Conservation of Gametes and Use during Assisted Reproductive Technologies in Equine

Abstract

The appropriate selections, modifications and preservation of gametes are essential for the effectiveness of assisted reproductive technologies (ARTs) in equine. Biotechnologies used in reproduction have an impact on equine production and the preservation of species. In order to address this challenge from a variety of angles and areas, sperm selection techniques, oocyte activation protocols, in vitro fertilization, improvements in oocyte maturation, artificial insemination, embryo transfer, and cryopreservation are all forms of ARTs that, when utilized properly, can help manage and conserve equids. Semen is highly individualistic since no two stallions have the same chemical makeup, which causes each to freeze differently. Other stallions might be able to tolerate the cryopreservation process well, while others might be hypersensitive to it. Since ice crystal formation is uncontrolled in the traditional vapour freezing procedure used to freeze semen straws, the crystals could potentially disturb and harm the sample’s cells. Cryopreserved semen by programmable freezer had higher forward sperm progressive motility than semen frozen in liquid nitrogen vapour, both immediately after thawing and again after 3 hours of incubation. This chapter will present the importance of the selection and manipulation of gametes in equine species.

1. Introduction

Conservation of semen and use is essential in equine industry. The current assisted reproductive technologies (ARTs) have an impact on equine production and the preservation of species. Approximately 20% of semen do not resist cryopreservation process well and do not meet the minimum quality requirements after thawing, despite the high quality of fresh stallion semen [1]. The term “poor freezers” refers to stallions whose semen has a low level of freezing stability. The development of an improved cryopreservation method for stallion semen cryopreservation is the subject of increasing numbers of studies [2], although it is unknown why the cryopreservation of equine sperm is less successful than that of other species. The effectiveness of cryopreservation technologies and procedures in equine has to be studied further. The efficacy of the approaches depends on understanding the mechanisms necessary for cryopreservation [3], such as seminal plasma removal, cooling, and freezing rates. These procedures have the potential to harm sperm permanently if not used properly. Sperm cryodamage is caused by inadequate cooling and freezing curves, as well as the toxicity of cryoprotectants (CPAs) [4].

Oocytes and embryos cryopreservation is a critical stage for the widespread conservation of animal genetic resources. The ideal protocol has not yet been developed since oocytes and early embryos are extremely susceptible to cryopreservation steps, despite recent advancements [5]. The embryo morphological criteria, including inner cell number, viability, degree of cellular fragmentation, blastomere symmetry and cleavage stage. The magnitude of the harm, as well as variations in survival and developing rates [6], can be very diverse depending on the species, developmental stage, quality, origin of the oocytes and embryos. All oocytes and embryos sustain significant morphological and functional damage during cryopreservation (for example, in vitro produced or in vivo derived, micro-manipulated or not). Analysis of cumulus oophorus, cytoplasmic and nuclear aspects are criteria used for oocytes quality evaluation.

In the equine industry, embryo transfer has typically been used to produce offspring from mares with limited reproductive potential (mares with undiagnosed subfertility, uterine disease, or simply older mares) [7]. Additionally, embryo transfer is widely used as an effective method of getting foals from mares (donors) without interfering with their competitive careers [8]. In this procedure, a mare (donor) is bred to a stallion or with the use of artificial insemination (AI) and the resulting embryo is then transferred into a mare (recipient), which is reproductively capable and carries the foal to term and until weaning [9]. Initially, a breeding soundness examination is performed on the donor mare to make sure is in good reproductive health, etc. [10].

This book chapter reviewed some of the available literature on the importance of obtaining stallion semen, evaluation of semen and sperm motility parameters, semen cryopreservation, equine oocytes retrieval techniques, in vitro embryo production, different permitting CPAs, oocytes and embryos cryopreservation, warming/thawing of oocytes, embryos and semen and application of assisted reproduction biotechnologies.

