Percent of embryos recovered in mares and jennies relative to the day of ovulation

There is a high degree of variation in the percent of embryos recovered in mares when taking into consideration the day of the recovery attempt. For instance; on the sixth, seventh, or eighth PO day the reported recovery rate varies from zero (^{Battut et al., 1997}), sixty-two (^{Camillo et al., 2003}), to one hundred percent (^{Hochi et al., 1995}) respectively. Still, recovery rates close to or higher than fifty percent are indicated by most studies (^{Allen et al., 1985}; ^{Iuliano et al., 1985}; ^{Squires et al., 1985}; ^{Hinrichs, 1990}; ^{Freeman et al., 1991}; ^{Hochi et al., 1995}; ^{Maclellan et al., 2002}; ^{Eldridge Panuska et al., 2005}; ^{Moussa et al., 2005}; ^{Derbala & Abdou, 2017}; ^{Silva et al., 2018}; ^{Roser et al., 2020}). When embryo collection is attempted early (sixth PO day), the recovery rate ranges from zero (^{Battut et al., 1997}) to eighty-two percent (^{Hochi et al., 1995}). The previous dramatic difference between reports could be partially attributed to the professional’s expertise since embryo searching and observation are more difficult during this early stage of development (day six PO) when the embryo size is usually ≤ 300 µmØ. Though is not possible to make a direct comparison of the average recovery rate due to differences in methods and number of animals used in each study, at large; they show that successful collection rate tends to be lower when performed early (day six PO compared to later days). Interestingly, it has been possible to increase the embryo recovery rate by modifications adapted to the collection technique; for example, allowing the recovery media or flushing solution to stay a few minutes longer inside the uterus (^{Hinrichs, 1990}; ^{Alvarenga et al., 1993}).

Besides showing a high degree of variation, the data related to the per day embryo recovery rate in jennies are limited (^{Allen et al., 1985}; ^{Vendramini et al., 1997}; ^{Camillo et al., 2010}; ^{Peña-Alfaro et al., 2014}; ^{Bottrel et al., 2017}; ^{Pérez- Marín et al., 2017}; ^{Ottmann et al., 2018}; ^{Dorado et al., 2020}). During the sixth PO day, the percentage of successful embryo collection ranges from forty-three (^{Vendramini et al., 1997}) to seventy-five (^{Dorado et al., 2020}). On the seventh PO day, it goes from twelve (^{Camillo et al., 2010}) to eighty percent (^{Dorado et al., 2020}). Alternatively, if attempted from the eighth to the ninth PO day, the successful embryo collection rate is usually higher than fifty percent (^{Camillo et al., 2010}; (^{Peña-Alfaro et al., 2014}; ^{Dorado et al., 2020}). As previously mentioned, the percent of recovered embryos in equids can be attributed to multiple factors like the number of cycles within each donor (^{Dorado et al., 2020}), type of semen (^{Meadows et al., 2000}), insemination and ovulation time (^{Eldridge-Panuska et al., 2005}), seasonality in mares but not in jennies (^{Peréz-Marín et al., 2017}; ^{Dorado et al., 2020}), ovulations per cycle (^{Squires et al., 1985}), fertility of males (^{Peréz-Marín et al., 2017}; ^{Dorado et al., 2020}) and others (^{Allen et al., 1985}).

The relationship between day of recovery and embryo size

In mares, embryo size is radically influenced by the PO day selected to implement its recovery (^{Betteridge et al., 1982}; ^{Hochi et al., 1995}; ^{Eldridge-Panuska et al., 2005}; ^{Choi et al., 2010}; ^{McCue et al., 2010}; ^{Díaz et al., 2016}; ^{Guignot et al., 2016}; ^{Wilsher et al., 2019}). For the most part, if the embryo is collected either during the sixth or sixth and a half PO day, it is typically ≤ 300 µmØ (^{Iuliano et al., 1985}; ^{Freeman et al., 1991}; ^{Eldridge-Panuska et al., 2005}; ^{Hudson et al., 2006}; ^{Choi et al., 2010}; ^{Guignot et al., 2015}; ^{Pérez- Marín et al., 2017}; ^{Pérez-Marín et al., 2018}; ^{Silva et al., 2018}). In contrast, when the embryo recovery is done on days seventh, seventh and a half; eight, eight and a half; or nine, it is generally >300 µmØ (^{Hochi et al., 1995}; ^{McCue et al., 2010}; ^{Díaz et al., 2016}; ^{Pérez- Marín et al., 2017}; ^{Wilsher et al., 2019}).

