CRYOPRESERVATION OF IN VITRO -PRODUCED BOVINE EMBRYOS BY VITRIFICATION: IN PURSUIT OF A SIMPLIFIED, STANDARDIZED PROCEDURE THAT IMPROVES PREGNANCY RATES TO PROMOTE CATTLE INDUSTRY USE

: The goal of cryopreservation is to retain the original stage of gametes and embryos after they have endured cooling and warming. Slow freezing is a standard method for in vivo -derived bovine embryo cryopreservation, three-fifths of such embryos being frozen by this method globally. However, it is evident that slow freezing is not efficient for cryopreserving in vitro -produced bovine embryos. Hence, only one-third of in vitro -produced bovine embryos are cryopreserved. Vitrification is a preferred method for storage of human embryos; consequently, it has been explored as a novel means to store in vitro -produced bovine embryos, for which it shows considerable promise as an alternative to slow freezing. This is due to several reasons: vitrification is often less time-consuming than slow freezing; it does not need expensive slow rate freezing machines; and it has been proven to have comparatively higher survival rates. Yet, in the cattle industry vitrification continues to present shortcomings, such as possible toxicity of vitrification solutions and failure to standardize methods, which pose a challenge for its application to in vitro -produced bovine embryos. Therefore, determining the most suitable procedure is crucial to make vitrification more practical in commercial settings.


Introduction
In commercial breeding of farmed livestock embryo transfer is an important tool to increase the number of elite animals in a herd or for herd replacement. In the cattle industry embryos are generated by means of either in vivo or in vitro production. With the in vivo method, bovine embryos are generated by a superovulation technique in which donors are given exogenous gonadotropin hormones in order to stimulate the activity of their ovaries. As a result, more oocytes are able to develop to maturity, when they are receptive to fertilization by sperm. When oocytes are fertilized in the oviduct of a cow, they migrate to the uterus and develop to the morula and blastocyst stage; hence these are known as in vivo-derived embryos. The alternative route of embryo production, in vitro to in vivo, involves the aspiration of either mature or immature oocytes from donor ovaries, in vitro fertilization, usually with frozen sperm, and subsequent in vitro culture to the blastocyst stage. This means of embryo production has been established since the 1980s but as late as the early 2000s the great majority of bovine embryos were still generated by the in vivo method (Merton et al., 2009).
The last decade has witnessed a pronounced shift in the practice of bovine embryo transfer such that the most recently published statistics indicate that globally the number of in vitro-produced embryos exceeded that of their in vivoderived counterparts (742,908 to 386,133 recorded transfers, respectively) (Viana et al., 2019). This trend particularly reflects the increasing reliance on in vitro technologies in North America and South America, the two continents that are together responsible for the majority of the world's bovine assisted reproduction industry. In all regions, in vitro-produced embryos were transferred predominantly fresh (overall 73.2%) (Viana et al., 2019). Bovine embryos can be transferred into the uterus of recipients on the same day that they are harvested, when they are termed 'fresh embryos'. Typically, remaining embryos that are not transferred immediately are retained by cryopreservation for future use.
Embryo cryopreservation has become an essential aspect of assisted reproductive technology (Leibo, 2008). The proportion of frozen in vitro-produced bovine embryos transferred in 2018 decreased compared to 2017 (26.8 vs. 33.9%, respectively), breaking an annual rise observed since 2013. On the other hand, more in vivo-derived frozen-thawed embryos were transferred (60.1%) compared to in vivo-derived fresh embryos (Viana et al., 2019). Development of the cryopreservation procedure in cattle, as for all mammals, is facilitated by, and is closed associated with, the success of cryopreservation of sperm, oocytes and embryos (Vajta and Nagy, 2006). Slow freezing and vitrification are the two recognized methods for cryopreservation of each of in vivo-derived and in vitroproduced bovine embryos. Among beef cattle, animals produced from in vitroproduced embryos greatly exceed those from in vivo-derived embryos. This is due to the intrinsically large number of follicles in beef breeds such as Nelore (Pontes et al., 2009).
It is apparent that understanding the cryopreservation process and identifying a simple but reliable method for in vitro-produced bovine embryos is necessary in order to enhance the efficiency of their production and transfer in on-farm contexts. While the published literature covers a broad scope of bovine embryo cryopreservation, including the standard slow freezing procedure, this review focuses primarily on vitrification as a method to cryopreserve in vitro-produced bovine embryos. It also examines theoretical aspects in regard to ice crystal formation, cooling/warming rates, and roles of cryoprotectants in protecting cells from the chilling process. Furthermore, the specific demands for storage of in vitroproduced bovine embryos are considered. Efforts to raise the efficiency of vitrification through modifications to in vitro culture and enhancements in the capacity of in vitro-produced embryos are also discussed. Finally, biosafety issues relating to vitrification are addressed.

