Intrafollicular Oocyte Transfer (IFOT) of Abattoir-Derived and In Vitro-Matured Oocytes Results in Viable Blastocysts and Birth of Healthy Calves

Abstract

There are still major differences between in vitro production (IVP)-derived and in vivo-derived bovine blastocysts. Therefore, intrafollicular oocyte transfer (IFOT) was used in the present study to allow early embryonic development within the physiological oviductal environment, in order to avoid subsequent harmful effects of the in vitro culture environment. Using modified ovum pickup equipment, in vitro-matured oocytes were transferred into the preovulatory follicle of synchronized heifers (follicular recipients), enabling subsequent ovulation, in vivo fertilization, and in vivo development. When 1646 in vitro-matured oocytes were transferred to 28 follicular recipients, a total of 583 embryos (35.2%) were recovered in excess after uterine flushing at Day 7. Although numbers of generated extra embryos were highly variable, preovulatory follicles with a diameter of 13-14 mm delivered significantly (P < 0.05) larger amounts of extra embryos (34.3 vs. 7.3), as well as extra morulae and blastocysts (8.3 vs. 0.8), compared with follicles with a diameter of 9-10 mm. Nevertheless, the developmental rate to the blastocyst stage was lower in IFOT compared with in vitro-derived control (Vitro) embryos at Day 7 (8.0% vs. 36.5%). Likewise, cumulative developmental rates to the morula or blastocyst stage until Day 7 were lower in IFOT-derived embryos when related to the number of transferred (8.4% vs. 51.7%) or flushed (22.8% vs. 51.7%) embryos. Of the latter, IFOT-derived embryos yielded significantly lower cleavage rates compared with the Vitro controls (63.2% vs. 88.8%), and developmental rate to the morula or blastocyst stage were lower even when related to the proportion of cleaved embryos (36.8% vs. 58.2%). In contrast, lipid content and cryotolerance did not differ between IFOT and fully IVP embryos; but IFOT-derived embryos showed significantly lower lipid content (P < 0.05) and significantly higher cryotolerance compared with IVP-derived embryos cultured in CR1aa medium supplemented with estrus cow serum (ECS), but not when cultured in SOFaa medium supplemented with fatty acid-free BSA (BSA-FFA). Finally, transfer of 19 frozen-thawed IFOT-derived blastocysts to synchronized recipients (uterine recipients) resulted in pregnancy rates comparable with those obtained after transfer of fully in vivo-derived embryos or IVP-derived embryos cultured in SOFaa + BSA-FFA, whereas pregnancy rate following transfer of IVP-derived blastocysts was significantly lower when they were cultured in CR1aa + ECS (42.1% vs. 13.8%). All in all, seven pregnancies presumed to be IFOT derived went to term, and microsatellite analysis confirmed that five calves were indeed derived from IFOT. To our knowledge, these are the first calves born after IFOT in cattle. Interestingly, the average birth weight of IFOT-derived calves was lower than that of IVP-derived calves, even when embryos were cultured in SOFaa + BSA-FFA, indicating that the environment during early embryo development might cause fetal overgrowth. Taken together, for the first ti...

Intrafollicular oocyte transfer in cattle – a technical report

“…In a further step, IFOT was performed. The synchronization scheme was used as previously described (Kassens et al 2015). Briefly, heifers (n = 21) which had a corpus luteum on the day of rectal examination were synchronized by i.m.…”

“…numbers of embryos obtained were highly variable, it was shown that IFOT of in vitro matured oocytes represents a feasible technique for generating bovine blastocysts closely resembling IVD blastocysts in terms of lipid content and cryotolerance (Kassens et al 2015). In the following study, immature oocytes were transferred into growing follicles prior to ovulation.…”

“…Since oocyte collection and oocyte transfer do not differ substantially, OPU systems have been slightly modified in order to perform first studies on IFOT. Equipment described in all mentioned IFOT studies (Kassens et al 2015;Sprícigo et al 2016;Hoelker et al 2017) consists of a plastic holder and an ultrasound transducer (7.5-10 MHz). A long tubing system was connected with a disposable needle (G26, G27) at one end and with either a laboratory pipette or a 1 ml syringe at the other end.…”

“…The tubing system was filled with fluid (phosphate-buffered saline or follicular fluid). Oocytes were loaded into the needle at a fluid volume of 60 or 200 µl (Kassens et al 2015;Sprícigo et al 2016;Hoelker et al 2017). Handling required at least two people: one person performed rectal manipulation and navigated the follicle bearing ovary in front of the ultrasound; the second person introduced IFOT set and took care of the careful oocyte transfer into the follicle.…”

Grand Challenge Animal Reproduction-Theriogenology: From the Bench to Application to Animal Production and Reproductive Medicine

“…However, in recent years and with the development of ultrasound-guided follicular aspiration, oocytes are now routinely and repeatedly collected from valuable females and either stored or fertilized. Fertilization in vivo (oocyte transfer) or in vitro through techniques such as intracytoplasmic sperm injection or in vitro fertilization (IVF) is now commonplace in ruminants (48). Development of these technologies has seen a surge in the equine recently (49, 50).…”