Studies in jennies describing a relationship between the size of the embryo and the day of its collection are scarce. Yet, it seems that contrary to mares a high percent of donkey embryos collected during the seventh, or seventh and a half PO days are still < 300 µmØ (^{Vendramini et al., 1997}; ^{Panzani et al., 2012b}; ^{Bottrel et al., 2017}; ^{Pérez- Marín et al., 2017}; ^{Pérez-Marín et al., 2018}; ^{Bottrel et al., 2019}; ^{Bottrel et al., 2020b}; ^{Dorado et al., 2020}; ^{Fanelli et al., 2020}). When the collection of the donkey embryo is attempted from the eighth PO day or later, its diameter is typically close to or larger than 500 µmØ (^{Panzani et al., 2012a}; ^{Pérez- Marín et al., 2017}; ^{Bottrel et al., 2019}; ^{Dorado et al., 2020}).

Protocols including or excluding embryo capsule (EC) puncture before vitrification

The capsule does not require to be punctured before the vitrification process if a horse embryo size is ≤300 µmØ, and its PVSPR is usually higher than thirty percent (^{Eldridge-Panuska et al., 2005}; ^{Hudson et al., 2006}; ^{Choi et al., 2011}; Reviewed by ^{Urías-Castro & Boeta, 2020}). Most reports about the vitrification of equine embryos indicate that when the horse embryo is ˃300 µmØ it might not only be punctured to perforate its EC but its blastocoelic fluid might be withdrawn as well to optimize the PVSPR (^{Choi et al., 2011}; ^{Guignot et al., 2015}; ^{Díaz et al., 2016}; ^{Ferris et al., 2016}; S^{ánchez et al., 2017}; ^{Wilsher et al., 2019}; Reviewed by ^{Urías-Castro & Boeta, 2020}). However when further divisions in terms of embryo size are taken into consideration; it is observed that the vitrification of equine embryos ranging from 300 to 550 µmØ in size (≤ 550 µmØ); the perforation of their embryo capsule without the aspiration of their blastocoelic fluid results in pregnancy rates higher than 75% (^{Wilsher et al., 2021}).

Studies reporting the PVSPR of donkey embryos are scarce. The work available shows that the vitrification of donkey embryos ≤300 µmØ result in pregnancy rates that ranges from thirty-six percent at day fourteen of pregnancy (D14), to twenty-seven percent at day twenty-five (D25); suggesting that if the embryo size is ≤300 µmØ, EC puncture and blastocoelic fluid extraction might not be essential (^{Panzani et al., 2012}b). Furthermore, research projects describing the puncture of the donkey EC and its blastocoelic liquid aspiration are limited (^{Ottmann et al., 2018}). Whether large donkey embryos ˃300 µmØ require EC puncture and blastocoelic fluid extraction to increase their PVSPR has been indirectly tested only, since EC puncture and blastocoelic extraction resulted in a high percentage of in vitro survival (^{Ottmann et al., 2018}; Reviewed by ^{Urías-Castro & Boeta, 2020}).

Intrauterine infusion of liquid solutions to collect equine embryos

Some of the first studies describing the collection of embryos in equids reported the employment of a physiological saline solution containing two percent of gelatin (^{Oguri & Tsutsumi, 1974}). Nowadays, the use of Ringer lactate (^{Alvarenga et al., 1993}; ^{Barfield et al., 2008}; ^{Sánchez et al., 2017}; ^{Wilsher et al., 2019}); which composition is close to the Hartmann’s solution has been described (^{Guignot et al., 2015}). Other media utilized to recover equine embryos include Dulbecco’s phosphate-buffered saline DPBS/PBS (^{Alvarenga et al., 1993}; ^{Landim E Alvarenga et al., 1993}). A Dulbecco’s modified phosphate-buffered saline (DMPBS) combined with fetal calf serum (FCS) has been reported by ^{Hinrichs (1990}). DMPBS can be supplemented with heat-inactivated steer serum (^{McKinnon & Squires, 1988}); or bovine serum albumin (BSA) plus antibiotics (^{Battut et al., 1997}). The DMPBS mixed with either one percent of FCS or bovine calf serum, plus glucose and pyruvate has also been described as an embryo recovery medium (^{Carnavale et al., 2000}). The commercial Emcare® complete flush solution (^{Eldridge-Panuska et al., 2005}) and the Vigro® flush medium (^{Hudson et al., 2006}) are other alternatives reported as embryo recovery media.

