Cryopreservation of Porcine Embryos: Recent Updates and Progress

Abstract

Cryopreservation of embryos is important for long-distance embryo transfer and conservation of genetic resources. Porcine research is important for animal husbandry and biomedical research. However, porcine embryos are difficult to cryopreserve because of their high cytoplasmic lipid content and sensitivity to chilling stress. Vitrification is more efficient than slow freezing, and vitrification is mostly used in embryo cryopreservation. So far, the vitrification process of porcine embryos has been continuously improved, resulting in improved survival rates of warmed embryos and farrowing rates after the transplant procedure. It is worth noting that automatic vitrification has made great progress, which is expected to promote the standardization and application of vitrification.

In this article, the vitrification process of porcine embryos at the blastula stage and early development stages is reviewed in detail. In addition, the efficiency of different vitrification systems was compared. In addition, we summarize technology that can improve the survival rate of cryopreserved porcine embryos, such as delipidation methods (including physical delipidation and chemical delipidation) and medium improvements (including chemically defined media and adding antioxidants). Meanwhile, gene expression changes during cryopreservation are also elaborated.

Introduction

The significance of embryo cryopreservation lies in adapting to the industrialization of embryo transfer so that embryo transfer is not restricted by time and place; facilitating embryo transport as an alternative to the introduction of live breeding stock; and establishing animal gene banks to preserve good domestic and endangered animal resources.^1,2 Assisted reproductive technologies are also increasingly using frozen embryos.^3–6 Embryos are more likely to be fully dehydrated after cooling than oocytes.⁷ During embryo cryopreservation, although some cells are damaged or die, the remaining cells are sufficient for normal development of the embryo. In addition, the membrane lipid composition of the embryo contains more polyunsaturated fatty acids than that of the oocyte.⁸ In general, embryos are easier to cryopreserve than oocytes. Porcine research is important for agricultural production and biomedical research. However, porcine embryos have a higher cytoplasmic lipid content than sheep, bovine, and mouse embryos.⁹ Therefore, establishing an effective method to cryopreserve porcine embryos is more difficult.

Injuries during embryo cryopreservation and embryo thawing include chill injury, ice crystal formation, fracture injury, osmotic stress, and multiple aster formation, and these cryogenic injuries can lead to damage to the cytoskeleton, zona pellucida, and mitochondria.¹⁰ These cryopreservation injuries can therefore affect the survival and subsequent development of embryos after thawing.^11–13 Improved cryopreservation procedures are aimed at reducing these cryoinjuries and increasing success rates.

Cryopreservation of embryos can be achieved with two methods: slow freezing and vitrification.¹⁴ Slow freezing is easy to standardize and commercialize, but is compromised with more cryodamage due to the low cooling rate.¹⁵ On the contrary, vitrification is devoted to increasing the cooling rate to produce a glass-like solidification, which reduces the formation of disordered ice crystals so that the injury to the embryo can be reduced.^16,17 In 1985, Rall and Fahy vitrified mouse embryos successfully for the first time.¹⁸ The main features of vitrification are the extremely high concentration of the cryoprotectant (5–8 M) and a fast cooling/warming rate.¹⁹ Three critical factors affecting vitrification are the cooling/warming rate, viscosity of the cryoprotectant, and volume.^20,21 There are two types of cryoprotectants. The permeable cryoprotectant, such as ethylene glycol (EG), glycerol (Gly), dimethyl sulfoxide (DMSO), and 1, 2-propanediol (PrOH), has a small molecular weight and can easily permeate through the cell membrane into the cytoplasm, replacing most of the water in the cell, which allows cells to quickly reach the concentration needed for vitrification, thus preventing formation of lethal ice crystals in the cell. The impermeable cryoprotectant, such as sugar, glucose, ficoll, and trehalose, cannot enter the cytoplasm. Its main function is to regulate the osmotic pressure inside and outside cells, to promote the formation of vitrification, and to help stabilize the membrane by stabilizing the phospholipid headgroups.^22–25 The permeable cryoprotectants have some cytotoxicity, with EG having the least, followed by PrOH, Gly, and DMSO.²⁶ Therefore, the vitrification solution needs to balance the permeability and cytotoxicity of various cryoprotectants.

