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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION |
* USDA-Agricultural Research Service-National Center for Genetic Resources Preservation, National Animal Germplasm Program, 1111 S. Mason St., Fort Collins, CO 80521-4500; and
Agriculture and Agri-Food Canada, Agassiz Research Centre, PO Box 1000-6947, #7 Highway, Agassiz, British Columbia, Canada, V0M 1A0
4 Corresponding author: phil.purdy{at}ars.usda.gov
A series of experiments was designed to evaluate the quality of cryopreserved rooster sperm and its fertility so that programs needing to bank germplasm and recreate animals can do so utilizing a minimal amount of cryopreserved semen. In experiment 1, rooster semen from the National Animal Germplasm Program genebank was thawed and glycerol was removed using a discontinuous Accudenz column or by stepwise dilution. The postthaw sperm motilities, plasma membrane integrity, and concentration were determined before and after deglycerolization. Line differences in postthaw sperm concentration and progressive motility were observed before deglycerolization (P < 0.05). After glycerol removal, the sperm that was centrifuged through Accudenz had greater total motility (37 vs. 33% sperm; P < 0.05), but use of the stepwise dilution method recovered more sperm per milliliter (320.4 x 106) compared with the Accudenz method (239.2 x 106 sperm; P < 0.05; range across 6 lines of 165.7 to 581.0 x 106 sperm/mL). In experiment 2, rooster semen was cryopreserved using Lake’s diluent containing either dimethyl acetamide (DMA) or glycerol as the cryoprotectants. Postthaw analysis revealed that the samples cryopreserved with glycerol survived freezing better, determined by total motility (47.8 and 15.1% glycerol and DMA samples, respectively; P < 0.05) and annexin V analyses (1.6 and 11.3% membrane-damaged sperm for glycerol and DMA samples, respectively; P < 0.05). Differences in sperm motilities (total and progressive motility) and velocities (path velocity, straight-line velocity, curvilinear velocity) were observed between the 2 cryoprotectant treatments once the glycerol had been removed from those samples cryopreserved with glycerol, of which the glycerol samples had significantly more motile sperm and higher velocities (P < 0.05). The fertility of the samples frozen using the 2 cryoprotectants was tested using a single insemination (intravaginal or intramagnal) of 200 x 106 sperm and the fertility (number of live embryos) was evaluated over 18 d. Overall, the intravaginal inseminations had lower fertility than the intramagnal inseminations (P < 0.05). In the intravaginal inseminations, the sperm cryopreserved using DMA resulted in lower fertility, but there were no differences in fertility in the intramagnal inseminations due to cryoprotectant (P > 0.05). These results indicate that reasonable postthaw sperm quality and fertility can be derived using cryopreserved rooster semen. By utilizing this information, estimations can be made for storing sufficient material for line or breed, or both, recreation programs.
Key Words: rooster • spermatozoa • cryopreservation • deglycerolization • artificial insemination
1 Agassiz Research Centre Publication 760.
2 Mention of trade name, proprietary products, or specified equipment does not constitute a guarantee by the USDA and does not imply approval to the exclusion of other products that may be suitable. The USDA, Agricultural Research Service, Northern Plains Area, is an equal opportunity-affirmative action employer. All agency services are available without discrimination.
3 All methods used were approved by the Animal Care Committees of the USDA-Agricultural Research Service Avian Disease and Oncology Laboratory and the Agassiz Research Centre and followed principles described by the Canadian Council of Animal Care for the research performed at these facilities.
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