Use of the hypo-osmotic swelling test and aniline blue staining to improve the evaluation of seasonal sperm variation in native Spanish free-range poultry.

The season may affect the values of fresh semen variables and therefore influence the success of cryopreservation. The aim of this study was to improve the evaluation of seasonal changes in semen quality in Spanish Black Castellana roosters maintained under natural environmental conditions. Semen was collected from 11 Black Castellana roosters (housed under natural photoperiod and temperature conditions) by massage twice every month for 12 mo. In addition to determining ejaculate volume, sperm concentration, and sperm motility (the classic sperm variables), we used the hypo-osmotic swelling test to examine the membrane integrity of the spermatozoa. Further, morphological abnormalities and acrosome integrity were assessed via aniline blue staining. Semen volume (P < 0.05), sperm concentration (P < 0.01), and the percentage of spermatozoa with an intact acrosome (P < 0.01) were significantly affected by the season of the year. The annual profile of the percentage of spermatozoa showing acrosome integrity followed a trend roughly parallel to annual variations in temperature (Spearman rank correlation = 0.77, P < 0.01). According to the hypo-osmotic swelling test, membrane integrity fell in July (P < 0.05 compared with all other months), the month of highest temperatures. Aniline blue staining and the hypo-osmotic swelling test provide an easy and useful means of evaluating sperm abnormalities, including acrosome morphology and membrane integrity, and could be easily introduced into routine avian semen quality assessments. The results show that high semen quality is associated with long day photoperiods. Extreme heat or cold appear to exert a negative influence on sperm quality.