Caihong HAO, Guizhi TONG, Xueying SONG, Xintao LI, Likun WANG, Zhenhua JIN, Jiahou WANG, Wenkai GUO, Ping LI, Li LI, Yan ZHANG
Abstract Embryo cryopreservation is an important part of embryo biotechnology. It plays a key role in the development of quality animal husbandry. However, the effect of in-vivo (in-vitro) embryo freezing of cattle and sheep in China has not reached an expected level, which limits the commercial application of embryo transfer technology. The technical bottleneck that needs to be broken through is that embryos are subject to mechanical damage and protective agent toxicity during freezing and thawing. This paper summarized the successful application of plant antifreeze protein in vitrification preservation of pig embryos, which makes this technology widely used in animal husbandry production, and is of great significance in accelerating the large-scale breeding of improved livestock and saving endangered animals.
Key words Sheep; Mutton sheep; Embryo; Freezing
Received: September 21, 2022 Accepted: November 28, 2022
Supported by Key Project of Qiqihar Science and Technology Program (ZDTG-202201); Qiqihar Innovation Incentive Project (CNGG-2021015).
Caihong HAO (1982-), female, P. R. China, assistant research fellow, devoted to research about sheep raising and breeding.
*Corresponding author.
In embryo cryopreservation, various cryoprotectants and freezing methods should be combined to compensate with each other. To screen appropriate cryoprotectants and cryopreservation methods for embryos at different development stages and simplify the freezing and thawing procedure, we should look for methods that do not require expensive freezing equipment, have short operation time, and are easy to promote. The phenomenon of high abortion rate, low pregnancy rate and low farrowing rate of frozen embryo transfer needs further study. The successful application of plant antifreeze protein in vitrification preservation of pig embryos makes this technology widely used in animal husbandry, and the research content is of great significance.
Effects on Embryo Quality
Low quality of the inner cell mass of embryos before freezing and many cell fragments will make the integrity of each blastomere poor, and the balance between cell dehydration and the penetration of cryoprotectants into the cell process will be destroyed. Therefore, mastering embryo quality grading technology according to embryo age before freezing is one of the key technologies. Embryo cell morphology is a key factor affecting the quality of frozen embryos, as well as an important factor directly affecting the survival rate and pregnancy rate of embryos after transplantation[1].
Different Stages of Embryo Development
Some scholars cryopreserved sheep embryos at the age of 6.0-8.5 d old in liquid nitrogen, and the normal rate of expanded blastocysts and hatched embryos at 7 d after thawing was the highest. Scholars believe that the development rate of newly hatched pig embryos after low-temperature freezing and thawing is higher than that of pig embryos at other development stages. It has been reported that when the embryos of cattle and sheep were frozen with glycerin cryogen by the conventional slow freezing method, the transfer conception rate and farrowing rate of blastocysts and expanded blastocysts after thawing were significantly higher than those of morulae, and there was no significant difference between blastocysts and expanded blastocysts[2-3]. It indicated that glycerol cryogen was more suitable for freezing embryos at blastocyst and expanded blastocyst stages. For morulae, due to the small molecular weight of ethylene glycol cryoprotectant, it can quickly penetrate into embryonic cells, and the blastomeres of frozen embryos maintain a high degree of consistency, without any change in external morphological characteristics; and when glycerin having a large molecular weight is used as an embryo cryoprotectant, the position of blastomeres of morulae is related to the degree of protection to the blastomeres, and the morphology of the blastomeres of morulae located outside the morulae is normal and intact, while the blastomeres located inside the morulae are easy to be damaged.
Therefore, it is recommended to use different cryoprotectants for sheep embryos at different development stages. Ethylene glycol is appropriate as the cryoprotectant for embryos at blastocyst and expanded blastocyst stages, and glycerin is appropriate for morulae as the cryoprotectant.
Methods of Embryo Freezing
Conventional slow freezing method
This method was first invented by Whittingham and used for mouse embryo freezing[4]. Later, Wilmut and Rowson used it for bovine embryo freezing[5]. Willadsen improved the slow freezing method in 1977[6] and changed it into the fast freezing method, which still needs the aid of a control rate freezer, and has cumbersome steps, but consumes a shorter time, so it becomes a commonly used method in current production. In this method, first, embryos are cooled down to 0 ℃ at a rate of 1 ℃/min, and balanced at this temperature for 10 min. Then, cooling is Continued, and ice seeding is performed at -7 ℃. Next, the temperature is cooled down to -30 ℃, and the embryo are finally stored in liquid nitrogen.
