Abstract
Since the 1980s, equine embryos have been successfully cryopreserved. Embryos are mainly obtained by flushing the uterus of the mare. Cryopreservation of these embryos allows for the embryos to be stored more easily and used when needed. At the present time, two different techniques for the cryopreserving of embryos are available: the method of slow-freezing and vitrification. Both techniques have advantages as well as disadvantages, among which are cellular damage and complexity of the procedure. Size seems to be a limiting factor in the cryopreservation of equine embryos; smaller, early embryos (<300 µm) are generally better for cryopreservation but are more difficult to obtain due to low recovery rates while flushing. Larger, expanded embryos (>300 µm) are more easily obtained but are difficult to cryopreserve due to their size, volume, and capsule, and yield lower pregnancy rates after transfer than the smaller, earlier embryos. Micromanipulation of said obstructing capsule may provide a way to successfully cryopreserve larger embryos. This review discusses the results of multiple researches on micromanipulation, most of which report normal pregnancy rates after micromanipulation of the capsule and aspiration of the blastocoel fluid. Therefore, micromanipulation of the capsule seems to be a good way to increase the pregnancy rate in expanded equine embryos. More research on this subject should, however, be done to confirm this. In the future, micromanipulation, along with aspiration of the blastocoel fluid, could become part of the standard cryopreservation protocol for equine embryos exceeding 300 µm in diameter.
Keywords: equine, cryopreservation, embryos, slow-freezing, vitrification, micromanipulation
Introduction
The cryopreservation of embryos is a process used in the conservation of embryos in many species. The process allows for the embryos to be stored for a prolonged period of time, which is convenient for transport and when implantation of the embryo cannot happen immediately, for, for example, when the recipient mare is not ready to receive the embryo, due to the donor mare and recipient mare not being on the same cycle. Embryos can be cryopreserved by either slow-freezing or vitrification, both of which methods have been proven successful with equine embryos and yielding acceptable pregnancy rates. There are, however, some limitations regarding the cryopreservation of the larger (>300 µm in diameter) embryos due to the size, volume and presence of a capsule. These factors are limiting for the penetration of cryoprotectants in expanded embryos, thus making the cryopreservation process be significantly less successful.
The objective of this review is to compare the results of multiple studies to research whether micromanipulation of the embryonic capsule of in vivo produced, expanded embryos of >300 µm in diameter embryos will yield the same pregnancy rates as in vivo produced, early, <300 µm in diameter sized embryos. The results of multiple articles will be discussed to come to an informed conclusion. The hypothesis tested in this review is that the micromanipulation of the capsule of expanded equine embryos improve the pregnancy rate after transfer of said embryos.
The literature used in the review was found by using the search engines PubMed, Google Scholar and CAB Abstracts. For finding the articles, the following keywords, or combination of keywords, were used: equine, embryos, cryopreservation, blastocyst, vitrification, slow-freezing, biopsy, micromanipulation, recovery, uterine, flushing, transfer, history.
Upon selecting articles for reviewing, articles that were less than 10 years old had preference over older articles, unless they were used to give historical examples. Some of the articles used in this review were not found but were instead recommended by the supervisor of this review.
History of embryo transfer and cryopreservation
The first successful embryo transfer was performed by Walter Heape, who successfully transferred embryos in rabbits in 18901,2 George John Romanes has done the same to research whether embryo transfer would result in birth and if it would have any effect on the children1. In the 1970s, better techniques on embryo collection and transfer started to develop3, mainly as a result of high demand in European beef cows which were hard to import due to the risk of diseases1. The discovery of glycerol as a competent cryoprotectant for spermatozoa in 19491,2 gave rise to researches on the possibility of cryopreservation of embryos. In the 1950s, the first oocytes were frozen, and in 1960, the first mice were born from oocysts originating from frozen-thawed transplanted ovaries1. The first successful cryopreservation of embryos was recorded in 1972 by Whittingham et al1, in this case it were mouse embryos. About a year later, the first calf was born after cryopreservation of the embryo it developed from1. The first foal to come from a cryopreserved embryo was born in 1982 in Japan1,3.
