Evaluation of glycerol removal techniques, cryoprotectants, and insemination methods for cryopreserving rooster sperm with implications of regeneration of breed or line or both

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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 × 106) compared with the Accudenz method (239.2 × 106 sperm; P < 0.05; range across 6 lines of 165.7 to 581.0 × 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 × 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.