There are many studies on cryopreservation of porcine embryos, but they lack a transverse and systematic comparison. In this review, the efficiencies of different vitrification methods and different stages of embryos were compared. In addition, we summarize some technology that can improve the survival rate of cryopreserved porcine embryos, such as delipidation methods and medium improvements. Meanwhile, we will focus more on the mechanism of cryopreservation of porcine embryos by discussing the intrinsic properties of embryos and the effects of cryopreservation on transcriptome alteration to help us better understand and advance research on porcine embryo cryopreservation.

Effects of Different Embryonic Development Stages on Cryopreservation

Vitrification of blastocysts

The developmental stage of the embryo is considered to be a key factor in the survival rate after cryopreservation. The porcine embryo in the blastocyst expansion stage has higher permeability for EG and glycerin than early blastocysts, so it has the highest cryotolerance.²⁷

In 2004, in vitro produced (IVP) embryos at the expanded blastocyst stage were vitrified using the minimum volume cooling (MVC) method and the embryo survival rate was 41.2%. When treated with noninvasive delipidation before vitrification, the survival rate improved to 83.3%.²⁸ Blastocysts from in vitro culture of two- to four-cell embryos were vitrified using superfine open pulled straw (SOPS) and Vit-Master, for which the cooling rate is ∼80,000°C/min with a survival rate of 75%.²⁹ In addition, porcine blastocysts derived from delipated oocytes by parthenogenetic activation (PA) or handmade cloning could be vitrified effectively by the Cryotop method.³⁰ In 2008, porcine expanding blastocysts were vitrified using the metal mesh vitrification (MMV) method, for which the cooling rate can be up to 180,000°C/min, and the survival rate was 84.4%. The MMV method using the mesh has a better cooling rate than the plastic plate vitrification method, for which the survival rate was 53.1%.³¹ Moreover, porcine blastocysts were vitrified using a pullulan film, and the oxygen consumption rate and cell viability were similar to those of embryos vitrified by the MVC method.³² The pullulan film is soluble in warm water, so the vitrification solution can be diluted in a straw, and then the embryos can be directly transferred into a recipient.

It is well known that there is a risk of contamination with pathological microorganisms from liquid nitrogen (LN₂). A study identified 32 bacterial species and 1 fungal species from samples randomly drawn from LN₂.³³ In vivo produced porcine blastocysts were vitrified using the cryologic vitrification method, where without any contact with LN₂, the survival rate was 94% and the farrowing rate was 17.2% after transplation.³⁴ Similarly, expanded blastocysts were vitrified using a microvolume air cooling method, which avoids direct contact with the undefined factors of serum and LN₂, where the survival rate was 88.9% and farrowing rate was 12.5%.³⁵ Furthermore, Arav et al. invented a device that can produce clean liquid air (CLAir) with temperature similar to LN₂ and a sterile, storage canister device (Esther), which can keep samples sealed in their own compartment to prevent cross contamination.³⁶

Some studies collapsed the blastocoel cavity before vitrification to avoid intrablastocoelic ice formation and to improve the post-thaw survival of blastocyst-stage embryos.^37,38 In porcine embryos, a study reported that manually induced collapse of the blastocoel increased reexpansion rates of blastocysts after Cryotop vitrification,³⁹ while another study reported that collapsing the blastocoel of embryos before vitrification did not increase cryosurvival.⁴⁰ The different results may be attributed to the blastocoel collapse methods, in vitro origin of embryos, and/or vitrification methods. In addition, the oxygen consumption rate can reflect the mitochondrial activity, and fresh in vivo embryos with a greater oxygen consumption rate have better hatching ability.⁴¹ In 2016, a study proposed that the measurement of oxygen consumption after vitrification may be useful for estimating embryo survivability.⁴²

In some cases, we need to transfer embryos into a recipient after being thawed, but the complex anatomy of the cervix and uterine horn of pigs makes it difficult to transfer embryos into the recipient.⁴³ Cuello et al. achieved the birth of piglets by using nonsurgical deep uterine embryo transfer (NsDU-ET) from vitrified/warmed blastocysts.⁴⁴ There was also a study that reported that NsDU-ET results were better when warming embryos on a plate rather than in a syringe.⁴⁵ In addition, the farrowing rates of vitrified/warmed embryos were 50%–75% through the improvement of NsDU-ET.⁴⁶ In general, this method can reduce the stress of the recipient during the transplant procedure and has higher efficiency.^47,48 In some circumstances during ET, such as when the number of thawed embryos exceeds the number of embryos that need to be transferred, embryos could be revitrified and stored again. A study demonstrated that >60% of vitrified embryos could be successfully revitrified and rewarmed. In addition, a dry shipper (DS) could store vitrified porcine embryos for at least three 3 days, which can provide enough time for the international air transport of embryos.⁴⁹

