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Research Update

Embryo Cryopreservation

Objective : To evaluate and compare Partnar’s emP3 1.5M Ethylene glycol with a commercially available product using in vitro produced embryos.

Secondary Objective : Freeze-thaw of in vivo produced bovine embryos with Partnar’s emP3 1.5M Ethylene glycol or a commercially available product (Bioniche’s Vigro Freezing).

Methodology:

In vitro embryos
Cumulus oocyte complexes were aspirated from slaughter house ovaries over 2 IVF runs. At Day 7 of in vitro culture, embryos were evaluated and classified according to their stage and quality of development (based on IETS classification specifications). A total of 124 Stage 6 or 7 Quality 1 or 2 blastocysts were frozen in either emP3 or Vigro.

In vivo embryos
In vivo produced embryos were also used in this study. Briefly, standard superovulatory protocols were used to produce 36 Stage 4,5 6 or 7 Quality 1 or 2 morulaes or blastocysts and were also frozen in either emP3 or Vigro Freezing Protocol
All embryos were frozen using conventional stepwise freezing methods normally used for Direct Transfers. Briefly, embryos were placed in Holding medium for evaluation and transferred in groups of 2 or 3 in either of the ethylene glycol solutions under testing (emP3 or Vigro). Embryos were then mounted in straws and placed in a Digicool freezing apparatus. After 1 minute of equilibration, crystallization was induced and the freezing protocol was started. Temperature is lowered 0.5 C per minute up to -30 C and the embryos are then plunged in liquid nitrogen.

Thawing Protocol
Straws were thawed by removing them from the liquid nitrogen, keeping them in air for 10 seconds and plunging them in a 35 C water bath for 30 seconds. Straws were then emptied in a petri dish letting the medium equilibrate for 1 minute. Embryos were then transferred in Holding medium for 5 minutes and then transferred in to individual drops (10 μl droplet under oil) of BRL (Buffalo Rat Liver) cell-condition medium. The droplets were cultured for 48 hours in an incubator set at 38.5 C 5% CO2 in 100% humidified air.

Embryo classification post-thaw
Viability is the major indication of embryo survival to the freezing process. Briefly, any embryo resenting a Quality 1 or 2 was considered as viable. Quality 3 or degenerate embryos were considered non-viable. Different times of observations were performed: 0 H (few minutes after thawing); 3 H, 24 H and 48 H after thawing. For the in vitro produced embryos, all observations are the result on the mean of the 2 experiments except for the 3 H observation where this data set was collected only from the 2nd IVF run. For the in vivo embryos, the embryos frozen with emP3 originated from 7 different embryo flushes (7 different donors). Results:

In vitro Embryos

Table 1 presents the post-thaw survival rates of in vitro-produced embryos frozen in either emP3 or Vigro media. Data is also represented in Figure 1. It is clear, step-wise freezing is not optimal for in vitro produced embryos.

Table 1Survival rate (% ± SEM) of in vitro produced embryos frozen-thawed using different EG solutions.
Medium Number of embryos % survived at 0 H % survived at 3 H* % survived at 24 H % survived at 48 H
Vigro 60 (42 Q1 + 18 Q2) 77.8 ± 2.8 16.7 10.4 ± 6.3 0
emP3 64 (44 Q1 + 20 Q2) 63.8 ± 9.9 26.9 16.9 ± 1.5 0
*Note: For the 3 H observation, only the embryos of experiment no. 2 were analyzed: Vigro = 24 embryos and emP3 = 26 embryos.

Figure 1
Figure 1. Survival rate (% ± SEM) of in vitro produced embryos frozen-thawed using different EG solutions

In vivo Embryos

Table 2 presents the post-thaw survival rates of in vivo-produced embryos frozen in either Vigro or emP3 media. Data is also represented in Figure 2.

Table 2 Survival rate (%) of in vivo-produced embryos frozen-thawed using different EG solutions.
Medium Number of embryos % survived at 0 H % survived at 3 H* % survived at 24 H % survived at 48 H
Vigro 6 (3 Q1 + 3 Q2) 100.0 66.7 33.3 0
emP3 30 (14 Q1 + 16 Q2) 96.7 71.4 33.3 16.7
*Note: For the 3 H observation, only the embryos of experiment no. 2 were analyzed: Vigro = 6 embryos and emP3 = 7 embryos.

Figure 2
Figure 2. Survival rate (%) of in vivo produced embryos frozen-thawed using different EG solutions

This was not a perfectly structured study as only 6 embryos from 1 donor were frozen in Vigro and 30 embryos from 7 donors were frozen in emP3. The results in Table 2 and Figure 2 indicate clearly that both media are very comparable. Interestingly, included in Table 3 below are the embryos from the same donor frozen in either Vigro or emP3. Again, both media perform as well.

Table 3 Survival rate (%) of in vivo produced embryos from the same donor frozen-thawed using different EG solutions.
Medium Number of embryos % survived at 0 H % survived at 3 H* % survived at 24 H % survived at 48 H
Vigro 6 100.0 66.7 33.3 0
emP3 7 85.7 71.4 14.3 14.3

Another interesting note is that the embryologist that analyzed all embryos after thawing noticed that in vivo produced embryos frozen in emP3 seem to conserve their stage of development and initial quality more effectively than Vigro. This may be simply anecdotal information but the emP3 medium seems to perform quite well.

Conclusion:

Despite the small sample sizes, it would appear that Partnar’s emP3 EG medium works as well as as the Vigro EG medium to protect embryos through the step-wise cryopreservation technique.

This research was sponsored by Partnar Animal Health Inc and was conducted in a independent research facility.