Extender osmolality, glycerol and egg yolk on the cryopreservation of epididymal spermatozoa for gamete banking of the Cantabric Chamois (Rupicapra pyrenaica parva).

Germplasm banking is a key technology enabling the ex-situ conservation of wild species. However, cryopreservation protocols must be tested to assure the applicability of the banked material. The objective of this study was defining a range of parameters for the composition of a semen extender for Cantabrian chamois epididymal spermatozoa (post-mortem collection). The freezing extender was based in a TES-Tris-fructose buffer, modifying its composition in three experiments: Osmolality of the buffer (320, 380 or 430 mOsm/kg, 8% glycerol, 15% egg yolk), glycerol (4 or 8%, 430 mOsm/kg, 15% egg yolk), egg yolk (10 or 15%, 430 mOsm/kg, 4% glycerol). Sperm was extended at 100 mill. spermatozoa/ml, cooled at 5 °C and frozen at -20 °C/min. Sperm quality was assessed pre and post-thawing (CASA, HOS test, abnormal forms, cytoplasmic droplets, and viability and acrosomal damage by flow cytometry). Freezability was good overall, with total motility of 65.5% ±â€¯2.4 initial and 55.8% ±â€¯2.4 post-thawing. The extenders affected the post-thaw sperm quality marginally. Whereas osmolalities and glycerol concentrations seemed not to differ, 430 mOsm/kg and 4% glycerol might be preferred. Egg yolk concentrations only differed on sperm velocity (VCL: 84.0 ±â€¯6.7 µm/s in 10% vs. 70.7 ±â€¯6.2 µm/s in 15%, P < 0.05). Our results suggest a good cryotolerance of chamois epididymal spermatozoa, with a preferred extender composition of hyperosmotic buffer, glycerol in the 4% range and lower egg yolk (10% range) than other ruminants.

Ram spermatozoa migrating through artificial mucus in vitro have reduced mitochondrial membrane potential but retain their viability.

Sperm motility in vitro is one of the most common predictors of fertility in male screening. We propose that a mucus-penetration assay can isolate a cellular subpopulation critical to reproductive success. To this end, a device was designed with three modules (sample, test and collection) and its conditions of use evaluated (length of mucus, incubation time, mucus medium, sperm concentration and position in relation to the horizontal). The number of spermatozoa migrating and the viability and acrosomal status of the spermatozoa not migrating were calculated. The second objective was to evaluate the qualitative parameters of the spermatozoa migrating in 1.6% polyacrylamide for 30min. The number of spermatozoa migrating and the sperm motility, viability and the acrosomal and mitochondrial status of three sperm populations (fresh, not migrating and migrating) were determined. A higher number of migrating spermatozoa were observed after 60min of incubation, but this situation adversely affected sperm quality. The methylcellulose-based test showed a significantly lower number of migrating spermatozoa than the polyacrylamide test. The position at an angle of 45° resulted in a higher number of migrating spermatozoa in the polyacrylamide-based test. The sperm counts for three consecutive assays indicated an acceptable repeatability of the method. The viability and acrosomal status of the migrating spermatozoa showed no significant changes with regard to the control when the device was placed at 45°, whereas these parameters showed lower values at 0°. The percentage of high mitochondrial membrane potential spermatozoa was significantly reduced in the population of migrating spermatozoa.

Seasonal changes in sperm chromatin condensation in ram (Ovis aries), Iberian red deer (Cervus elaphus hispanicus), and brown bear (Ursus arctos).

The effect of seasonality (temperate environment, Spain) on the chromatin status of ovine (Churra breed), Iberian red deer, and brown bear spermatozoa was studied. This work aims to improve genetic resource banks (GRBs) by enhancing existing knowledge of the effect of season on sperm quality. Samples were obtained by electroejaculation in Iberian red deer and brown bear and by artificial vagina in ram. We used the sperm chromatin structure assay (SCSA) to study the level of chromatin condensation of the spermatozoa in each studied period. These periods were: ram, breeding season (from September to January), nonbreeding season (from February to June), and summer (July and August); red deer, breeding season (September and October), postbreeding (November and December), and nonbreeding (the rest of the year); brown bear, prebreeding (March and April), breeding (May and June), postbreeding (July and August), and nonbreeding (September to February). Chromatin in ram was more decondensated in summer, and no differences were observed between the breeding and nonbreeding season. However, in red deer, spermatozoa obtained during the nonbreeding season showed more condensed chromatin than those obtained in the rut and postrut periods. Similarly, brown bear rendered sperm with loose chromatin in the prebreeding and breeding seasons. Less condensed chromatin in the breeding season may be related to faster epididymal transit due to enhanced spermatogenesis.