Effect of Culture System on Developmental Competence, Cryosurvival and DNA-Fragmentation of In Vitro Bovine Blastocysts

In vitro maturation and fertilization of abattoir-derived bovine oocytes were carried out as described elsewhere (10). At 18-24 hours post insemination (pi), presumptive zygotes were dissected from surrounding cumulus cells and then cultured in either a co-culture system of vero cells in B2 medium (CCD, Paris, France) containing 10% fetal calf serum (FCS) or sequential synthetic oviductal fluid media (SOF1 and 2) as described by Thompson et al. (21). For each experiment, embryos were developed in 50 μl drops in 60 mm plates covered with 10 ml of paraffin oil. Embryos (10/50 μl droplets) were first cultured for 72 hours (phase 1) when the ratios of cleavage, 8-16 and morula were recorded and then refreshed into new culture droplets each 48 hours until day 8 (phase 2) when the ratios of days 6-7-8 blastulation were compared. On day 8 pi, good quality grades 1 and 2 blastocysts developed in both sequential SOF and B2 co-culture systems were selected and counted. Half of these embryos were randomly cultured until hatching for the following quality assessments: total cell number (TCN), viability assay and TUNEL measurement of DNA fragmentation. Meanwhile, the remaining half of the expanded blastocysts in both groups were removed from culture medium, vitrified, thawed within 30 minutes and further cultured for two additional days. The ratios of hatching/ degeneration of non-vitrified embryos along with the ratios of immediate/total degeneration, re-expansion and hatchability of vitrified embryos developed under different culture conditions were determined. Hatched embryos were used for quality assay as mentioned for non-vitrified embryos.

The vitrification protocol used in this study was a modified technique of Martínez et al. (22) carried out as described previously (16). Briefly, embryos were pre-equilibrated in a solution of 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (DMSO) in Dulbecco Modified Eagles' Medium (DMEM) supplemented with 20% FCS until the expanded blastocysts endured a period of shrinkage and returning (up to 8 minutes, at 38.5°C). Equilibrated embryos were then exposed to a vitrification solution consisting of 15% EG + 15% DMSO in holding medium (DMEM-F12 with 20% FCS) at room temperature for one min and then loaded into the tips of the cryotops (Cryologic; CVM™, Fibreplug & Sleeve, Australia) with a minimum amount of the vitrification solution and immediately plunged into liquid nitrogen (LN2). For thawing, embryos were removed from LN2 and quickly exposed to DMEM + 20% FCS supplemented with 1 M sucrose for 1 minutes on a warm plate (38.5°C). Then, embryos were transferred to DMEM + 20% FCS containing 0.25 M sucrose for 5 min and finally washed thoroughly in DMEM + 20%FCS. Viable embryos in each group were cultured in the corresponding culture system. The percentages of re-expansion, hatching and degeneration were determined at 12 hours and 48 hours post-thawing and hatched embryos were used for assessment of TCN, viability and DNA fragmentation.

The viability of hatched blastocysts developed in vitrified and non-vitrified groups was determined by the differential staining method of Hosseini et al. (23). In brief, hatched blastocysts were incubated for 30 minutes in pre-equilibrated culture medium containing propidium iodide (PI; 300 μg/ml) and bisbenzimide (Hoechst: H33342; 5 μg/ml). Embryos were then washed in phosphate buffer saline (PBS) free of calcium and magnesium and then fixed in 2.5% glutharaldehyde. Fixed embryos were then washed in PBS before being mounted on microscopic slides. The stained embryos were examined under an epifluorescent microscope [excitation wavelength of 330-385 nm and barrier filter (400 nm)] to detect necrosed versus viable blastomers as red and blue in appearance, respectively. Moreover, determination of DNA-fragmentation within the embryonic mass was performed by Hosseini et al. (9).

