The interest on donkey knowledge has showed an important increase in recent years. New uses, such as onotherapy, ecotourism, silviculture, or as companion animals, are increasing. On the other hand, there has been an increased global demand for donkey products, donkey milk and milk products as alternative in milk allergic infants or adults, donkey milk-based cosmetics, meat, or skin to obtain a donkey-hide gelatin (ejiao) used in the traditional Chinese medicine.

The Catalonian donkey breed has suffered a substantial reduction in its numbers, a consequence of the intense mechanization of agriculture (Aranguren et al., 2001). This could lead to high levels of inbreeding, which would only increase the risk of the breed’s extinction. The use of reproductive technologies and the setting up of gene banks can, however, contribute to the preservation of endangered species, and might help the Catalonian donkey. In this respect, the optimization of the cooling of collected semen for transport would facilitate the involvement of animals far from facilities where artificial insemination or semen cryopreservation are performed.

Cooling semen reduces the metabolic activity of the spermatozoa it contains, reduces microbial growth, and helps to extend the time over which the viability of sperm is maintained. However, cooling between 18 and 8ºC is a critical step that can lead to “cold shock”, a problem associated with damage to sperm cell plasma membranes (Amann & Pickett, 1987). Indeed, semen processing involves a number of factors that can cause such damage, including the addition of semen extender, centrifugation and storage (Aurich, 2005). Damage to the plasma membrane results in the irreversible loss of its functions. A loss of motility and fertilizing capacity can be caused, at least in part, by the peroxidation of lipids in the membrane (Papas et al., 2019). Injuries can be reduced if the cooling rate is slow (<0.3ºC/min), but there is no real consensus regarding the optimal final storage temperature for the liquid preservation of equine spermatozoa. Temperatures as low as 4-6°C have been reported to provide better environments for the maintenance of motility (Varner et al., 1989; Moran et al., 1992) and fertility (Squires et al., 1988), but some authors (Province et al., 1985) indicate temperatures of 15 or even 20ºC to better maintain motility and fertility (if storage is no longer than 12 h).

The centrifugation of equine semen is necessary to limit the harmful effects of seminal plasma on sperm motility during storage (Miró & Papas, 2018). It is also necessary for the addition of cryoprotectants and the adjustment of sperm concentrations before freezing (Vidament et al., 2000). Centrifugation has to be performed with great care since damage can result from the mechanical forces induced by the close packing of the spermatozoa. This can be manifested as structural damage of the sperm acrosome and an important loss of motility and enzymatic activity (Matás et al., 2007). Some studies have assessed the use of different extenders, storage temperatures and the elimination of seminal plasma by centrifugation as means of better preserving donkey semen (Mello et al., 2000; Cottorello et al., 2002; Serres et al., 2002; Rota et al., 2008; Miró et al., 2009).

The aim of the present study was to determine the effect of three storage temperatures (5, 15 and 20ºC for 2 h) with and without centrifugation on donkey sperm survival and motility, and to study the changes induced by these treatments in the different sperm subpopulations.

Equine semen is commonly cooled to reduce the metabolic activity of the spermatozoa, to reduce microbial growth, and to maintain the viability of the sperm for extended periods of time. However, semen processing is known to damage the plasma membrane, contributing significantly to a loss of motility and fertilizing ability. Sometimes only a short storage period is required before artificial insemination or semen freezing, but no data are available on the best shortterm storage conditions for donkey semen. Success in the use of cooled/stored semen depends on damage being avoided as far as possible since mature spermatozoa can no longer call upon repair mechanisms (Eddy & O’Brien, 1994). The decision was therefore made to evaluate sperm motility and survival after short-term storage at different temperatures followed by centrifugation (or not) at the same temperatures.