Standard techniques of flushing and cryopreservation of embryos
Flushing is the most common method used to recover embryos in horses. This is performed by inserting a catheter through the cervix of the donor mare. Through this catheter, 1 to 2 litres of medium enters the uterus of the mare and is drained through a filtering cup. This procedure is repeated three or more times. After filtering, the found embryos are measured and classified in to different categories of quality, grade 1 being excellent, 5 being dead or degenerate.
For cryopreservation, there are two ways of cryopreserving an embryo: slow-freezing and vitrification. In the process of slow-freezing, the embryos are being exposed to low concentrations of cryoprotectants, usually (ethylene) glycerol, and cooled from room temperature to -30°C to -65°C at 0.3-0.5°C per minute, after which the embryos are immersed in liquid nitrogen for storage4,5. For vitrification, the embryo is exposed to relatively high concentrations of cryoprotectants before being submerged in liquid nitrogen, thus being cooled by a much higher rate.
There are several advantages and disadvantages regarding both cryopreservation processes. The advantages with slow-freezing are reasonable pregnancy rates with equine embryos that are less than 300 µm in diameter6 and the embryos are exposed to relatively low concentrations of cryoprotectants7. The disadvantages are, however, the time that is required for the process of both freezing and thawing and the fact that it also requires an expensive programmable freezer6. Contrary to the slow-freezing technique, vitrification is a rapid process and does not require sophisticated or expensive equipment. With this process, the liquids transform in to a glass-like substance. It does, however, requires high concentrations of cryoprotectants, resulting in possible embryonic damages if the procedure is not performed correctly6,8. Timing is the key in this process8, which can make the procedure stressful and which also means skills are needed to successfully complete this process.
These techniques not only differ in the process, but also differ in results. In the research by Hendriks et al.7, slow-freezing of both small (<300 µm) and large (>300 µm) resulted in lower percentage of dead cells than vitrification. This could reduce the quality of the cryopreserved embryos.
There are also different ways of containing the embryo during the vitrification process. An “open” or “closed” can be used. In a closed system, the embryo is contained in a straw, or similar, and trapped in vitrification solution between two air bubbles. In an open system, embryos are contained in a vitrification solution an open ended straw. This last method utilizes less fluid, making it freeze more rapidly9.
Cryopreservation of embryos in other species
Cryopreservation started with mouse embryos but is also done in many other species like cows, sheep, and humans, for a variety of reasons. The cryopreservation of bovine embryos is often easily achieved since the embryos are small of size when they enter the uterus, averaging at 160 µm in diameter according to one study10. These techniques are well developed in cattle and the possibility of superovulation, which involves administering a dose of hormones to superstimulate ovulation, makes embryo cryopreservation very effective10.
The cryopreservation of canine embryos is sometimes done to be able to preserve the better embryos for better reproduction of assistance dogs, such as guide dogs11.
In humans, cryopreservation of embryos is mainly used to store embryos created by in vitro fertilization (IVF)12. This way, multiple embryos produced in the IVF procedure do not have to be placed immediately. While transferring more than two embryos at the same time increases the pregnancy rate, chances of multiple births also increase, as well as the chances of abnormalities13. While the cryopreserved human embryos do not seem to yield a higher birth rate then fresh embryos like previously thought, it does seem to lower the risk of ovarian hyperstimulation syndrome12, which is portrayed by the cystic growth of the ovaries of women.
Limitations regarding cryopreservation of equine embryos
While cryopreservation of embryos allows for long transport of embryos and other benefits, there are still some limitations attached to the process of cryopreservation. The major obstacle in the cryopreservation of equine embryos is the rapid growth in diameter of the embryo and the optimal time to recover them. The optimal size for the cryopreservation of an embryo is a diameter of 300 µm or less. The embryo does not reach the uterus until day 6 to 6.510, at which time it is still <300 µm in diameter. However, the optimal time to recover the embryos is around day 7-8, since recovery rates at this time are higher than those on day 6 to 6.514,15. This complicates the cryopreservation of equine embryos since at day 7-8, the embryos have already exceeded the 300 µm diameter by 100-500 µm16,17, thus being too big for conventional cryopreservation methods.
