Cryopreservation of llama semen using a combination of permeable cryoprotectants

Abstract

Semen cryopreservation is not available for massive use in South American Camelids (SACs) due to the lack of an efficient protocol and the low pregnancy rates obtained with artificial insemination (AI). The use of a single cryoprotectant (CP) is commonly used in SACs frozen semen. The objective of the study was to evaluate the combined cryoprotective capacity of two permeable CPs at different stages of the cryopreservation protocol in llama semen. Sixteen ejaculates from 4 llama males were analysed, and sperm quality was assayed in raw semen, at 5°C, after equilibration of samples with the CPs and when samples were thawed. The following CPs and combination were used: 6% glycerol (GL), 6% dimethylformamide (DMF) and the combination of both CPs: 3% GL and 3% DMF. A Kruskal-Wallis test and an experimental factorial design, considering one factor with four levels (raw semen, 6% GL, 6% DMF and GL/DMF), were used. Total sperm motility and live sperm with intact acrosomes remained unchanged after equilibration of samples (p > .05). A significant decrease in the percentage of functional membrane, motile and live sperm with intact acrosomes was observed when samples were thawed (GL, DMF and GL/DMF). Nevertheless, the cryopreservation protocols used preserved sperm DNA quality; thus, sperm chromatin condensation and DNA fragmentation were unaffected (p > .05) when GL, DMF and GL/DMF were used. To conclude, no superiority was found between the use of a single or a combination of permeable cryoprotectants to freeze llama semen.

Use of dimethylformamide to cryopreserve alpaca semen previously incubated with collagenase

“…Even though, Fumuso et al. (2021) study did not detect a compromise in live sperm with intact acrosomes on equilibrated samples (1 hr at 5°C with 6% DMF), they found similar results as ours in frozen‐thawed llama samples with a significant decrease in this parameter after cryopreservation. Also, Santiani Acosta et al.…”

“…Nevertheless, due to the lack of success obtaining pregnancies with frozen‐thawed semen in SACs, this biotechnology is not commercially viable in these species (Aller et al., 2003; Bravo et al., 2000; Fumuso et al., 2018; Stuart et al., 2019). This situation leads to conclude that SACs sperm are very susceptible to cryopreservation and, although a clear descent in frozen‐thawed sperm motility have been reported in these species (Carretero et al., 2015; Contreras et al, 2020, Fumuso et al., 2021; Santiani et al., 2005, Santiani Acosta et al., 2013), other sperm undetectable sub‐lethal changes can occur.…”

“…Artificial insemination (AI) with cryopreserved semen is used to reproduce genetically valuable animals and to improve domestic species productivity. In South American camelids (SACs), different diluents have been used, several cryoprotectants have been tried at different concentrations and diverse cooling, and freezing rates have been tested (Carretero et al., 2015; Contreras et al., 2020; Fumuso et al., 2021; Kershaw et al., 2017; Morton et al., 2007, 2010; Santiani et al., 2005; Santiani Acosta et al., 2013; Stuart et al., 2019, amongst others). Nevertheless, due to the lack of success obtaining pregnancies with frozen‐thawed semen in SACs, this biotechnology is not commercially viable in these species (Aller et al., 2003; Bravo et al., 2000; Fumuso et al., 2018; Stuart et al., 2019).…”

“…Finally, Fumuso et al. (2021) reported no superiority between the use of a single or a combination of permeable cryoprotectants (glycerol and DMF) to freeze llama semen.…”

“…Recently, Stuart et al (2019) reported no difference in post-thaw sperm motility and viability amongst three glycerol concentrations (2%, 5% and 10%) used to frozen alpaca semen treated with papain; however, no pregnancy was observed with these samples. Finally, Fumuso et al (2021) reported no superiority between the use of a single or a combination of permeable cryoprotectants (glycerol and DMF) to freeze llama semen.…”