316 SUPPLEMENTAL CYSTEINE PRESENCE DURING THE DECONDENSATION OF SPERM CHROMATIN IMPROVES FERTILIZATION AND BLASTOCYST FORMATION AFTER INTRACYTOPLASMIC SPERM INJECTION IN PIGS

The effect of a cysteine supplement in culture media for oocytes matured in vitro after intracytoplasmic sperm injection (ICSI) on fertilization and embryo development were examined. In the first experiment, sperm injected oocytes were cultured in NCSU23 (control) or NCSU23 supplemented with 0.57–3.71 mM cysteine (0.57–3.71 Cys) for 12 h after ICSI, and then fixed to observe pronuclear formation. In the second experiment, to examine the appropriate duration time of cysteine supplement to support fertilization, sperm-injected oocytes were transferred into NCSU23 following culture in NCSU23 supplemented with 1.71 mM cysteine for 1, 2, 3, 4, 5, 6, or 9 h after ICSI, and then fixed at 12 h. At the same time, morphological changes of sperm heads in oocytes cultured in NCSU23 (1.71 Cys) were observed. In the third experiment, to examine the developmental ability of ICSI embryos fertilized in NCSU23 (1.71 Cys), sperm injected oocytes were cultured under the following conditions for a total of 168 h; NCSU23 (control), NCSU23 (1.71 Cys) for 3 h followed by transfer into NCSU23 (1.71 Cys-3 h), NCSU23 (1.71 Cys) for 12 h followed by transfer in NCSU23 (1.71 Cys-12 h), or NCSU23 (1.71 Cys) (1.71 Cys). Data were pooled from at least five replicates. Values in each replicate were analyzed using one-way ANOVA. Significance of differences was assessed by Student's t-test. Culture with several concentrations of cysteine for 12 h showed that 1.71–3.71 Cys significantly (P < 0.05) increased fertilization rates above controls or 0.57 Cys (56–60%, 35%, or 48%, respectively). Culture for several duration times with 1.71 Cys showed that fertilization rates increased as the duration time increased to 3 h which was significantly (P < 0.05) higher than controls (68% and 34%, respectively), and culture times of greater than 3 h did not increase fertilization rates (58–68%). At 3 h, 59% of oocytes cultured in NCSU23 (1.71 Cys) had decondensed sperm heads and 16% of those had enlarged sperm heads. At 6 h, 50% of oocytes cultured in NCSU23 (1.71 Cys) had male pronuclei. Blastocyst formation rate in 1.71 Cys-3 h was 29% which was higher than for controls (20%). On the other hand, 1.71 Cys-12 h cultures showed low blastocyst formation rates, and continuous culture in NCSU23 (1.71 Cys) for 168 h (1.71 Cys) significantly (P < 0.05) decreased blastocyst rates (16% and 7%, respectively). We found that the supplement of 1.71 mM cysteine to NCSU23 for culture of oocytes after ICSI improved fertilization rates. However, the presence of 1.71 mM cysteine for 12 h or longer after ICSI had adverse effects on embryo development. Since 1.71 mM cysteine supplement for 3 h after ICSI improved blastocyst formation with the same fertilization rates as when supplemented for 12 h, the presence of cysteine only during the decondensation of sperm chromatin was found to be associated with the improvement of fertilization and also the promotion of blastocyst formation.