Under the normal process of cryopreservation, early equine embryos, those with a diameter of 300 µm or less, yield a pregnancy rate ranging from 45% to 88%5,8,18-20. A research done by Sanchez et al.9, however, reported that embryos with diameters between 180 and 300 µm that were vitrified, yielded a pregnancy rate of 0%, meaning that not every size is suitable for vitrification or cryopreservation at all.
The implantation of cryopreserved expanded embryos, yields a pregnancy rate of 0-57%5,6,8,20,21. This is explained by the fact that an expanded embryo contains a large volume of blastocoel fluid8 and is encapsulated by capsule made of glycoproteins10,22. This capsule has a negative effect on the penetration of the cryoprotectants used in the cryopreservation process8,10,23. Even though it is not yet clear what the function of the capsule is, removal of the capsule fails to result in pregnancy after transfer in to the recipient mare24.
New techniques
In 1997, the first successful pregnancies after biopsy on equine embryos were recorded25. This discovery led to multiple studies on the effectiveness of micromanipulation of expanded equine embryos before cryopreserving them. In a study on preimplantation genetic diagnosis, the researchers found that one mare was still pregnant at day 30, after implantation of a biopsied, cryopreserved embryo with a diameter >300 µm18. Similar results were already found in a study on the viability of biopsied equine embryos22.
The process of micromanipulation can be done in various ways. One of these ways is microblade dissection, which involves the pressing of a blade on an embryo and moving it laterally25. This method, however, seems to yield a rather low pregnancy rate due to unrepairable damage to the embryo26. Another much used method of micromanipulation is with the use of a micropipette, at times assisted by the use of a small drill10,17-19,21,22. Both of these techniques puncture the capsule, but the latter usually also involves aspiration of the blastocoel fluid.
Results of reviewed articles
In a research done by J. Scherzer et al.10, in total, 5 embryos ranging from 805 to 1286 µm in diameter were taken. Two of these embryos were micromanipulated with the help of a laser and in one of the two embryos, about 20% of the blastocoelic fluid was extracted. After this, the embryos were vitrified using (ethylene) glycerol. For thawing, the embryos were exposes to room temperature for 5 seconds after which the embryos were transferred to a warm water bath of 30°C for 15 seconds. After thawing, the embryos were transferred into a recipient mare with the use of a AI pipette.
This research yielded a pregnancy rate of 50% a week after transfer to the recipient mares, with the embryo that had 20% of its blastocoelic fluid removed being the only surviving embryo. At the examination of day 28, however, ultrasonography showed that the embryo was resorbed.
In the research done by Y.H. Choi et al.22, embryos were micromanipulated by the use of a biopsy pipette and a Piezo drill after which, trophoblast cells were biopsied which caused them to collapse. After this process, the day 7 embryos were held in an overnight treatment. This consisted of a culture at a temperature of 38.2°C, dropping gradually to a temperature of 28°C. The day 8 embryos, however, where either biopsied and placed in the same medium, but starting at 30°C, gradually dropping to 25°C over the course of 6 hours, or held overnight in the medium and then biopsied and shipped.
After transferring the biopsied embryos, pregnancy rates of 83% for the day 7 embryos (297 – 611 µm in diameter), both at the first check and at the heartbeat stage, and 75% for the day 8 embryos (790 – 1350 µm in diameter) at the first check and a 50% rate at the heartbeat stage.
Another research done by J. Scherzer et al.27 reported a 44% pregnancy rate with the use of embryos ranging from 360 to 1170 µm (n = 11), but none of the mares were still pregnant at the heartbeat stage. The embryos in this study were micromanipulated with the use of a laser and the blastocoel fluid was replaced with cryopreservative through diffusion. After this process, the embryos where vitrified with the use of ethylene glycerol as a cryoprotectant. The embryos were loaded in a open system (CryoLeaf) and immersed in liquid nitrogen for 90 seconds for vitrification. For the thawing process, each CryoLeaf was immersed in a thawing solution for 3 minutes at 30°C and transferred twice to other solutions.
