91 MOTILITY (CASA) AND VIABILITY OF FROZEN SPERM AFTER DIFFERENT TIMES OF EXPOSURE AT 15°C

Abstract

One of the key factors for successful long-term cryopreservation in liquid nitrogen is maintaining the samples at -130°C or lower at all times to avoid cell damage (Barth A 1991 Proc. 10th Annu. Conv. Am. ET Assoc., 20-26). Previous data reported that exposure of semen straw to ambient temperature for more than 15 s can raise the temperature above -130°C and could reduce sperm motility by subjective evaluation (Berndston et al. 1976 Proc. 6th NAAB Tech. Conf. Artif. Insem. Reprod., 51-60). The CASA system provides an opportunity to assess multiple motility characteristics on a semen sample objectively and with high repeatability. Two experiments were designed to evaluate the effect of exposing frozen semen in 0.5-mL straws for 15, 30, or 60 s to room temperature on motility characteristics assessed by CASA and viability parameters by vital stain, HOS test, and acrosome integrity. Thirty-three ejaculates from different bulls (88% British breeds) were used for CASA evaluation, and 12 ejaculates were from other bulls (7 Bos indicus and 4 Bos taurus) were used for viability evaluation. All ejaculates were diluted using a chemically semi-defined media (Andromed, Minitub, Germany) and frozen in an automatic freezer (Digicool, IMV, France). Five frozen straws per bull were used, one for each time of exposure and one as control (0 s = 0 time). Straws were exposed to room temperature (15°C ± 1.78) for different times and then placed back into liquid nitrogen. Semen thawing was conduced in a water bath at 37°C during 1 min. Motility characteristics were evaluated by the IVOS Sperm Analyzer (Hamilton Thorne Research). Two chambers of 20 μm depth and 5 fields per chamber were analyzed (30 frames/0.5 s for each field). Six motility parameters were evaluated: % of motile sperm; % of progressive sperm; VAP (path velocity, μm s^-1); ALH (lateral amplitude, μm); BCF (beat frequency, Hz); and LIN (linearity, %). Viability characteristics were evaluated by % of live sperm (eosin-nigrosin); % positive to HOS test, and % of intact acrosome (Giemsa stain). A nonparametric AOV (Kruskal Wallis) test was used to compare variables among groups, and results are shown in Table 1. There was a reduction (P < 0.05) in the percentage of motile and progressive sperm when exposure to 15°C was longer than 30 s. The alive cells have similar motile characteristics as VAP, VCL, ALH, BCF, and LIN. The viability of spermatozoa was reduced (P < 0.05) when they were exposed to room temperature beyond 30 s. Also, a lower proportion of positive spermatozoa for HOS test was detected for exposures beyond 15 s. In conclusion, these results suggest sperm motility and viability would not be affected if straws are exposed up to 30 s to 15°C. Further study should be done regarding viability tests.

Table 1.  Motility and viability parameters of exposed frozen semen