Effects of Radiographic Contrast Dye on Domestic Cat Spermatozoa.

Abstract

Laparoscopic artificial insemination has played an important role in felid conservation, but requires minor surgery. The less invasive technique of transcervical artificial insemination has not been heavily pursued because of concerns that sperm is not adequately transported through the uterine horns. The use of radiographic contrast dyes to visualize and verify gamete placement within the uterine horns could ensure the success rate of this less invasive procedure. Two radiographic dyes (Omnipaque and MD76) were diluted 1:1.1 (Omnipaque) and 1:4.4 (MD76) with ultra-purified water to a final osmolarity of 320-330±3 mOsm, consistent with the osmolarity of domestic cat spermatozoa. Each diluted dye was tested for radio-opacity by injecting aliquots into uterine horns recovered from spayed cats and radiographing the tissue. Gamete rescued freshly collected and frozen-thawed spermatozoa were extended 1:1 with feline in vitro fertilization media (control) or with diluted radiographic dyes. Motility, forward progression, and acrosomal integrity were recorded every 30 minutes for four hours to analyze felid sperm activity after the addition of the experimental media, while morphology data was taken to determine if radiographic dye induced abnormalities in the sperm cells. To assess sperm penetration abilities, spermatozoa (fresh: 2×105cells/100µl droplets; frozen: 5×105cells/100µl droplets) extended with treatment media were coincubated with conspecific in vitro matured oocytes for 18-20h. All presumptive embryos were fixed and stained three days post insemination to determine fertilization and cleavage rates based on DNA content. Morphology analysis confirmed radiographic dyes did not induce abnormalities in felid spermatozoa. Motility and forward progression were converted into sperm motility indices, and results demonstrated no significant differences between media for freshly collected spermatozoa. For frozen-thawed spermatozoa, a lower sperm motility index was calculated for Omnipaque vs control, but not compared with MD76. Motility index decreased over time for all treatments for both freshly collected and frozen-thawed sperm. Acrosomal integrity for freshly collected spermatozoa was significantly reduced when treated with Omnipaque vs MD76 but not compared with control media. Differences in acrosomal integrity were not seen within frozen-thawed treatment groups. Acrosomal integrity for freshly collected and frozen-thawed sperm cells also significantly decreased with time. Preliminary data also indicate sperm exposed to radiographic contrast media maintained their ability to penetrate and fertilize conspecific oocytes. These findings propose radiographic dye can be added to domestic cat spermatozoa with minimal reduction to sperm fitness, thus creating a foundation for developing improved transcervical insemination techniques for non-domestic felids. (poster)