Vitrification of early stage embryo

The vitrification of embryos at the pronuclear (PN) stage is very important because they can provide genomic host cells to be targeted by TALENs or CRISPR/Cas9. Kamoshita et al. achieved the birth of live piglets when they vitrified PN-stage embryos using a novel cryoprotective agent, carboxylated ɛ-poly-L-lysine (COOH-PLL).⁵⁰ COOH-PLL can inhibit ice crystallization and recrystallization during freezing and thawing with antifreezing protein-like activities in vitro. In 2018, a study revealed that both the MVC and microdrop methods were effective for zygote vitrification.⁵¹ The donor is also an important factor for successful vitrification of porcine zygotes, morulae, and blastocysts.^52,53 In addition, a study reported that the porcine parthenogenetic embryos vitrified by the Cryotop method at the two-cell stage showed a higher blastocyst formation rate than those vitrified at the four-cell stage.⁵⁴

The zona pellucida is a barrier that obstructs the free movement of water and cryoprotectant. Therefore, some studies perforated the zona pellucida using lasers or micromanipulation.^55,56 However, an intact zona pellucida is a prerequisite for minimizing disease risk and a requirement for the international transport of embryos. In 2001, the first production of piglets from zona pellucida-intact compacted morulae vitrified by the open pulled straw (OPS) method was achieved.⁵⁷ Moreover, porcine embryos at the compacted morula stage to the early blastocyst stage can survive after vitrification by the microdroplet method without any special intracellular manipulation or treatment.⁵⁸ Porcine morulae also were vitrified by the MVC method combined with delipidation, with a survival rate of 95%.⁵⁹ In addition, cloned pigs were produced from somatic cell nuclear transfer embryos at the morula stage and were subjected to delipidation and vitrification.⁶⁰ Recently, researchers invented a new vitrification method called hollow fiber vitrification (HFV). The blastocyst formation rate achieved was 73.8% when parthenogenetic morulae were vitrified by this method.⁶¹ This method also worked for porcine in vitro maturation (IVM) or in vitro fertilization-derived morulae.⁶² Both zona-free porcine morulae and their isolated blastomeres still remained viable after HFV.⁶³ In 2020, in IVP porcine morulae vitrified by HFV, the blastocyst formation rate was up to 90.3%.⁶⁴ These studies indicated that the HFV method is excellent for cryopreservation of embryos. Embryos loaded in a volume less than 0.1 μL would generate surface tension, which would be detrimental. However, the unique properties of the hollow fiber device seem to be able to avoid that result, which improves post-thaw success. In addition, porcine PA embryos could be successfully vitrified by the Cryotop method without delipidation or other treatment, and the optimal time for vitrification was day 4 after activation.⁶⁵ In 2017, a study found that IVP PA embryos could provide the possibility of assisted pregnancy with cryopreserved embryos.⁶⁶ The vitrification process of porcine embryos at different stages is summarized in Table 1.

Table 1.

Vitrification of Porcine Embryos at Different Stages

Embryo stage	Source	Vitrification method	Survival rate (%)	Blastocyst formation (%)	Hatching rate (%)	Farrowing rate (%)	Reference
Zygote	IVP	MD	98.5 ± 0.7	21.1 ± 1.5			⁵¹
Zygote	IVP	MVC	90.1 ± 5.8	19.1 ± 4.1			⁵¹
Zygote	IVF	SSV	93.4 ± 63.5	15.8 ± 62.1			⁶⁷
2-Cell	Vivo	SOPS		66.1 ± 6.0	4.9 ± 3.4		⁶⁸
2-Cell		SSV		76.5 ± 5.2	3.6 ± 2.5
4-Cell		SOPS		53.8 ± 6.9	9.1 ± 6.2
4-Cell		SSV		34.5 ± 6.3	5.0 ± 5.0
Morula		SOPS		94.6 ± 3.0	58.5 ± 6.8
Morula		SSV		96.6 ± 2.3	48.3 ± 6.6
2-Cell	PA	Cryotop	90.4 ± 2.7	38.5 ± 3.3			⁵⁴
4-Cell	PA	Cryotop	88.3 ± 2.9	27.1 ± 2.2
Morula	Vivo	OPS				13	⁵⁷
Morula	Vivo	MVC	95	74			⁵⁹
Morula	PA	HFV		73.8	32.3	40.3	⁶¹
Morula	IVF	HFV		77.6	10.6	19.3	⁶²
Morula	PA	HFV		90.3	40.9		⁶⁶
Morula	SCNT	MVC		31.5		1.6	⁶⁰
Blastocyst	Vivo	SOPS	81.0 ± 3.7		46.2 ± 0.5		⁶⁹
Blastocyst	Vivo	MVAC	85.0	45.0			⁷⁰
Blastocyst	HMC	Cryotop	79 ± 6	21 ± 6			³⁰
Blastocyst	IVP	MD	26.8 ± 2.7		2.5 ± 2.5		⁵¹
Blastocyst	IVP	MVC	52.2 ± 6.1		2.3 ± 1.6
EB	Vivo	OPS	86.2		74.1		⁷¹
EB	Vivo	MMV	84.4			18.5	³¹
EB	Vivo	MVAC	88.9 ± 3.9		69.4 ± 7.3	12.5 ± 6.5	³⁵