Different methods of freezing embryos
Of the two methods of bovine embryo cryopreservation slow freezing was developed in the 1970s whereas vitrification was realized a decade later and has seen a rapid period of more recent development (Hasler, 2014). The majority of in vivo-derived embryos are generated in donors with the aid of exogenous gonadotropin hormones and are then frozen. van Wagtendonk-de Leeuw et al. (1997) reported that these two methods of cryopreservation led to similar conception rates of recipients. Hasler (2014) affirmed that on occasion vitrification is recommended for cryopreservation of in vivo-derived embryos yet maintains that slow freezing yields similar pregnancy rates among recipients.
A major objective of cryopreservation is to minimize intracellular ice formation. Kleinhans and Mazur (2009) proposed that during the slow freezing process, survivability of cells is dependent on a cooling rate slow enough for unfrozen intracellular water to dehydrate by osmosis to near the equilibrium level before reaching the temperature at which intracellular nucleation occurs. In contrast, Kim et al. (2012) andLawson et al. (2012) each argued that vitrification negates any risk of intracellular ice crystals forming.

(a) Slow freezing
In North America 70% of superovulated bovine embryos are slow frozen, indicating that if a programmable freezer is available this method is preferred (Hasler, 2014). As an example, Hasler (2001) demonstrated a high overall pregnancy rate of 56.1% for recipients implanted with in vivo frozen embryos. Furthermore, Lopatarova et al. (2010) reported a conception rate of 48.8% for recipients implanted with frozen and biopsied in vivo embryos; for recipients of intact and frozen in vivo embryos the conception rate was 50.7%. Bovine embryos are standardly frozen and stored in a straw with 1.8 M ethylene glycol (EG) as cryoprotectant so enabling the possibility upon thawing of direct transfer to a recipient cow (Dochi et al., 1995;Voelkel and Hu, 1992). The success of this method for in vivo-derived embryos has paved the way for its commercial use on cattle herds worldwide.
Although slow freezing is considered the conventional method of storing in vivo-derived embryos, several studies have shown it to be inefficient at cryopreserving in vitro-produced bovine embryos (Do et al., 2017;Enright et al., 2000;Mahmoudzadeh et al., 1994;Mucci et al., 2006;Nedambale et al., 2004;Yu et al., 2010). Prevention of intracellular ice formation is a key factor in the survival of cryopreserved cells (Mazur et al., 2005;Mazur et al., 2007). Ice crystals forming during cooling and warming processes are the main cause of cell damage and death Mazur and Seki, 2011). Seki et al. (2014) observed extracellular ice crystal formation initially during the slow freezing process. An additional difficulty of cryopreserving in vitro-produced bovine embryos is that they contain more lipid droplets in their cytoplasm than do their in vivo counterparts (Abe et al., 1999;Pryor et al., 2011), leading to greater susceptibility to the freezing process (Pereira et al., 2008;Seidel, 2006).

