59 EFFECT OF STORAGE DURATION ON POST-THAW PARAMETERS OF BULL SEMEN

Abstract

It has been proposed that once good-quality sperm are collected, extended, and cryopreserved that the post-thaw quality will remain high regardless of the duration of storage. A previous study has suggested that there is no effect on post-thaw sperm motility of bovine semen stored in liquid nitrogen up to 13 years. However, no studies have reported the effect of extended storage of bovine semen in LN₂. In this study, cryopreserved semen from Angus bulls (n = 25) was utilized. Time frames were assigned to each bull based on the year of collection and cryopreservation: time frame 1 (1960–1975), time frame 2 (1976–1991), and time frame 3 (1992–2006). Due to differences in packaging methods across time periods (glass ampules and plastic straws), thawing methods utilized were different and based on the packaging type. Semen packaged in glass ampules (n = 7) were thawed in a 37°C water bath for 80 s, while those in plastic straws (n = 18) were thawed in a 37°C water bath for 30 s. Once thawed all packages were wiped dry and either scribed (glass ampule) or the end cut (plastic straw) and the semen expelled into a 1.5-mL microcentrifuge tube and maintained at 37°C for evaluation. For sperm motility, a 20-µL sample was placed onto a prewarmed microscope slide for analysis. Sperm motility parameters (total motility and progressive motility) were evaluated by 2 experienced technicians and averaged. A hemocytometer was used to determine sperm concentration in a final dilution of 1 : 100. An eosin/nigrosin stain was used to determine sperm morphology. Parameters were analyzed using one-way ANOVA. There was no difference in sperm concentration between time frames 1 and 2 (53 × 10⁶ mL^–1 and 59 × 10⁶ mL^–1, respectively) or time frames 1 and 3 (53 × 10⁶ mL^–1 and 37 × 10⁶ mL^–1, respectively). However, time frame 2 exhibited a higher (P < 0.05) sperm concentration compared with time frame 3 (59 × 10⁶ mL^–1 v. 37 × 10⁶ mL^–1, respectively). There were no differences among time frames 1, 2, or 3 for total (42, 51, and 55%, respectively) and progressive sperm motility (29, 38, and 41%, respectively). Similarly, there were no differences among time frames 1, 2, and 3 for percent normal sperm (80 ± 3.6, 76 ± 5.0, and 71 ± 4.0%, respectively) and abnormal sperm (19 ± 3.6, 23 ± 5.0, and 28 ± 4.0%, respectively). Furthermore, time frames 1, 2, and 3 did not exhibit a difference in primary (9 ± 2.5, 7 ± 2.6, and 6 ± 1.0%, respectively), secondary (2 ± 0.8, 5 ± 1.4, 6 ± 2.0%, respectively), or tertiary abnormalities (7 ± 2.4, 10 ± 2.7, and 16 ± 2.7%, respectively). These data suggest that once good-quality bovine sperm is cryopreserved, the duration of storage (up to 43 years) has no effect on Angus bull post-thaw semen parameters.