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Artificial insemination in ram is scarcely widespread comparing with other domestic species. This has been due not only to fertility results being irregular and low but also because of the difficulty in the application of enhancements such as the use of frozen-thawed sperm. Although there is a lot of information on the use of different options to improve these AI results (such as transcervical application, the use of thawed sperm, etc.) commercial programmes can be classified on two general categories: those using refrigerated semen (15 degrees C) by superficial intracervical deposition (vaginal), and, more restricted, those using thawed sperm by intrauterine deposition (laparoscopy). In the present work, we have summarized our viewpoint on three general research lines for the improvement of AI results in sheep: semen preservation, AI procedures and semen assessment. Briefly, in ram it is necessary to develop a medium term methodology of sperm refrigeration (3-5 days), which would allow the distribution of sperm doses to a widespread area. Nevertheless, it is also necessary to develop an intrauterine transcervical AI technique, which allows thawed semen to be applied by vaginal insemination. Besides, the low predictive value of classic assessment techniques limits the ability to adjust the number of spermatozoa per dose according to its actual fertility.

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We have carried out a study on the effect of postmortem time (PT) in some characteristics of epididymal sperm salvaged from hunted Iberian red deer and roe deer. Testis were collected, identified, refrigerated down to 5 degrees C, and sent to our laboratory by the wardens of the hunting reserves. This way, samples were delivered at different times postmortem. Sperm were extracted from the cauda epididymis by means of cuts. Analyzed parameters were: osmolality, pH, motility-both subjectively and with CASA, HOS test reactivity, acrosomal status and viability (assessed with propidium iodide). Osmolality and pH rose with prolonged postmortem time, possibly due to tissue decomposition. Most sperm quality parameters negatively correlated with PT. Besides, when comparing PT classes (groups of 24 h for red deer and 30 h for roe deer), we could appreciate that motility was more affected by PT than other quality variables. Progressive motility was especially impaired. We also classified the samples in high, medium and low quality for each PT group (considering progressive motility, intact acrosomes and reactivity to the HOS test), and it was clear that after 2 days the number of high quality samples was testimonial, and after several days, we almost found only low quality samples. In conclusion, epididymal sperm from Iberian red deer and roe deer undergo a decrease of quality with PT, but it could stay acceptable within many hours postmortem. There are implications for wildlife conservation programs, as epididymal sperm is a good source of germplasm. If valuable animals die and it is not possible to process their sperm immediately, it may still be possible to obtain viable spermatozoa many hours later.

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 optimization of cryopreservation extenders is a fundamental issue for adequately performing germplasm banking on wild species. We have tested two glycerol concentrations (4 and 8%), and three extender osmolalities (320, 380 and 430 mOsm/kg; before adding cryoprotectants), for cryopreservation of epididymal and ejaculated sperm samples from Iberian red deer. All the extenders were based on Tes-Tris and fructose (for osmolality adjustment), and complemented with 20% egg yolk. Epididymal and ejaculated sperm samples were obtained from the cauda epididymis (post-mortem) and using electroejaculation, respectively. Samples were diluted 1:1 with each extender and equilibrated for 2 h at 5 degrees C. Then, they were diluted down to 100x10(6) sperm/mL and frozen at -20 degrees C/min. Post-thawed samples were assessed for motility (CASA), HOS test, proportion of swollen (osmotically challenged) cells in the untreated sample, viability and acrosomal status. For epididymal samples, 8% glycerol rendered a slightly higher proportion of intact acrosomes on viable spermatozoa than 4%; regarding extender osmolality, 380 and 430 mOsm/kg rendered higher motility results, and the 430 mOsm/kg yielded the lowest proportion of swollen spermatozoa. For ejaculated samples, 4% glycerol yielded more viable spermatozoa than 8%; for extender osmolality, 320 mOsm/kg rendered the highest percentages of progressively motile and viable spermatozoa, although 380 mOsm/kg extender was not significantly different. These results show that sample source influences extender suitability, and that extenders should be isoosmotic or rather slightly hyperosmotic. Future studies should test multiple glycerol concentrations and extender osmolalities in order to adjust them to these kinds of sample.