EB, expanded blastocyst; HFV, hollow fiber vitrification; HMC, handmade cloning; IVF, in vitro fertilization; IVP, in vitro produced; OPS, open pulled straw; PA, parthenogenetic activation; SCNT, somatic cell nuclear transfer; MD, microdroplet; MMV, metal mesh vitrification; MVAC, microvolume air cooling; MVC, minimum volume cooling; SOPS, superfine open pulled straw; SSV, solid surface vitrification.

Removal of Intracellular Lipids Increases Cryosurvival

Lipids can be an energy substrate for the oocyte and embryo development.^72–74 However, they can damage embryos during freezing. Lipid phase transition (LPT) is a major source of cellular damage during vitrification. This is because LPT can turn the cell membrane into a solid ordered phase, which then decreases the permeability of the membrane.⁷⁵ It has been shown that both invasive removal by postcentrifugation micromanipulation and noninvasive lipid removal using 4% trypsin combined with centrifugation can significantly improve the survival of vitrified embryos.^28,76,77 The best stage to perform delipidation is the morula embryo, when there is minimal damage to the embryo.⁷⁸ Changing the lipid composition of the embryo or reducing the lipid content of the embryo by optimizing the culture medium is theoretically less disruptive to the embryo. Forskolin is an activator of adenylyl cyclase, which increases intracellular cAMP levels, subsequently stimulating the lipolysis of triacylglycerols.^79,80 The supplementation of 10 μM of forskolin during porcine oocyte maturation in vitro significantly increased the cryotolerance of oocytes.⁸¹ Likewise, treatment of 2–4-cell embryos with forskolin up to 24 hours before freezing significantly improved their freezing tolerance.⁶⁸ Increasing cAMP levels can also be achieved by adding the β-adrenoceptor agonist, isoprenaline (ISO), during in vitro culture, which positively affects the IVM of porcine oocytes.⁸² Another similar supplement is L-carnitine in fatty acid metabolism, which as a key cofactor involved in the carnitine shuttle and can affect β-oxidation rates and adenosine triphosphate (ATP) levels in the oocyte. A study demonstrated that exposure of oocytes to 3 mM L-carnitine for 1 hour before fertilization increased the cleavage rate and improved the cryotolerance of resulting blastocysts.⁸³ In addition, phenelzine ethanesulfonic acid acts as a regulator of cellular metabolism, increasing the activity of the pentose phosphate pathway and then decreasing lipid droplet levels (LDs).⁵⁵

Swelling of the zona pellucida through partial enzymatic digestion and condensing the volume of the embryo by high osmolality are the two methods to break the bridge-like structure in the perivitelline space to prevent lipid droplets from redistributing into the embryo. In a study from 2013, in vivo-derived embryos treated with high osmolality and centrifugation to polarize intercellular lipids increased the reexpansion rate.⁸⁴ Furthermore, Tatsumi et al. developed a forced selective autophagy system to induce forced lipophagy, which reduced the LD size and number.⁸⁵ Aizawa et al. used two-step centrifugation to remove almost all LDs from mouse MII oocytes.⁸⁶ In 2019, Somfai et al. found that oocytes of the indigenous Vietnamese Ban pig had low intracellular lipid levels and showed high survival and maturation rates after vitrification.⁸⁷ The level of lipid content in cells is related to genetic differences and environmental factors. Therefore, further research on an indigenous pig breed may help identify oocytes or embryos with high cryotolerance.

