Alpaca sperm chromatin evaluation using Toluidine Blue

Abstract

Alpacas are a domestic species of South American camelids (SAC), which are internationally appreciated because they have a high-quality fibre. Toluidine Blue (TB) is a cationic stain that binds to DNA permitting differentiation between sperm heads according to the degree of chromatin decondensation. This technique was used to evaluate sperm chromatin in alpacas, determine chromatin condensation patterns in alpacas and determine if it is possible to use dithiothreitol (DTT) as a positive control for the stain. A total of 15 ejaculates were collected from 9 alpacas using electroejaculation. TB stain was carried out according to Carretero et al. (2009) on smears of raw semen. Spermatozoa were classified according to the degree of chromatin decondensation. Descriptive statistics and analysis of variance for evaluating male differences were performed. Three patterns of staining with TB were observed in alpaca sperm: light blue (negative, without alteration of chromatin condensation), light violet (intermediate, some degree of decondensation), dark blue-violet (positive, high degree of decondensation). The percentages observed were (mean ± SD): TB positive 7.55 ± 5.22, TB intermediate 21.67 ± 6.81 and TB negative 70.78 ± 10.80. In the spermatozoa incubated with 1% DTT, 2 main categories were observed: reacted (positive with TB) and non-reacted (negative with TB). In reacted sperm, 3 sub-categories were observed according to sperm head morphology and presence of vacuoles. Variability between males was observed for positive (P = 0.03) but not for intermediate TB stained sperm. It is possible to use TB to evaluate the degree of chromatin decondensation in alpaca spermatozoa, showing the same TB patterns as in other SAC; also 1% DTT can be used as a positive control for this technique.