|Abstract:||In several species, cryopreservation of sperm has played a major role in genetic advancement as well as conservation of genetic resources. However, due to variability in post-thaw quality of sperm and decreased pregnancy rates and litter sizes, the swine industry has not been able to fully utilize the potential of frozen-thawed boar sperm (FTS). Two experiments were performed to evaluate factors affecting the fertility of FTS.
Experiment one determined the effect of thawing rate of FTS and its effect on post-thaw quality. Ejaculates (n = 15) used were obtained from 13 boars and frozen from March 2010 to March 2011. Samples were frozen in 0.5 mL straws at a concentration of 1.4 x 109 sperm/mL. To test for the effects of thawing temperature and duration of thawing, sub-samples from individual straws from each ejaculate were assigned to each treatment. The treatments included straws (n = 15/treatment) thawed at 50 °C for 10, 20, and 30 s or thawed at 70°C for 5, 10, and 20 s. Following thawing, samples were evaluated for post-thaw motility (PTM), viability (VIA), and intact acrosomes (IA) at 5, 30, and 60 min. Data were analyzed using the MIXED procedures of SAS for the effects of temperature, duration of thawing, and boar. There was an effect of treatment (P < 0.0001) on PTM, VIA, and IA, and an effect of storage time (P < 0.0001) on PTM and IA. Treatment influenced PTM with thawing at 70 °C for 20 s having reduced PTM (3.4 ± 0.5%) compared to the other treatments (41.3 ± 0.7%). There was also an effect of treatment (P < 0.0001) on VIA with 70 °C for 20 s resulting in the lowest (5.8 ± 0.8%) and 70 °C for 5 s also showing lower VIA (50.2 ± 1.6%) compared to all other treatments (55.8 ± 1.3%). Treatment also influenced IA (P < 0.0001) with 70 °C for 20 s showing reduced IA (61.9 ± 1.8%) compared to the other treatments (79.9 ± 1.1%). These results indicate that when thawing boar sperm in 0.5 mL straws with the cryoprotectant used, 70 °C for 20 s and 5 s reduced sperm
fertility, while thawing at 70 °C for 10 s and 50 °C for 10 to 30 s resulted in no effect on PTM, VIA, and IA. However, it would appear that 50 °C for 20 s provided the greatest safety margin with the highest fertility measures.
Experiment two was performed to determine the effect of variation in the post-thaw motility of FTS on pregnancy rate, litter size, and fetal paternity when used in the 1st or 2nd insemination. Ejaculates from 38 boars were collected and frozen in 0.5 mL straws. Upon thawing at 50 C for 20 s, samples were classified (mean ± SEM) by motility as Poor (P, 20.2 1.1%), Moderate (M, 31.3 0.9%), or Good (G, 43.5 0.8%). In 7 replicates, mature gilts were synchronized and checked for estrus at 12 h intervals and assigned at estrus (n = 207) to receive 4.0 billion total sperm in each AI at 24 and 36 h after onset of estrus using the treatments: 1) P and M (P-M); 2) M and P (M-P); 3) G and M (G-M); and 4) M and G (M-G). For each treatment combination, a set of three boars were randomly selected within motility class for their allelic distinction with M sperm from a single boar represented across all treatments and sires used in both 1st and 2nd inseminations. Inseminations occurred at 24 and 36 h following estrus, and insemination to ovulation interval (IOI) was determined using ultrasound at 12 h intervals. Reproductive tracts were collected at 32 1.0 d following AI. Treatment did not interact with IOI (P > 0.10) and did not affect (P > 0.10) pregnancy rate (57, 67, 71, 76 7.2%, pooled SEM) or total number of fetuses (9.2, 9.1, 9.5, 10.0 0.8) for P-M, M-P, G-M, and M-G treatments, respectively. Treatment did affect (P < 0.05) the number of fetuses sired from the 1st AI (3.1, 7.2, 6.4, 6.3 ± 1.2) and 2nd AI (5.7, 2.6, 3.0, 3.6 ± 0.9) for the P-M, M-P, G-M, and M-G treatments, respectively. The IOI also affected (P < 0.05) the proportion of offspring sired by the 2nd AI (30.0, 57.7, 51.3, 18.3, 6.5%), as well as the number of fetuses sired by each AI.
The results of these studies show that thawing frozen boar sperm in 0.5 mL straws can be accomplished at 70 °C for 10 s or 50 °C for 10, 20, or 30 s, however we recommend thawing at 50 °C for 20 s to provide a window for less than optimal temperature and time of thawing. In addition, our data suggest that use of poor motility sperm in the first or second AI will sire fewer offspring than higher quality sperm, but moderate and good motility sperm optimized the number of pigs sired and offspring produced. It was also shown that the first AI sired a greater number of pigs in most cases when FTS was used in a double insemination in mature gilts.