2. Semen collection in equine

Semen collection is a tool for breeding soundness evaluation of stallions before or after purchase to diagnose suspected or known infertility. However, if done incorrectly, the procedure might really be the root of low fertility or poor semen quality. It is known that the stallion’s tail (cauda) epididymis can hold enough functionally matured sperm for 10 ejaculates, which are stored there in a metabolic inactive condition to prevent premature activation [11, 12]. All stallions intended for semen collection should get a yearly test for venereal diseases that could be spread way before any semen collection. A wide range of factors influences the ability to collect high-quality semen for semen cryopreservation program or AI in stallions. These factors may be of hereditary or environmental, however, majority are greatly influenced by the management of stallion. Semen quality is an important marker of fertility and reproductive health [13]. Current equine fertility measures are affected by factors not related to the stallion, such as the incidence of conception, pregnancy, and foaling [14, 15]. Conventional sperm evaluations for semen quality include sperm concentration, motility, viability, and morphology despite being important, however, offer limited information about the sperm quality and are not fully predictive of fertilizing capability [16, 17]. Moreover, semen quality may be a reflection of managerial issues as well as individual variance [18].

2.1 Methods of semen collection in equine

In collecting semen from stallions, there are various methods including (a) recovering sperm from the epididymis, (b) artificial vagina, and (c) electro ejaculation [19, 20] for obtaining ejaculates that are acceptable for semen evaluation and processing. Some methods involve just the stallion, while others involves the combination of the stallion and a mare.

2.1.1 Epididymal semen

The epididymis of a stallion can be used to collect semen in the event of injuries necessitating euthanasia, unexpected death, emergency castration or catastrophic illness to preserve genetic material of valued breeding stallions [21, 22]. Method of retrieving cauda epididymal sperm involves aspiration [23], retrograde flushed [24] or sliced [25]. The epididymis tube can be separated from the testis once the tunica vaginalis has been removed, and rinsed with Dulbecco’s phosphate buffered saline (DPBS) at 37°C [26, 27]. Post-mortem testicles can be maintained at a temperature between 4 and 8°C or room temperature (22°C) and transported to the laboratory within 2 hours, without altering the sperm quality [28]. However, it is possible to store and transport the gonads for up to 24 hours prior to performing cryopreservation since most unexpected deaths and emergency castrations take place in locations that are geographically far from the cryopreservation laboratories [29].

2.1.2 Artificial vagina method

The artificial vagina method also has some concerns with animal welfare for stallions (training stress from isolation) and mares (restraining as dummies during collection) [28], yet the animals still need training [30]. This training is necessary for a stallion’s future success as a semen donor. Semen collection using an artificial vagina when the stallion is mounted on a mare in heat or a dummy mount is the most popular method for semen collection from stallions [31]. Semen collection with artificial vagina depends on the collector’s preferred lubricant and challenges faced throughout the semen collection process. Following exposure to lubricant, either of these conditions might have a negative effect on the semen/sperm quality [32].

2.1.3 Electro ejaculator method

A transrectal probe fitted with an electrode is used in the electro-ejaculator method to provide low voltage, current electrical impulses to the male rectum, which causes penile erection, expulsion, and consequent ejaculation [33, 34]. In some species, such as bovine and ovine, electro ejaculation is a common and successful method for collecting semen, however, using it on stallions is not advised due to the risks involved for both the animal and the operator [35]. A similar electrical ejaculator to that used on the bull can be used to collect semen from the stallion if he is anesthetized. This method may be beneficial if an accident strikes and semen needs to be taken from an injured stallion before being euthanized or undergoing risky surgery. However, when used often, these methods trigger a stress response that has negative impacts on animal welfare [36].

2.2 Semen and sperm parameters evaluation

2.2.1 Semen volume

The amount of semen in equine is approximately 50 mL or greater [37]. However, the semen volume tends to vary with species, breed, age and environment [38]. Semen volume and sperm concentration in stallions vary according to season and photoperiod [39]. When breeding mares using AI, an adequate fertility can be obtained with as little as 1/2 mL of semen [9]. Despite the rising desire for breeding stallions to compete at the highest levels, little research has been undertaken to determine how equestrian disciplines and competition level affect semen quality and quantity [40]. Moreover, semen volume is not thought to have a direct impact on fertility [41].

