Improvement Strategies in Ovine Artificial Insemination

P. 30-42Artificial 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°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

Decay of sperm obtained from epididymes of wild ruminants depending on postmortem time

P. 24-40We 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 °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.S

Post mortem time and season alter subpopulation characteristics of Iberian red deer epididymal sperm

P. 958-974We have studied the effect of post mortem time and season on sperm subpopulation pattern and characteristics. We used epididymal samples from free-ranging Iberian red deers harvested during the hunting season. We studied samples at different moments of the year (rut, transition period and post-rut), and at different times post mortem (up to 4 days). Sperm were extracted from the cauda epididymis and their motility was evaluated by means of a CASA system. A principal component and clustering analysis were carried out to identify subpopulations. Post mortem time caused a significant decrease in motility quality, and a general deterioration in subpopulation characteristics. We found three subpopulations the first day, and the one indicating good sperm quality decreased with post mortem time until it disappeared on the fourth day. This may indicate considerable impairment of the samples after 72 h post mortem, which could compromise their use in AI programs. With regard to season, subpopulation pattern and characteristics were better in the transition and post-rut periods. Moreover, we found one subpopulation formed by mature spermatozoa, which increased from rut to post-rut. This might be a negative fact, because samples collected after the rut may undergo hypermaturation, which possibly impairs fertility. Our results are of interest for the management of wildlife germplasm banks based on post mortem sperm recovery.S

Effect of the interval between estrus onset 4 and artificial insemination on sex ratio 5 and fertility in cattle: a field study

P. 1264-1270We have carried out a field trial in cattle to study the effect of the interval between the onset of estrus and AI on sex ratio and fertility. Data were obtained from 716 cows that had been inseminated at different times between 8 and 44 h from the visual detection of estrus. Before analyzing the data, it was grouped in three intervals considering the time between estrus onset and AI (8–18, 18–30, and ≥30 h). Our results show that the percentage of calved females (73.05%) is significantly superior for early inseminations (8–18 h), and it decreases 1.85% per hour from the onset of estrus. Delayed AIs (≥30 h) produce a significant deviation of the sex ratio towards the males (72.06%); nevertheless, fertility (percentage of successful pregnancies) diminishes significantly, from 66.19% (8–18 h) to 45.35% (≥30 h). In conclusion, variations in the interval between the onset of estrus and AI modify sex ratio. However, we must consider its effect on fertility.S

Spermatozoa recovery and post-thawing quality of brown bear ejaculates is affected for centrifugation regimes

P. 77–84Sperm cryopreservation protocols for brown bear (Ursus arctos) require the centrifugation of semen samples to increase sperm concentration and to clean urine in contaminated samples. We evaluated the effect of centrifugation regimes (time and relative centrifugal force—RCF) on the quantity of sperm recovered and the quality of post-thawed sperm. Thirteen brown bears were electroejaculated. The ejaculates were diluted 1:1 in Tris–citric acid–glucose (TCG) extender and centrifuged with different RCF/time combinations: 600×g, 1,200×g and 2,400×g, for 3, 6 or 12 min. After centrifugation, spermatozoa were diluted in TES–Tris–fructose extender with egg yolk and glycerol (final glycerol concentration of 8%) and frozen in 0.25-mL straws. In the post-thawed semen, motility was assessed by CASA, and acrosomal status (PNA-FITC), viability (SYBR-14 with propidium iodide) and chromatin status (SCSA) were determined by flow cytometry. The longest centrifugation time (12 min) significantly decreased some motility parameters. Sperm recovery significantly decreased in brown bear at 600×g. Our results suggest that brown bear spermatozoa are more sensitive to long centrifugation times than to high RCF. Centrifugation regimes showed no effects on the post-thawing chromatin status. We recommend preparing the brown bear semen for freezing by centrifugation 1,200×g or 2,400×g for 6 min, after electroejaculation and dilution 1:1 in TCG extender, since these procedures increase the spermatozoa recovery without harmful effects on the post-thawed quality of brown bear spermatozoa.S

DNA fragmentation assessment by flow cytometry and Sperm–Bos–Halomax (bright‐field microscopy and fluorescence microscopy) in bull sperm

