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 sp ermatozoa treated with the nuclear fluorescent dye bisbenzimide, ovidu ctal tissue was collected from hens and homogenized. Samples of homoge nate were dried, and the number of spermatozoa mm(-2) was determined w ith the use of a fluorescence microscope. When spermatozoa were added to excised uterovaginal junction tissue before homogenization, results indicated a I:I linear relationship between actual numbers of spermat ozoa added to the tissue and calculated numbers of spermatozoa added t o the tissue. This new technique was used to show that insemination of hens with 25, 50 or 100 x 10(6) spermatozoa resulted in a linear incr ease in the number of spermatozoa stored in the uterovaginal junction. Insemination of hens with 328 x 10(6) spermatozoa produced no increas e in uterovaginal junction storage of spermatozoa over insemination wi th 100 x 10(6) spermatozoa. At the maximum sperm storage tubule fillin g dose of 100 x 10(6) spermatozoa, only 0.22% of the spermatozoa insem inated were found in the uterovaginal junction 24 h after insemination . Treatment of spermatozoa with bisbenzimide had no detrimental effect s on fertility or penetration rates when compared with untreated (cont rol) spermatozoa. However, when spermatozoa were treated with bisbenzi mide, hatchability of fertile eggs was reduced. In conclusion, this ne w fluorescence technique appears to be valuable in determining the tot al number of spermatozoa stored in the uterovaginal junction of hens.