; D002469:Cell Separation; D002970:Cleavage Stage, Ovum / Q000187:drug effects*; D046508:Culture Techniques; D005260:Female; D005306:Fertilization; D005307:Fertilization in Vitro / Q000662:veterinary; D005434:Flow Cytometry; D006493:Heparin / Q000008:administration & dosage*; D008297:Male; D009865:Oocytes / Q000187:drug effects / Q000254:growth & development*; D013076:Sperm Count; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology
D006493:Heparin