313 IMPROVEMENT OF FERTILIZATION IN VITRO BY PRE-INCUBATION OF FROZEN–THAWED RAT SPERMATOZOA WITH CAFFEINE-SUPPLEMENTED MEDIUM

Abstract

We previously reported the successful production of live offspring from cryopreserved rat spermatozoa through IVF (2006 Reprod. Fertil. Dev. 18, 256). The objective of the present study was to improve the IVF system with frozen–thawed spermatozoa for more efficient reproduction of live offspring. We examined the effect of caffeine-supplemented medium on post-thaw sperm fertility in vitro. Epididymal spermatozoa of Wistar rats were cryopreserved in 2.0 mL of freezing medium containing 23% (v/v) egg yolk, 8.0% (w/v) lactose monohydrate, and 0.7% (v/v) Equex STM. Post-thaw spermatozoa were diluted to 0.5 to 1.5 × 10⁶ sperm mL^-1 in a droplet of 200 µL of R1ECM as the pre-incubation medium. In Experiment 1, the effect of caffeine-supplemented medium on fertility in vitro was examined. The post-thaw spermatozoa were incubated for 5 h in pre-incubation medium with added caffeine at concentrations of 0, 1, 2, and 4 mM. Ovulated oocytes were introduced into droplets of the pre-incubation medium and co-cultured for 10 h. The oocytes were denuded and fixed to examine for 2 pronuclei formation (2PNF). In Experiment 2, the effect of caffeine-supplemented medium on post-thaw sperm function was examined using the chlortetracycline (CTC) fluorescence assessment. The post-thaw spermatozoa were incubated in pre-incubation medium with added caffeine at concentrations of 0 (Caf+) and 2 mM (Caf-), and CTC patterns were examined hourly until 6 h later. Statistical analyses of the results were carried out by one-way ANOVA. In Experiment 1, the percentages of 2PNF were 14 ± 6, 32 ± 8, 42 ± 4, and 27 ± 5% for the 0, 1, 2, and 4-mM groups, respectively, and only the 2-mM group was different from the 0-mM group (P < 0.05). In Experiment 2, the rate of capacitated spermatozoa was not significantly different (P > 0.05) among incubation times in the Caf+ group, but the rates at 5 and 6 h were lower (P < 0.05) than the rate at 1 h in the Caf- group. Although the rates of acrosome-reacted spermatozoa were not significantly different (P > 0.05) in the Caf+ group after 3 h of incubation, the accelerated trend of the acrosome reaction was observed in the Caf- group with increasing incubation time. The results demonstrated that frozen–thawed rat spermatozoa pre-incubated in medium containing 2 mM caffeine could improve in vitro fertility. This positive effect of caffeine would be provided to induce and keep sperm capacitation, and to inhibit the spontaneous sperm acrosome reaction in frozen–thawed rat spermatozoa.