(b) Vitrification
Characterized by rapid cooling/warming rates and high concentrations of cyoprotectants (Jin et al., 2008), vitrification is an alternative procedure to slow freezing for in vitro-produced embryo cryopreservation. Wowk (2010) stated that cells -cryopreserved by slow freezing -survive in glass between ice crystals, while cells -cryopreserved by vitrification -survive in glass as the whole sample is vitrified. In fact, since vitrification has become the main cryopreservation method to store human embryos (Vajta et al., 2015), it has also been recommended for cryopreservation of in vitro-produced bovine embryos (Mucci et al., 2006;Hasler, 2014;Taylor-Robinson et al., 2014).
Vitrification is a technically simple method (Do et al., 2016a;Rios et al., 2010), which does not require the use of a programmable freezer (Hasler, 2014;Sinha, 2009;Vajta and Nagy, 2006). Laboratory experiments have indicated that in vitro-produced bovine embryos cryopreserved by vitrification achieve a better survival rate than do those cryopreserved by slow freezing (Do et al., 2017;Gómez et al., 2008;Mucci et al., 2006;Yu et al., 2010). Vitrification has not yet, however, achieved widespread application in commercial cattle production. This is because most embryos are produced by superovulation, and conventional slow freezing has already become a standard method to store in vivo embryos, while well over half of the transfers in both Europe and the USA are from frozen embryos (Table 1). In contrast, there is a smaller proportion of embryos produced by in vitro fertilization, especially in Europe (Table 1). Most fresh in vitro-produced embryos are transferred due to the lack of a reliable cryopreservation method (Pontes et al., 2011), and of an efficient method that allows the directly transfer of those embryos after being thawed (Sanches et al., 2016).

Limitations of current vitrification protocols
Cryobiologists have progressively modified vitrification procedures to achieve incremental improvement but shortcomings remain, and, unlike with the slow freezing technique, no standardized method has been agreed (Leibo, 2008;Seidel, 2006). There is a profusion of different vitrification protocols utilizing varied timings, temperatures and cryoprotectants, so the outcome is multifactorial. In addition, the operator has a choice of vitrification carriers (for example, plastic straw, electron microscope grid microdrop, open pulled straw, cryotop, cryohook), with drop-size and straw barriers affecting cooling and warming rates. Warming protocols can differ too, which adds further to the complexity of choice for an efficient vitrification method (Vajta et al., 2015); this is a particular concern when vitrified samples are exchanged between laboratories. However, perhaps the most influential factor to consider when developing a highly successful vitrification method is the skill of the operator. Although described as a simple method, not requiring slow-rate freezing machines, in our experience vitrification actually requires much more skill and dexterity of the operator compared to slow freezing (Do et al., 2019).
While vitrification appears more favourable than slow freezing, further investigation is required to compare its efficacy with the slow freezing method (Sinha, 2009). Moreover, although limited data on pregnancy rates are available (Gutnisky et al., 2013;Kim et al., 2012;Nedambale et al., 2004), the reported sample sizes were relatively small. For instance, Gutnisky et al. (2013) transferred vitrified embryos to recipients and obtained conception rates of 46.8%. However, the number of embryos transferred, 96, was modest and thus may not reflect the Van Huong Do et al. 256 potential efficacy of the vitrification method. Of 45 confirmed pregnancies 33 calves were born (34.3%) (Gutnisky et al., 2013), which is not a promising outcome under commercial conditions where the ultimate goal of an embryo transfer program is to achieve a high number of healthy calves (Do et al., 2018a). Previously, Enright et al. (2000) reported that abortion rates of surrogate cattle receiving in vitro-produced embryos are raised, and that care should be taken with pregnancy interpretation.
Considerable attempts aimed at improving the quality of in vitro-produced embryos before cryopreservation have been made in various ways, such as modification of in vitro culture systems (Do et al., 2016b;Sanches et al., 2013;Sudano et al., 2011), enhancement of the intrinsic capacity of embryos (Filho et al., 2011), and other means of pretreatment of embryos before vitrification (Min et al., 2013). Nonetheless, a significant advancement has so far proved elusive.