2.2.2 Semen colour

The collected semen sample can be milky white, evenly turbid, free of clots and odorless [37]. Consistency changes of semen colour, from creamy or milky to watery, considered is an indicator of low sperm count/ concentration (oligozoospermia) or the complete lack of sperm (azoospermia) [42]. The physiological colour of the stallion semen is pale or whitish-grey.

2.2.3 Sperm motility and velocity parameters

To improve the sensitivity of diagnostic tests for stallion fertility, emphasis has been placed on assessing the functionality of these sperm cells in vitro with specific reference to their motility and their capacity to engage in the complex cascade of cellular interactions that culminate in fertilization [43]. The introduction of computer aided semen analysis (CASA) systems to objectively quantify the various elements of sperm movement has improved the diagnostic power of sperm motility assessments [44]. An analysis of sperm motility plays a significant role in determining fertilizing potential, as sperm motility is only one attribute of the sperm [45]. The objective of estimating sperm motility is to determine the percentage of motile and the proportion of progressively moving sperm [46]. The CASA technology was developed in 1980s for analyzing sperm motility and velocity parameters and has been successfully utilized in enabling this field of research focus [44]. Therefore, sperm motility should be evaluated alongside other parameters such as sperm velocity, morphology and viability, when calculating the fertility potential of the sperm [47].

2.2.4 Sperm morphology

An analysis of sperm morphology plays a significant role in any breeding soundness study [48]. Sperm morphology analysis requires specialist tools, the technician’s knowledge, experience and frequent time and patience [49]. Due to its moderate-to-high connection with stallion fertility, sperm morphology stands out as one of the most significant sperm assays [50]. The current classification approach involves keeping track of the prevalence of sperm morphologic abnormalities [47]. However, the presence of several abnormalities on a single sperm is a sign of a more serious disruption of spermatogenesis and may indicate a worsening reproductive outlook [51]. The increase in number of head defects, pyriform heads, nuclear vacuoles, midpiece defects and droplets in sperm is abnormal [52].

Threshold values for the various categories of sperm morphology analysis often fall into one of the following ranges: <30% sperm head abnormalities, <25% proximal cytoplasmic droplets, <10% premature germ cells, 30% morphologically normal sperm [53]. The Society for Theriogenology’s most recent recommendations for evaluating the breeding soundness of stallions are used to pick stallions, when bred artificially or spontaneously and may produce at least 75% of 40 or more pregnant mares or 120 or more mares. Some authors have acknowledged the division of sperm abnormalities into major and minor categories, claiming that major flaws lead to early embryonic mortality or preclude conception [54].

2.3 Semen extenders and composition

The improvement in cryopreservation techniques can be obtained by optimizing temperature curves, incubation duration and also extender composition. Currently, the majority of the ingredients in the semen extenders currently in use include micellar milk proteins, egg yolk as a source of phospholipids, antibiotics, and glycerol as CPA (Table 1) [59]. With a maximum concentration of 3.5% glycerol is still the CPA that is typically utilized for better outcomes [1]. Glycerol turns harmful to equine sperm once it exceeds this threshold and interest in amides has grown recently [60]. Despite the lower permeability and higher molecular weight of glycerol, formamide and dimethylformamide have a lower molecular weight and higher permeability [61]. On the viability and motility of stallion sperm, fast addition and removal of CPAs have detrimental effects. In other studies, the use of protein-based treatments or modified amino acids in place of glycerol produced positive results [62, 63].

HF-20 [55]	Skim milk-egg yolk [56]	Modified glucose-EDTA-lactose [57]	Tris [58]	EquiPlus [58]
Glucose 5 g	Sucrose 9.3 g	Glucose 6.0 g	Tris 2.42 g	Part A: 95 mL medium
Lactose 0.3 g	Skim milk 2.4 g	Sodium citrate dehydrate 0.37 g	Citric acid 1.34 g	Part B: 5 mL egg yolk-glycerol component
Raffinose 0.3 g	Streptomycin 0.025 g	Disodium EDTA 0.37 g	Fructose 1 g	Minitube-Animal Reproduction Technologies (Germany)
Sodium citrate 0.15 g	Penicillin 25,000 IU	Sodium bicarbonate 0.12 g	Streptomycin 0.08 g	—
Sodium phosphate 0.05 g	Egg yolk 8 mL	Streptomycin 0.08 g	Glycerol 7 mL	—
Potassium sodium tartrate 0.05 g	Glycerol 3.5%	Benzyl penicillin 0.08 g	Egg yolk 20 mL	—
Egg yolk 10%	—	Double distilled water up to 100 mL	Deionized water (made up to 100 mL)	—
Penicillin 25.000 IU	—	Egg yolk 20 mL	—	—
Streptomycin 0.08 μg	—	Glycerol 5%	—	—
Glycerol 3%	—	—	—	—
Deionized water (made up to 100 mL)	—	—	—	—

Table 1.

