67 The growth and development of in vivo-derived dromedary embryos during short-term incubation: Use of embryo holding medium and the effect of embryonic morphology

Abstract

Production of in vivo embryos for transfer in dromedary camel is a well-established practice, whereas freezing of these embryos is still an ongoing challenge. A common approach in evaluation of freeze–thawing method is achieved by studying in vitro development of frozen–thawed embryos. However, not much is known about the development pattern of fresh dromedary embryos during incubation. The objectives of this study were to evaluate the usefulness of commercial holding media for easy short-term culture of these embryos and to provide preliminary insights on the growth and development of hatched blastocysts with different shapes. Recovered hatched blastocysts from superovulated donors were graded as transferable and non-transferable. Embryos with significant folding or crinkliness were further categorized as collapsed, whereas those with a round or oval appearance were categorized as spherical. Culture was performed in 500-μL drops at 38.5°C, 5% O₂, 0–6% CO₂, and maximum humidity in groups of 2 to 4. The 4 experimental media included culture medium (CM; TCM-199, 10% fetal calf serum (FCS), 0.3 mM sodium pyruvate, 2.2 mg mL⁻¹ sodium bicarbonate), serum-supplemented holding medium (SSH; Syngro + 10% FCS), serum-free holding medium (Syngro) and V-Onestep (Vitromed). In experiment 1, a total of 36 embryos were assigned to 4 groups and further development of the embryos was monitored up to 96 h by morphological evaluations, identifying static and degenerating embryos on daily basis. In experiment 2, a total of 16 spherical and 16 collapsed embryos were cultured in SSH and CM and two-thirds of the culture drop was replaced with fresh medium at 72 h. The proportion of developing embryos and their size expansion was compared between treatments by Fisher’s test and Mann–Whitney U test, respectively. Statistically similar proportions of embryos continued to develop in all media within the first 48 h despite a numeric advantage in CM group; at 72 h, the proportion of growing embryos was significantly higher in CM (77.8%) and SSH (66.6%) compared with SFH (11.1%) and OneStep (22.2%) (P < 0.05). None of the embryos in SFH and only 1 embryo in the V-Onestep group survived beyond 72 h, whereas 3/9 embryos in SSH and 7/9 embryos in CM continued to expand. In experiment 2, the proportion of spherical embryos that developed was higher compared with their collapsed counterparts (8/8 in both groups vs. 5/8 and 4/8 in CM and SSH, respectively) at 24 h. However, remaining collapsed embryos grew and expanded at similar rates to spherical ones in each group (P > 0.05). Replacing the medium did not favour continuation of embryonic growth in SSH beyond 72 h; only 5/16 embryos survived to 96 h compared with 12/16 in CM. In conclusion, serum-supplemented commercial holding preparations provide comparable results to culture medium for short-term incubation of in vivo dromedary embryos. Natural collapsing of hatched blastocysts might be associated with lower developmental competence.