Optimization of the In Vitro Culture Medium Can Improve the Cryopreservation Tolerance of Embryos

A culture medium containing animal serum leads to deposition of lipid components in cells, which significantly reduces the cold tolerance of the embryo. In addition, the results of vitrification and media from different batches of animal serum are inconsistent and it is difficult to eliminate these effects. It is therefore important to develop an effective chemically defined medium for the vitrification process. A study using polyvinyl alcohol (PVA) instead of serum for vitrification and warming has shown that PVA has no detrimental effects on in vitro development of embryos.⁸⁸ In 2016, Cuello et al. used Tyrode's lactate–HEPES–polyvinyl alcohol medium (TL-PVA) as a basic medium in vitrification and warming procedures, obtaining a high percentage (>90%) of embryo survival rates.⁸⁹ Meanwhile, as a pH-stable medium, TL-PVA could simplify the vitrification procedure and facilitate the application of embryo transfer in practice. In addition, a study reported that knockout serum replacement could be used to replace fetal bovine serum as a defined serum supplement for postwarming recovery culture of vitrified blastocysts.⁹⁰

L-ascorbic acid (AC) is a nonenzymatic antioxidant that can protect cells from harmful oxidative products. Castillo-Martín et al. added AC to the culture and vitrification–warming media, which increased gene expression of SOD1 and GPX1 and reduced HSPA1A expression and peroxide levels, thus improving survival rates of vitrified porcine blastocysts.^91,92 In 2018, ascorbic acid was added to chemically defined vitrification and warming media, which increased the IVP porcine blastocyst survival rate by decreasing reactive oxygen species (ROS) production.⁹³ Moreover, vitrified–warmed Germinal Vesicle mouse oocytes matured with 10⁻¹¹ mol/L melatonin were shown to decrease mitochondrial heat production and ROS levels, increase ATP levels, and reduce the aneuploidy rate.^94,95 Mouse immature vitrified/warmed oocytes incubated in milrinone 3 hours before IVM led to recovered ROS levels and mitochondrial membrane potential and recovered the blastocyst formation rates.⁹⁶ Vitrification could induce abnormal mitochondrial distribution, and milrinone as a cAMP degradation inhibitor may stabilize mitochondrial functional activity and oxidative stress to reduce cryoinjury.^97,98

The substrate and temperature of the medium are also important. IVP embryos cultured in PBM (PZM-5 with glucose and glycine) from day 5 to 6 achieved higher warming survival and hatching rates than those cultured in PZM-5.⁹⁹ Somfai et al. found that appropriate warming temperature was a critical factor for achieving high survival rates, and adjusting the hotplate temperature to 42°C during warming resulted in higher survival rates of vitrified oocytes when compared with 38°C.¹⁰⁰ Another study suggested that cryoprotectant toxicity exerted by media at 22°C was lower than that at 38°C and improved the postwarm survival rate.¹⁰¹

Gene Expression Alterations Caused by Cryopreservation of Porcine Embryo

ROS, cryoprotectants, and low temperature during cryopreservation affect the embryo's genome.^102,103 Research on changes in gene expression during vitrification can help us to better understand the effects of cryopreservation on embryos. A study indicated that gene expression was downregulated for POU5F1 and upregulated for HSPA1A in vitrified blastocysts, but expression levels of BAX, BCL2L1, SOD1, and SOD2 were not affected.¹⁰⁴ In 2018, a study demonstrated that the rate of apoptotic cells in vitrified blastocysts increased, and the pan-caspase, caspase-3, caspase-8, and caspase-9 activities also increased, but the relative abundance of Bcl-2 and SOD-1 mRNA decreased.¹⁰⁵ Z-VAD-FMK can reversibly bind to the catalytic sites of caspase-3, -8, and -9 to inhibit apoptosis and improve the survival rates of vitrified embryos.^106,107 In 2019, a study indicated that vitrification did not influence the gene expression of PCNA, CDX2, and CPT1, but enhanced mRNA levels of POU5F1 and uPA.⁹⁰ The expression of aquaporins increased permeability to specific cryoprotectants. A study reported that expression of the T85A mutant of zebrafish aquaporin 3b improved the survival rates of cryopreserved early mouse embryos.¹⁰⁸ Moreover, vitrification decreased the expression of the imprinted genes, IGF2 and IGF2R, which is very important for embryo development.¹⁰⁹ These studies indicate that cryopreservation can affect the expression of genes related to pluripotency, stress level, peroxide level, apoptosis level, and epigenetic modification of embryos. The RNA-Seq transcriptome profiling of mouse oocytes demonstrated that the influence of vitrification on the transcriptome was negligible, while IVM affects the mitochondrial membrane protein gene expression.¹¹⁰ The RNA-Seq transcriptome profiling of porcine oocytes demonstrated that there were 19 upregulated and 18 downregulated genes in the vitrification group.¹¹¹ In addition, single-cell transcriptomic analyses revealed that vitrification reduced the survival rate of porcine cloned blastocysts and caused the altered expression of 540 genes.¹¹²

