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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.

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A sperm cryopreservation protocol requiring dimethylacetamide (DMA, 6%) as a cryoprotectant was optimized via assays involving different prefreezing equilibration times (1, 10, 30, 60, and 120 min at 5°C) and different freezing rates achieved by the following: 1) using nitrogen vapor to reduce the temperature from 5°C to -85°C at 10°C/min (slow freezing rate); 2) using a biological freezer unit in a 2-step method to reduce the temperature from 5°C to -35°C at 7°C/min and then from -35°C to -140°C at 60°C/min (medium freezing rate); or 3) using a biological freezer unit in a 1-step freezing method to reduce the temperature from 5°C to -180°C at 60°C/min (rapid freezing rate). Heterospermic semen samples from chicken breeds raised as part of a Spanish genetic resource conservation program were used in all assays. The 1-min equilibration treatment was associated with a lower percentage of viable thawed spermatozoa than the 30-min treatment (P < 0.05). The remaining sperm variables studied were not affected by equilibration time. The medium-rate 2-step freezing method was associated with a higher percentage of motile spermatozoa after thawing and with greater acrosome integrity (P < 0.05) than the slow nitrogen vapor or rapid 1-step methods. Thawed sperm movement quality and plasma membrane integrity (as assessed by the hypoosmotic swelling test) were better (P < 0.05) in samples frozen by the medium-rate 2-step freezing method than in those subjected to the slow nitrogen vapor method. Fertility was not influenced by freezing method, although that achieved with the medium rate 2-step freezing method showed a trend toward being greater than that achieved with the rapid 1-step method (P = 0.07). Together, the present results suggest that slow cooling rates are not recommendable when using dimethylacetamide. The 2-step freezing method may be useful in the establishment of a germplasm bank for Spanish chicken breeds.

The purpose of this study was to analyze the effect of housing system and cold stress on the heterophil-to-lymphocyte ratio, the fluctuating asymmetry, and the tonic immobility duration of chickens. In experiment 1, hens (n=120; 36 wk old) from 5 Spanish breeds and a White Leghorn population, which had been housed in pens with or without access to an outdoor area from 20 wk of age, were used. The effect of housing system on heterophil-to-lymphocyte ratio varied from breed to breed, differences between housing systems being significant (P<0.05) in 2 breeds. In these breeds (Red-Barred Vasca and Birchen Leonesa), heterophil-to-lymphocyte ratio was significantly greater in hens housed in deep litter. Housing effect was significant for the relative asymmetry of leg length (P<0.01), wattle length (P<0.05), and the combined relative asymmetry (P<0.05), the relative asymmetry of hens housed in deep litter being larger. There was no significant difference for the duration of tonic immobility between hens housed in deep litter or free range. Thus, hens with access to an outdoor area were less stressed than hens without access to an outdoor area, although the fearfulness was similar in both groups of birds. In experiment 2, cocks (n=120; 36 wk old) from 4 Spanish breeds, a synthetic breed, and the White Leghorn population, which had been housed in cages with or without a cold stress (0 to 10 degrees C) from 24 wk of age, were used. Cold x breed interaction was significant for heterophil-to-lymphocyte ratio (P<0.05), differences between cold-stressed and control birds being significant in 2 breeds. In these breeds (Red-Barred Vasca and Buff Prat), heterophil-to-lymphocyte ratio was significantly greater in cold-stressed birds. Cold stress effect was significant for the relative asymmetry of toe length (P<0.001) and the combined relative asymmetry (P<0.05), the relative asymmetry of birds with cold stress being larger than that of control birds. Thus, cold stress seriously negatively affects the welfare of cocks.