308 GUANACO SPERM CHROMATIN EVALUATION USING TOLUIDINE BLUE

Abstract

Guanacos, a wild species of South American camelids, have a high-quality fiber with great economic potential. To evaluate reproductive aptitude in guanacos, our laboratory has developed a reliable semen collection technique using electroejaculation and has applied various methods for evaluating semen characteristics. Studies for evaluating the state of sperm chromatin have also been initiated. Toluidine blue (TB) is a cationic stain that unites with the phosphate groups in the DNA, thus permitting differentiation between sperm heads according to the degree of chromatin decondensation. The objectives of this study were to determine the TB staining patterns of guanaco sperm chromatin, establish a positive control for the stain, and evaluate the effect of collagenase on sperm chromatin condensation. Semen was collected from 4 guanacos, between 6 and 9 years old, using electroejaculation. In Experiment 1, to establish a positive control for the stain, equal quantities of 1% dithiothreitol (DTT) and raw semen were incubated at room temperature for 30 s, 1.5 min, and 3 min. After incubation, smears were made and then dried, to avoid continuing the reaction, and finally were stained with 0.02% TB. A split-plot design was used with time as the splitting factor and considering the males as a block. In Experiment 2, raw semen was divided into 2 aliquots, one diluted 4 : 1 in 0.1% collagenase in HEPES-TALP-BSA medium and the other left without enzyme. Both aliquots were incubated 4 min at 37°C and, after centrifugation to remove the enzyme, smears were made and stained with TB. Spermatozoa were classified according to the degree of chromatin decondensation. Analysis of variance was performed using the males as a blocking factor and the treatment as a fixed factor. According to the degree of chromatin decondensation, three patterns of staining with TB were observed: light blue (negative, without alteration of chromatin condensation), light violet (intermediate, some degree of decondensation), and dark violet (positive, high degree of decondensation). A significant increase (P < 0.05) of sperm with highly decondensed chromatin was observed in semen incubated for 3 min with DTT when compared to 30 s of incubation. Therefore, 3 min of incubation with DTT was chosen as the positive control for Experiment 2. No significant differences in any of the 3 patterns of TB staining were observed between semen incubated with or without 0.1% collagenase. In conclusion, it is possible to use TB to evaluate the degree of chromatin decondensation in guanaco spermatozoa and to use DTT as a positive control for the stain. Treatment of guanaco semen with 0.1% collagenase did not affect sperm chromatin condensation; therefore, this enzyme can be used to decrease semen viscosity and aid handling in the laboratory.