A novel experimental design for boar sperm cryopreservation

Lay Summary Freezing of sperm is a routine in many livestock species, but not in pigs, because it results in lower conception in small litter sizes. This study aims to identify factors in pig sperm freezing protocols that affect sperm quality using an efficient and relatively rapid approach. The Plackett-Burman experimental design is one such method used to rapidly screen factors and was the method used for this study. Using commercial pig semen, freezing factors were tested to determine their impact on sperm quality after freezing. Through this method, glycerol concentration (prevents cell damage), cooling rate (speed used to cool sperm to refrigerator temperature), antioxidants, and straw size (straws in which sperm are packaged for freezing) were identified as highly influential factors that affect sperm health. Extender type (the chemical base used to freeze sperm), starting osmolality (the concentration of salts and sugars in a solution), sodium dodecyl sulfate addition (detergent), and stepwise addition of glycerol (to prevent sperm damage) were also influential for some but not all sperm health measures tested. Using the Plackett-Burman design, it can be concluded that four of the nine factors warrant detailed investigation in the development of boar sperm freezing extenders. Cryopreservation of sperm is a routine technology in many livestock species, but not in swine. Frozen sperm must result in acceptable conception rates and produce 11 to 12 piglets/litter to be competitive with traditional cooled semen. The development of an extender that results in high post-thaw sperm quality and acceptable litter size requires the identification of factors that markedly affect post-thaw semen quality. The present study aims to first identify factors in boar sperm cryopreservation that significantly affect post-thaw sperm quality using an efficient, cost-effective, and relatively rapid approach. The Plackett-Burman experimental design is ideal for the screening of factors at their extreme, greatly reducing the amount of time and resources needed for a follow-up, full factorial design. Using commercial semen, a 9-factor, 12-run Plackett-Burman design was used on 10 boars split between 12 treatments. Through this method, glycerol concentration, cooling rate, antioxidant supplementation with GameteGuard (Membrane Protective Technologies, Inc. Fort Collins, CO), and straw size were identified as highly influential factors that affect post-thaw sperm quality. Extender type, starting osmolality, sodium dodecyl sulfate addition, and stepwise addition of glycerol were also influential for some but not all post-thaw sperm parameters (P < 0.05). Equilibration time in the straws before freezing was determined to have no impact on post-thaw sperm quality parameters. Using the Plackett-Burman design, it can be concluded that four of the nine factors warrant detailed investigation in full factorial experiments in the development of boar sperm cryopreservation extenders. Using the Plackett-Burman design, we identified four out of nine factors in the boar sperm cryopreservation procedure that affect cryosurvival. This aids in the identification of factors that should receive prioritized focus in full factorial experiments when developing novel cryopreservation extenders. Aside from the finding in this study, we feel this experimental design may be useful in other areas of animal science as a cost-effective and timely approach to screen variables.

Automated summary

This study identified important factors that affect post-thaw sperm quality in boar sperm cryopreservation using the efficient Plackett-Burman experimental design method, including glycerol concentration, cooling rate, antioxidant supplementation, and straw size.