Impact of cooling rates
During slow rate cooling of embryos the temperature drops at a rate of approximately 0.5°C per minute to -30°C or -35°C, at which point they are plunged into liquid nitrogen (Leibo, 2008). For vitrification, by comparison, cooling rates of thousands of degrees Celsius are attained, such that a sample containing embryos can reach extremely rapidly a glassy state. Two crucial characteristics of vitrification are extreme cooling rates and high concentration of cryoprotective additives (Seki and Mazur, 2014).
Minimum volume vitrification theory pertains to the rate of cooling and vitrification carriers. When plastic straws are used as the principal carrier to vitrify and store embryos, their survival rates of embryos following vitrification process (Enright et al., 2000). For example, in all experiments in which 0.25 ml plastic mini-straws were used by Palasz et al. (1997), only around 20% of vitrified embryos were recovered. Hence, cryobiologists have aimed to establish a carrier that contains the minimum volume of vitrification solution needed to raise cooling rates. Kuwayama (2007) determined that this strategy prevents breakage of zonae pellucidae. Vajta et al. (1998) pioneered a novel vitrification carrier -open pulled straw -made from a standard plastic straw (typically used in slow freezing of embryos) heat-softened and pulled to reduce its internal diameter so that 1-2 μl vitrification solution containing embryos loads spontaneously due to capillary action. Cooling rates as high as 25,000°C/min were recorded over a temperature range of -25°C to -175°C (Vajta et al., 1998). These compare favourably to 2,250°C/min, ten times less, when using a larger diameter plastic straw (5 μl, typically utilized for sperm freezing). More recently, Malenko et al. (2017) modified this protocol, replacing the plastic straw with a glass capillary tube. The heat-pulled conical tip can be moulded into a protective sheath that may be moved into either a closed or open position. Initial results for survival and hatching rates post-warming were comparable to those gained using various vitrification carriers. Further research is required to achieve high and stable survival rates with different developmental stages of in vitro-produced bovine embryos.
Kuwayama (2007) described a purpose-built carrier, named Cryotop®, a narrow and thin strip of film affixed to a hard plastic holder. This simple device enables loading of < 0.1 μl volume of vitrification solution containing the sample. As a result, cooling rates of 69,250°C/min may be achieved (Seki and Mazur, 2014). However, Rios et al. (2010) suggested that despite the contention that minimum volume vitrification enhances thermal conductivity and cooling rates, it does not improve hatching rates of embryos upon vitrifying/warming and subsequent culture in vitro (Rios et al., 2010).

Impact of warming rates
Recent research has highlighted the significance of warming rates to cryopreservation because recrystallization influences vitrification outcomes (Zhou et al., 2010). This starts at -109°C during the warming process , whereupon devitrification can cause cryo-injury to the cell (Sansinena et al., 2014). Hopkins et al. (2012) observed that warming rates are closely associated with cooling rates due to the presence of ice fractions inside vitrified cells. In accord with this, Seki et al. (2014) reported that during the cooling process, small ice crystals form; this causes recrystallization. Water molecules move from smaller to larger crystal size, ultimately reaching a size that proves lethal to the cell.
Faster cooling rates therefore need more rapid warming rates in order to block recrystallization (Mazur and Seki, 2011). Seki et al. (2014) found that combining moderate cooling rates with high warming rates of 117,500°C/min can avert recrystallization. Although it is apparent that rapid warming allows cells to avoid devitrification, insufficient cryoprotectant concentrations can promote recrystallization during the warming process (Jin et al., 2008). In concurrence, Seki et al. (2014) suggested that the short time, 1-2 minutes, for which embryos are exposed to equilibration solution is not enough for permeable solutes to penetrate cells to replace water molecules (by dehydration). (2014) showed that keeping the warming solution of vitrified embryos at 45°C facilitates an improved survival rate compared to at 50°C, 60°C or 70°C. Corroborating this, Caamaño et al. (2015) considered that 41°C is the most suitable temperature to warm vitrified embryos.