In another research done by Y.H. Choi et al.17, day 7-8 embryos were micromanipulated in various ways. One group of embryos (n = 19), ranging from 300 to 730 µm, were biopsied and immediately vitrified using two different methods, with dimethylsulfoxide (DM), or by ethylene glycol (EG/s). The biopsy in this group was done by removing cells of the trophoblast layer with the suction of a biopsy pipette and with the assistance of the Piezo drill when it was deemed necessary. After this, the embryos were vitrified and thawed before either being shipped in a portable incubator for 4 to 6 hours, or cultured for 6 hours. In this group, of the 29 transferred embryos, 8 of the mares with embryos of the DM group and 6 of EG/s group were pregnant after transfer, yielding a 48% pregnancy rate. At the heartbeat stage, this percentage decreased to 28% due to the loss of 4 embryos in the DM group.
In another group (n = 15), embryos ranging from 300 to 710 µm, the embryos were biopsied in a different way than the previous group. In this group, the capsule, as well as the trophoblast layer were punctured by the pipette and the inserted into the centre of the blastocoel, where the blastocoel fluid was suctioned out for at least 70% after which cell could be obtained from the inside of the blastocyst. The embryos were vitrified using the EG/s method. After the warming process the embryo was either immediately transferred (n = 7) or transferred after culturing for 6 hours (n = 8). The embryos that were immediately transferred yielded a 57% pregnancy rate at both the first check and at the heartbeat stage, but one foal was lost at 8 months without an identified cause. The cultured group yielded a 13% pregnancy rate which dropped to 0% at day 90. The other 3 pregnancies were carried to term and produced 3 healthy foals.
In a third group (n = 8), the embryos, ranging from 407 to 780 µm in diameter, the embryos were biopsied in a similar way as the previous group, except that the cells are biopsied from the collapsed trophoblast. After biopsy, the embryos were vitrified using the EG/s procedure. The embryos were warmed before shipped in a warmed container for 4 to 6 hours. This process yielded a pregnancy rate of 75% after transfer, which decreased to 63% at the heartbeat stage.
In a research by F. Guignot et al. on genetic diagnosis18, 27 embryos were divided over 3 group in one of the performed studies; control, biopsied fresh and biopsied frozen. 8 embryos were biopsied and transferred, and 7 were biopsied and frozen before transfer to recipient mares. The embryos that needed to be frozen were vitrified in an open system and with the use of ethylene glycerol as a cryoprotectant, after which they were immersed in liquid nitrogen. Of the “biopsied and transferred” group, 6 embryos were bigger than 300 µm and yielded a 33% pregnancy rate at day 30. In the “biopsied and frozen” group, 5 embryos had a diameter exceeding 300 µm, and resulted in a pregnancy rate of 40%. The biopsies were also, like in aforementioned researches, performed by penetrating the capsule and inserting the pipette, followed by removing 70% of the blastocoel fluid. After this, several cells were biopsied from the trophoblast.
In an experiment performed in the research done by F. Diaz et al.21, 6 expanded embryos, ranging from 448 to 1168 µm, were micromanipulated with the use of a pipette and then vitrified with the use of both glycerol and ethylene glycerol in an open system. During the process of micromanipulation, the embryonic capsule was pierced by a pipette, after which 95 to 99% of the blastocoel fluid was aspirated. After transfer into recipient mares, the pregnancy rate was 83% and continued to stay that way up to the heartbeat stage. Two of these pregnancies were terminated while the other three were carried to term and produced three healthy foals.
Y.H. Choi et al.19 reported a pregnancy rate of 55% after transfer and 45% at the heartbeat stage in a study on vitrification of equine blastocysts, all of which resulted in foals. The embryos used in this experiment were sized between 303 and 608 µm and were subjected to micromanipulation by micropipette to remove ≥70% of their blastocoel fluid. Afterwards, the embryos were vitrified in a medium that contained ethylene glycerol as a cryoprotectant before being plunged into liquid nitrogen. For warming, a medium containing sucrose with a temperature of 38.2°C was used.
