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Development of a novel fluorescence technique for quantifying the total number of spermatozoa stored in the uterovaginal junction of hens

A technique was developed to determine the total number of spermatozoa stored in the uterovaginal junction of hens. After insemination of spermatozoa treated with the nuclear fluorescent dye bisbenzimide, oviductal tissue was collected from hens and homogenized. Samples of homogenate were dried, and the number of spermatozoa mm^–2 was determined with the use of a fluorescence microscope. When spermatozoa were added to excised uterovaginal junction tissue before homogenization, results indicated a 1:1 linear relationship between actual numbers of spermatozoa added to the tissue and calculated numbers of spermatozoa added to the tissue. This new technique was used to show that insemination of hens with 25, 50 or 100 x 10⁶ spermatozoa resulted in a linear increase in the number of spermatozoa stored in the uterovaginal junction. Insemination of hens with 328 x 10⁶ spermatozoa produced no increase in uterovaginal junction storage of spermatozoa over insemination with 100 x 10⁶ spermatozoa. At the maximum sperm storage tubule filling dose of 100 x 10⁶ spermatozoa, only 0.22% of the spermatozoa inseminated were found in the uterovaginal junction 24 h after insemination. Treatment of spermatozoa with bisbenzimide had no detrimental effects on fertility or penetration rates when compared with untreated (control) spermatozoa. However, when spermatozoa were treated with bisbenzimide hatchability of fertile eggs was reduced. In conclusion, this new fluorescence technique appears to be valuable in determining the total number of spermatozoa stored in the uterovaginal junction of hens.