The purpose of this study was to evaluate the spermatozoal viability, acrosome integrity, mitochondrial activity and fertility in the frozen-thawed fowl semen by different cryoprotective agents and to figure out their correlations with rate of fertilization.
The experiment was carried out on 10 sexually adult roosters of Ogye. The semen was collected twice a week and pooled semen was diluted 1:1 EK extender containing no cryoprotectant at 5℃. After equilibration for 30 minutes, diluted semen was used by mixing an equal volume of semen diluent containing either 7% dimethylacetamide (DMA), 7% dimethylformamide (DMF) or 7.5% methylacetamide (MA) at final concentration. Diluted semen was put in 0.5mL plastic straws and frozen for 30 minutes by exposure to liquid nitrogen vapor 4cm above the surface of liquid nitrogen, followed by plunging into liquid nitrogen at -196℃. Frozen semen was thawed in water bath at 5℃ for 2 minutes. For cytometric analysis, the frozen-thawed semen was diluted with EK extender to a final concentration of 90 million spermatozoa per mL. Sperm membrane integrity was evaluated as SYBR-14 and propidium iodide (PI). Acrosome integrity was assessed as fluorescein isothiocyanate-labeled PSA and PI. The percentage of mitochondrial function was estimated by using Rhodamine123 (R123) and PI. Intravaginal artificial insemination was performed twice a week by injecting 0.2mL of thawed semen directly into the vagina within 2 min after thawing.
As the result of freezing rooster''s semen, when using DMF, survival rate was 52.1±5.52% and it was significantly higher than that of using DMA(46.94±5.06%) and MA(36.56±4.66%)(p<0.05), respectively. For the rates of acrosomal membrane and death of sperm, semen using MA was highest with 61.16±1.86% and then followed by samples with DMA(47.48±1.9%) and DMF(36.56±1.42%), respectively with significant differences(p<0.05). For rate of sperm survival with intact mitochondrial membrane, semen frozen by using DMF was highest with 52.68±1.07% and then followed by samples with DMA(44.46±1.04%) and MA(38.36±1.88%), respectively with significant differences(p<0.05). Samples with DMF and DMA showed similar rates of fertilization 67.85% and 68.35%, respectively while Sample with MA showed somewhat lower rate of fertilization 60.95% but there were no significant differences. In summarizing the results of this study, when freezing rooster semen by using 7% DMF as cryoprotective agent, there were no significant difference between rates of fertilization but this sample was significantly highest in rates of survival and mitochondrial function while its rate of damage of acrosome was significantly lowest. As a result, DMF is the cryoprotective agent which have the lowest influences on cell membranes and acrosome integrity of sperm and could be used to freezing conservation method for poultry genetic resources.