Optimization of the Thawing Protocol for Iberian Boar Sperm

Abstract

Thawing protocols have been barely studied, and their modifications may lead to a substantial improvement in post-thawing sperm quality, which could be of great relevance to existing sperm banks, such as those for Iberian pig breeds with varieties in danger of extinction. For that, the study aimed to evaluate different thawing rates and to evaluate modifications in the composition of the thawing extender (basic pH to 8–8.2, incorporation of cyclodextrins loaded with cholesterol [CLC] and the incorporation of ion chelators [EDTA and EGTA]). After thawing, overall sperm motility and kinematic parameters, acrosome status and sperm membrane integrity were evaluated. The most optimal results were obtained with the thawing rate reaching 70 °C for 8 s with the inclusion of 12.5 mg of CLC/500 × 106 spermatozoa in the thawing extender, which showed an improvement compared to the control at 70 °C. In conclusion, to adapt the thawing conditions may be relevant, especially for endangered species or breeds such as some varieties of Iberian pig, since this process could also be used in samples cryopreserved in gene banks.

Evaluation of Different Thawing Protocols on Iberian Boar Sperm Preserved for 10 Years at Different Liquid Nitrogen Levels

“…Sperm plasma membrane integrity was evaluated using phase contrast microscopy (Nikon Eclipse E400, Tokyo, Japan) coupled with a fluorescence system (Nikon C-SHG1) equipped with a mercury lamp (100 W) and a Nikon filter (510/590 nm), following a previous technique [ 15 ]. An aliquot was diluted with BTS to 30 × 10 6 sperm/mL.…”

“…Briefly, samples were fixed in 2% glutaraldehyde, and a minimum of 200 acrosomes were examined per sample. The damage to the acrosome cap was classified by the scoring system previously reported by [ 15 ], and the data are presented in the results as a percentage of spermatozoa with a normal acrosomal ridge (NAR, %)…”

“…The routine procedure involves plunging a straw in a circulating water bath at 37 °C for 20 s and dilution 1:2 in Beltsville Thawing Solution (BTS). A previous study tested modifications in the composition of thawing extenders and in thawing rates that lead to enhancements in post-thawing quality, improving the results from conventional thawing protocols [ 15 ].…”

Cryopreservation of Semen in Domestic Animals: A Review of Current Challenges, Applications, and Prospective Strategies

“…From a fundamental cryobiology point of view, samples that are cooled quickly must be warmed quickly, because samples that are cooled in a quasi equilibrium or nonequilibrium manner will be subject to deleterious recrystallization upon warming, so warming rates must be sufficient to outrun this process [ 174 ]. Semen is no exception [ 175 ]. Hernández found that more-rapid warming resulted in increased motion parameters in boar sperm, with significant interactions among warming rates and glycerol concentrations [ 176 ].…”

“…However, rapid warming rates are more difficult to achieve. Hernández et al, and subsequently Tomás-Almenar and de Mercado, used a 70 °C water bath to achieve this and noted sensitivity on the scale of seconds between successful warming and killing sperm [ 175 , 176 ].…”