The optimum storage temperature for maintaining the motility and fertility of horse semen has previously been reported as 4-6ºC (Varner et al., 1989; Moran et al., 1992). However, other authors have reported 15 or 20ºC to be better than 5ºC for maintaining motility and fertility – although the duration of storage was between 4 and 12 h (Francl et al., 1987; Province et al., 1995). Cottorello et al. (2002) concluded that a 5ºC maintenance temperature and the use of modified Baken extender (10% egg yolk) was more appropriate than a 10ºC or 0ºC storage temperature for the preservation of donkey sperm motility in vitro, while Serres et al. (2002) reported it to be better preserved at 4ºC or 15ºC in the presence of INRA82 extender than at 20ºC. The latter authors also observed that the storage of donkey semen diluted with INRA 82 under aerobic conditions at 15ºC best maintained the integrity of the plasma membrane. The results of the present study indicate that, after 2 h of cool storage, survival, total motility and progressive motility were maintained at all the temperatures investigated, although 5ºC or 20ºC seem to be the best for maintaining the mean motion characteristics as determined by the CASA system. The discrepancies between the present results and those of other authors might be due to the use of different storage times, different diluents, aerobic conditions, or differences in the accuracy of sperm motility assessments.

Stallion semen is routinely centrifuged to reduce the harmful effect of seminal plasma and to adjust the sperm cell concentration before freezing. Certainly, an improvement in the maintenance of sperm motility in cooled stallion and indeed donkey semen has been observed after reducing the concentration of seminal plasma (Varner et al., 1987; Pruitt et al., 1993; Serres et al., 2002; Miró et al., 2009). However, Rota et al. (2008) reported higher total motility values and percentages of rapid spermatozoa in non-centrifuged donkey samples. These authors also observed that the removal of seminal plasma increased the number of spermatozoa showing high progressive motility, a consequence of the greater straightness of their tracks after 48 h of storage. In the present study, the FRESH+CENTRIFUGATION treatment had a significant, positive effect on sperm linearity and reduced the curvilinear velocity, in agreement with other authors (Rota et al., 2008).

Several authors (Cochran et al., 1984; Vidament et al., 2000; Crockett et al., 2001) have reported better post-thaw motility and fertility for stallion semen centrifuged at 22-25ºC and then cooled to 5ºC before freezing than for semen centrifuged at 5ºC. Backman et al. (2004) compared the post-thaw motility of ejaculates first cooled to 5ºC for 18 h and then centrifuged to that of ejaculates centrifuged at room temperature and then cooled to 5ºC for 18 h, and reported no differences with respect to post-thaw total and progressive motility. The present results show that after all STORAGE+CENTRIFUGATION treatments, total motility was significantly reduced, while progressive motility was not affected. Moreover, sperm viability decreased in the STORAGE 15ºC+CENTRIFUGATION treatment. The sperm in the STORAGE 5ºC+CENTRIFUGATION treatment showed mean motion values more similar to those of FRESH samples. These results are not comparable with those of other authors since, in the present study, post-thaw motility was not evaluated (Backman et al., 2004). Further studies are needed to determine the effect of storing and centrifuging donkey sperm at different temperatures on post-thaw motility and fertility.

Motile sperm subpopulations have been described in a large number of mammals, including the donkey (Abaigar et al., 1999; Rigau et al., 2001; Quintero-Moreno et al., 2003; Martínez-Pastor et al., 2005; Miró et al., 2005; Flores et al., 2008). Previous studies have established that motility changes induced by centrifugation, cooling or freezing/thawing procedures are linked to changes in specific motion variables and the percentage of sperm cells belonging to each Subpopulation (Flores et al., 2008; Miró et al., 2009). In the present study, the Subpopulation 4 structure of donkey semen was maintained after cooling and centrifugation at all the temperatures investigated. The STORAGE 15ºC+CENTRIFUGATION treatment was associated with important changes in the sperm motion characteristics, especially in Subpopulations 2 and 4. However, the STORAGE 5/20ºC+CENTRIFUGATION treatments induced only slight changes on the mean motion characteristics of each Subpopulation. No differences were seen with respect to FRESH sperm in terms of the percentage of sperm cells belonging to each Subpopulation when the semen was subjected to the STORAGE 20ºC+CENTRIFUGATION treatment; significant changes were only seen in the percentage of spermatozoa belonging to Subpopulation 4 after the STORAGE 5/15ºC+CENTRIFUGATION treatments. In conclusion, fresh donkey semen can be maintained in adequate condition for around 2 h if kept at 20ºC and then centrifuged. However, 5ºC would also appear to be an adequate storage temperature before centrifugation if, for some reason, such conditions were necessary or, possibly, if more semen transportation time were needed.