Pros and cons of cryoprotectants
In cryobiology cryoprotectants perform a key function in preventing cryodamage to cells. Permeating and non-permeating cryoprotectants are used frequently to cryopreserve gametes and embryos. Replacing intracellular water with a permeating compound protects a cell from ice crystals forming; the cell is then rehydrated upon warming (Palasz and Mapletoft, 1996). Low molecular weight, non-electrolyte cryoprotectants penetrate cells at different rates to replace cytoplasmic water (Leibo, 2008). Embryos are equilibrated with each of the five most common permeable cryoprotectants -dimethyl sulfoxide (Me 2 SO), glycerol, EG, methanol and propylene glycol -at half the strength of the final vitrification solution. For instance, using a Cryotop® embryos are first exposed to an equilibration solution containing 7.5% v/v EG and 7.5% v/v Me 2 SO, followed by a vitrification solution (15% v/v EG and 15% v/v Me 2 SO).
Permeability of cells varies with the type of cryoprotectant. EG is more permeable to bovine embryos than is glycerol (Morató and Mogas, 2014), so EG is typically the cryoprotectant of choice in the field using a one-step dilution (Dochi et al., 1998). A high concentration of cryoprotectant is required to prevent intracellular ice crystal formation (Seki et al., 2014), and to attain vitrification (Lawson et al., 2012). However, this risks causing osmotic shock to cells (Morató and Mogas, 2014). Vanderzwalmen et al. (2013) argued that high concentrations of cryoprotectant intracellularly and extracellularly are necessary to minimize embryo swelling, which is a consequence of water permeation during the warming process. Therefore, non-permeable cryoprotectants such as the sugars sucrose and trehalose are used to reduce osmotic stress. While cryoprotectants preserve the integrity of cellular organelles during cooling and warming (Dobrinsky, 2002), their toxic characteristics may be detrimental (Fahey, 2010;Wu et al., 2013).
Toxicity is a major drawback of cryopreservation regardless of whether slow freezing or vitrification is used, so lessening the effects on cell viability should be an ongoing priority of cryopreservation methods. In regard to bovine assisted reproduction, it is a commonly held belief that exposure to a high concentration of cryoprotectant during vitrification may be harmful to embryos. However, Vanderzwalmen et al. (2013) maintained that intracellular levels of cryoprotectant in vitrified murine embryos are significantly lower than those detected within slow frozen embryos. In supporting this perspective, Do et al. (2018b) indicated that vitrification may not be toxic to bovine embryos since no miscarriage is observed among recipients that are implanted with vitrified in vitro-derived embryos.

Impact of cytoplasmic lipid droplets
The presence of cytoplasmic lipid droplets in embryos is suggested to have no apparent effect on their viability after cryopreservation (Block et al., 2010); however, it is also argued that the survivability of cryopreserved embryos is related to their lipid contents (Cagnone and Sirard, 2014;Seidel, 2006). Pryor et al. (2011) andSeidel (2006) identified a correlation between lipid droplets and embryo cryotolerance. Furthermore, Cagnone and Sirard (2014) noted that in vitro culture affects the embryonic lipid content of embryos and their subsequent cryo-survival. Previously, in a preliminary study Abe et al. (1999) had discovered that the sensitivity of in vitro embryos to cryopreservation is due to the substantial cytoplasmic accumulation of lipid droplets. Embryos cultured in serum-free medium contain predominantly lipid droplets of < 2 µm; in contrast, embryos cultured in serum-supplemented medium have lipid droplets > 62 µm. Such large lipid droplets were frequently observed in morulae and blastocysts cultured in media containing 5% v/v calf serum (Abe et al., 1999). The lipid content of in vitro-produced embryos cultured in a serum-free medium was similar to their in vivo-derived counterparts, but the lipid volume in embryos cultured with serum was almost double that of in vivo embryos (Ferguson and Leese, 1999).
The abnormal accumulation of lipid in the cytoplasm of in vitro embryos resulting from culture together with serum is the main factor that makes them unsuitable for cryopreservation (Pereira et al., 2008). Mucci et al. (2006) contended that raised numbers of lipid droplets in an embryo affect its cellular repair after cryopreservation. Moreover, Sudano et al. (2011) maintained that high concentration of fetal calf serum in in vitro culture media causes a rise in accumulated lipid in blastocysts, and in apoptosis, but reduces expansion of blastocoels after vitrification.