In a research on the influence of embryonic size and manipulation by R. Sanchez et al.9, embryos larger than 300 µm were relieved of their blastocoel fluid and vitrified in one of two ways. 5 embryos were with a Stripper-Tip while 10 others were vitrified using a hemi-straw. The pregnancy rate for these groups were 0% and 70%, respectively, on both the day 16 check as the day 45 check.
This study also found that embryos ranging between 180 and 300 µm in diameter had a low pregnancy rate when either subjected to the slow-freezing procedure (14.3% at day 16 to birth) or the vitrification procedure (0%).
W.K. Hendriks et al.7 found in a research comparing vitrification and slow-freezing techniques that vitrification of embryos results in a higher percentage of dead cells for both small (<300 µm) embryos as well as large (>300 µm) ones, with percentages of 19.9% and 53.3% respectively. The embryos in this study were either slow-frozen or vitrified using both glycerol and ethylene glycerol as a cryoprotectant. Thawing of the vitrified embryos was done by first keeping the embryos at room temperature for 10 seconds and afterwards the embryos were immersed in a warm water bath of 20°C for 10 seconds and were then incubated for 5 minutes. The slow-frozen embryos were first subjected to room temperature for 10 seconds, after which the embryos were submerged in a water bath of 35°C for 1 minute. Afterwards, the cryoprotectant was removed. This study also researched whether or not exposure to cryoprotectants alone would cause cell damage, but no such connection was found.
Discussion
Previous studies have shown that expanded (>300 µm) equine embryos are more difficult to freeze due to their size, their capsule, and the volume of their blastocoel fluid5,6,8,20,21. These factors prevent the cryoprotectants to enter the embryo fully and do their work properly, resulting in damaging the embryos. Breaching the capsule of the expanded embryo might be a way to yield higher pregnancy rates with these embryos, since this would allow the cryoprotectants to be able to enter the embryos better and therefore protecting the embryo during the cryopreserving process. Also, aspiration of the blastocoel fluid might help with ensuring that the cryoprotectants are able to do their job since the aspiration will remove the limiting volume and fluid.
The studies included in this review show that the micromanipulation of expanded (>300 µm) embryos yield pregnancy rates ranging from 0 to 83%. Most of these studies are done in a similar way; the embryo is punctured with the use of a micropipette after which the blastocoel fluid is aspirated. Often, 70% or more of the fluid is removed, and the embryos are exposed to cryoprotectants before vitrification. The puncturing of the capsule was in some cases assisted by the use of a small drill, to make penetrating the capsule easier. In some studies a laser was used to assist with the micromanipulation of the embryonic capsule.
The pregnancy rates found in most studies discussed in this review appear to be similar to the pregnancy rates of early (<300 µm) embryos. This would mean that micromanipulation of the capsule would be a good technique for the successful cryopreservation of expanded equine embryos. Some studies, however, reported pregnancy rates that did not differ from those of intact expanded embryos. The research done by J. Scherzer et al.27 on laser-assisted vitrification reported a 44% pregnancy rate with embryos with a diameter above 300 µm. This percentage dropped to 0 at day 23. They speculated that the embryoblasts were damaged during the process which would mean that most of the detected pregnancies between day 11 and 14 were actually empty vesicles and no real pregnancies. They also stated that replacing the blastocoel fluid with cryopreservative solution with a micropipette might improve viability but further research was needed.
Two of the three experiments done by Y.H. Choi et al.17 with embryos larger than 300 µm also resulted in lower pregnancy rates than those of embryos smaller than 300 µm. In one experiment they reported a normal pregnancy rate of 57% in the group that was immediately transferred after warming but reported a 13% pregnancy rate with embryos that were cultured for 6 hours before transfer. This seems to imply that the process of culturing could have a negative impact on the pregnancy rate.
This research also discovered that removing 70% or more of the blastocoel fluid resulted in higher pregnancy rates, which might also explain the fact that the pregnancy rate in the previous mentioned study was lower than the expected rate. In that study, it is unclear how much of the blastocoel fluid was actually replaced by the cryopreservative, which means there is a possibility that less than 70% of the blastocoel fluid was replaced, negatively impacting the pregnancy rate.