The main disadvantage of this method is that the freezing procedure is tedious and time-consuming, and expensive control rate freezers must be provided. Moreover, the effect of freezing bovine and sheep oocytes and embryos in vitro is poor. Due to the need to artificially induce ice formation and then cool down at a slow cooling rate, this method of embryo cryopreservation is reliable, but it requires professional embryo freezers and equipment, and the operation procedure is complex and takes a long time. And it must be operated by professional technicians, and requires special thawing solutions for removing cryoprotectants, stereomicroscopes and sterile laboratory conditions, which are not conducive to operation under site conditions. Therefore, it restricts the promotion and application of bovine and sheep embryo transfer technology and the industrialization process of embryo biotechnology[7].
Vitrification preservation
In cryobiology, a certain amount of compounds is added to cell preservation solutions to protect cells. During the rapid cooling process, the viscosity of the solutions increases sharply. After quick freezing, it is easy to form a vitrification state, which can prevent the formation of ice crystals in cells, cell dehydration, the increase of solute concentration and protein denaturation, and reduce the physical damage to membrane structures and cytoplasm caused by the formation of ice crystals. Research has proved[8] that vitrification preservation of mammalian embryos such as cattle and sheep is an effective and practical method among various freezing methods. After nearly 20 years of research, a variety of vitrification preservation solutions (thawing solutions) and preservation methods for bovine and sheep embryos in vitro have been invented, and the effect is significantly better than that of conventional freezing methods. With the one-step vitrification method for embryo freezing, the pregnancy rate of cattle was 40%-55% after thawing and transferring, and that of sheep was 52%[9].
The vitrification preservation technology does not require expensive freezing equipment, has simple operation procedures and saves time. Compared with traditional embryo freezing methods, it has obvious advantages, which is conducive to accelerating the rapid propagation of good cattle and sheep breeds and the process of variety improvement, and shortening generation interval. As a new method of embryo vitrification preservation, OPS method further reduces the damage of freezing to embryos by greatly increasing the speed of freezing and thawing, and significantly improves the effect of embryo freezing. Its characteristics such as simple operation, stable effect and wide application range are increasingly valued.
Cryoprotectants
Under practical freezing speeds, high solution concentrations (above 30%) can form vitrification. Therefore, the toxicity of the solute used must be considered. Nowadays, reducing the toxicity of high concentration solutions to embryos mainly includes the following aspects: ① balancing embryos through two steps, ② shortening the balance time, ③ reducing the equilibrium temperature, and ④ using anti-toxic substances such as sucrose and trehalose. The survival rate of embryos fertilized in vivo was 62.9% (39/62) after vitrification with 25% glycerol+25% ethylene glycol-modified PBS solution and embryo transfer in sheep. In freezing, ROS oxidative stress is also the cause of cytoplasmic peroxidation and organelle damage[10].
It has been reported that there was no significant difference in the transfer conception rates of frozen embryos of cattle and sheep using glycerol and ethylene glycol as cryoprotectants. The transfer conception rate of the glycerol cryoprotectant group was slightly better than that of the ethylene glycol cryoprotectant group, but both of them were feasible as cryoprotectants for embryos[11]. Glycerin can be used as the first choice for freezing sheep embryos, because frozen sheep embryos need to be added into tubes when they are transferred. At present, an antifreeze protein found in cold-resistant fish and insects can combine with the cell membrane to protect the cell membrane at low temperature. Moreover, the addition of the antifreeze protein to culture media can also inhibit the formation of ice crystals during freezing and thawing, thereby protecting embryos.
In a word, in the process of embryo freezing and thawing, we should explore the best combination of cryoprotectants, one-step release of cryoprotectants after thawing or direct transfer method, ensure the viability of frozen embryos, improve the conception rate and farrowing rate of frozen embryo transfer, and establish a simple and fast procedure of embryo freezing, thawing and transfer, so as to accelerate the promotion of livestock breeding process, and provide technical support for cattle and sheep breeding with embryo transfer supporting technology.
Conclusions
Embryo cryopreservation is one of the important links in the process of embryo transfer. It reduces the damage to embryos caused by low temperature during freezing and thawing through certain protective measures and cooling procedures, so embryos can be preserved for a long time under low temperature. This technology can reduce the waste of high-quality embryos, so embryo transfer is not limited by time and space and can be widely used, which is of great significance for accelerating large-scale breeding of improved livestock and saving endangered animals. However, in general, there are still many problems in embryo cryopreservation technology, such as the toxicity of protective agents used to embryos, and the low conception rate of embryo transfer after freezing and thawing.
Caihong HAO et al. High-efficiency Embryo Cryopreservation Technology for Mutton Sheep
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