Vitrification of embryos at various developmental stages
Vitrification of embryos at different stages is controversial (Asgari et al., 2012). Cryopreservation of the blastocyst stage enables immediate embryo transfer after warming and to skip the repeated in vitro culture of embryos; moreover, advanced embryos can overcome the period of genome embryonic transition that occurs at or between the fourth and fifth cell cycle in bovine embryos (Aono et al., 2013;Garcia-Garcia et al., 2006). Asgari et al. (2012) revealed that a large number of 5-8 cell stage bovine embryos were arrested at the morula stage due to the negative effect of cryopreservation on the embryonic activation transition period. Shirazi et al. (2009) showed that early blastocysts have a greater cryotolerance than do expanded ones. However, Sommerfeld and Niemann (1999) observed that expanded blastocysts survive vitrification better than do nonexpanded blastocysts; hatching rates of 42% and 12%, respectively. On the other hand, early stage embryo cryopreservation delays the in vitro embryo production procedure. Hence, it is important that after warming these embryos are cultured further in vitro to develop into blastocysts at which point they can be used as fresh embryos.

Direct transfer of vitrified in vitro-produced bovine embryos
Most vessels used for vitrification are not designed specifically for this purpose so they are ill suited to the direct transfer of embryos with a standard 0.25 mL plastic mini-straw (Ha et al., 2014;Inaba et al., 2011). Furthermore, several steps are taken when warming in order to gradually reduce the cryoprotectants left over from placing embryos in equilibrium and vitrification media before vitrification. Simplification of this process through a single warming step is required to enable the direct transfer of vitrified bovine embryos. It is thought that Van Huong Do et al. 260 warming by single or multiple steps achieves comparable results (Caamaño et al., 2015;Morató and Mogas, 2014). Vajta et al. (1999) designed an in-straw dilution procedure whereby a French mini-straw is loaded with holding medium and 0.2 M sucrose; however, this requires a skilled operator (Vieira et al., 2007). Another drawback of this modification is embryo loss during warming. Vieira et al. (2007) considered that unintentional warming in air for a few seconds before an embryo is loaded possibly reduces the warming rate, which is a critical factor in its survival (Mazur and Seki, 2011). Although direct transfer has provided promising results (Caamaño et al., 2015;Morató and Mogas, 2014;Vieira et al., 2007), the loss of embryos, evaluated by transfer, is limiting to successful outcomes.

Refined techniques to enhance cryopreservation outcomes
Prior to vitrification of in vitro-fertilized embryos micro-manipulation could increase their survival rates following cryopreservation. As an example, it is suggested that dehydrating blastocysts sufficiently before vitrification, through artificially collapsing the blastocoel, increases their implantation potential (Liebermann et al., 2012). By using this technique Min et al. (2013) observed improved survival rates of bovine in vitro embryos and cloned embryos after postwarming and incubation for a further 24 hours (Min et al., 2013). The survival of in vitro embryos following blastocoel forced treatments was significantly greater than for untreated controls (81.9% versus 69.8%). However, this technique is complicated due to time constraints and requires a highly skilled embryologist. Pryor et al. (2011) used a laser-assisted hatching method in which a laser microbeam drills a hole in the zona pellucida surrounding the embryo, through which lipid droplets can exit the cytoplasm. While technically feasible, this appears a complex method.
Enhancing the intrinsic characteristics of in vitro-fertilized bovine embryos is another way to raise their quality prior to cryopreservation. According to Pribenszky et al. (2010) and to Pribenszky and Vajta (2011), exposing gametes and embryos to sublethal hydrostatic pressure elevates their intrinsic developmental competence to overcome physiological stress such as experienced during cooling. This improved cell performance may be due to high hydrostatic pressure causing a minimum stressful condition under which cells can synthesize heat shock proteins (Díez et al., 2012;2008). In support of this, pretreating bovine blastocysts with high hydrostatic pressure, Filho et al. (2011) reported that reexpansion and hatching rates of treated embryos are significantly higher than those of untreated embryos. However, pregnancy rates were not detailed. This promising approach to enhancing the survivability of embryos requires further investigation.

