This document provides an overview of short term maintenance and culture of embryos. It discusses key events in the history of embryo transfer including the first successful transfers in rabbits in 1890 and cattle in 1951. It then covers topics like superovulation, embryo evaluation, transfer procedures, cryopreservation techniques including slow freezing and vitrification for various species like cattle, horses, sheep, goats and pigs. It provides details on the cryopreservation protocols and outcomes for these different animal embryos. Key references are also cited at the end.

1. SHORT TERM
MAINTENANCE
& CULTURE OF
EMBRYO
SUBMITTED BY
SUSHIL NEUPANE
MVSc Theriogenology
1st semester

2. INTRODUCTION
 In 1890 Heape carried out the first successful embryo transfer in
rabbits (Heape, 1890) and the first calf after embryo transfer was
born in 1951 (Willett et al., 1953).
 The first lambs were born after embryo transfer in 1949 (Warwick
& Berry, 1949).
 The first child conceived by in vitro fertilization (IVF) was born in
1978 (Steptoe & Edwards, 1978), followed in 1981 by the first calf
(Brackett et al., 1982).
 In 2015 the number of transfers of in vitro produced bovine
embryos to recipients worldwide exceeded 400,000, mostly in
South and North America (Perry, 2016).

3. INTRODUCTION
 Superovulation is a prerequisite for the successful application of
embryo transfer in species with a physiologically low ovulation
rate (cattle, sheep/goats, and horses).
 A close synchrony of estrus between the donor and the recipient is
crucial for obtaining optimal pregnancy rates and is an important
part of the entire embryo transfer planning process.
 Embryo evaluation is performed in a dish that should contain no
blood or other debris.
 The morphological evaluation of embryos includes assessment of
stage and quality of the embryo and follows the guidelines from the
International Embryo Technology Society (Stringfellow & Givens,
2010).

4. TRANSFER OF EMBRYO
 Each embryo is then loaded into a 0.25 ml sterile insemination straw.
 The straw is placed in an ordinary insemination pipette or a specially
designed embryo transfer catheter, which is a thinner and longer
version of the ordinary insemination pipette.
 The oestrous cycle of the recipients must be synchronised with that of
the donor, and before transfer, it must be ascertained that oestrus has
occurred within ± 24 hours of the donor (Hasler, 2003) and that a
functional CL is present.
 The synchrony between recipient and embryo developmental stage
may be a more reliable parameter than oestrous synchrony. Thus
morulae should, for example, be transferred to recipients that are at
Day 6 after oestrus, and blastocysts to recipients at Day 7.

5. CRYOPRESERVATION OF
EMBRYOS
 The general cryopreservation principles are similar for all
mammalian cells.
 The loading of cryo-protectants and the intracellular dehydration
and rehydration processes are different compared with other cell
types (Leibo 1977, Lehn-Jensen & Rall, 1983).
 The first successful freezing of cattle embryos was reported by
Wilmut and Rowson (1973), applying the principles developed in
mice by Whittingham et al. (1972).
 Day 6 to 7 sheep and cattle embryos at the morula, early blastocyst,
or blastocyst stage are more tolerant to low temperature
preservation than earlier and later developmental stages.

6. CRYOPRESERVATION OF
EMBRYOS
 ‘Slow rate freezing–thawing procedure’ is to use low concentrations of
the intracellular cryoprotective agent, normally 1.5 mol/L glycerol or 1.5
mol/L ethylene glycol in Dulbecco’s PBS supplemented with 20% FCS,
or using a commercially available medium.
 The embryos are equilibrated for 10 to 15 minutes, then loaded into a
straw and placed in the chamber of a manual or automatic freezer at
−6°C.
 After approximately 5 minutes, ice crystal formation (‘seeding’) is
induced with supercooled forceps, and a slow freezing rate is used until
−35°C, at which stage the embryos are plunged directly into liquid
nitrogen at −196°C.

7. CRYOPRESERVATION OF
EMBRYOS
 Exposing the straw for a short period of time to air and then
submerging it in a water bath at 25° to 30°C for a few minutes is
the best method of thawing, irrespective of species.
 When embryos are frozen/thawed in ethylene glycol, they can be
transferred directly whereas embryos frozen/thawed in glycerol
must be submitted to a procedure in which the cryoprotectant is
removed stepwise before transfer.
 Sucrose, a nonpermeating cryo-protectant, may be used to dilute
the glycerol, and this seems to improve the embryo survival rates.
 Obviously, injuries may occur during freezing and thawing, and
these include osmotic shock, intracellular ice crystal formation,
fracture damage, and chilling injuries.

