Ovum Pick Up and In Vitro Maturation of Jennies Oocytes Toward the Setting Up of Efficient In Vitro Fertilization and In Vitro Embryos Culture Procedures in Donkey ( Equus asinus )

Abstract

Most wild and domestic donkey breeds are currently endangered or threatened. Their preservation includes the creation of a Genome Resource Bank. Embryo cryopreservation allows preservation of genetics from both male and female and is the fastest method to restore a breed. Embryo production in vivo is limited in equids. We recently established a technique of ovum pick up (OPU) in the donkey. Conditions of in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture of zygotes have been evaluated. Equine abattoirederived oocytes were used as controls. Donkey cumulus-oocyte complexes (COCs) collected by OPU were matured in vitro in TCM199 with fetal calf serum and epidermal growth factor for 24, 30, 34, or 38 hours. Forty-four percent were in metaphase 2 after 34 hours. In our conditions, IVM of donkey oocytes was slower than that of equine oocytes and the optimal duration for donkey oocytes IVM may be 34 hours. Oocytes we co-incubated with frozen-thawed donkey semen treated with procaine for 18 hours and cultured for 30 hours in a Dulbecco Modified Eagle Medium-F12based medium. Only 15% of jennies oocytes contained 2 pronuclei after co-incubation, and none of them developed further after 48 hours after IVF. Treatment of donkey sperm with procaine may not be efficient for IVF. Some parthenogenetic activation occurred. In conclusion, we confirm that our conditions for OPU in jennies yielded high recovery rates that improved with operator experience. Maturation rates of 44% can be achieved using the IVM medium routinely used for equine oocytes in our lab. Further studies are in progress to establish efficient conditions for IVF and development of donkey zygotes.

Metabolic profiling of preovulatory follicular fluid in jennies

“…The time interval between animal death and harvesting of PFF samples could lead to postmortem accumulation of lactate (Tews et al, 1963), as reported by a previous study carried out with equine oviductal fluid (González-Fernández et al, 2020). TCM-199 is the basic culture medium most used for the formulation of IVM media in horses and in the attempts carried out so far in donkeys (Goudet et al, 2015;Deleuze et al, 2018;Zhao et al, 2011;Abdoon et al, 2014;Douet et al, 2019). When comparing our results to the metabolites described in TCM-199 culture medium (see Table 2), we detected that only 11 of the 27 metabolites found in jenny PFF were present in TCM-199 medium.…”

“…In equids, the production of in vivo-derived embryos from donors is limited by the lack of efficiency in superovulation treatments (Squires et al, 2003;Smits et al, 2012;Meyers-Brown et al, 2011), thus reducing the number of embryos available to one per oestrus cycle (Smits et al, 2012;Goudet et al, 2015;Deleuze et al, 2018). In addition, the large size of recovered in vivo embryos makes them "bad candidates" for cryopreservation (Choi and Hinrichs, 2017;Herrera et al, 2015).…”

“…In addition, the large size of recovered in vivo embryos makes them "bad candidates" for cryopreservation (Choi and Hinrichs, 2017;Herrera et al, 2015). In vivo-derived embryos are normally recovered after 7-8 days from ovulation in the blastocyst stage and have a diameter of >300 μm, due to a blastocoelic cavity full of fluid that makes more difficult the exchange of water by cryoprotectants (Squires et al, 2003;Deleuze et al, 2018;Choi and Hinrichs, 2017;Herrera et al, 2015;Hinrichs and Hinrichs, 2012). For this reason, the very first step for IVP of embryos from donors is widely documented in horses: the ultrasound-guided oocyte retrieval, or Ovum Pick Up (OPU) (Kanitz et al, 1995;Purcell et al, 2007;Jacobson et al, 2010;Galli et al, 2007;Spacek and Carnevale, 2018;Dell'Aquila et al, 1997;Caillaud et al, 2008;Douet et al, 2017).…”

“…In addition, IVP of embryos and their cryopreservation would allow the preservation of the male and female genetics, being a fast method to restore a breed (Douet et al, 2017;Woelders et al, 2012). Although the first experiments of oocyte IVM (Goudet et al, 2015;Deleuze et al, 2018;Zhao et al, 2011;Abdoon et al, 2014), in vitro fertilization (IVF) (Goudet et al, 2015;Deleuze et al, 2018) and even vitrification (Douet et al, 2019) were performed in jennies, maturation and sperm penetration rates are still very low and no IVP embryos have been obtained yet.…”

Retrograde Flushing Followed by Slicing Float-Up as an Approach to Optimize Epididymal Sperm Recovery for the Purpose of Cryopreservation in Equids

“…The superior sperm recovery obtained herein using retrograde flushing and then slicing float-up methods can have an even more impactful effect in the equid breeding industry than previously; nowadays, with the widespread use of intracytoplasmic sperm injection (ICSI) in horses [ 18 ] and more recently in donkeys [ 19 ], it has allowed maximum use of frozen sperm as 1000–10,000-fold less sperm is needed to result in a pregnancy in comparison with artificial insemination. As a result, many veterinary practices and clinicians are now simultaneously freezing sperm for use via artificial insemination and ICSI.…”