9 Endoscopy-Mediated Intratubal Insemination in the Cow – A Preliminary Report About the Application of a Novel Minimally Invasive Insemination Technique

Abstract

On their long path through the female reproductive tract to the fertilization site, spermatozoa are exposed to diverse influences and hazards of the cervical, uterine, and oviducal environment that naturally select viable sperms for the following fertilization. Consequently, this results in a reduction from several billions of sperms in the ejaculate to a functional sperm reservoir within the range of 102 in the isthmus of the Fallopian tube. A technique to deposit spermatozoa directly into the ampulla, thus bypassing most of the reproductive tract, enables a rigorous reduction in number of sperms deposited. Furthermore, it provides a direct assessment of sperm fertility. The aim of our study was to establish an endoscopy-assisted intratubal insemination technique using different sperm dosages, fresh or cryopreserved, to determine adequate conditions for optimal fertilization. Eighteen Simmental heifers were inseminated with fresh semen, and 9 heifers were inseminated with frozen semen using this novel technique. The heifers were synchronized using a modified Ovsynch protocol, and insemination was conducted 18 to 20 h after the second gonadotropin-releasing hormone application. Insemination of heifers was performed under epidural anaesthesia. A tubing system bearing the endoscope and an insemination device was introduced through the vaginal wall into the peritoneal cavity. The insemination device consisted of a tube connected to a curved glass capillary tube loaded with semen. After a visual examination of the ovaries for the presence of an ovulatory Graafian follicle, the capillary tube was inserted directly via the infundibulum into the ipsilateral ampulla and the semen dose was deposited. The entire procedure took ~10 min. Two days later the oviduct was flushed by the same technique. A tubing system connected to a metal catheter served for flushing the embryos and unfertilized oocytes from the oviduct into the uterine horn. Afterward, embryos and oocytes were collected by flushing the uterine horn using an embryo flushing catheter and an embryo filter (EmCon). Embryos were stained using a Hoechst dye to visualise the numbers of attached spermatozoa to the zonae pellucidae. From 18 inseminations with fresh semen doses of 7 to 28 million sperms, 7 embryos at the 2- to 8-cell stage were found. Two of these embryos had more than 10 accessory sperms (AS), 3 had 3 to 6 AS, and 2 were without AS. From 9 inseminations with frozen semen doses containing 1.5 million sperms, we obtained 2 embryos, one at the 4-cell stage without AS and one at the 8-cell stage with 5 AS. Additionally, 3 unfertilized oocytes were collected. In conclusion, these preliminary results demonstrate a promising technique for intratubal AI, which has to be further optimized by studying numbers and treatment of spermatozoa and time of insemination.