Semen extenders applied to equine semen during cryopreservation.

EDTA = Ethylenediaminetetraacetic acid.

2.4 Type of cryoprotectants used for cryopreservation of equine semen

Many different CPAs and commercially accessible CPAs have been developed in response to market demands (Table 2). According to the latest theory, CPAs function by reducing exposure to osmotic stress, stabilizing biomolecules and their structures and limiting the impacts of reactive oxidative species (ROS) [65, 66]. The majority of CPAs exhibit some sperm toxicity. Changes in sperm motility after addition and removal of glycerol CPA may be associated with disruption of plasma membrane integrity and disruption or loss of mitochondrial membrane potential [65]. Numerous steps can be taken to reduce this toxicity; by reducing the CPA concentration as much as possible [67]. Penetrating CPAs work inside cells to replace cellular water when it is pushed out into the extracellular space, thereby preventing the production of internal ice crystals that might potentially burst the membrane [64]. The initial phase of freezing, which typically ranges from −10 to −20°C, is when non-penetrating substances or macromolecules capitalize on the elevated concentrations in the extracellular regions to osmotically drain water from the cells [68].

Penetrating cryoprotectants	Non-penetrating cryoprotectants
Dimethylsulphoxide	Egg yolk
Glycerol	Sugars
Methylformamide	Liposomes
Dimethylformamide	Milk proteins
—	Polymers

Table 2.

Examples of penetrating and non-penetrating CPAs [64].

3. Cryopreservation methods for equine semen

3.1 Controlled-rate method

The controlled-rate method (programmable freezer) is the alternative approach since it preserves cellular integrity by achieving the ideal freezing rate [69]. To prevent the formation of intracellular ice crystals, an ideal rate must be slow enough to allow for sufficient cellular dehydration while being quick enough to prevent sperm from being exposed for an extended period to solutions that are overly saturated with the extracellular environment [70]. Although automated systems for maintaining temperature during cryopreservation have been developed, they are might be expensive [71]. In this regard, several procedures combining various storage quantities and freezing rates have been created and put to the test to increase the effectiveness of cryopreserved equine semen and lessen the harm caused by cryopreservation [72].

3.2 Liquid nitrogen vapour method

The Styrofoam^® box might have offered a more variable freezing rate than the programmed freezer because the liquid nitrogen level inside the box is commonly assessed subjectively, subject to evaporation, and challenging to standardize for each freezing rate [73]. The quantity of straws used and the removal of the lid may both affect the temperature of the vapour inside the Styrofoam^® box and consequently, the cooling rate during subsequent freezing cycles [74]. The key benefits of the Styrofoam^® box freezing technology include affordability, usability, low liquid nitrogen requirements, and portability. In comparison, the programmable freezer is costly, consumes a lot of liquid nitrogen, and it might be permanently installed in the lab. For the other sperm quality factors looked at, there were no differences between the freezing techniques.

3.3 Thawing temperatures for equine semen

For stallion semen, various thawing techniques are utilized, however, it is uncertain which technique (46°C for 20 s for 0.5 mL straws, 46°C for 12 s for 0.25 mL straws, 37°C for 30 s for 0.5 mL straws, 37°C for 60 s and 38°C for 60 s, etc.) is best [75]. Different thawing temperatures must be evaluated in order to determine the optimal temperature for equine sperm and whether the thawing temperature affects sperm quality. The lower temperature (37°C) is more manageable because thawing at the higher temperature (46°C) needs specific equipment and must be done at precisely the right interval to prevent harm to the sperm [73].