Cryopreservation-related biosafety considerations
Biosafety is of paramount importance when importing and exporting bovine embryos internationally. Therefore, the threat posed by cryopreserved embryos of pathogen transmission demands evaluation. The two systems of vitrification, closed and open, present different risks. Theoretically at least, there is a reduced risk of microbial contamination using a closed vitrification system, as for slow freezing in straws, compared to methods that require direct contact of embryos with liquid nitrogen (Vajta et al., 2015). It has been argued that it is a false assumption to consider liquid nitrogen or cryopreserved samples to be sterile (Morris, 2005). Mirabet et al. (2012) indicated that some viruses can survive at subzero temperatures without the need for a cryoprotectant or cryopreservation. For instance, Bielanski et al. (2000) investigated possible contamination from liquid nitrogen of three species of virus; bovine viral diarrhea virus, bovine herpesvirus and bovine immunodeficiency virus. Unsealed carriers tested either positive or negative for these viruses but no sealed source was contaminated. While 32 bacterial and one fungal species were identified in various liquid nitrogen cannisters storing semen and embryos for between 6-35 years, bovine viral diarrhea virus and bovine herpesvirus were not detected in clean semen and embryo straws kept alongside infected straws (Bielanski et al., 2003). Nonetheless, it is not easy to maintain long-term cleanliness of samples that are contained in the same liquid nitrogen cannister as unclean ones.
Despite the apparent low risk of microbial contamination of samples through contact with liquid nitrogen, it remains a potential hazard (Vajta and Kuwayama, 2006). An open system achieves sufficiently high cooling rates, but a closed system may not; hence, striking a balance between technical and sterility considerations presents a challenge to the operator.

Conclusion
It is evident that formation of intracellular ice crystals during cooling and warming processes is the main cause of damage to cryopreserved cells. Slow freezing and vitrification both aim to reduce to a minimum, if not prevent, ice accumulation. While cryoprotectants are necessary to protect cells from chilling they are possibly cytotoxic. Although slow freezing and vitrification are each efficient for in vivo-derived bovine embryo cryopreservation, slow freezing may not be suited to cryopreserving in vitro-produced bovine embryos. This is perhaps due to the cytoplasmic lipid content of such embryos being greater than that of their in vivo counterparts, although improved culture conditions may mitigate this effect. Collective research indicates the advantages of vitrification as the method of choice for storing in vitro-produced embryos. A better understanding of cooling and warming processes, cryoprotectants, modifications to in vitro culture media, and to treatment of cells prior to cryopreservation is each necessary in order to improve outcomes. Furthermore, embryo biosafety should be safeguarded.
Vitrification appears a superior procedure to slow freezing for cryopreserving in vitro-produced bovine embryos, yet non-standardization of methods between laboratories and, consequently, inconsistent results currently hamper its applicability to the international trade in cattle genetics and the management of embryo biobanks (Mogas, 2018). Thus, it is important to optimize in vitro production of blastocysts with fewer lipid droplets, together with identifying the most efficient means to vitrify in vitro-produced bovine embryos. In turn, such technical advancements will both contribute to a greater understanding of bovine cryobiology and facilitate increased use of in vitro embryos in commercial livestock settings.