Análise computacional da cromatina de espermatozóides de galo (Gallus gallus) e determinação do período de armazenamento dos espermatozóides nas espermatecas de galinhas

Abstract

The objectives of this work were to evaluate new methodological variants using toluidina blue (AT), to establish a trustworthy protocol for the computational evaluation of the chromatin compaction of the fowl s spermatozoa, to find out alterations in the chromatin compactation during the storage in the sperm storage tubules of fowl hens, to observe the storage time and to estimate the variation in the amount of stored spermatozoa in the cranial, medium and caudal sections of the uterovaginal junction during 23 days. Twenty semen samples were used: 10 from fowls that were 35 weeks old and had high fertility (young fowls) and 10 from fowls that were 60 weeks and had low fertility (Old fowls). To the analysis of the spermatozoa during the storage in the sperm storage tubules of hens, 48 thirty-six- week-old meat chickens (44 hens and 4 fowls) of the Cobb Avian 48 strain were also used. A sample from the uterovaginal junction of the hens was collected for 23 days after the match with the fowls, 6 hens being sacrificed every four days to make histological microscope slides. Different methods of denaturation and staining were tested. The best method with AT was the hydrolysis with 1N HCl for 10 minutes, staining in bucket with 0.025% AT, pH 4,0, for 20 minutes, dehydration in alcohol, clearing in xylol and mounted with Canada balsam. All the semen samples were submitted to this protocol and later evaluated through computational image analysis. Area, length, width, perimeter and the chromatin compaction homogeneity of head spermatozoa were measured. The sperm of old fowls presented more chromatin changes than the ones of young fowls. The spermatozoa of the young fowls presented bigger heads than the ones of old fowls. It was not possible to determine the chromatin compaction alterations of the fowl sperm after their storage in the sperm storage tubules through the used method. Spermatozoa were found through out the area of the sperm storage tubules. Most of then gathered in the medium section of the uterovaginal junction. There was not meaningful statistical difference in the amount of sperm in the sperm storage tubules over the days of the experiment. A greater amount of sperm cells were observed until the 23rd day of assessment. Therefore, the storage time of the spermatozoa in hens lasts at least 23 days.