4. Synchronization and artificial insemination in equine

Oestrous synchronization is important in equine AI programs. The use of hormonal protocols for synchronization of oestrus and ovulation has increased as a result of AI in the past [76].

4.1 Oestrous synchronization programs

Synchronization of mare oestrus using prostaglandin F2α [77] or progesterone releasing intravaginal devices insertion [78] or in combination with estradiol lowers financial costs and improves the effectiveness of AI by enabling AI to be applied on specified days and times. These protocols usually include the injection of human chorionic gonadotrophin (hCG) or a gonadotrophin-releasing hormone counterpart. The hCG can be utilized to induce ovulation in mares, since it stimulates luteinizing hormone (LH) receptors in the granulosa cells of the follicles [79]. The efficiency and use of hCG as an ovulation trigger have been proven since it stimulates the final follicular maturation and ovulation and exhibits an LH-like function [80, 81].

Mares undergo seasonal cycling in the spring and summer as a result of the longest period of daylight since they are polyestrous [82, 83]. With one to two follicular waves every cycle developing. The period between ovulations in equine species is around 22 days [77, 83]. Which might be influenced by breed and reproductive stage (e.g., lactation days vs. non-lactating days). From a financial point of view, the breeder’s most efficient strategy is to induce and detect ovulation, inseminate at the time closer to ovulation, and use the lowest semen doses (one straw of 0.5 mL of frozen semen or one vial of 20 mL of cooled semen) for less than the cost of one pregnancy, per successful insemination [84].

4.2 Artificial insemination

One of the most effective reproductive technologies for equine breeding is which improves the genetics of the herd and the usage of stallions [76]. An effective use of AI in equine breeding programs can enhance bloodlines available for successful competition horses and significantly improve operating efficiency. The timing of insemination is critical for breeding profitability when using fresh or frozen-thawed semen. Since semen deposition in the mare reproductive tract can occur either too early or too late, the process not only waste important time but also the costs to purchase semen and mare preparation for insemination [84]. Therefore, insemination should take place right before or after ovulation to enhance pregnancy rates [35].

When fresh and cryopreserved sperm are used for AI in mares, a normal physiological inflammatory response takes place that is characterized by rapid migration of polymorphonuclear neutrophils within the uterine to clear bacteria and semen [85, 86]. Moreover, it is of great importance that AI using stallion epididymal sperm results in satisfied pregnancy rates while using low doses of sperm, to make use of all the semen that is in limited supply [35]. Cryopreserved semen has previously been reported to have lower pregnancy rates than fresh or cooled semen. According to more recent research, pregnancy rates with cooled semen are comparable to or even better than those with fresh semen, with little evidence of additional problems [87]. However, stallions are usually selected only on their performance in competitions, without consideration of fertility or semen quality. Therefore, the variable pregnancy rates related to equine AI are explained by this [88].

5. Equine oocytes collection

Oocytes may be collected from ovaries excised from mares in which ovariectomy has been performed, or who have died, in order to preserve valuable genetics that would otherwise be lost [89]. There are currently two approaches to oocyte collection. One approach is the collection of mature oocytes. This is done by aspirating a large dominant follicle right before it ovulates and typically only one follicle is aspirated per cycle. The other approach is the collection of immature oocytes. In this approach all the follicles that are present on the ovary at the time of the procedure can be aspirated regardless of size because the mare does not need to be cycling [89]. Oocyte collection from mares can involve either ovum pick-up (OPU)/ transvaginal ultrasound guided follicular, aspiration and slicing method.

5.1 Ovum pick-up method/transvaginal ultrasound guided follicular aspiration

Despite being a pricey process, ovarian pick-up is increasingly preferred for the collection of equine oocytes. The OPU often referred to as oocyte aspiration or oocyte collection, is the procedure used to transvaginally aspirate oocytes straight from a mare’s ovaries. In this procedure, the ovary is visualized using a transvaginal ultrasound probe, and a long needle is passed through a guide in the ultrasound handle, through the wall of the vagina, and into the follicle. The follicle is then flushed several times and the oocyte is aspirated out of the follicle.

