Hypoosmotic Test Combined with Coomassie Blue Staining in Llama Sperm

Abstract

To stablish sperm fertilization capacity membrane integrity and functionality are important characteristics to determine. The objectives of the present study were: 1) to evaluate the functionality of the plasma membrane over time by the hypoosmotic test (HOS) and 2) combined the HOS test with Coomassie Blue (CB) staining to simultaneously evaluate the functionality of membrane and acrosomal integrity in raw llama sperm. A total of 12 ejaculates from 6 adult llama males were collected using electroejaculation under general anesthesia. Hypoosmotic swelling test was performed with and without fixation in paraformaldehyde and the samples were evaluated over time (0 h without fixation and 0; 2; 4; and 24 h when fixed). To perform the CB stain one aliquot of the fixed samples was used. No significant differences (p>0.05) were observed in the percentage of sperm with membrane function when comparing fixed samples analyzed over time versus the samples not fixed. Also, no statistical differences (p>0.05) were observed on membrane functionality between the two protocols used; HOS test versus HOS combined with the CB stain. It is possible to differ the moment of the evaluation of the membrane functionality of llama spermatozoa using the HOS technique from the semen collection. The use of the dual technique (HOS + CB) allow the evaluation of sperm membrane function and acrosome status in raw llama semen with only one technique that can be applied on the field.

Keywords

• acrosome
• llama
• Coomassie Blue
• HOS – test