Vitrification of in vitro-derived bovine embryos: targeting enhancement of quality by refining technology and standardising procedures.

Bovine invitro fertilisation technology has been widely exploited in commercial settings. The majority of invitro-derived cattle embryos are transferred into recipient cows as recently collected (i.e. 'fresh') embryos due to the lack of a reliable cryopreservation method that results in favourable pregnancy rates following transfer of thawed embryos. This is a primary reason for the poor industry uptake of this extreme temperature freezing process. Numerous investigations into vitrification have revealed the importance of rapid cooling and warming rates, enhancing embryo viability after cryopreservation compared with conventional slow freezing. Those studies spawned a considerable assortment of cryovessels and diversity of procedures, delivering variable rates of success, which makes performing vitrification consistently a practical challenge. Hence, further research is required in order to both optimise and standardise vitrification methodology and to design a cryovessel that enables direct transfer of vitrified embryos to recipients after warming. In parallel with improvements in vitrification, it is important to continue to raise the quality of invitro-derived cattle embryos through modifications in laboratory culture techniques. The twin goals of methodology refinement and standardisation, leading to embryo quality enhancement, are each imperative if invitro fertilisation technology is to be adopted in the field.

Does the Addition of Salubrinal to in vitro Maturation Medium Enhance Bovine Blastocyst Yields and Embryo Cryotolerance?

BACKGROUND: Modifications to in vitro maturation (IVM) media are made to improve rates of blastocyst formation and quality of mammalian embryos. Embryo quality is an important factor in the viability of embryos following cryopreservation. Salubrinal is a specific inhibitor of endoplasmic reticulum stress-induced apoptosis in eukaryotic cells. Here, it was added to IVM medium in an attempt to increase blastocyst formation and to enhance embryo quality in cattle. OBJECTIVE: To assess the effect on blastocyst formation and cryotolerance of the supplementation of salubrinal to bovine IVM medium. MATERIALS AND METHODS: Cumulus-oocyte complexes (COCs) collected from slaughterhouse ovaries were assigned randomly to two groups, either cultured in IVM medium that was supplemented with 400 mM salubrinal (treated, 262 COCs) or that was not supplemented (control, 263 COCs). All oocytes of the matured COCs were fertilized in vitro with sperm from the same proven bull and cultured for 6-7 d. At the time of blastocyst collection, expanded blastocysts were chosen for cryopreservation, while early, hatching and hatched blastocysts and those of poor quality were not used. There were 83 expanded blastocysts classified to be of good quality in both the control and salubrinal-treated groups that were subjected to vitrification. After 5 to 10 months of cold storage, the embryos were warmed and cultured in vitro for 24 h to assess the survival rate and for 48 h to assess the hatching rate. RESULTS: The blastocyst developmental rate in the salubrinal-treated group was similar to that in the control group, 61.5% compared with 62.7% (P > 0.05). The survival rate of blastocysts after vitrification was also similar, at or very close to 100%. In addition, there was no statistically significant difference in the hatching rate of expanded blastocysts derived from the COCs cultured with (treated) and without (control) addition of salubrinal to the IVM medium (91.6% compared with 85.5%; P > 0.05). CONCLUSION: Supplementation of salubrinal to the IVM medium neither improved nor reduced rates of bovine blastocyst formation and of embryo cryotolerance as determined by post-warming viability.

An in vitro assessment of the value of sucrose supplementation to warming media for the vitrification of bovine embryos.

　　BACKGROUND: In order to thaw slow-cooled bovine embryos it is standard practice to draw out permeating cryoprotectants by passing embryos through successively decreasing osmotic solutions. However, recently it has been suggested that sucrose may not be needed in the warming media. OBJECTIVE: The aim of this experiment was to compare the effect of warming media prepared with or without the inclusion of sucrose on the survival and hatching capacity of vitrified in vitro-derived bovine embryos. MATERIALS AND METHODS: Expanded blastocysts were produced in vitro and vitrified. Vitrified embryos were warmed either successively through 0.5, 0.3 and 0.2 M sucrose solutions ('stepwise'), or by placing directly into the blastocyst solution without the addition of sucrose ('direct'). A total of 93 expanded blastocysts were assigned randomly to two treatment groups, respectively. RESULTS: The re-expansion rates of vitrified embryos warmed after 24h in vitro culture were similar between the two groups (46/46, 100%; 46/47, 97.9%). From those vitrified embryos that expanded at 24 h there was also no significant difference in hatching rates after 48 h in vitro culture (42/46, 91.3%; 40/46, 87.0%). CONCLUSION: The findings indicate that stepwise warming through sucrose solutions is not required for continued embryo development. Hence, a more time-efficient warming method for vitrified embryos may be followed when conducting cattle embryo transfers.