In donkeys, the Ringer’s lactate solution is the most frequently reported as embryo recovery medium (^{Panzani et al., 2012}a; ^{Peña-Alfaro et al., 2014}; ^{Panzani et al., 2016}; ^{Pérez-Marín et al., 2018}; ^{Bottrel et al., 2019}; ^{Bottrel et al., 2020a}; ^{Bottrel et al., 2020b}). Another reported alternative is the DPBS (^{Camillo et al., 2010}).

Liquid solutions used to hold recovered equine embryos

The simplest embryo holding media in terms of composition are either the saline medium containing two percent of gelatin, or the Ringer’s lactate solution with mare serum (1:1, v/v) and one thousand international units of penicillin per milliliter (^{Oguri & Tsutsumi, 1974}). A more complete in terms of composition is the Ham’s F10 medium, complemented with ten percent FCS plus one percent penicillin/streptomycin; this medium is gassed with five percent of carbon dioxide, oxygen, and ninety percent of nitrogen (^{Carnavale et al., 2000}). The commercial embryo holding media Vigro® (^{Hudson et al., 2006}) and Syngro® (^{Barfield et al., 2009}) have also been described. French investigators have worked with the EHM medium, which is a PBS-based liquid solution containing four grams of BSA per liter (^{Guignot et al., 2015}). Researchers at the Colorado State University have reported the utilization of the DMEM/F12 medium (inorganic salts, amino acids, vitamins, antibiotics and others) supplemented with twenty percent of fetal bovine serum - DMEM/F12/FBS (^{Ferris et al., 2016}). A DMPBS lacking calcium and magnesium, complemented with ten percent FBS and fifty micrograms of gentamicin per milliliter has been employed by ^{Sánchez et al. (2017}). Alternatively, the M-199 Hepes medium supplemented with ten percent FBS plus one percent antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin) has also been described as an embryo-holding solution (^{Wilsher et al., 2019}).

PBS and lactated Ringer have been reported as holding media for donkey embryos (^{Camillo et al., 2010}). The use of the commercial Emcare Holding Solution® is also documented by several researchers (^{Camillo et al., 2010}; ^{Panzani et al., 2012}a). Another alternative to hold donkey embryos is the Holding Vitrocell^®, which contains zero-point four percent of BSA (^{Peña-Alfaro et al., 2014}). The Syngro® holding medium has been employed with donkey embryos before transferring them into the mule uterus (^{Bottrel et al., 2017}). The use of an isotonic holding medium (IVM-Technologies) has been reported by ^{Pérez-Marín et al. (2018}). Recently, Bottrell and his team employed TCM-199 HEPES complemented with twenty percent FCS as an embryo holding medium (^{Bottrel et al., 2019}).

Embryo washing and manipulation media

A liquid solution that has been employed in mares to wash and manipulate the collected embryos is PBS; it is usually supplemented with a serum of animal origin, like the fetal calf serum FCS (^{Alvarenga et al., 1993}). The use of PBS without calcium and magnesium, supplemented with sodium pyruvate, glucose, and FCS has been documented by ^{Eldridge-Panuska et al. (2005}). A PBS solution containing bovine serum albumin (BSA) is also reported as an embryo washing/manipulating medium (^{Guignot et al., 2015}). Other washing solutions include the Vigro® holding solution (^{Hudson et al., 2006}; ^{Barfield et al., 2008}) and the Syngro® holding medium (^{Barfield et al., 2009}). The use of DMEM/F12 supplemented with twenty percent of fetal bovine serum (FBS) has also been reported by ^{Ferris et al. (2016}). A solution of Dulbecco’s PBS (DPBS) with ten percent FCS has been employed to wash equine embryos by researchers at the Colorado State University (^{Carnavale et al., 2000}). Another washing medium that has been documented is the M-199 HEPES with ten percent FBS, penicillin, and streptomycin (^{Wilsher et al., 2019}).