8. CRYOPRESERVATION OF
EMBRYOS
 An alternative to slow freezing is an ‘ultrarapid freezing and warming
procedure’, the vitrification method that is defined as a glass-like solidification
of solutions at low temperature without ice formation.
 Vitrification requires addition of high concentrations of the cryoprotectant
followed by ultrarapid freezing and warming, with an inherent risk of osmotic
and toxic injuries.
 High concentrations of cryoprotectant, as with vitrification, may result in
irreversible depolymerisation of the microfilaments and microtubules.
 To use the method commercially, each embryo must be handled and the straws
marked individually, so the time required for vitrification is likely to be at
least as long as when using slow rate freezing.

9. CRYOPRESERVATION OF
CATTLE EMBRYOS
 Approximately 60% of all transferred in vivo-derived cattle embryos have been frozen
(Perry 2016), mostly using the slow rate freezing method, although vitrification is also
used.
 Embryonic loss after transfer of frozen–thawed embryos is slightly higher than that
reported for fresh embryos, and poor quality frozen–thawed embryos will even
experience a slightly increased gestation length (Callesen et al., 1996).
 Vitrification seems to be a promising alternative to preserve IVP embryos (Donnay et
al., 1998).
 For In Vitro Produced (IVP) embryos, Vajta et al. (1998) achieved high hatching rates
when culturing 6- to 7-day-old blastocyst in vitro, and nonsurgical transfer of warmed
OPS vitrified embryos has given pregnancy rates of about 50% to 60%.

10. CRYOPRESERVATION OF
HORSE EMBRYOS
 The cryopreservation of equine embryos has been more complex and less successful.
 The survival of equine blastocysts after cryopreservation is complicated by the
development of the capsule, which apparently hinders the permeability and equilibration
of cryoprotectants (Legrand et al. , 2001).
 Partial removal of the capsule with trypsin treatment has led to a decreased percentage of
necrotic cells after cryopreservation, although there was no clear advantage in the term of
pregnancy rates (Legrand et al. 2000, Legrand et al. 2002, Maclellan et al. 2002).
 Puncturing, followed by partial removal of the blastocoele before vitrification using
micromanipulation equipment or manually with a 25-gauge needle, has been used with
good results in embryos larger than 300 and up to 650 μm in diameter, even though
pregnancy and foaling rates are still lower than for embryos smaller than 300 μm (Ferris
et al. 2016, Choi & Hinrichs 2017).

11. CRYOPRESERVATION OF
HORSE EMBRYOS
 Glycerol and ethylene glycol are generally used, having resulted in birth of live foals
when the conventional slow freezing procedure has been employed (Slade et al. 1985,
Seidel et al. 1989, Hochi et al. 1996).
 Equine blastocysts exhibit a higher permeability to ethylene glycol than to glycerol and
ethylene glycol or a combination of glycerol.
 Increasing concentrations of ethylene glycol combined with vitrification resulted in
acceptable pregnancy rates.
 The most practical commercial approach in veterinary practice without the availability of
micromanipulation equipment is to collect embryos for cryopreservation just when they
enter the uterus 5.5 to 6 days after ovulation.

12. CRYOPRESERVATION OF
SHEEPAND GOAT EMBRYOS
 Slow rate freezing of small ruminant embryos follows the principles
outlined for cattle and has been used extensively (Tervit et al.1986).
 Several vitrification studies have shown that it is possible to achieve
pregnancy rates of 50% to 60% after direct transfer (Baril et al. 2001,
Hong et al. 2007).
 Direct laparoscopic transfer of vitrified sheep embryos (Okado et al.
2002, Isachenko et al. 2003) yields lower rates (20%).

13. CRYOPRESERVATION OF PIG
EMBRYOS
 Pig embryos at most stages of development are sensitive to cooling and freezing
(Polge 1977), probably because of their high lipid content.
 Nevertheless, it was possible to produce offspring from slow freezing of expanded
blastocysts (Hayashi et al. 1989).
 It seems advantageous to use perihatching stages.
 Removal of the lipid droplets seems to improve the survival rates considerably.
 Vitrification has resulted in high in vitro survival rates.

14. REFERENCES
Baril, G., Traldi, A. L., Cognié, Y., Leboeuf, B., Beckers, J. F., & Mermillod, P. (2001). Successful direct transfer of vitrified
sheep embryos. Theriogenology, 56(2), 299-305.
Brackett, B. G., Bousquet, D., Boice, M. L., Donawick, W. J., Evans, J. F., & Dressel, M. A. (1982). Normal development
following in vitro fertilization in the cow. Biology of reproduction, 27(1), 147-158.
Callesen, H., Liboriussen, T., & Greve, T. (1996). Practical aspects of multiple ovulation-embryo transfer in cattle. Animal
Reproduction Science, 42(1-4), 215-226.
Choi, Y. H., & Hinrichs, K. (2017). Vitrification of in vitro-produced and in vivo-recovered equine blastocysts in a clinical
program. Theriogenology, 87, 48-54.
Donnay, I., Auquier, P., Kaidi, S., Carolan, C., Lonergan, P., Mermillod, P., & Massip, A. (1998). Vitrification of in vitro produced
bovine blastocysts: methodological studies and developmental capacity. Animal reproduction science, 52(2), 93-104.
Ferris, J., Mahboubi, K., MacLusky, N., King, W. A., & Favetta, L. A. (2016). BPA exposure during in vitro oocyte maturation
results in dose-dependent alterations to embryo development rates, apoptosis rate, sex ratio and gene expression. Reproductive
toxicology, 59, 128-138.
Hasler, John F. "The current status and future of commercial embryo transfer in cattle." Animal reproduction science 79, no. 3-4
(2003): 245-264.
Hochi, S., Maruyama, K., & Oguri, N. (1996). Direct transfer of equine blastocysts frozen-thawed in the presence of ethylene
glycol and sucrose. Theriogenology, 46(7), 1217-1224.