5.2 Slicing method

The most popular method for getting more oocytes of higher quality per ovary is by slicing the ovaries. With the aid of forceps, ovaries are typically put in a petri dish containing a collection medium. Using a scalpel blade, incisions were made along the entire ovarian surface. After that, the medium and the collected cumulus oocyte complexes (COCs) are transferred to a petri plate. When collecting the COCs by puncture and slicing methods, the ovaries are kept completely dipped in the medium.

6. In vitro embryo production in equine

6.1 In vitro maturation

In vitro oocyte maturation (IVM) may be simply performed by placing immature oocytes into the culture. The oocyte’s developmental competence (ability to produce blastocysts in vitro) following fertilization, however, indicates that a variety of factors influence both the rate of nuclear maturation and the acquisition of cytoplasmic maturation. Oocytes retrieved after a delay of 5–9 hours require more time to culture and have lower developmental competency than those recovered immediately after slaughter [90]. It has to be noted that the base media more commonly used for equine IVM are tissue culture medium 199 or Dulbecco’s modified eagle medium/nutrient mixture F-12 (DMEM/F-12), which are generally chosen depending on the preferences of the laboratory where IVM is performed, and core differences exist among them [91]. Furthermore, none of these media have been developed specifically for equine IVM; instead, they were developed for cell culture, albeit equine COCs are capable of maturing with similar efficiency in either medium [92]. To try to better understand the physiological conditions that equine COCs require and improve current IVM conditions, several reports have tried to address the metabolic requirements of equine COCs in vitro, the differences between the proteomic profiles of equine COCs maturated in vivo or in vitro [91], or the differential expression and localization of glycosidic residues in equine COCs matured in vitro vs. in vivo, among other approaches. All these reports have revealed a specific metabolic profile of equine COCs matured in vitro and important differences between equine COCs matured in vitro vs. in vivo [93].

6.2 In vitro fertilization

In terms of assisted reproductive technology, in vitro fertilization (IVF) is the most successful method. In IVF, mature oocytes are removed from ovaries and fertilized in a laboratory using sperm. The most popular treatments for infertile animals are intracytoplasmic sperm injection (ICSI) and IVF. However, the transfer of many embryos and multiple births, undermine the success of IVF/ICSI treatments. Since we now understand a large number of the genes that control COCs growth, we may be able to use these genes as markers to distinguish between healthy and damaged oocytes during IVF. Oocyte IVM has promise as a substitute for IVF, but oocyte quality is still a problem, necessitating the development of new methods in the coming years. Establishment of optimal methods for equine embryo culture has been slow when compared to some domestic species [94]. In part, this delay was caused by the failure of standard IVF techniques in mares and also the scarce availability of abattoir ovaries and the lack of interest from horse breeders and breed associations have been the main reasons for this delay [94].

The fertilization rate is measured 16–18 hours following insemination or ICSI. Presumptive zygotes are cultivated in a specifically designed culture medium to support their growth. On the second and third days after retrieval, they will be assessed. If there are enough embryos that show good growth and development, they can be chosen to develop in a specially created culture medium until they reach the blastocyst stage. The benefits of blastocyst culture are numerous. Fewer embryos can be transferred on day 5 to lower the possibility of multiple pregnancies because the potential for implantation of embryos at this point is higher. Low number of embryo quality reduces the odds for good blastocyst development. Therefore, cycles with a day 3 embryo transfer are advised [95].

6.3 In vitro culture

The development of ICSI for the assisted fertilization of equine oocytes has resulted in a renewed interest in establishing optimal methods for embryo culture [94]. Currently, ICSI-produced equine embryos are cultured using media designed for other species or other cell cultures and typically, with the addition of serum. Although systems specifically for equine embryo culture still have not been established, ICSI-produced embryos are developmentally competent and capable of producing live offspring [96]. To promote the development of an embryo, two different types of culture media are available: sequential, or two-step, media and single step media. The key distinction between the two methods is whether the culture media is changed or cryopreserved between the fertilization check and embryo transfer. There are variations amongst laboratories in terms of preferred media, medium additions and IVF techniques [96]. A culture media system is employed in the lab to support the metabolic and physiological phases of embryo development in vitro prior to implantation. Equine blastocyst production can be obtained by initial culture in a modified synthetic oviductal fluid, followed by transfer to DMEM/F-12 at approximately day 5.

