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Pregnancy rates in donkeys after artificial insemination with cryopreserved semen are still low, compared to the horse species. Addition of autologous seminal plasma to frozen-thawed semen appeared to improve pregnancy rates. The aims of this study were to evaluate (1) sperm motility and plasma membrane integrity after thawing (T0) and after one and 2 h (T1 and T2) of post-thaw incubation in either 0% (SP0) or 70% (SP70) autologous seminal plasma and (2) sperm motility, plasma membrane integrity and DNA quality (%COMP-αt) after thawing (T0) and after 2 and 4 h (T2 and T4) of post-thaw incubation in either 0% (SP0), 5% (SP5) or 20% (SP20) homologous seminal plasma. In experiment 1, seminal plasma decreased total and progressive sperm motility and plasma membrane intact spermatozoa immediately after dilution and at all following time points (p < 0.05). In experiment 2, total and progressive motility did not differ between treatments immediately after dilution and between SP0 and SP5 at T2, while they were lower in both SP5 and SP20 than in SP0 at T4. Plasma membrane intact sperm cells did not differ between SP0 and SP5 and were lower in SP20 at all time points. DNA quality was not affected by treatment immediately after dilution and was significantly worse for SP20 after 4 h of incubation (p < 0.05). The post-thaw addition of seminal plasma at the tested concentrations did not improve donkey frozen semen characteristics in vitro over time., (© 2014 Blackwell Verlag GmbH.)

The aim of the study was to develop a hypo-osmotic swelling test (HOS-test) for evaluating plasma membrane integrity in donkey spermatozoa. In the first study, six different hypo-osmotic solutions (fructose or fructose/sodium citrate, 75 or 150 mOsm, or bi-distilled water at 1 : 10 semen : solution ratio, or bi-distilled water at 1 : 3) were compared. The 75 mOsm fructose solution (1 : 10) and bi-distilled water (1 : 3) were chosen for study 2, where two incubation times (5 or 45 min) were tested. Bi-distilled water showed a significantly higher proportion of plasma membrane intact spermatozoa than the fructose solution (p < 0.05), it was thus concluded that the simple incubation for 5 or 45 min at 37 degrees C of one part of semen with three parts of bi-distilled water is an applicable HOS-test in the semen analysis of donkey spermatozoa. Regression analyses showed a significant correspondence of the latest method with Sybr 14- propidium iodide staining.

In the donkey species, the application of cooled semen artificial insemination could aid the survival of endangered breeds. Fifteen ejaculates collected from three Amiata donkeys were used to evaluate the effect of three extenders on spermatozoal motility characteristics after cooling and preservation for up to 72 h. Semen was diluted at a 1:4 semen:extender ratio in INRA96, INRA82 and INRA82 added of 2% centrifuged egg yolk (INRA82-Y) and motility was evaluated by the computerized analyzer CEROS 12.1 at hours 0, 24, 48 and 72. Total motility, and rapid spermatozoa after 24, 48 and 72 h of preservation were higher in INRA82-Y than in INRA96 or INRA82, as was progressive motility after 72 h. INRA82-Y was thus used in a second study, where the effects of centrifugation and of removal of seminal plasma on cooled donkey semen were evaluated on 12 ejaculates from four males. Rapid spermatozoa after 24 and 72 h, and total motility after 72 h were better preserved in the non-centrifuged samples than when seminal plasma was removed, the contrary was true for the proportion of spermatozoa keeping their progressive motility at hour 48. In conclusion, INRA82-Y kept sperm motility characteristics during cooled storage better than INRA82 or INRA96, and removal of seminal plasma during in vitro preservation did not seemed advantageous. Further studies are needed to better understand the changes in motility patterns of donkey spermatozoa caused by seminal plasma and semen extenders, and their relation to fertility.

This study on extended, cooled stallion spermatozoa aimed to compare the ability of three extenders to maintain sperm motility during 24 h of preservation, and to describe pregnancy and foaling rates after artificial insemination (AI) of stallion spermatozoa stored and transported in the extender chosen from the in vitro study. After 6 and 24 h of preservation, motility, both subjective and evaluated by the motility analyzer (total, progressive and rapid), was lower in non-fat, dried skim milk-glucose than in both other extenders: dried skim milk-glucose added to 2% centrifuged egg yolk, and ultra high temperature treated skim milk-sugar-saline solution added to 2% centrifuged egg yolk (INRA82-Y). Rapid spermatozoa and sperm velocity parameters, after 24 h, were significantly higher in INRA82-Y. In the fertility trial, semen collected from three Maremmano stallions, diluted in INRA82-Y, and transported in a refrigerated Styrofoam box, was used to inseminate 56 mares of the same breed. Pregnancy rates after the first cycle and per breeding season were significantly higher for the 31 mares inseminated in three AI centres (54.8 and 80.6%, respectively) than for the 25 mares inseminated at the breeder's facilities (28.0 and 52.0%). Foaling rates were not significantly different between the AI centres mares (54.8%) and the other mares (44.0%). In conclusion, INRA82-Y yielded satisfactory pregnancy and foaling rates, especially when employed in the more controlled situation of an AI centre, and can therefore be included among those available for cooled stallion semen preservation.

• Concentrations of AMH was similar in pre-pubertal and post-pubertal male donkeys. • Serum AMH concentrations peaked significantly in male donkeys at puberty. • Serum AMH changes were mirrored in seminal plasma 2 months later. • AMH secretion was redirected from the blood tream to seminal plasma at puberty. • The serum AMH peak at puberty was associated with testicular width at 24 months. Sertoli cells produce anti-Müllerian hormone (AMH) and number of these cells is associated with numbers of sperm produced. The study aim was to quantify AMH concentrations in serum and seminal plasma of donkeys during puberty, and to correlate the values with those for testicular width and semen quality of sexually mature males. Blood was collected from five donkeys every second month from 4 to 24 months of age, and then once at 40 months of age. Semen was collected once monthly, from 13 to 19 and 23–25 months of age. There was quantification of AMH concentrations in serum and seminal plasma. During puberty, there was a redirection of AMH secretion from the blood stream into seminal plasma. In serum, AMH concentrations increased during puberty with a maximal concentration at 16 months and the changes were similar for seminal plasma with a maximal concentration at 18 months of age. Serum AMH concentrations from 14–20 were greater than at 12 or 22 months of age. Maximal serum AMH concentrations were associated with testicular width at 24 months (r = 0.97, P = 0.005), but not with sperm count, sperm motility or percentage of sperm with normal morphology at 42 months of age. There were no significant correlations among values for AMH concentrations in seminal plasma during puberty and values for any of the seminal variables. [ABSTRACT FROM AUTHOR]