Sperm selection in cooled llama semen with egg yolk extender

Abstract

The objective of the study was to separate motile and with functional membrane integrity sperm from diluents in cooled llama semen. A total of 21 ejaculates from 7 llama males were obtained and cooled with the follow methodology. Each sample was incubated with collagenase 0.1% and then divided in two aliquots: R- diluted 1:2 with a lactose-egg yolk diluent (L-E) and Rc- centrifugated at 800 g and the pellet resuspended in L-E diluent. Both samples where them cooled at 5 ºC during 24 h. Three different methodologies were used to separate sperm from diluents; Androcoll-E-Large, Percoll® (45%, 45%-70%, 45%-80%, 45%-90% y 60%-90%) and swim up. Motile llama sperm with lower amount of egg yolk was obtain only when using Percoll® 45%, 45%-70%, 45%-80%. After this the remaining samples (14 ejaculates) where processed with the following methods. Raw semen was cooled with seminal plasma (R) and without it (Rc), with the following final treatments; Percoll® 45/70 (R70), 45/80 (R80) and Percoll® 45/70 (Rc70) y 45/80 (Rc80). When sperm variables showed statistical normality results were analyzed with a factorial design, if not a Friedman test was used. The Percoll® Rc80 was the one that separated higher motile and alive sperm free from the diluent compared with the other treatments evaluated. To conclude, this protocol can be used to process cooled llama sperm to apply in assisted reproduction biotechnologies.

Keywords

• cooled semen
• sperm selection
• llama