7. Oocytes and embryos cryopreservation in equine

7.1 Oocytes cryopreservation

There are many ways to evaluate the success of oocyte vitrification, including recovery rate (the number of oocytes discovered after thawing/warming), survival rate (the number of oocytes with an intact plasma membrane and zona after thawing/warming), maturation rate if they were vitrified at an immature stage, cleavage and blastocyst rates after IVF [97]. When equine oocytes are vitrified at the germinal vesicle stage, it appears that a variety of vitrification techniques can support high maturation rates for example, 61% [98], 48% [99], and 46% [100]. Blastocyst rates per injected metaphase II oocyte have only been 7% [99], 10% [100], and 15%, after ICSI of vitrified-warmed equine immature oocytes that have matured in vitro after warming. However, very few studies have shown blastocyst production following ICSI [101].

7.2 Embryos cryopreservation

Small in vivo-derived equine embryos (under 300 mm) can be successfully cryopreserved by gradual freezing or by vitrification, with pregnancy rates of 45–67% following thawing/warming and transfer [102]. The usual size of equine embryos created by ICSI and embryo culture is similarly around 300 mm, and gradual freezing or vitrification of these embryos yields continuous pregnancy rates comparable to those for fresh in vitro production embryos [103].

7.3 Cryopreservation methods for equine oocytes and embryos

7.3.1 Slow-freezing method

During the slow-freezing method, the embryos are exposed to a cryoprotective substance gradually (i.e. stepwise) before being properly cooled in stages (Table 3). Glycerol (10%: 1.36 M) is the “traditional” CPA for slow-freezing equine embryos, and the embryo is equilibrated to it by incubating it in 2–4 solutions in increasing concentration [5]. There are two threats that the embryo is vulnerable to during slow-freezing which are ice crystal formation and dehydration. These approaches slowly dehydrate cells during freezing and prevent the development of intracellular ice crystals by cooling at controlled rates, typically between 0.3 and 0.5°C/min [104]. The extracellular fluid that is left behind becomes more osmolarity as cooling progresses because extracellular ice slowly develops while leaving behind solutes. Since the intracellular fluid has not yet frozen, water that flows out of the cells dehydrates them to balance the quantities of intracellular and external solutes. Slow-cooling techniques have not been routinely used in commercial programs for equine embryo transfer, except for Hinrichs [105], who successfully transferred frozen and thawed embryos of less than 220 m in diameter and recorded pregnancy.

Conventional slow-freezing method	Vitrification
Standard 0.25 mL freezing straws	Numerous devices for loading embryos and oocytes (conventional straws, OPS, cryoloops, cryoleaf)
Minimal cryoprotectant concentrations	High cryoprotectant concentrations/reduced volume and time with vitrification solution
Seeding at −5 to −7°C, controlled slow cooling (0.1–0.3°C/min)	Ultrarapid cooling rates (−2500°C/min or 20,000°C/min using OPS or cryoloop)
Plunging at −30 to −70°C and storage in liquid nitrogen (−196°C)	Plunging into liquid nitrogen (−196°C)

Table 3.

Oocyte and embryo cryopreservation methods [6].

OPS = Open pulled straw.

7.3.2 Vitrification method

The main alternative to slow-freezing is vitrification, or ultra-fast freezing, which causes an immediate change in the liquid state of both intracellular and extracellular fluids to a solid, glass-like phase (or “solidification”) without ice formation [5]. However, solidification is only possible when very high CPA concentrations are used (about 4–5 times greater than for slow-freezing) and when the temperature is dropped drastically, i.e. via submersion in liquid nitrogen [106]. Standard 0.25 mL straws generate a cooling rate of about 2500°C/min when submerged in liquid nitrogen, but the freezing rate can be increased even further to about 20,000°C/min [5], by lowering the volume of the medium surrounding the embryo (open-pulled straw technique), stretching the straw to reduce its internal diameter, or using cryotops or cryoloops [107]. Rapid passing through the severe cryoinjury zones is ensured by the faster cooling rate, which also lowers the necessary CPA concentration. Oocyte and embryo cryopreservation protocol are expressed on Table 3.