For donkeys, the Emcare Holding Solution^® has been frequently described to wash/manipulate embryos (^{Camillo et al., 2010}; ^{Panzani et al., 2012a}; ^{Panzani et al., 2012b}; ^{Panzani et al., 2016}). The Syngro® holding medium is employed as an embryo washing solution by several researchers (^{Bottrel et al., 2017}; ^{Pérez- Marín et al., 2017}; ^{Bottrel et al., 2019}; ^{Bottrel et al., 2020a}; ^{Bottrel et al., 2020b}). Alternatively, the PBS (^{Camillo et al., 2010}), the Ringer’s lactate (^{Camillo et al., 2010}), and the isotonic holding media have been described as embryo washing solutions too (^{Pérez-Marín et al., 2018}).

Micropipette size and puncture of the embryo capsule (EC)

No studies in mares formally correlate the diameter of the micropipette tip used to perforate the EC and its association with the PVSPR. Most experiments where the EC puncture is performed are designed with micropipettes tips equal to or smaller than sixteen micrometers in diameter (umØ); as described by several studies (^{Choi et al., 2010}; ^{Choi et al., 2011}; ^{Hinrichs & Choi, 2012}; ^{Guignot et al., 2015}; ^{Díaz et al., 2016}; ^{Sánchez et al., 2017}). Only a couple of researchers demonstrate the survival of embryos as reflected by pregnancy rates, after puncturing the EC with micropipette tips larger than sixteen umØ (^{Wilsher et al., 2019}). Pregnancies have been reported even after puncturing the EC with twenty-five-gauge needles (^{Ferris et al., 2016}). On the other hand; when working with embryos derived from jennies, they are generally vitrified at the ≤ 300 umØ stage, and hence they required neither punction nor size reduction (^{Panzani et al., 2012}b; ^{Pérez-Marín et al., 2018}; ^{Bottrel et al., 2019}). The Piezo drill micromanipulation system has been described in one study experimenting with large donkey embryos >300 µmØ (^{Ottmann et al., 2018}), and this system employs micropipettes tips in the 7-15 umØ range (^{Choi et al., 2011}; ^{Hinrichs & Choi, 2012}; ^{Hiraoka & Kitamura, 2015}). However, in the work by Ottman and his colleagues, the embryos were not transferred after their warming and only their in vitro survival rate was reported (^{Ottmann et al., 2018}). A recent study has shown the puncture of equine blastocyst with a (< 1µm tip) ultra fine micro-needle (^{Wilsher et al., 2021}).

Cryopreservative solutions employed to vitrify equine embryos

Most protocols employed for the vitrification of horse embryos utilize a mix of penetrating cryoprotectants such as dimethyl sulfoxide (DMSO), ethylene glycol and glycerol. Other protocols include a final vitrification step where penetrating cryoprotectants such as ethylene glycol, or ethylene glycol and DMSO; are mixed with non penetrating cryoprotectants such as galactose or sucrose. For instance, the embryo is placed first in a solution containing a low concentration of glycerol, and then it is removed from this solution and gradually placed in solutions containing increased concentrations of glycerol and ethylene glycol (^{Eldridge-Panuska et al., 2005}; ^{Díaz et al., 2016}; ^{Sánchez et al., 2017}; ^{Pérez-Marín et al., 2018}). On the other hand, the vitrification of horse embryos with media containing ethylene glycol and DMSO is also described (^{Choi et al., 2011}; ^{Sánchez et al., 2017}; ^{Wilsher et al., 2019}). These protocols are started by putting the embryo in a solution with a low concentration of ethylene glycol and DMSO. After the previous initial step, the embryo is subsequently moved into a solution containing higher concentrations of these cryoprotectants. Other cryopreservative liquid solutions used for the vitrification of horse embryos contain a low concentration of ethylene glycol (initial incubating solution), and a final or vitrifying solution. The final solution is composed of galactose with a higher concentration of ethylene glycol (^{Choi et al., 2011}; ^{Guignot et al., 2015}; ^{Ferris et al., 2016}; ^{Guignot et al., 2016}).

