The aim of this study was to find the relationship between fertility (as 90-day non-return rates) and DNA fragmentation assessed by two techniques [sperm chromatin structure assay (SCSA) and Sperm-Bos-Halomax (SBH)]. Furthermore, other quality parameters were achieved (motility, morphological abnormalities, cytoplasmic droplets, viability, capacitation and acrosomal and mitochondrial status) and their correlations with fertility were analysed. Bulls were divided into three fertility groups: high [non-return rate (NRR) >or= 80], medium (80 < NRR >or= 70) and low (70 < NRR > 40). The results of this study indicate that there is a good correlation between fertility and different parameters of sperm quality (SBH and SCSA parameters, % of spermatozoa with head, neck and total abnormalities, and % of spermatozoa with proximal cytoplasmic droplets) and differences between fertility groups were observed in some of them (SBH and SCSA parameters and % of spermatozoa with head, neck and total abnormalities). In this sense, SBH parameters rendered good correlations with fertility (r = -0.42 using bright light microscope and r = -0.47 with fluorescence). Also, standard deviation of DNA fragmentation index (SD-DFI) and DFIh (cells with High DNA fragmentation index) showed good correlations with fertility (r = -0.41 and r = -0.29). No correlations were observed between SCSA and SBH parameters. A multiple regression shows that four parameters (% of proximal cytoplasmic droplets, % of intact acrosomes in total population, SD-DFI and percentage of fragmented DNA detected by bright light microscope) present a good predictive value of the fertility of sperm samples (r(2) = 0.34, p < 0.001).

The objective of this study was to identify possible specificity in the extender formulation for the cryopreservation of ram spermatozoa recovered from three origins (ejaculate, electroejaculate or epididymis), by evaluating post-thawing sperm quality and fertility. Ejaculated, electroejaculated or epididymal spermatozoa samples obtained from identical rams (8) were cryopreserved in four different extenders (TES-Tris-fructose with one of two egg yolk concentrations: 10% Y10 and 20% Y20, and with one of two glycerol rates: 4% G4 and 8% G8). Samples were analyzed before and after cryopreservation by CASA (motility) and flow cytometry (viability with SYBR-14/PI and acrosomal status with PNA/PI). Spermatozoa obtained by electroejaculation were of poorer quality after freezing/thawing, demonstrating that protocols for these samples need to be optimized. Egg yolk at 20% was more appropriate for freezing sperm from any of the sources. In general, 4% glycerol improved the quality of post-thawing samples recovered from ejaculate and electroejaculate, while 8% glycerol was more appropriate for samples recovered from the epididymis. Based on these results, an analysis of fertility was conducted. Fertility rates were similar between ewe groups inseminated with post-thawed sperm obtained from two sources: ejaculate (cryopreserved in Y20+G4), and cauda epididymis (Y20+G8), and this rate was less in the electroejaculated sample (Y20+G4).

The improvement of freezing extenders is critical when defining sperm cryopreservation protocols for wild species, in order to create germplasm banks. The aim of this study was to evaluate the effect of additives (Equex Paste and EDTA) supplementation, egg-yolk (10 and 20%) and glycerol (4 and 8%) concentrations and extender osmolality (300 and 320 mOsm/kg) on the post-thawing quality of brown bear semen. Semen was obtained from 20 adult males by electroejaculation, and centrifugated individually (600 x g for 6 min). The pellets were diluted 1:1 in the corresponding extender TTF (TES-Tris-Fructose with the aforementioned variants) and cooled to 5 degrees C. Then, it was diluted down to 100 x 10(6) spz/mL, loaded in 0.25 mL straws and frozen at -20 degrees C/min. After thawing (in water at 65 degrees C for 6s), the semen samples were assessed for motility (CASA), viability (SYBR-14 with propidium iodide), acrosomal status (PNA-FITC with propidium iodide) and mitochondrial activity (JC-1). Extender supplementation with additives rendered significantly higher results for these sperm parameters. Comparing the two percentages of egg yolk, 20% egg yolk showed the highest motility results, percentages of viable spermatozoa and viable spermatozoa with intact acrosome. No differences were detected among samples frozen using 4 or 8% glycerol. For extender osmolality, 300 mOsm/kg showed higher values of VAP, VCL, VSL, and ALH than 320 mOsm/kg. Based on the best performance of sperm motility, viability and acrosome status, we conclude that the most suitable extender to cryopreserve brown bear spermatozoa was TTF adjusted to 300 mOsm/kg, supplemented with 20% egg yolk, 4-8% glycerol, and the additives 1% Equex paste and 2% EDTA.

The objective of this study was to determine how the extender and dilution ratio used during centrifugation affect bear spermatozoa quality before and after freezingthawing. Semen was collected from 15 brown bears by electroejaculation. In experiment 1, semen was divided into five aliquots and diluted using one of the following extenders: Tris-citric-glucose (TCG), Tris-citric-glucose-3% BSA, Tris-citric-glucose-1% egg yolk or CaninePro. In experiment 2, semen was divided into five aliquots and diluted 1:1, 1:4, 1:8 or 1:16 (semen:extender) with Triscitric-glucose. In both experiments, one aliquot was left undiluted and it was used as a control. All the aliquots were centrifuged at 600×g for 6 min and frozen. Samples were analysed by post-thawing for motility (CASA) and, by flow cytometry, for viability (YO-PRO-1), acrosomal status (PNA-FITC/PI) and mitochondrial status (JC-1). CaninePro rendered the highest motility with respect to the undiluted control (total motility, 53.1% vs. 38.5%, P<0.001), and CaninePro and TCG significantly increased the percentage of viable and acrosome-intact spermatozoa (43.2 and 43.4, respectively, vs. 39.4, P<0.05). In experiment 2, dilution 1:4 yielded the highest value of total motility (78.8 vs. 67.2, P<0.05) and proportion of spermatozoa with intact membrane and acrosome (64.5 vs. 54.4, P<0.01). In general, diluting 1:4 or 1:8 brown bear semen prior to centrifugation improved the motility and acrosome status of the thawed spermatozoa.

We have tested several freezing protocols for brown bear semen, modifying the time when glycerol was added (before and after cooling to 5 °C). No differences were found among protocols, indicating a good tolerance of brown bear semen to glycerol. This finding indicates that freezing protocols for brown bear semen could be modified to fit practical solutions which would facilitate preparation of the seminal samples in the field with the addition of glycerol at ambient temperature.