The purpose of this study was to evaluate the sperm viability, normal acrosome and mitochondrial activity in the frozen-thawed fowl semen by different cryoprotectants. 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 chicken semen was diluted 1:1 extender containing either 7% dimethylacetamide (DMA), 7% dimethylformamide (DMF) or 7.5% methylacetamide (MA) at final concentration and was put in 0.5 mL plastic straws and frozen for 30 minutes by exposure to liquid nitrogen vapor 4 cm above the surface of liquid nitrogen, followed by plunging into liquid nitrogen. 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 with fluorescein isothiocyanate-labeled PSA and PI. The percentage of mitochondrial function was estimated by using Rhodamine123 (R123) and PI. In conclusion, freezing rooster semen by using 7% DMF as cryoprotectant 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 cryoprotectant that has the lowest influences on sperm membranes and acrosome integrity. Therefore it could be used for freezing method of animal genetic conservation method for poultry diversity.
The use of methylacetamide (MA) as a cryoprotective agent for freezing Korean Native Black rooster Ogye semen was examined with artificial insemination. The diluted Ogye semen with HS-1 was subjected for 2 step dilution method of cryopreservation in which the final concentration of MA was adjusted to 7.5%. The sperm viability after thawing was reduced from 95.17 ± 0.93% to 55.93 ± 1.38% which was confirmed by live-death analysis based on Fluorescence-Activated Cell Sorting (FACS). The rates of fertilized eggs with fresh or frozen-thawed semen were reduced from 94.98 ± 3.93% to 66.36 ± 8.43% at day 7 with significant difference. However, the hatching rates of experiments at day 21 did not shown difference between 92.64 ± 2.33% and 90.45 ± 8.05% (P<0.05). With these results, the utilization of MA for freezing of Ogye spermatozoa could affect on viability of frozen-thawed semen but not on the fertility of lain eggs and hatchability of fertilized eggs and also provide possible tools of freezing for poultry genetic resource conservation.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.