الملخص الإنجليزي
Dromedary camels are economically and culturally valuable animals in the Arabian Peninsula. However, they face many in vivo and in vitro reproductive challenges. Oocyte quality plays a key role in maximizing the success of embryo development and production. Therefore, this study is conducted to evaluate camel oocytes quality using multiparametric assessment; enzymatic, molecular, fluorescent and structural analysis. Dromedary camel ovaries (n=80, between 4 to 14 years old) were obtained from local slaughterhouses (Sinaw and Salalah) during the breeding season (December to April) and non-breeding season (May to October). Camel oocytes were recovered by aspiration and/or slicing of ovaries. Cumulus oocyte complexes (COCs) were stained with 26 UM BCB for 90 min. Unstained COCs were used as a control (BCBc). Stained COCs were categorized according to cytoplasmic coloration into BCB+ and BCB- and their diameter were measured under a stereomicroscope. Marker genes for competency were used in this study (COX2, COX3, ATP6, NDS, CYTB, TNFa, HSPAIB, GSTT1 and ZP3). GAPDH was used as a housekeeping gene for normalization, Quantitative gene expression analysis was carried out by total RNA extraction from denuded oocytes and cumulus cells (CCs) from all three groups (BCBC, BCB+ and BCB-) with the use of RNeasy® Mini Kit. Reverse transcription was performed by Omniscript followed by cDNA amplification with Taq PCR Core Kit. Fluorescent assessment of the mitochondrial distribution and membrane potential was carried out with Rhodamine 123 and JC-1 staining, respectively. Zona pellucida surface structure was assessed by scanning electron microscopy and the pore size was measured by CorelDRAW software. The data were analyzed with independent t-test and one way ANOVA using PRISM software. Total numbers of BCB- and BCB+ oocytes were 399 and 276 respectively. During the reproductive season, the mean = SE of BCB+ oocytes collected after 2h post collection were significantly lower (P<0.05) from those recovered after 24h. BCB staining showed no difference in the number of BCB+ oocytes in neither reproductive nor non reproductive seasons (P>0.05). BCB+ oocytes mean diameter (um) was significantly larger (P < 0.05) in BCB+ oocytes. Camel oocytes and their surrounding CCs displayed down-regulation pattern in two mitochondrial genes (COX3 & CYTB). Similar expression pattern was observed in nuclear genes, whereas stress related genes, TNFa and GSTT1 were down-regulated in BCB- and BCB+ oocytes compared to the control. Mitochondrial distribution pattern of BCB and BCB+ oocytes exhibit good maturation levels. Mitochondrial membrane potential was equal in BCB groups. Three zona pellucida surface morphology types were found in camel oocyte; ZP type I for BCB+ oocytes, ZP type II for BCB oocytes and ZP type III for BCBC oocytes. Pore sizes were small and not significantly different in BCBC, BCB- and BCB+ oocytes. In conclusion, oocytes selected by BCB staining and validated by multi-assessments failed to provide a clear distinction between BCB- and BCB+. Therefore, similar to pigs and horses, BCB staining is unsuitable test for camel oocytes selection. Conversely however, gene expression pattern and mitochondrial potential and distribution as well surface structure of ZP improve oocytes cytoplasmic and nuclear maturation prediction. The future of embryo assessment will rely on objective evaluation associated with genomics, proteomics and metabolomics technologies.