P. 88-98The 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) ≥ 80], medium (80  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 (r2 = 0.34, p < 0.001

Undiluted or extended storage of ram epididymal spermatozoa as alternatives to refrigerating the whole epididymes

P. 76-82The effect of storage procedure at 5 °C on the quality of ram spermatozoa from the cauda epididymis was analyzed. Two strategies were tested at 0, 24, 48 and 72 h post-mortem: (1) spermatozoa held in the epididymal fluid and stored either in the cauda epididymis (In-EPID) or in vitro (Ex-EPID), (2) epididymal spermatozoa extended in three media at 320, 370 and 420 mOsm/kg (D320, D370, D420). Analyzed parameters were: osmolality, pH, motility, acrosomal status and viability. In experiment 1, osmolality of the In-EPID samples, but not in Ex-EPID, increased with post-mortem time. Motility of In-EPID spermatozoa in samples, after 24 h post-mortem, was higher compared to the Ex-EPID samples, although differences decreased at 48 and 72 h. In experiment 2, total (TM) and progressive motility (PM) were not significantly affected by storage time for D320 and In-EPID samples. However, the motility of D370 and D420 samples significantly decreased with time. TM and PM of D320 were significantly higher than D370 and D420 at 72 h. At 24 h, sperm viability was higher for In-EPID (80.7 ± 3.4%) than for the extended samples (44.8 ± 2.9%, 37.7 ± 3.9% and 48.6 ± 6.0% for D320, D370 and D420, respectively), which also decreased faster with time. At 24 h, the percentage of damaged acrosomes was low and similar for the four methods of storage, but damaged acrosomes increased with time for D320 and D370. Storing the spermatozoa in the epididymis is a good strategy for maintaining sperm quality in ram, at least for 48 h. The D320 extender preserve motility of epididymal spermatozoa but does not protect the status of the acrosome.S

Effect of basic factors of extender composition on post-thawing quality of brown bear electroejaculated spermatozoa

P. 643-651The 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 × 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 °C. Then, it was diluted down to 100 × 106 spz/mL, loaded in 0.25 mL straws and frozen at −20°C/min. After thawing (in water at 65 °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.S

Sperm concentration at freezing affects post-thaw quality and fertility of ram semen

P. 1111-1118We have investigated the effect of sperm concentration in the freezing doses 200, 400, 800, and 1600 × 106 mL−1 on the post-thaw quality and fertility of ram semen. Semen was collected from seven adult Churra rams by artificial vagina during the breeding season. The semen was diluted in an extender (TES-Tris-fructose, 20% egg yolk, and 4% glycerol), to a final concentration of 200, 400, 800, or 1600 × 106 mL−1 and frozen. Doses were analyzed post-thawing for motility (computer-assisted sperm analysis system [CASA]), viability, and acrosomal status (fluorescence probes propidium iodide [PI]/peanut agglutinin conjugated with fluorescein thiocyanate (PNA-FITC), SYBR-14/PI [Invitrogen; Barcelona, Spain] and YO-PRO-1/PI [Invitrogen; Barcelona, Spain]). Total motility and velocity were lower for 1600 × 106 mL−1 doses, while progressive motility and viability were lower both for 800 and 1600 × 106 mL−1. The proportion of viable spermatozoa showing increased membrane permeability (YO-PRO-1+) rose in 800 and 1200 × 106 mL−1. Intrauterine inseminations were performed with the 200, 400, and 800 × 106 mL−1 doses at a fixed sperm number (25 × 106 per uterine horn) in synchronized ewes. Fertility (lambing rate) was similar for semen frozen at 200 (57.5%) or 400 × 106 mL−1 (54.4%), whereas it was significantly lower for 800 × 106 mL−1 (45.5%). In conclusion, increasing sperm concentration in cryopreserved semen, at least at 800 × 106 mL−1 and more, adversely affects the postthawing quality and fertility of ram semen.S

A pilot study on post-thawing quality of Iberian red deer spermatozoa (epididymal and electroejaculated) depending on glycerol concentration and extender osmolality

P. 1165-1172The 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 °C. Then, they were diluted down to 100 × 106 sperm/mL and frozen at −20 °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.S