Summary1. Sex differences in testosterone levels and sex-biased sensitivity to testosterone are the basis of some ideas postulated to account for sex-linked environmental vulnerability during early life. However, sex variation in circulating testosterone levels has been scarcely explored and never manipulated at post-natal stages of birds in the wild. 2. We measured and experimentally increased circulating testosterone levels in nestling Eurasian kestrels Falco tinnunculus . We investigated, possible sexual differences in testosterone levels and the effect of this hormone on growth (body mass and tarsus length) and cell-mediated immunity in males and females. We also explored testosterone effects on rump coloration, a highly variable melanin-based trait in male nestlings. We analysed data on circulating testosterone levels of nestlings in 15 additional bird species. 3. Increased levels of testosterone tended to negatively affect body condition, reduced cell-mediated immune responses in male and female nestlings and also diminished the expression of grey rump coloration in male nestlings. No sex differences were observed in testosterone levels in either control or increased testosterone group nestlings, and no interactions were found between sex and treatment. However, male nestlings showed a lower cell-mediated immune response than females in both groups. 4. Our results indicate first, that a high level of testosterone in all nestlings in a brood entails costs, at least in terms of immunity, coloration and probably growth. Secondly, sex differences in post-natal cell-mediated immunity, and consequently in the capacity to prevent diseases, cannot be explained by sex differences in circulating testosterone levels. Finally, by comparing published data at an interspecific level, contradictory sex patterns in circulating testosterone levels have been found, supporting the idea that circulating testosterone might not be a proximate factor causing sex-dependent vulnerability in bird species.

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.