The main disadvantage of vitrification is the toxicity due to the high quantities of CPA utilized in embryos or oocytes [108]; therefore, it is crucial to strictly follow the suggested intervals for immersion in the different solutions, especially the final solution with the highest concentration of CPA [107]. This is more challenging than it appears since solutions with high CPA concentrations are highly dense and the embryo sinks unexpectedly slowly; success with vitrification consequently requires a skilled personnel in identifying and manipulating embryos in dense solutions [109].

7.4 Thawing temperature for equine oocytes and embryos

To avoid water recrystallizing during thawing, which could lead to damage to ice crystals, a rapid temperature change is desirable. Great care must be taken to prevent osmotic shock from the penetrating CPA, which is now present in the intracellular region in very high concentrations [106]. To avoid this, a second non-penetrating CPA is being utilized. The concentration of the non-penetrating CPA steadily increases as the penetrating CPA diffuses out of the oocyte and this process continues until the oocyte is placed back into the usual culture media [6].

8. Embryo transfer in equine

During embryo flushing, the entire uterine lumen is allowed to fill by gravity flow with flushing medium, such as DPBS either supplemented with a protein source (fetal calf serum or bovine serum albumin) to prevent the embryo from sticking to the silicone or plastics of the flushing tool. Currently there is commercially ready-to-use equine embryo flushing media. After filling with flushing medium, the uterus is gently massaged on the rectum to ensure that the entire lumen is flushed.

On day 7 after ovulation, the donor mare’s embryos are typically removed [7]. According to Brinsko et al. [47], embryos are harvested 1 day later if the mare was bred to frozen semen as opposed to fresh or cooled semen because the mare embryo grows incredibly quickly from a morula to an expanding blastocyst (day 5–7), which results in unique characteristics defining embryo development. The use of surgical transfer to ARTs is restricted, according to studies, because nonsurgical transfer has resulted in pregnancy rates greater than 75% per embryo transferred at day 15 for more than 8 years [9]. Additionally, on day 9 or 10 of gestation, the recipient mare is checked for pregnancy for the first time, and on day 16 of gestation (9 days after transfer), a clear positive or negative result is known [110]. Moreover, recipient mares have pregnancy rates, which are roughly 75–80% at 14 days and 6–70% at 50 days [96]. The succeeding of mare embryo transfers from 1998 to 2022 are shown in Tables 4 and 5.

Grade	Pregnancy/transfer	Success rate (%)
1–2	2.921/3.426	85.3
3	99/154	64.3
Total	3.020/3.580	84.4

Table 4.

Success for in-clinic (on-site) transfers, 1998–2022 [111].

Grade	Pregnancy/transfer	Success rate (%)
1–2	3.266/3.866	84.0
3	212/399	53.1
Total	3.478/4.285	81.2

Table 5.

Success for transported transfers, 1998–2022 [111].

Young maiden mares or mares who have recently given birth to a foal to term are the ideal recipient mares; an older maiden mare or a mare with a history of subfertility would not be good choices as a recipient for a valuable embryo [7]. In addition, embryo transfer can be done on mares that are 2 or 3 years old or yearlings to help them start producing 1 or 2 years earlier than with traditional breeding [47]. The recipient mare should have ovulated 1 day before the donor mare to 3 days after the donor mare when choosing the right recipients for a given donor mare [10]. Prior to transfer, the recipient mare should be examined by palpation and ultrasonography per rectum; the mare should have healthy uterine tone and a tightly closed cervix, which are signs of an acceptable level of circulating progesterone [47].

9. Conclusion

Cryopreservation of embryos has the potential to significantly improve equine embryo transfer’s adaptability and lower its expenses. Furthermore, given sufficient operator skill and either gradual freezing or vitrification, cryopreservation of tiny embryos can be reasonably successful. Other stallions’ sperm might be able to tolerate the cryopreservation process effectively, while others might not.

Acknowledgments

The Agricultural Research Council is acknowledged for funding the running costs and the Germplasm Conservation, and Reproductive Biotechnologies for support.

Conflict of interest

There are no conflicts of interest.