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United States Department of Agriculture

Agricultural Research Service

Research Project: NATIONAL ANIMAL GERMPLASM PROGRAM (NAGP)

Location: Plant And Animal Genetic Resources Preservation Research Unit

Title: Evaluation of Glycerol Removal Techniques, Cryoprotectants, and Insemination Methods for Cryopreserving Rooster Sperm with Implications for Breed and/or Line Regeneration

Authors
item Purdy, Phil
item Song, Y - AGRICULTURE & AGRIFOODS
item Silversides, F - AGRICULTURE & AGRIFOODS
item Blackburn, Harvey

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 19, 2009
Publication Date: October 1, 2009
Citation: Purdy, P.H., Song, Y., Silversides, F.G., Blackburn, H.D. 2009. Evaluation of Glycerol Removal Techniques, Cryoprotectants, and Insemination Methods for Cryopreserving Rooster Sperm with Implications for Breed and/or Line Regeneration. Poultry Science. 88:2184-2191

Interpretive Summary: 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, semen from roosters was collected and cryopreserved using Lake’s diluent containing glycerol and glycerol was removed post-thaw using an Accudenz column, or by dilution. The post-thaw sperm motilities, plasma membrane integrity and concentration were determined before and after glycerol removal. Line differences in post-thaw sperm concentration, total motility and progressive motility were observed prior to glycerol removal. Following glycerol removal, the sperm that was centrifuged through Accudenz had greater total motility but use of the dilution method recovered more sperm per semen straw compared with the Accudenz method. In Experiment 2, rooster semen was cryopreserved using Lake’s diluent containing either dimethyl acetamide (DMA) or glycerol as the cryoprotectants. Post-thaw analysis revealed that the samples cryopreserved with glycerol survived freezing better, determined by motility and membrane quality analyses. Differences in sperm motilities were observed between the two cryoprotectant treatments once the glycerol had been removed from those samples cryopreserved with glycerol. The fertility of the samples frozen using the two cryoprotectants was tested using a single insemination (intravaginal or intramagnal) and the fertility (number of live embryos) was evaluated over 18 days. Overall the intravaginal inseminations had lower fertility than the intramagnal inseminations. 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. These results indicate that reasonable post-thaw sperm quality and fertility can be derived using cryopreserved rooster semen. By utilizing this information, predictions can be made for storing sufficient material for line and/or breed recreation programs.

Technical Abstract: 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, semen from roosters was collected and cryopreserved using Lake’s diluent containing glycerol and glycerol was removed post-thaw using a discontinuous Accudenz column, or by step-wise dilution. The post-thaw sperm motilities, plasma membrane integrity and concentration were determined before and after deglycerolation. Line differences in post-thaw sperm concentration, total motility and progressive motility were observed prior to deglycerolation (P < 0.05). Following glycerol removal, the sperm that was centrifuged through Accudenz had greater total motility (31% vs. 27% sperm; P < 0.05) but use of the step-wise dilution method recovered more sperm (277.5 x 106 sperm) per semen straw compared with the Accudenz method (208.8 x 106 sperm; P < 0.05). In Experiment 2, rooster semen was cryopreserved using Lake’s diluent containing either dimethyl acetamide (DMA) or glycerol as the cryoprotectants. Post-thaw analysis revealed that the samples cryopreserved with glycerol survived freezing better, determined by motility and Annexin V analyses. Differences in sperm motilities were observed between the two cryoprotectant treatments once the glycerol had been removed from those samples cryopreserved with glycerol. The fertility of the samples frozen using the two 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 days. Overall the intravaginal inseminations had lower fertility than the intramagnal inseminations. 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. These results indicate that reasonable post-thaw sperm quality and fertility can be derived using cryopreserved rooster semen. By utilizing this information, realistic predictions can be made for storing sufficient material for line and/or breed recreation programs.

Last Modified: 4/17/2014