New protocol to separate llama sperm without enzymatic treatment using Androcoll‐E^™

Abstract

The objective of this study was to design a protocol to separate spermatozoa from seminal plasma of raw llama semen without prior enzymatic treatment using a single‐layer centrifugation with Androcoll‐E™ (AE). Two experiments were performed: (a) samples were divided into three aliquots (1 ml) that were deposited on the top of 4, 5 or 6 ml of AE and were centrifuged at 800g for 20 min and (b) samples were divided into two aliquots (1 ml) that were deposited on the top of 4 ml of AE and were centrifuged at 600g or 1,000g for 20 min. Columns of 5 and 6 ml of AE showed a total sperm motility (TM) significantly lower, while in the 4 ml column, this parameter was not different from the TM of samples before the AE treatment. The percentage of spermatozoa with intact and functional membranes, normal morphology and intact acrosomes, as well as the percentages of sperm with highly condensed chromatin, was conserved (p ˃ .05) in the three column heights and in the two centrifugation speeds evaluated. In conclusion, the different column heights of AE (4, 5 and 6 ml) and the different centrifugation speeds used (600, 800 and 1,000g) allow separating spermatozoa of raw llama semen without enzymatic treatment, preserving the evaluated sperm characteristics. However, of all the studied treatments, centrifugation in the 4 ml column of AE at 800g would be the method of choice to process raw llama semen samples destined for reproductive biotechnologies.

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

“…In contrast, Giuliano et al (47) observed that the use of AE colloid was unable to separate cooled llama sperm from the egg yolk extender used to preserve the samples. Bertuzzi et al (32), on the other hand, successfully achieved the separation of llama sperm from SP in raw semen without previous enzymatic treatment using AE, but similarly to the present study, obtained low sperm recovery. In our opinion, it would be possible to obtain greater numbers of recovered sperm by increasing the number of columns used and process the whole ejaculate (and not only an aliquot).…”

“…In this study, a small volume of sample (100 µl) was placed over the AE column, but considering that the total volume of llama frozen-thawed semen varies between 0.25 and 5 ml, multiple columns could be used to process the whole frozen ejaculate, thus increasing the efficiency of the protocol in terms of sperm recovery. Furthermore, it has been suggested that the initial presence of thread formation in llama ejaculates would seem to play an essential role in the percentage of sperm recovery after the AE treatment (32). These authors observed a lower sperm recovery in samples that showed thread formation compared to the ones that did not.…”

“…We decided that testing a higher centrifugation speed was a good strategy because, taking into account the rheological characteristics of SAC semen (high structural viscosity and thread formation) and the fact that we did not treat the samples enzymatically prior to centrifugation through Androcoll, we considered that a higher speed could counteract the possible interference of these seminal characteristics with the separation of sperm from SP. Besides, in a previous experiment, we evaluated a lower centrifugation speed (600 g) to treat raw llama semen with AE without improving the quality of the sperm obtained and with less sperm recovery compared to 800 g (32). These results would seem to indicate that not only is the use of a species-specific Androcoll important when processing the samples but, for the protocol be efficient, the colloid:semen ratio and the centrifugation speed used perhaps also need to be varied according to the species.…”

“…Recently, AE allowed the Abbreviations: SACs, South American camelids; SP, seminal plasma; AE, Androcoll-E TM . separation of llama sperm from SP in raw semen samples, avoiding the use of enzymatic treatments (32). However, this colloid has not yet been used with llama frozen-thawed semen samples.…”

Impact of papain on the treatment of raw diluted dromedary semen

“…Llama SP influences the motility pattern of spermatozoa, but not by mechanical action but by biochemical and/or molecular signals. For example, oscillatory sperm motility was observed in samples without thread formation (Bertuzzi et al., 2020). In turn, the addition of egg yolk to cryopreservation diluents induces progressive sperm motility in samples with thread formation (Fumuso et al., 2019).…”