For the vitrification of donkey embryos, a protocol has been adapted from the early study in horse embryo vitrification by ^{Eldridge-Panuska et al. (2005}); it typically consists of three steps, with one specific solution for each step. For instance, solution one contains glycerol only; solution two has a mix of glycerol and ethylene glycol; and solution three contains higher concentrations of glycerol and ethylene glycol (^{Panzani et al., 2012}b; ^{Panzani et al., 2016}; ^{Pérez-Marín et al., 2018}). Other donkey embryo vitrification studies were designed taking into consideration the research by ^{Campos Chillon et al. (2009)}. They utilized solutions containing ethylene glycol, and ethylene glycol complemented with galactose (^{Ottmann et al., 2018}; ^{Pérez-Marín et al., 2018}). Others have used an initial solution with a mix of ethylene glycol and DMSO; subsequently, a second solution containing ethylene glycol, DMSO, and sucrose is employed (^{Bottrel et al., 2019}; ^{Bottrel et al., 2020a}; ^{Bottrel et al., 2020b}).

Embryo carriers and loading volumes employed during the vitrification process

The zero-point twenty-five milliliter (mL) capacity non irradiated polyvinyl chloride straw was one of the first embryo carriers described in equine species and has been frequently used thereafter (^{Oberstein et al., 2001}; ^{Eldridge-Panuska et al., 2005}; ^{Hudson et al., 2006}; ^{Hendriks & Stout, 2010}; ^{Choi et al., 2011}; ^{Hendriks et al., 2015}; ^{Sánchez et al., 2017}). Contrary to the polyvinyl straw which uses a large volume of liquid solution during the final vitrification step; the cryolock carrier system allowed researchers to vitrify horse embryos into a small liquid volume (^{Díaz et al., 2016}; ^{Ferris et al., 2016}; ^{Wilsher et al., 2019}). The vitrification of equine embryos using small liquid volumes is achieved with devices such as the open pulled straw (^{Oberstein et al., 2001}; ^{Moussa et al., 2005}; ^{Guignot et al., 2015}; ^{Guignot et al., 2016}), the fibreplug (^{Pérez Marín et al., 2018}), cryoloop (^{Oberstein et al., 2001}), the fine diameter microloader pipette tip (^{Choi et al., 2011}), cryoleaf (^{Scherzer et al., 2011}), hemi straw (^{Sánchez et al., 2017}) and the stripper tip (^{Sánchez et al., 2017}).

Horse embryos can be vitrified utilizing different volumes of liquid solutions. The embryo loading volumes range from fifty (^{Oberstein et al., 2001}) to thirty uL (^{Eldridge Panuska et al., 2005}; ^{Hudson et al., 2006}; ^{Hendriks & Stout, 2010}; ^{Choi et al., 2011}; ^{Hendriks et al., 2015}). Volumes around the three or two uL range have been reported (^{Moussa et al., 2005}; ^{Guignot et al., 2015}; ^{Sánchez et al., 2017}; ^{Pérez-Marín et al., 2018}; ^{Wilsher et al., 2019}). Embryo loading volumes close to or smaller than one uL are described as well (^{Oberstein et al., 2001}; ^{Díaz et al., 2016}; ^{Sánchez et al., 2017}). In donkeys, the zero-point twenty-five mL capacity non-irradiated polyvinyl chloride straw is also reported as an embryo carrier (^{Panzani et al., 2012}b; ^{Fanelli et al., 2020}). Several small volume capacity devices have been recently described for the vitrification of donkey embryos; they include the cryotop (^{Bottrel et al., 2019}; ^{Bottrel et al., 2020a}; ^{Bottrel et al., 2020b}), the open pulled straw (^{Ottmann et al., 2018}) and the fibreplug (^{Pérez-Marín et al., 2018}). Thirty uL of a liquid solution have been used to vitrify donkey embryos (^{Panzani et al., 2012b}); but low volume embryo carriers, employing volumes ranging from three to less than one uL have also been utilized (^{Pérez-Marín et al., 2018}; ^{Bottrel et al., 2019}; ^{Bottrel et al., 2020a}; ^{Bottrel et al., 2020b}).