All experiments in this study were repeated at least three times. To compare rates of blastocyst development and hatching between treatments, chi square analysis was applied. The analysis of variance (ANOVA) was used for vitrification groups and the t test was used for comparisons of means. When the ANOVA test showed statistical differences, the Student- Neumann-Keuls test was used to discriminate between groups. All statistical evaluations were carried out using the Statistical Package for Social Sciences (SPSS), version 11. All data were presented as means ± SEM and differences were significant at p<0.05.

From 420 bovine ovaries obtained from an abattoir, 2540 COCs were cultured for in vitro maturation (IVM), 2230 were subjected to in vitro fertilization (IVF) and 1840 presumptive zygotes were subsequently cultured in sequential SOF (n=900) and vero B2 co-culture systems (n=1250) for 8 days when the ratios of cleavage, morula, and days 6-8 blastocyst formation were determined. Table 1 compares detailed results of developmental competence in addition to the quality of the resultant blastocysts developed under both culture systems. As shown in Table 1, there was no significant difference in cleavage rate of embryos cultured in the co-culture (92.0%) versus SOF (86.5%) groups (p<0.05). However, zygotic development in the co-culture conditions resulted in significantly greater development to the 8-16 (87.0%) and morula (75.0%) stages compared to SOF culture media (47.3% and 54.4%, respectively; p<0.05). On the other hand, while 37.1% of morula stage embryos in SOF medium developed to blastocysts at day 6 of in vitro production (IVP), none of the embryos (0.0%) in the co-culture system developed into blastocysts at the same day. Similarly, the blastocyst yield rate at days 7 and 8 were significantly higher when embryo development occurred in SOF versus the coculture system (49.3% and 51.2% vs. 28.3% and 40.1%, respectively; p<0.05; Table 1).

Hatching ability of the blastocysts in both groups were non-significantly different (p<0.05). Comparing the quality of hatched blastocysts developed in the two culture systems, although the culture upon monolayer significantly increased total cell number (TCN) of the blastocysts that developed compared to SOF (248.0 vs. 221.0), the inner cell mass / trophectoderm (ICM/TE) ratio of the SOF-cultured embryos (41.5%) was significantly higher than the related rate of co-cultured embryos (33.1%). However, there was no significant difference in the viability and DNA fragmentation index (DFI) of the blastocysts developed in both groups.

After vitrification/warming of co-culture versus SOF developed blastocysts, there was no significant difference in the proportion of immediate degenerated blastocysts (15.7% vs. 16.7%) nor the proportion of embryos re-expanded when assessed 12 hours post warming (84.3% vs. 83.3%; p<0.05). However, the hatching rate of vitrified/ warmed blastocysts developed in SOF medium (65.0%) was significantly lower than the rate of the co-culture condition (74.3%). Consequently, the post-warming total degeneration rate of the coculture group (25.7%) was significantly lower than the SOF (35.0%) group.

After vitrification-warming, blastocysts developed in SOF medium had signifcantly lower quality in terms of TCN and ICM/TE ratio than those developed in the co-culture system. Although analysis of viability indicated no significant difference between the hatched blastocysts of the two groups, DFI value of the co-culture group (10.0%) was significantly lower than the SOF group (13.5%).

In table 3 the results of quality assessment of nonvitrified (Table 1) versus vitrified (Table 2) blastocysts which have been cultured under the same conditions were paired and compared.

The levels of difference (LD) between the values of each comparison were also compared (based on statistical analysis of the replicate data). As shown, irrespective of the culture conditions employed, overall quality of vitrified-warmed IVP embryos (as measured by the rates of hatching, TCN and DFI) were significantly lower than the corresponding rates of non-vitrified embryos. Moreover, by looking into the LD between vitrified/non-vitrified embryos cultured in the same condition (Table 3), the culture of embryos in SOF versus the co-culture system resulted in different LD values which approached significance for hatching, TCN and DFI percentages (15.8%, 32.6% and 6.0% vs. 9.0%, 77.6% and 3.9%, respectively).