Donkeys (Equus africanus asinus) share the same genus with horses and zebras, the only extant members of the family Equidae. Interestingly, despite their phylogenetical proximity, Equus members exhibit very divergent karyotypes [1], genetic plasticity, and significant differences in reproductive physiology [2,3]. Although it is still uncertain when donkeys were domesticated [4,5], most authors underline that domestication started approximately 6000 years ago, allowing large-scale food redistribution and transportation with an essential role in the overland trade in Africa and western Asia [6]. Presently, domestic donkeys are still employed for transportation in undeveloped countries, for meat and milk production, as companion animals, and, the jack, to produce hybrids when crossing with mares (mules). There are six extant species of donkeys including the Somali wild asses (Equus africanus somaliensis) and Nubian wild asses (Equus africanus africanus) considered critical endangered, and the Asiatic Wild Ass, represented by Hemiones (Equus hemionus) and Tibetan Kiangs (Equus kiang) considered near threatened and less concern respectively [7] (International Union for Conservation of Nature's Red List). Moreover, among domestic donkey breeds, seven out of the 28 European breeds are in a critical status, and 20 endangered [8]. Therefore, there is an urge to improve donkey conservation programs to increase the animal population, distribution, and the genetic pool among these individuals.

With a few exceptions, the horse breeding industry has allowed the implementation of assisted reproductive technologies (ARTs) to maximize reproduction efficiency and genetic preservation [9]. Particularly, in vitro embryo production (IVEP) has gained relevance in the last few years [10]. Currently, somatic cell nuclear transfer (SCNT) and intracytoplasmic sperm injection (ICSI) are the main techniques used worldwide to produce equine embryos in vitro [[11], [12], [13]], since conventional in vitro fertilization has been recently reported as a successful technique [14]. Moreover, successful reports of zebra SCNT embryos and hybrids ICSI blastocyst (horse egg – zebra sperm) has been recently achieved [15] supporting the use of IVEP for the preservation of wild equids genetics. Only a few reports have used domestic donkeys as a model for IVEP, but none achieved blastocyst development stage in vitro or pregnancies [[16], [17], [18], [19]].

Ovum pick up (OPU) and in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) are needed for in vitro embryo production. OPU has been implemented in jennies in a similar way as in mares [17,20,21] yielding COCs recovery rates between 34 and 76%. While IVM of horse oocytes has been studied by many researchers, donkey oocyte IVM needs further investigation. In most eutherian mammals, oocyte maturation naturally occurs within a growing follicle context [22]. Thus, follicular fluid (FF) has been used to support in vitro maturation in different species. In bovine, the use of FF improved oocyte cumulus expansion, blastocyst developmental kinetics and their cells number [23]. Particularly in the Equus genus, preovulatory follicular fluid (PFF) was reported to support IVM in horses [24,25] and donkey oocytes [18], with nuclear maturation rates ranging from 50 to 80%. No reports of ICSI or SCNT in donkey have been published yet. Instead, oocyte chemical activation and conventional in vitro fertilization were attempted achieving cleavage and early stages of preimplantation embryo development [18,20]. Time-lapse imaging is a useful non-invasive tool to screen embryo morphokinetics and quality during their in vitro development, and recently used in human IVEP to obtain data and parameters to develop embryo selection algorithms [26]. Some of these morphokinetic parameters were first described for horse ICSI embryos in 2019 [27,28]. The use of this technology had not been reported for donkey embryos at the time of writing this manuscript.

This paper explores the hypothesis that supplementation with jenny PFF during in vitro maturation could improve oocyte developmental competence in the domestic donkey. Thus, we assessed in vitro nuclear maturation rates, cumulus cell expansion, and embryo developmental after ICSI of COCs matured in media supplemented with fetal bovine serum (FBS) or donkey PFF. Donkey PFF was subject to nuclear magnetic resonance to determine its metabolic profile. Horse oocytes were used as a control group. For a deeper understanding of preimplantation development, time-lapse imagining was performed in donkey and horse ICSI embryos.