Modelling for the inheritance of multiple births and fertility in endangered equids: Determining risk factors and genetic parameters in donkeys (Equus asinus).

Multiple births or twinning in equids are dangerous, undesirable situations that compromise the life of the dam and resulting offspring. However, embryo vitrification and freezing techniques take advantage of individuals whose multiple ovulations allow flushing more fertilised embryos from the oviduct to be collected, increasing the productivity and profitability of reproductive techniques. Embryo preservation is especially important in highly endangered populations such as certain donkey (Equus asinus) breeds; for which conventional reproductive techniques have previously been deemed inefficient. For instance, becoming an effective alternative to artificial insemination with frozen semen to preserve the individuals' genetic material. The objective of this study was to examine the historical foaling records of Andalusian donkeys to estimate prevalence, risk factors, phenotypic and genetic parameters for multiple births, assessing the cumulative foal number born per animal, maximum foal number per birth and multiple birth number per animal. We designed a Bayesian General Animal Mixed Model with single records considering the 'fixed' effects of birth year, birth season, birth month, sex, farm, location, and husbandry system. Age was considered and included as a linear and quadratic covariate. Gibbs sampling reported heritability estimates ranging from 0.18 ±â€¯0.101 to 0.24 ±â€¯0.078. Genetic and phenotypic correlations ranged from 0.496 ±â€¯0.298 to 0.846 ±â€¯0.152 and 0.206 ±â€¯0.063 to 0.607 ±â€¯0.054, respectively. Predicted breeding values obtained enable the potential selection against/for these traits, offering a new perspective for donkey breeding and conservation.

Effect of different olive oil-derived antioxidants (hydroxytyrosol and 3,4-dihydroxyphenylglycol) on the quality of frozen-thawed ram sperm.

The aim of the present study was to evaluate the effect of the addition of different concentrations of two olive oil-derived antioxidants, hydroxytyrosol (3,4-dihydroxyphenylethanol, HT) and 3,4-dihydroxyphenylglycol (DHPG), on ovine semen during the freezing-thawing process. Sperm was collected, pooled and diluted with commercial extenders and then divided into aliquots supplemented with different concentrations (10 µg/ml, 30 µg/ml, 50 µg/ml and 70 µg/ml) of HT, DHPG and a mixture (MIX) of both antioxidants. A control group, without antioxidant, was also prepared. Sperm motility, viability, acrosome integrity, mitochondrial membrane potential and lipid peroxidation (LPO) were assessed. The results showed that frozen-thawed ram spermatozoa exhibited lower values for motility, membrane integrity, acrosome and mitochondrial membrane potential than fresh samples (P ≤ 0.01). However, when antioxidants were added, thawed spermatozoa exhibited relatively low LPO, recording values similar to fresh spermatozoa; by contrast, the control group of frozen-thawed spermatozoa without antioxidants exhibited significantly higher LPO (P ≤ 0.01). The addition of a HT+DHPG mixture (MIX) had a negative impact on sperm membrane and acrosome integrity, suggesting that a pure antioxidant supplementation has the potential to offer superior results. In conclusion, HT and DHPG exhibited a positive effect on the frozen-thawed spermatozoa inasmuch as they reduced the LPO. These olive oil-derived antioxidants have the potential to improve frozen-thawed sperm quality, although further studies should be carried out to analyse the antioxidant effect at different times after thawing.

Short-term tolerance of equine spermatozoa to various abiotic factors.

The aim of this study was to determine the effects of various abiotic factors, such as light, physical stress (pipetting) and thermal shock, on the quality of fresh and cooled equine sperm. In experiment I, four sperm aliquots were subjected to different light exposures: (i) protected control samples (CTRL), (ii) exposed to UV light at 10 cm (UV10), (iii) exposed to UV light at 20 cm (UV20) and (iv) exposed to laboratory lighting (LAB). In experiment II, four semen aliquots were subjected to repeated pipetting for 0, 10, 20 and 30 times (CTRL, P10, P20 and P30, respectively). In experiment III, four semen aliquots at 15°C were subjected to thermal oscillations: (i) cooled control sperm at 15°C (CTRL), (ii) oscillations of 1.9°C/min to a temperature of 30°C (T30), (iii) oscillations of 1.4°C/min, with the temperature rapidly falling until reaching 1.3°C (T0R) and (iv) oscillations of 1.1°C/min, with the temperature slowly falling until reaching 4.2°C (T0S). The results revealed that after 30 min, UV10 and UV20 sperm samples showed significantly (p < .05) lower total and progressive motility values, sperm kinematic parameters and mitochondrial potential. After 45 min of exposure, differences were highly significant (p < .001). No significant differences (p > .05) were found for pipetting or thermal oscillations. The results suggest that, even if equine sperm samples are not handled in the laboratory under optimal conditions, fresh and cooled equine spermatozoa are able to resist the impact of various abiotic stimuli without any reduction in their quality. This study analyses the effect on normospermic samples, but future research could look at the tolerance that asthenozoospermic equine samples have to these abiotic influences.

