Short term incubation of frozen/thawed bovine embryos

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Published on: August 13, 2020

Dalena Hobbs, Colton Holcomb, and John Gibbons, College of Veterinary Medicine, Lincoln Memorial University, Harrogate, TN, 37752


Embryo transfer is an assisted reproductive technique that enables progressive cattle producers to reach their financial, reproductive, and genetic goals and the process began to gain considerable traction in the late 1970s to early 1980s as non-surgical methods to collect embryos were developed (Troxel, 2013).  Currently, conventional embryo collection techniques require ovarian hyper-stimulation of donor cows with exogenous FSH, artificial insemination, and trans-cervical uterine lavage to recover embryos about 7 days post insemination.  Another approach that has gained popularity is in vitro fertilization (Mapletoft, 2013).  In vitro embryo production involves recovering the ova directly from the ovaries using an ultrasound guided trans-vaginal follicular aspiration technique.  The recovered oocytes are matured, fertilized, and cultured in vitro and this approach has become substantially popular recently despite the increased cost associated with specialized equipment, training, staff, etc.  According to AETA reports (AETA, 2017; 2019) there has been a 120% increase in the number of in vitro produced embryos transferred over the most recent two years of available AETA data (AETA, 2017; 2019).  In vitro embryo production seems to be gaining popularity over conventional embryo transfer techniques because of the potential to produce more calves per year (Stadheim, 2015), may require fewer hormone injections, and does not require synchronization.  In conventional in vivo embryo collections, almost one half of recovered ova are non-viable, and that percentage has not changed substantially in many years (AETA, 2010; 2019).  Many of these non-viable ova are considered degenerate embryos that have not developed to the appropriate stage relative to the other embryos in the cohort. Salvaging these degenerate embryos that would otherwise be discarded may translate to additional embryo transfers or calves per embryo collection.  This experiment evaluated short term incubation environments and the potential damage to the embryo and zona pellucida associated with the freeze / thaw process.  As many practitioners have become involved in in vitro embryo production, the equipment, supplies, and staff are likely in place to consider an in vitro culture approach of degenerate or poor quality embryos (fresh or post-thaw) to enable the development of these embryos.  

Methods and Materials:

Frozen / thawed bovine (Day 7) in vivo derived embryos processed for direct transfer (Ethylene Glycol) were thawed for 30 seconds in a 30°C water bath. Thawed embryos (total of 30 / group over three replicates) were placed in commercially available holding media temporarily to be graded and staged (according to the International Embryo Technology Society rubric) and then placed into either holding media, phosphate buffered saline (PBS) supplemented with 15% fetal bovine serum (FBS; v/v) and antibiotic / antimycotic (gentamicin; 2 mL/ml; v/v), or a commercially available in vitro culture media for approximately 18 hours at 38.5°C. Embryos in the holding media and PBS (+15% FBS) were loaded (individually) into ¼ cc plastic straws which were sealed and submerged in a water bath (38.5°C). Embryos in the in vitro culture media group were rinsed and placed (individually) in equilibrated 25 mL culture drops on tissue coated plastic 60 mm dishes overlaid with lightweight mineral oil and were incubated in 5% CO2 and 100% humidified air (18 hours). Following the incubation period, embryos were recovered, rinsed, and graded and staged again. The numerical change in the embryo grade (1 through 4) and stage (3 through 8) from the pre-freeze information on the straw label, the post-thaw and post-incubation evaluation were recorded and analyzed with ANOVA.


Statistically, there was no difference between the pre-freeze, post-thaw, and post-incubation grades or stages between the holding media and PBS+FBS group; however, there was a decrease in the quality grade (P<0.001) following incubation in all groups (Figure 1).  The decline in the quality grade following incubation in the holding media and PBS+FBS groups was significantly lower than the decline in the quality grade in the in vitro culture group (Figure 1).  There was also a significant decline in the quality grade associated with the freeze / thaw cycle among all the groups (Figure 1).  The developmental stage pre-freeze and post-thaw and post incubation was unaffected in the holding media and PBS+FBS groups; however, in the in vitro culture group, on average embryos developed from the morula to the early blastocyst (P<0.001) stage indicating that on average, viability was maintained. This experiment also indicated that approximately 29% of embryos experience some form of damage to the zona pellucida following the freeze / thaw process.


Cryopreservation of bovine embryos – although critically important to the embryo transfer industry (Mapletoft, 2013), is detrimental to the quality of the embryo and zona pellucida.  With the advent of the direct transfer technology, this decrease in quality is not obvious as embryos are seldom observed post thaw.  Practically, incubation of poor quality embryos for some time may be a mechanism to salvage a few embryos that have not reached the developmental stage of other embryos in the collection and are normally discarded.  The in vitro culture media and system provided a substantially more effective environment to enable embryos to develop further, although the quality of those embryos was negatively affected.  It is difficult to determine if the damaging effects of the freeze / thaw cycle can be overcome during an incubation period; however, the damage was apparently suppressed using an in vitro culture approach.  Holding media and PBS+FBS while useful as a temporary storage device for bovine embryos is not an adequate short-term incubation media and apparently did not mitigate any damage due to the freeze / thaw process.  Future research will involve short-term incubation of fresh embryos in order to eliminate the negative effects associated with cryopreservation.  In conclusion, these results and future research may be useful in the bovine embryo industry, and for cattle producers alike, by increasing the number of transferable quality embryos that would otherwise be discarded.


AETA Statistics Committee Report – 2010

AETA Statistics Committee Report – 2017

AETA Statistics Committee Report – 2019

Mapletoft, R. (2013, September). History and Perspectives on Bovine Embryo Transfer. Retrieved June 22, 2020, from

Stadheim, J. A. (2015, April 10). TEST TUBE TECHNOLOGY: Using IVF on cows has numerous pros, cons. Retrieved June 22, 2020, from

Troxel, T. R. (2013, February 11). Embryo Transfer in Cattle [Scholarly project]. Retrieved June 24, 2020, from

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