Research articleEstablishment of conditions for ovum pick up and IVM of jennies oocytes toward the setting up of efficient IVF and in vitro embryos culture procedures in donkey (Equus asinus)
Introduction
Most wild equids are currently endangered or threatened in the wild, and in particular the Asiatic wild ass and the African wild ass, as mentioned in the Red List of endangered animal species of the International Union for the Conservation of Nature [1]. Moreover, many domestic donkey breeds are vulnerable to extinction such as the Asinina de Miranda donkey in Portugal [2], the Martina Franca donkey in Italy [3], [4], the Zamorano-Leonés donkey in Spain [5], and the American Mammoth Jack donkey in the United States of America [6]. Several donkey breeds are even nearly extinct with fewer than 100 active breeding jennies, such as the ‘Grand Noir du Berry’ donkey or the ‘Normand’ donkey in France (communication from the Institut Français du Cheval et de l’Equitation: http://statscheval.haras-nationaux.fr/core/zone_menus.php?zone=229&r=1316), and so forth.
The actions that are currently undertaken to preserve endangered donkey breeds include the creation of a Genome Resource Bank. Genome resource banking requires cryopreservation of semen, oocytes, and/or embryos. Embryos cryopreservation allows the preservation of genetics from both male and female and is the fastest method to restore a breed [7].
In vivo donkey embryo production after natural mating or artificial insemination has been reported previously with an embryo recovery rate per cycle from 50% to 76% [8], [9]. In equids, embryo production in vivo is limited because experimental induction of multiple ovulations has a low efficiency [10] and routine induction of multiple ovulations is still ineffective [6]. To our knowledge, no efficient superovulation treatment is available in the donkey.
Providing the collection of several oocytes per ovary, embryo production in vitro would allow the production of several embryos per cycle that could withstand cryopreservation owing to their small size [11]. Thus, this is the choice technique for embryo production for preservation of genetics. In vitro production requires several steps. The first step is the collection of several immature oocytes on a donor female using ovum pick up (OPU). The OPU technique has been widely used in the equine [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21]. However, to our knowledge, OPU has never been described in jennies. The second step is IVM of the oocytes. To date, only two reports about IVM of abattoir derived donkey oocytes have been published [22], [23]. Moreover, the optimal duration of in vitro culture has not been evaluated. The third step is IVF of oocytes. Several attempts to establish an efficient conventional IVF technique in the equine have been performed, but IVF rates remained quite low and IVF techniques were not repeatable [24], [25], [26], [27], [28], [29], [30]. Intracytoplasmic Sperm Injection (ICSI) has been widely adopted to generate horse embryos in vitro, both for scientific purposes and in the horse breeding industry [11], [31]. However, ICSI requires expensive equipment and expertise in micromanipulation. In 2009, a 60% rate of equine IVF was reported after treatment of fresh spermatozoa with procaine [32]. However, procaine was reported to induce cytokinesis in horse oocytes [33]. To our knowledge, this IVF technique has never been tested in the donkey. The last step for in vitro embryo production is in vitro culture of the embryo to a developmental stage that allows cryopreservation. Up to now, no study has been published concerning in vitro culture and development of donkey embryos. Thus, there is a huge lack of knowledge concerning OPU, IVM of oocytes, IVF, and in vitro development of embryos in the donkey.
Because there is an urgent need of techniques for in vitro embryo production for the preservation of endangered donkey breeds, our objective was to establish conditions for OPU, IVM, IVF, and in vitro development of embryos in donkeys.
To ascertain that our in vitro conditions are appropriate for oocyte maturation and fertilization, equine oocytes were used as a control.
Section snippets
Materials and methods
All procedures on animals were conducted in accordance with the guidelines for the care and use of laboratory animals issued by the French Ministry of Agriculture and with the approval of the ethical review committee (Comité d’Ethique en Expérimentation Animale Val de Loire) under number 02701.01.
All chemicals were purchased from Sigma–Aldrich (St Quentin Fallavier, France) unless otherwise indicated.
