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.

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 the present study was to examine the seasonal variation in freezing damage in free-range rooster sperm. Over a period of 1 year, heterospermic semen samples were collected weekly by massage from the roosters of 14 Spanish chicken breeds, all housed under natural photoperiod and climatic conditions. All samples were frozen in straws using DMA as a cryoprotectant, placing them first in nitrogen vapour and then plunging them into liquid nitrogen. No seasonal effects on fresh sperm quality were found. Neither did season affect the percentage of viable frozen-thawed spermatozoa nor the percentage with an intact acrosome. However, the collection season influenced (p < 0.05) most frozen-thawed sperm motility values. The percentage of immotile frozen-thawed spermatozoa was lower (p < 0.05) in spring-collected sperm than in summer- or autumn-collected samples. The percentage of spermatozoa showing progressive motility was higher in spring-collected sperm compared with winter-, summer- or autumn-collected samples (p < 0.05). The curvilinear velocity (VCL), straight-line velocity (VSL) and average path velocity (VAP) values of spring-collected sperm were also higher (p < 0.05). In conclusion, spring would appear to be the best season for collecting and freezing the semen of free-range Mediterranean chicken breeds.

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Semen was collected from 10 Black Castellana roosters and the classic sperm variables (ejaculate volume, sperm concentration and sperm motility) examined. In addition, the hypo-osmotic swelling test was used to investigate sperm cell membrane integrity, and acidic aniline blue staining used to screen for morphological abnormalities (including acrosome integrity) and to examine the condensation status of the chromatin. The latter was also examined by Gram staining. Large and small semen volumes were associated high and low sperm concentrations respectively (R 2 =0.04, P<0.05). The percentage of motile spermatozoa correlated strongly with the percentage of sperm cells showing an intact acrosome (R 2 =0.13, P<0.001) and with the percentage of morphologically normal spermatozoa (R 2 =0.04, P<0.05). The percentage of Gram positive spermatozoa was positively correlated with semen appearance (R 2 =0.12, P<0.05), sperm cell concentration (R 2 =0.13, P<0.05), and with the sperm motility variables studied (R 2 =0.14, P<0.05 for percentage mobility, and R 2 =0.12, P<0.05 for quality of movement). Only three of the 10 roosters, all with fertilisation potentials of 80-90%, were considered potential sperm donors for genome resource banking purposes. The remaining birds were all of low fertility (≤ 50%); in fact, some produced semen volumes too small to perform fertility tests. Semen volume and membrane integrity were found to be the best variables for predicting the fertilisation potential of rooster ejaculates.Additional key words: aniline blue, artificial insemination, fertility, Gram staining, semen quality ResumenVariables espermáticas predictoras de fertilidad en gallos de raza Negra Castellana; uso en la selección de donantes en bancos de recursos genéticos Se utilizó semen de 10 gallos de raza Negra Castellana, en el que se realizó un espermiograma clásico (volumen eyaculado, concentración espermática, motilidad). Además, se usó el test de endósmosis para evaluar la integridad de membrana plasmática, y la tinción de azul de anilina para analizar morfoanomalías (incluyendo la integridad del acrosoma) y el estatus de condensación de la cromatina, el cual también se evaluó mediante la tinción de Gram. Los resultados muestran que el volumen seminal estaba correlacionado con la concentración espermática (R 2 =0,04; P<0,05). El porcentaje de espermatozoides mótiles mostró una correlación con el porcentaje de espermatozoides con acrosomas intactos (R 2 =0,13; P<0,001) y con el porcentaje de espermatozoides con morfología normal (R 2 =0,04; P<0,05). El porcentaje de células espermáticas Gram positivas estuvo correlacionado con el aspecto seminal (R 2 =0,12; P<0,05), la concentración espermática (R 2 =0,13; P<0.05) y variables de motilidad (R 2 =0,14; P<0,05 y R 2 =0,12; P<0,05, para el porcentaje de células mótiles y calidad del movimiento, respectivamente). Sólo tres de los 10 gallos estudiados mostraron altas tasas de fertilidad del 80-90%, y fueron considerados como los únicos donantes potenciales de semen en bancos...