A novel approach to study the bovine oviductal fluid proteome using transvaginal endoscopy.

The oviduct provides the optimal micro milieu for early embryo development. However, accessing the bovine oviductal fluid in vivo for analysis is still challenging and therefore the oviductal fluid is usually collected post mortem. In the study presented here we introduce a novel approach to gain minimal invasive access to the bovine oviductal fluid proteome in vivo by transvaginal endoscopy at different stages of the estrous cycle. The first experiment aimed at transferring C4 derivatised magnetic beads to bind the oviductal fluid proteome in situ. Protein carrying beads were recovered by flushing the oviduct and proteins were eluted. In the second experiment a flushing solution was injected into and aspirated from the oviduct repeatedly. The flushing solution was centrifuged to separate the fluid from the cellular debris. Proteins were identified by nano-LC-MS/MS. Two different stages of the estrous cycle (Day 1 and Day 3) were analyzed in samples from 30 heifers. Both methods were applied successfully and in total, more than 3000 proteins were identified, so far representing the most comprehensive OF proteome published. This new minimal invasive approach to access the bovine oviductal fluid proteome facilitates future innovative experimental designs to study the role of the oviductal micro environment during early embryo development.

Endoscopy-mediated intratubal insemination in the cow - Development of a novel minimally invasive AI technique.

Conventionally inseminated spermatozoa suffer a dramatic reduction in numbers during their long journey until fertilization. In addition sperm survival seems to be strongly affected by the reconstitution of the female reproductive tract in the post partum period. The purpose of this study was to develop a novel AI technique for cattle that allows the deposition of spermatozoa directly into the ampulla in the immediate vicinity of the fertilization site. This new reproductive biotechnique was investigated with focus on semen origin, sperm dosage, semen preparation and time of insemination. Finally, a first practical application was carried out by inseminating superovulated heifers with sex-sorted semen. In total, 49 Simmental heifers were used for 65 intratubal inseminations (ITI) with single ovulation and 8 ITIs after superovulation, respectively. Insemination into the oviduct was performed under epidural anesthesia via transvaginal endoscopy using a curved glass capillary loaded with semen. Two days later the oviduct and the adjacent uterine horn were endoscopically flushed and embryos or unfertilized oocytes were collected for determination of fertilization success. Across all experimental groups, tubal insemination successfully resulted in the collection of embryos; however, first tubal AI attempts and ITIs close to ovulation led to low recovery rates. In total, 109 complexes were flushed from ITIs in superstimulated heifers (n = 8) using sex sorted semen, of which 24 (22%) were at the embryo stage. In conclusion, it was shown that intratubal insemination can be successfully used for semen deposition, thus bypassing the lower female genital tract. Factors such as time of insemination, semen processing and semen quantity for superovulatory use should be further investigated.