Oocyte recovery
For jennies oocytes collection, five puncture session were performed on adult cyclic jennies during follicular phase (ovaries containing growing follicles without CL). From the five puncture sessions, 193 follicles were flushed and 92 COCs were collected (48%) with an increase of the recovery rate along the puncture sessions from 34% to 56% (Table 1). Globally, we collected an average of 4.2 COCs per female, with a maximum of 10 COCs per female in two jennies.
For equine oocytes collection, a
Discussion
Most wild donkeys are currently endangered or threatened and many domestic donkey breeds are at risk of extinction. Embryos cryopreservation allows the preservation of genetics from both male and female and is the fastest method to restore a breed [7]. Thus, there is an urgent need of techniques for in vitro embryo production for the preservation of endangered donkey breeds. Our objective was to establish conditions for OPU, IVM, IVF, and in vitro development of embryos in donkey.
Early attempts
Acknowledgments
The authors would like to thank the staff of the experimental stud at the “Unité Expérimentale de Physiologie Animale de l’Orfrasière” for technical help. This work has benefited from the facilities and expertise of the “Plateforme d'Imagerie Cellulaire” of the UMR 85 “Physiologie de la Reproduction et des Comportements”.
This work was financially supported by the “Institut Français du Cheval et de l’Equitation” and the French National Infrastructure of Research CRB Anim funded by
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Metabolic profiling of preovulatory follicular fluid in jennies
2022, Research in Veterinary ScienceCitation Excerpt :In addition, acetate and valine concentrations were considerably higher in donkey PFF than in TCM-199 medium, whereas glycine, isoleucine, leucine, and threonine were less concentrated in the former than in the latter. Although the TCM-199 medium is routinely enriched with fetal bovine serum (FBS) to perform oocyte IVM (Goudet et al., 2015; Deleuze et al., 2018; Zhao et al., 2011; Abdoon et al., 2014; Douet et al., 2019), this supplement only provides eight additional metabolites (Caseiro et al., 2018). According to the metabolites described in two available commercial FBS formulations by Caseiro et al. (Caseiro et al., 2018), supplementation with FBS would provide a series of metabolites.
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2020, TheriogenologyCitation Excerpt :A higher oocyte recovery rate per slaughtered jenny’s ovary was obtained by scraping (4.86 ± 0.67 COCs/ovary) compared to aspiration with a 18-G (3.14 ± 0.36 COCs/ovary) or 20-G (0.61 ± 0.16 COCs/ovary) needles [84]. A 48–57% oocyte recovery rate was obtained after transvaginal ultrasound guided follicle aspiration [85,86] employing a 12-G double-lumen needle, as described for the horse [88]. While oocyte maturation rate increased from 39 to 77% when COC were incubated using TCM199, DMEM-F12 or CR1aa based media [83–86], the first attempt of in vitro fertilization (IVF) of in vivo collected donkey oocytes was unsuccessful (15% of pronuclei formation) [87].
First attempts for vitrification of immature oocytes in donkey (Equus asinus): Comparison of two vitrification methods
2019, TheriogenologyCitation Excerpt :To our knowledge, no efficient superovulation treatment is available in the donkey. Moreover, in vitro equine embryo production using conventional in vitro fertilization is still inefficient [9,10], and attempts for in vitro fertilization of donkey oocytes were not successful [11]. Equine embryos can be produced via intracytoplasmic sperm injection (ICSI) but blastocyst development rates are low and this technique requires expensive equipment and expertise in micromanipulation [10,12].
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)
2018, Journal of Equine Veterinary ScienceCitation Excerpt :The first step toward in vitro embryo production (IVP) of donkeys is the availability of more oocytes. Retrieving several immature oocytes from a donor by ultrasound-guided ovum pick up (OPU) is widely reported in horses [10–20] and has been recently described in the donkey [21]. These immature oocytes collected by OPU require further in vitro maturation (IVM).