Liquid solutions used to warm embryos

With small variation between protocols, the media employed to warm horse embryos usually contain a concentration equal to or smaller than zero point three molar of sucrose. The horse embryos are warmed and held for around one minute in this previous medium. Thereafter, the embryos are placed in a second solution with a lower content of sucrose; which is usually close to or lower than zero point fifteen molar of sucrose; finally, the embryos are placed in a liquid medium containing no sucrose during the final step of the warming process (^{Choi et al., 2011}; ^{Guignot et al., 2015}; ^{Ferris et al., 2016}; ^{Guignot et al., 2016}; ^{Wilsher et al., 2019}). A single-step embryo warming protocol was first reported by ^{Eldridge-Panuska et al. (2005}) utilizing a zeropoint five molar galactose solution. The single-step warming protocol has been implemented in other studies also (^{Choi et al., 2011}; ^{Díaz et al., 2016}; ^{Sánchez et al., 2017}). Some researchers warm the embryos using a three-step protocol; in the first step, the embryo is placed in a one molar galactose solution; at the second, it stays in a zero-point five molar galactose; and during the third warming step, the embryo is placed in a liquid solution of zero-point twenty-five molar galactose (^{Campos-Chillon et al., 2009}; ^{Choi et al., 2011}).

As described in horse embryos (^{Eldridge-Panuska et al., 2005}), the warming of donkey embryos is performed with a zero-point five molar galactose medium in a direct onestep warming protocol (^{Panzani et al., 2012b}; ^{Panzani et al., 2016}). Other protocols employ a zero-point twenty-five molar galactose as a warming solution (^{Pérez-Marín et al., 2018}). In some warming protocols, the donkey embryo is consecutively placed in distinct liquid solutions with decreasing sucrose concentrations (^{Ottmann et al., 2018}; ^{Pérez-Marín et al., 2018}; ^{Bottrel et al., 2019}; ^{Bottrel et al., 2020a}). A recent study has compared the quality of post vitrified warmed donkey embryos when using either a three-step sucrose dilution or the one-step sucrose solution protocol (^{Bottrel et al., 2020b}).

Media employed for the intrauterine transfer of equine embryos

Some of the components of the liquid solutions used to transfer equine embryos were first described during the seventies by ^{Oguri & Tsutsumi (1974}). In this study, two different transfer solutions were reported: a saline solution containing two percent gelatin powder and a Ringer’s lactate supplemented with mare serum and antibiotics. There are two post-warming transfer techniques reported by ^{Eldridge-Panuska et al. (2005)}. The first uses PBS plus ten percent FCS as a transfer medium after removing the galactose contained in the warming solution. The second is a direct post-warming transfer technique. The latter consists in the mixing of the vitrification solution containing the embryo (thirty microliters of three-point four molar glycerol and fourpoint six molar ethylene glycol) with two columns of liquid (of zero point five molar galactose in PBS) ninety microliters each (^{Eldridge-Panuska et al., 2005}). In the previous technique, both the vitrification and the warming galactose solutions are mixed into the straw during the warming process, forming one diluted liquid solution containing the embryo that is subsequently transferred into the mare’s uterus (^{Hudson et al., 2006}). The Syngro® holding medium has been reported as an embryo transfer solution as well (^{Barfield et al., 2008}; ^{Sánchez et al., 2017}). French researchers experimented with a culture medium containing a synthetic modified oviductal fluid and used it as an embryo transfer medium (^{Guignot et al., 2015}).

The EmCare Holding Solution^® has been employed to transfer donkey embryos by Italians and Spanish investigators (^{Panzani et al., 2006}; Panzani et al., 2012a; ^{Camillo et al., 2010}). Other reported transfer media are either the PBS or the Ringer’s lactate (^{Camillo et al., 2003}; ^{Camillo et al., 2010}). The Holding Vitrocell^® containing zero point four percent of BSA has been used as a donkey embryo transfer medium by ^{Peña Alfaro et al. (2014)}. An isotonic holding medium (IMV technologies, L’Aigle, France) has been reported as an embryo transfer solution too (^{Pérez-Marín et al., 2018}).