This study aimed to characterize the sperm kinematic values with high frames per second, to define the subpopulation structure of a horse and a donkey and compare them. A total of 57 fresh semen ejaculates (26 Spanish and 16 Arabian horse breeds and 10 donkeys) were collected and subsequently analyzed for kinematic parameters using the Computer-aided sperm motility analysis ISAS^®v1.2 system and using a Spermtrack^® 10-μm depth counting chamber. Sequences were recorded at 250 frames per second, and eight kinematic parameters were automatically evaluated. All kinematic parameters showed significant differences between a donkey and a horse and between horse breeds. All ejaculates evaluated showed excellent semen motility characteristics, with significantly higher values for all kinematic parameters for donkeys compared with horses except for beat-cross frequency. Donkey sperm was faster and linear than the horse. Regarding horse breeds differences, the Spanish horse had higher average path velocity, curvilinear velocity, and beat-cross frequency compared with the Arabian horse. Spanish horse sperm was rapid, but Arab horse was more linear. The principal component analysis showed three sperm subpopulations in the ejaculate of donkeys and horses with a significantly different motility characteristic between them. The dominant subpopulation for both donkey and horse was for rapid, straight, and linear with a high beat sperm (38.2 and 41.7%, respectively), whereas the lowest subpopulation was for the slowest and non-linear sperms. This, plus slight differences in the distribution of these subpopulations between Arabian and Spanish horses, were found. In conclusion, higher frames permitted to have a new interpretation of motile subpopulations with species and breed differences. More so, future works on donkey and horse breed spermatozoa should take into account differences between breeds that may interfere and alter the real analysis performed.

The evaluation of the male fertility potential is based on the analysis of the basic spermatic characteristics of concentration, motility and morphology. Thus, the study of sperm morphology is a fundamental element in the seminal analysis, but its real meaning has been biased by the techniques used for its evaluation. These techniques involve dehydration phases and subsequent staining, which involves the production of artifacts. The aim of the study is to compare two methods for equid semen morphology evaluation, Trumorph^® using living sperm vs. eosin-nigrosine stain. A total of 49 ejaculates from stallions and donkeys were used. After semen collection and dilution, an aliquot was placed on the slide and introduced in the Trumorph^® device. Then observation was made with a 40x objective and negative phase-contrast microscope. Another aliquot was stained using eosin-nigrosine stain and viewed using 100× magnification. Well-formed sperm were observed, and different abnormalities were identified using Trumorph^®. The use of eosin-nigrosin staining method and Trumorph^® led to the same results and both techniques can be used for stallion and donkey sperm morphological analysis. However, considering the fact that Trumorph^® uses living sperm helps prevent sperm cell alteration during sample preparation. Therefore, Trumorph^® can be a good alternative to the conventional staining method, which provides a quick test on live sperm.

In order to optimize the donkey sperm motility analysis by the CASA (Computer Assisted Sperm Analysis)-Mot system, twelve ejaculates were collected from six jackasses. Capillary loaded chamber (CLC), ISAS^®D4C depths 10 and 20 µm, ISAS^®D4C Leja 20 and drop displacement chamber (DDC), Spermtrack^® (Spk) depths 10 and 20 µm were used. Sperm kinematic variables were evaluated using each chamber and a high-resolution camera capable of capturing a maximum of 500 frames/second (fps). The optimum frame rate (OFR) (defined according to curvilinear velocity—VCL) was dependent on chamber type. The highest OFR obtained was 278.46 fps by Spk20. Values for VCL, straight-line velocity (VSL), straightness (STR), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) were high in DDC and 10 µm depth. In both DDC 10 and 20 µm, the sperm velocities (VCL, VSL, VAP) and ALH values decreased significantly from the centre to the edges, while Wobble and BCF increased. No defined behavior was observed along the CLC. However, all the kinematic variables had a higher value in a highly concentrated sample, in both chamber types. In conclusion, analyzing a minimum of nine fields at 250 fps from the centre to the edges in Spk10 chamber using a dilution of 30 × 10⁶ sperm/mL offers the best choice for donkey computerised sperm motility analysis.