59 EVALUATION OF CRYOPRESERVED CHICKEN SEMEN WITH DIFFERENT CRYOPROTECTANTS

Abstract

The purpose of this study was to evaluate the viability, acrosome integrity, mitochondrial activity, and fertility of frozen-thawed fowl semen using different cryoprotective agents. The experiments were carried out on 10 sexually adult roosters of the Ogye breed (Korean native black chicken). The semen was collected twice per week and diluted in a 1 : 1 ratio with EK extender without cryoprotectant at 5°C. After equilibration for 30 min, diluted semen was added with an equal volume of diluents containing 14% dimethylacetamide (DMA), 14% dimethylformamide (DMF), or 15% methylacetamide (MA). The semen were packed into 0.5-mL plastic straws, frozen for 30 min by exposure to liquid nitrogen vapor 4 cm above the surface of liquid nitrogen, and then plunged into liquid nitrogen at –196°C. Frozen semen was thawed in a cold water bath at 5°C for 2 min. For fluorescence-assisted cell sorting (FACS) analysis, the frozen-thawed semen was adjusted to a final concentration of 90 million spermatozoa per milliliter with EK extender. Sperm membrane integrity was evaluated by the live-dead staining method with SYBR-14 dye and propidium iodide (PI). Acrosome integrity was measured with fluorescein isothiocyanate-labelled Pisum sativum agglutinin (PSA) and PI. The percentage of functional mitochondria was estimated using Rhodamine 123 (R123) dye and PI. After intravaginal AI was performed twice per week by injecting 0.2 mL of thawed semen directly into the vagina within 2 min after thawing, the resulting eggs were incubated for 7 days to confirm fertilization. The survival rates of cryopreserved sperm with DMF, DMA, and MA were 52.1 ± 5.5, 46.9 ± 5.1, and 36.6 ± 4.7%, respectively. The survival rate of DMF was significantly higher than those of DMA and MA (P < 0.05). The percentages of sperm that had damaged acrosomal membranes using DMF, DMA, and MA were 36.6 ± 1.4, 47.5 ± 1.9, and 61.2 ± 1.9% (P < 0.05), respectively. The percentages of live sperm with intact mitochondrial membranes cryopreserved with DMF, DMA, and MA were 52.7 ± 1.1, 44.5 ± 1.0, and 38.4 ± 1.9%, respectively, with significant differences (P < 0.05). The fertilization rates of resulting eggs after AI were 68.4% in DMF, 67.9% in DMA, and 61.9% in MA, without significant differences. These results indicate that cryopreserved rooster semen with 7% DMF showed a significantly higher survival rate and mitochondrial functionality but a lower rate of damaged acrosomes. As a cryoprotective agent, DMF has the lowest influences on Ogye rooster sperm membranes and acrosome integrity and thus could be used as a conservation method for poultry genetic resources.