Evaluation of the cryoprotective effect of seminal plasma on llama ( Lama glama) spermatozoa

Abstract

In South American camelids, sperm survival is low after thawing and poor results are obtained when artificial insemination is performed with cryopreserved semen. The aim of this study was to evaluate the effect of different percentages (10% and 50%) of seminal plasma added prior to the process of cryopreservation and also to evaluate the absence of seminal plasma on llama sperm survival after freezing and thawing. A total of 15 ejaculates from five adult llama males (n = 5; r = 3) were evaluated. A significant decrease in sperm motility, viability, membrane function and intact acrosomes was observed in thawed samples (0%, 10% and 50%) when compared to raw semen. Neither morphology nor chromatin condensation was altered in all thawed samples (p > 0.05), but a significant increase (p < 0.05) in the percentage of spermatozoa with fragmented DNA was observed after thawing all samples compared to raw semen. Higher percentages of total and progressive sperm motility were observed when 0% and 10% of seminal plasma were used compared to 50%. However, no statistical differences were established for sperm viability, membrane function, morphology, acrosome status and DNA quality between thawed treatments. To conclude, neither of the percentages of seminal plasma used showed superiority nor cryoprotective effect on llama sperm survival.

Incubation of frozen‐thawed llama sperm with seminal plasma

“…Similarly, Sabatini et al, (2014) evaluated the effect of the addition of SP on the quality of DNA (fragmentation) in donkey thawed semen samples, without observing a significant impact at time zero using 0%, 5% and 20% SP. With regard to sperm morphology, no effect of the treatments (0%, 10% and 50% SP) was observed and similar results were observed in llama thawed spermatozoa cryopreserved with different concentrations of SP (0%, 10% and 50%) (Fumuso et al, 2019).…”

“…Despite the multiple roles that have been attributed to SP in SACs reproductive physiology (Adams & Ratto, 2013; Apichela et al, 2014; Berland et al, 2016; Ratto, Huanca, Singh, & Adams, 2005; Silva et al, 2015; Tribulo, Bogle, Mapletoft, & Adams, 2015), there are very few studies that evaluate the effect of SP on cryopreserved spermatozoa in these species (Carretero, Fumuso, et al, 2017; Carretero, Giuliano, et al, 2017; Fumuso et al, 2019). Due to the rheological characteristics of SACs ejaculates, effective procedures to remove SP from the ejaculates are low‐speed centrifugation of enzyme‐treated samples (Giuliano et al, 2010) or high‐speed centrifugation (Fumuso et al, 2018; Kershaw‐Young & Maxwell, 2011).…”

“…Another explanation for the almost null OM could be the type of extender used for cryopreservation. The extender contains egg yolk, a component that has been proposed to be involved in the induction of PM in llama spermatozoa, and it was hypothesised that this could be by interfering with the action of factors present in SP (Fumuso et al, 2019; Giuliano SM, personal observation).…”

“…As mentioned above, having observed that a certain percentage of SP is necessary in the medium to avoid spontaneous acrosome reactions in raw semen it was interesting to explore the effect the addition of SP could have on post‐thaw llama sperm physiology. The addition of different percentages of SP to the media prior to freezing has been studied (Carretero, Fumuso, et al, 2017; Fumuso et al, 2019), but the effect of adding SP after thawing has not. For all the above reasons and with the aim of improving the use of this biotechnology in SACs, this study was designed to evaluate, over time, the effect of adding different percentages of SP (10% and 50%) to frozen‐thawed llama spermatozoa, to study its effect on sperm cryosurvival.…”

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

“…Regarding sperm morphology, in both experiments samples treated with AE showed a decrease in the percentage of spermatozoa with cytoplasmic droplets as compared to the raw semen. The same changes in sperm morphology have been observed in chilled and frozen semen that were not processed using colloids (Carretero, 2012; Fumuso et al., 2019), leading one to question whether a real selection occurred or if centrifugation or some other process provoked the loss of the cytoplasmic droplets to the surrounding medium. In the second experiment, a decrease in the percentage of spermatozoa with abnormal mid‐pieces was observed at both centrifugation speeds (600 g and 1,000 g ).…”