Cryopreservation of llama semen using a combination of permeable cryoprotectants.

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

It is not easy to separate frozen-thawed South American camelid sperm from seminal plasma (SP) and diluents to be used for in vitro embryo production. The objective of this study was to evaluate Androcoll-E™ (AE) efficiency to separate llama sperm from SP and freezing extender in frozen-thawed semen. A total of 22 ejaculates from five Lama glama males were collected using electroejaculation. After performing semen analysis (sperm motility, concentration, viability, membrane function, and acrosome integrity), samples were cryopreserved with a diluent containing lactose, ethylenediaminetetraacetic acid (EDTA), egg yolk, and 7% dimethylformamide. After thawing, samples were divided in aliquots, one of which was used as a control and the others processed by AE. Experiment 1 (12 ejaculates): 100 µl of frozen-thawed semen was placed on top of 1,000 µl AE column and centrifuged at 800 g for 10 min. Experiment 2 (10 ejaculates): two samples of 100 µl of frozen-thawed semen were placed on two columns of 500 µl AE each, and both were centrifuged at 800 g for 10 and 20 min, respectively. Pellets were resuspended in Tyrode's albumin lactate pyruvate (TALP) medium, and sperm parameters were evaluated. A significant decrease in all sperm parameters was observed in thawed samples compared to raw semen. AE allowed the separation of frozen-thawed sperm from SP and freezing extender independently from the height of the column used and time of centrifugation assayed. Although no significant differences were found between AE columns, higher sperm recovery was observed with 500 µl of AE coupled with 20 min of centrifugation. Despite the significant decrease observed in sperm motility in AE samples, no changes in sperm viability, membrane function, and acrosome integrity were observed when comparing control thawed semen with the sperm recovered after AE (p > 0.05). The use of AE columns, either 500 or 1,000 µl, allows the separation of frozen-thawed llama sperm from SP and freezing extender, preserving the viability, membrane function, and acrosome integrity. Of the protocols studied, 800 g centrifugation during 20 min using a 500 µl column of AE would be the method of choice to process frozen-thawed llama semen destined for reproductive biotechnologies.