15. REFERENCES
Hong, Q. H., Tian, S. J., Zhu, S. E., Feng, J. Z., Yan, C. L., Zhao, X. M., ... & Zheng, S. M. (2007). Vitrification of Boer Goat Morulae and
Early Blastocysts by Straw and Open‐Pulled Straw Method. Reproduction in domestic animals, 42(1), 34-38.
Isachenko, V., Montag, M., Isachenko, E., van der Ven, K., Dorn, C., Roesing, B., ... & van der Ven, H. (2006). Effective method for in-vitro
culture of cryopreserved human ovarian tissue. Reproductive biomedicine online, 13(2), 228-234.
Kobayashi, K., Hayashi, S., Ohtubo, Y., Honda, A., Mizuno, J., & Hirano, S. (1989). Nonsurgical embryo collection from sows with
surgically shortened uteri. Theriogenology, 32(1), 123-129.
Legrand, E., Krawiecki, J. M., Tainturier, D., Corniere, P., Delajarraud, H., & Bruyas, J. F. (2001). Does the embryonic capsule impede the
freezing of equine embryos. EQUINE EMBRYO TRANSFER, 62.
Lehn-Jensen, H., & Rall, W. F. (1983). Cryomicroscopic observations of cattle embryos during freezing and thawing. Theriogenology, 19(2),
263-277.
Leibo, S. P. (1977). Fundamental cryobiology of mouse ova and embryos. The Freezing of Mammalian Embryos, 69-92.
Maclellan, L. J., Carnevale, E. M., Da Silva, M. C., Scoggin, C. F., Bruemmer, J. E., & Squires, E. L. (2002). Pregnancies from vitrified
equine oocytes collected from super-stimulated and non-stimulated mares. Theriogenology, 58(5), 911-919.
OKADA, A., YOSHII, K., MIZUOCHI, Y., ANDOH, T., JOUJOU, S., WACHI, S., ... & FUKUI, Y. (2002). Viability of cryopreserved and
vitrified embryos and fertility after direct transfer in ewes. Journal of Reproduction and Development, 48(2), 189-195.
Perry G. 2016. 2015 statistics of embryo collection and transfer in domestic farm animals. Embryo Transfer Newsl, 34(4), 10-24.
Polge, C. (1977). The freezing of mammalian embryos: perspectives and possibilities. The freezing of mammalian embryos, 52, 3-12.
Slade, N. P., Takeda, T., Squires, E. L., Elsden, R. P., & Seidel Jr, G. E. (1985). A new procedure for the cryopreservation of equine
embryos. Theriogenology, 24(1), 45-58.
Squires, E. L., Seidel Jr, G. E., & McKinnon, A. O. (1989). Transfer of cryopreserved equine embryos to progestin‐treated ovariectomised
mares. Equine Veterinary Journal, 21(S8), 89-91.

16. REFERENCES
Steptoe, P. C., & Edwards, R. G. (1978). Birth after the reimplantation of a human embryo. The
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Stringfellow, D. A., and D. Givens. "Manual of the International Embryo Transfer Society, Champaign." IL:
International Embryo Transfer Society (2010).
Tervit, H. R., Goold, P. G., & McKenzie, R. D. (1986). Development of an effective goat embryo transfer
regime. In Proceedings of the New Zealand Society of Animal Production (Vol. 46, pp. 233-236).
Tervit, H. R., Whittingham, D. G., & Rowson, L. E. A. (1972). Successful culture in vitro of sheep and cattle
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Vajta, G., Holm, P., Kuwayama, M., Booth, P. J., Jacobsen, H., Greve, T., & Callesen, H. (1998). Open pulled
straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Molecular
Reproduction and Development: Incorporating Gamete Research, 51(1), 53-58.
WARWICK, B. L., & Berry, R. O. (1949). Inter-Generic and intra-specific embryo transfers: In Sheep and
Goats. Journal of Heredity, 40(11), 297-303.
Willett, E. L., P. J. Buckner, and G. L. Larson. "Three successful transplantations of fertilized bovine
eggs." Journal of Dairy Science 36, no. 5 (1953): 520-523.

17. THANK YOU