Moreover, great progress has been made in automatic vitrification. In 2014, the first semiautomatic vitrification machine, Gavi^®, was invented, which showed embryo survival rates that were comparable with the Cryotop method and resulted in two pregnancies following human blastocyst automated vitrification in 2019.^113–115 RoboVitri was the first robotic system for vitrification of mammalian embryos.¹¹⁶ This robotic system was embedded with two contact detection methods to determine the relative z positions of the vitrification micropipette, embryo, and vitrification straw. A three-dimensional tracking algorithm was developed to visually observe the embryo transfer and real-time monitoring of embryo volume changes during vitrification. The excess medium was automatically removed from around the vitrified embryo on the vitrification straw to achieve a high cooling rate. Tests on mouse embryos demonstrated that the system was capable of performing vitrification with a throughput at least three times that of manual operation and high survival (88.9%) and development (93.8%) rates. In 2018, a novel automatic vitrification device named Sarah^® was invented.¹¹⁷ This automatic device is used to vitrify oocytes and embryos and consists of a vertical robotic handle where a special straw holder that can load up to six straws is attached. This robotic arm moves in a vertical plane, at predetermined time intervals, and by so doing, carries the biological samples contained in the straws between different solutions arranged into nine cups placed in a temperature-controlled, metal carousel plate. The final station on the carousel plate is the one containing LN₂ where the straws are ultimately plunged and the entire cycle of vitrification is considered completed. Vitrified mouse blastocysts processed by Sarah had 97% survival rate and 81% of them hatched. Vitrified bovine embryos had a 100% survival rate, 54% cleavage, and 9% blastulation rate. These devices are breakthroughs for the vitrification process, which is more simplified and standardized. The combination of cryobiology and mechanical automation is a direction worthy of further study.

Conclusion and Future Perspectives

Cryopreservation of porcine embryos is important for animal husbandry. However, porcine embryos are difficult to cryopreserve. Cryogenic injuries can be decreased by increasing the freezing/thawing rate, changing components of the cryoprotectant, or decreasing the cryoprotectant volume. The cooling rates of OPS, Cryotop, and SOPS are increased. In future research, the combination of cryobiology and material science will help to develop new materials with better thermal conductivity and that are more suitable for vitrification. For example, microinjecting gold nanorods into zebrafish embryos and irradiation with a laser pulse increased the thawing rate up to 1.4 × 10⁷°C/min.¹¹⁸ Currently, the most widely used cryoprotectant is the combination of EG, DMSO, Ficoll, and sucrose, but it still has chemical toxicity, so the search for a less toxic and more efficient cryoprotectant is worth studying.

There have been research studies for improving the survival rate by removing lipid droplets from embryos, but the presence of lipid droplets has specific biological significance. We need to consider the effect of removing lipid droplets on the subsequent development of thawed embryos, and the amounts of removal and retention are points that need to be balanced. It is a good idea to add drugs such as antioxidants to the culture medium to improve survival, and it would be interesting to study the molecular mechanisms or pathway by which drugs improve survival. In addition, with development of RNA-Seq and single-cell sequencing, there have been studies reporting the effects of vitrification on porcine oocytes or embryo genomes.^111,112 If this approach is combined with proteomic technology, it will be more helpful for us to understand the effects of vitrification on cell physiology.

Finally, the automatic vitrification technology has made great progress, and the first automatic vitrification device has achieved a considerable survival rate.¹¹⁷ This is a promising direction because the application and extension of automatic vitrification will rapidly promote the applications of vitrification so that cryopreservation can produce greater benefits and influence.

Footnotes

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

This study was supported by the National Natural Science Foundation Project of China (32072736 and 31101714) and Key Research and Development Projects in Hebei province (18226604D).

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