Comparison of sperm selection techniques in donkeys: motile subpopulations from a practical point of view

Abstract

The aim of this study was to compare the postthaw distribution of motile sperm subpopulations, following simple or colloid centrifugation. A new analysis was used to evaluate the available number of sperm from each subpopulation after each centrifugation protocol. Frozen/thawed semen samples were divided into the following after-thawing treatments: uncentrifuged control (UDC), sperm washing (SW) and two colloid centrifugation procedures (Equipure, SLC-E, and Androcoll, SLC-A). Percentage of total and progressive motility (TM and PM), as well as sperm motility kinematics, distribution of motile sperm subpopulations, and recovery rates, were statistically compared among treatments. The SLC treatments showed higher (P < 0.001) TM and PM than UDC and SW. Following each SLC procedure, different percentages of the subpopulation with the most vigorous and progressive sperm (sP4) were obtained. SLC-A recovered a larger number of sperm belonging to sP4 than SLC-E, but not significantly higher than SW. From a practical point of view, sperm washing, the standard centrifugation procedure for equine semen processing, recovered the same amount of fast and progressive sperm as colloid centrifugation, apparently the best treatment according to traditional analysis. In conclusion, samples processed by SLC have higher motility percentages than SW and UDC but, after combining the available number of sperm, SLC and SW techniques are equally efficient in recovering sperm from the most vigorous, fast and progressive motile subpopulation (sP4).

A New Approach of Sperm Motility Subpopulation Structure in Donkey and Horse

“…To complete the classical studies based on the comparison of the median values of each parameter as independent variables, the multivariate statistical procedures, including a reduction of dimensionality by PCA followed by clustering analysis, were developed to define sperm subpopulations (44,45). During the last years, several studies have shown the universal presence of defined subpopulation structure inside the whole sperm population in the ejaculate (20,(46)(47)(48). This fact has changed the previously established paradigm that considered the ejaculate composed of "equivalent" cells competing for reaching the oocyte fertilization.…”

Use of Androcoll-ETM to Separate Frozen-Thawed Llama Sperm From Seminal Plasma and Diluent

“…A significant decrease in all sperm parameters (total and oscillatory motility, viability, sperm membrane function, and acrosome integrity) was observed after thawing all samples. Several authors have observed the same results, not only in llamas and alpaca ( 20 , 24 , 34 , 38 – 40 ) but also in thawed semen samples from other species [horse: ( 41 ); ram: ( 42 , 43 ); dog: ( 28 ); bull: ( 44 ); donkey: ( 29 , 30 ); brown bear: ( 45 )]. The main concern in SACs is the greater loss in sperm quality after thawing (between 65 and 85%) compared to other species, in which the expected decrease in sperm survival is around 50% of the initial value ( 46 ).…”

“…Regarding the colloid:semen relation, a similar proportion to the one used in Experiment 2 was tested in stallions (25,26), while in studies carried out in brown bears, the authors used columns with similar volumes to those used in our experiment 1 (31). On the other hand, in dogs and donkeys, higher volumes of semen were placed over the colloid compared to our study (27)(28)(29)(30). Finally, concerning centrifugation velocities, in the species where sperm quality improved after the use of Androcoll columns, lower centrifugation speeds were used (300 g 20 min and 600 g 10 min) compared to the one applied in the present study (800 g).…”

“…Notably, in the present study and the one by Giuliano et al ( 47 ), the use of AE and Percoll®, respectively, did not accomplish sperm selection, in consequence, the seminal quality of llama cryopreserved samples did not improve. In other species, greater percentages of motile, viable, and morphologically normal sperm with intact acrosomes and DNA have been reported in frozen-thawed samples treated with Androcoll [horse: ( 25 , 26 ); dog: ( 27 , 28 ); donkey: ( 29 , 30 ); brown bear: ( 31 )]. Differences in our results could be due to the fact that these authors used a species-specific Androcoll (Androcoll-E, Androcoll-P, Androcoll-C, and Androcoll-bear).…”

“…Considering all these facts, selection of suitable frozen-thawed sperm from samples where the majority are dead could be an alternative to SAC. In other species, greater percentages of motile, viable, and morphologically normal sperm with intact acrosomes and intact DNA have been reported in frozen-thawed samples treated with Androcoll [horse: ( 25 , 26 ); dog: ( 27 , 28 ); donkey: ( 29 , 30 ); brown bear: ( 31 )]. Recently, AE allowed the separation of llama sperm from SP in raw semen samples, avoiding the use of enzymatic treatments ( 32 ).…”