Reported in the study done by F. Guigot et al.18, collapse of the blastocyst might positively impact the pregnancy rate, since it appears to reduce cell damage and crystal forming. Since the studies with the highest pregnancy rates in this review17,21 both collapsed the embryos with the use of fluid aspiration, and other studies in this review also reported normal pregnancy rates after blastocyst collapse, this definitely seems to positively impact the pregnancy rate.
The research done by R. Sanchez et al.9 provided the information that micromanipulated embryos with a diameter exceeding 300µm would yield a higher pregnancy rate (0% vs. 70%) when vitrified in a hemi-straw instead of a Stripper-Tip. The difference between these two methods is that the former is regarded as an “open” technique while the latter as a “closed” one. This could signify that not only the capsule, size and blastocoel fluid affect the pregnancy rate, but also the manner in which the embryo is vitrified contributes to this. The study with the highest pregnancy rate (83%)21 in this review utilized an open system vitrification (CryoLock). Combined with the blastocyst collapse, it seems to be a perfect combination for high pregnancy rates.
This study by R. Sanchez et al. also showed that sometimes even smaller embryos (180 to 300 µm) might not be suitable for certain cryopreservation methods, proving that different methods should be used according to the size of the embryo since one procedure does not seem to be fit for all.
The results of the study on the viability of equine embryos after puncturing the capsule22 show that the viability of the embryos is not affected by the micromanipulation of the capsule. This means that the results of the studies mentioned are most likely not affected by the process of micromanipulation, ruling that out as a possible negative impact on pregnancy rates.
What could be a negative impact, however, is the vitrification process itself. The research conducted by W.K. Hendriks et al.7 reported a higher percentage of cell death in embryos that were vitrified compared to those that were subjected to the slow-freeze method. This study, however, did not work with micromanipulation and blastocoel fluid aspiration, which does seem to increase viability, according to other studies mentioned previously. They ruled out that the cryoprotectants had any effect on the viability of the embryos, since exposure to only cryoprotectants without any cryopreserving procedures gave normal pregnancy rates. .
Conclusion
Based on the reviewed articles, it seems that micromanipulation of the capsule of expanded equine embryos could indeed result in a normal pregnancy rate, thus supporting the hypothesis. However, a lot of other variables need to be taken into consideration. It appears that aspiration of 70% or more of the blastocoel fluid increases the pregnancy rate. The collapse of the blastocyst due to fluid aspiration seemingly has a positive effect on the pregnancy rate as well, seeing that in multiple studies collapse resulted in higher pregnancy rates, possibly due to the fact that collapse offers protection against cell damage and the forming of ice crystals.
The use of an open or closed vitrification system also has an impact on the pregnancy rate. Open systems seem to have a positive impact on the pregnancy rates but should be researched further since the studies that used open systems had varying pregnancy rates. The pregnancy rates were, however, in the range that is deemed to be normal for early equine embryos.
This review, however, did not look at the possible influence of different cryoprotectants and vitrification solutions, age of the mares from which the embryos were taken, the age of the mares in which the embryos were implanted, nor did it look at the overall length of the cryopreservation process. Thus, more research on more different variables is needed to create an ideal cryopreservation protocol for equine embryos with a diameter above 300 µm. Ideally, more embryos should be used in these researches to ensure more credible results.
Future of cryopreservation
An effective cryopreservation protocol for various sizes of equine embryos, <180 µm, 180 – 300 µm and >300 µm in diameter, yielding high pregnancy rates would ideally be developed in the years to come. At the present time, micromanipulation of the capsule, along with aspiration of the blastocoel fluid, seems to be a promising method to successfully cryopreserve equine embryos with a diameter exceeding 300 µm.
Ideally, a way to successfully superovulate mares would be developed in the coming years since this procedure would significantly simplify any research or procedure done with equine embryos. Superovulation has already been successful in cattle, but has not been effective in horses. Since superovulation is a great way to obtain multiple embryos at once from the same mare, it would embryo collection much more efficient. This would mean that more embryos would be available for research and the chances of producing a foal from an embryo of a certain mare would rise, since more embryos would be collected, thus giving rise to more chances of pregnancy.