Effect of trehalose- and sucrose-based extenders on equine sperm quality after vitrification: Preliminary results.

There has been a lack of research into equine sperm vitrification to date, but studies of other species suggest it may have significant potential. To evaluate the impact of various cryoprotectant agents (CPA) and vitrification on equine sperm quality, a controlled study was carried out. A total of 12 ejaculates were subjected to exposure to CPA and vitrification. Sperm was diluted in a range of CPA: fresh, control (BSA), sucrose (0.15M, 0.3M and 0.5M), trehalose (0.15M, 0.3M and 0.5M) and the combination of sucrose and trehalose (M1: 0.15M sucrose+0.5M trehalose; M2: 0.5M sucrose+0.15M trehalose). Sperm motility, viability, acrosome integrity and DNA fragmentation were assessed at the time of CPA exposure and after vitrification. The exposure of spermatozoa to various concentrations of sucrose and/or trehalose significantly reduced sperm motility, with lower concentrations resulting in higher sperm motility. Sperm viability and DNA fragmentation did not vary after exposure to CPA, but acrosome integrity fell significantly when spermatozoa were exposed to CPA with high osmolality. When spermatozoa were vitrified, motility values were significantly higher than those obtained during the exposure. Low concentrations of sucrose (0.15M and 0.3M) and trehalose (0.15M) showed the best progressive sperm motility. The vitrification-warmed procedure significantly reduced sperm viability and acrosome integrity, but DNA did not vary with any of CPA used. Equine sperm vitrification demonstrates a low capacity for preserving sperm motility, and extenders containing trehalose or sucrose at lower concentrations are associated with a better protective effect on sperm motility. After vitrification, acrosome and plasma membranes were severely impaired, while the DNA structure was maintained. Equine spermatozoa partially recover the motility after vitrification, but there is a need for further studies into the preservation of sperm membranes.

Storage temperature and sucrose concentrations affect ram sperm quality after vitrification.

The aim of this study was to analyse the characteristics of ram spermatozoa subjected to varying concentrations of sucrose, and the influence of storage temperature (22°C or 5°C) prior to vitrification. Ejaculated semen was diluted in TCFEY (tris-citric acid-fructose 20% egg yolk), and two aliquots were prepared at a final concentration of 100×106spz/ml, one maintained at room temperature (22°C) and the other at 5°C. In the first experiment, the toxicity of sucrose diluents on the sperm was analysed; sperm samples at different temperatures were diluted (1:2) in TCF-BSA 2% (control) or in the same extender supplemented with various sucrose concentrations (0.4M, 0.6M and 0.8M). The effects of vitrification were studied in the second experiment, where sperm samples were mixed with different concentrations of cryoprotectants (sucrose) and vitrified by being plunged directly into liquid nitrogen. In both experiments, the sperm quality was assessed by measuring motility, morphology, membrane functionality (HOST), viability, acrosome integrity and DNA fragmentation. The toxicity test revealed significant differences (p≤0.05) when different sucrose concentrations were used; lower total and progressive motility, normal morphology and membrane functionality were noted when sucrose concentration was higher, compared to the control treatment. Samples maintained at room temperature showed significantly (p≤0.05) higher viability than samples stored at 5°C. In contrast, although the quality of vitrified sperm was drastically decreased in comparison with fresh sperm, sucrose was associated with greater total motility, viability and membrane functionality. This improvement was closely linked to the temperature at which the sperm had been previously maintained, showing higher values when sperm was stored at 5°C. The main conclusions to be drawn from the study are therefore that sucrose shows promising potential as a cryoprotectant, and storing samples at 5°C is linked to improved sperm quality following vitrification.