الفهرس 
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Abstract
The Arabian Camel (Camelus dromedarius) is a versatile animal with high meat and milk yield. It has unique biological features such as low reproductive efficiency under their natural pastoral condition. Recently, there has been a surge in interest in developing assisted reproductive technologies (ART) and cryopreservation for the conservation of camel genetic resources. Despite the advancement of ART techniques, gametes and embryos are exposed to a variety of potential ROS-inducing stimuli during the procedure and the hazard impact of induced oxidative stress is significantly greater than in-vivo due to the lack of physiological defense mechanisms, absence of natural antioxidants and the presence of multiple potential sources of ROS. Supplementation of in vitro maturation media with antioxidants, such as vitamin C and Melatonin during in vitro embryo production may prevent the increase in ROS, improve embryo production efficiency and increase the viability of vitrified-thawed in-vitro matured dromedary camel oocytes. The epididymal spermatozoa were used for in-vitro fertilization (IVF). The present study aimed to improve the developmental competence and viability of vitrified-thawed in-vitro matured dromedary camel oocytes using antioxidants.
Camel ovaries (n=289) were collected from camels slaughtered at El-Warraq abattoir, Giza, Egypt. Only excellent and good quality aspirated oocytes were selected for further experiments. selected oocytes were matured in maturation media TCM-199 with or without antioxidants supplementation (50µg/ml Vitamin C, 100µM/ml Melatonin). Four experiments were conducted:
Experiment 1: Effect of addition of antioxidants on the in-vitro maturation media on maturation rate of Dromedary camel oocytes.
Maturation of camel oocytes was assessed at the end of incubation. Assessment of cytoplasmic maturation of camel oocytes was carried through 4 grades (G0, GI, GII, GIII). Nuclear maturation rate (MII) was calculated based on number of oocytes with 1stpolar body. The mean of Glll and Gll cumulus expansion of matured oocytes in TCM-199 supplemented with Vitamin C and melatonin was higher than their mean in the control group which showed high mean of oocytes with Gl expansion 10.94±0.47 and G0 expansion 7.44±0.44 compared to the treated groups. There was a significance between the nuclear maturation rate among groups (P<0.001). The mean of matured oocytes in the control group with 1st polar body was significantly different (74.42 ± 0.69) compared to vitamin C (80.62 ± 0.62) and Melatonin treated groups (85.53 ± 0.62).
Experiment 2: Effect of addition of antioxidants on the in-vitro maturation media on the developmental competence of fresh in-vitro matured dromedary camel oocytes.
No of in-vitro matured dromedary camel oocytes entered fresh IVF (n=767). Matured oocytes were fertilized using epididymal spermatozoa. The matured oocytes in vitamin C group and melatonin group undergone fertilization and showed extrusion of 2nd polar body were significantly higher than the control group (P<0.001). The fertilization rate of in-vitro matured oocytes in vitamin C and melatonin treated groups was 70.88 ± 0.87 and 76.63 ± 0.82, respectively compared to the control group 56.26 ± 0.63
Experiment 3: Effect of antioxidants on the viability of vitrified-thawed in vitro matured Dromedary Camel oocytes.
Number of in-vitro matured dromedary camel oocytes exposed to vitrification (n= 1176). Following vitrification-thawing procedure, the oocytes changes. Normal survived thawed oocytes were fertilized using epididymal spermatozoa.
Experiment 3.1: Effect of antioxidants on the recovery rate of vitrified-thawed in-vitro matured dromedary camel oocytes.
No of recovered oocytes was 1029 from the total no of matured oocytes with 1st polar body that undergone vitrification 1176. There was no significant difference in the recovery rate of oocytes among groups (P=0.632)
Experiment 3.2: Effect of antioxidants on the morphology of vitrified-thawed in-vitro matured Dromedary Camel oocytes.
There was a significant (p=0.005) in the normal and abnormal morphology of vitrified-thawed oocytes between the control and the treated groups. The mean of normal oocytes in the control group was 71.51 ± 2.18 compared to vitamin c and melatonin treated groups 77.68 ± 0.80 and 79.94 ± 1.21 respectively. On the other hand, the control group showed high mean of abnormal morphology of vitrified-thawed oocytes (28.49 ± 2.18) than treated groups. Abnormal vitrified-thawed oocytes with fragmented cytoplasm was higher in the control group 50.25±4.80 than others while oocytes with shrinkage cytoplasm was detected to be high in the vitamin C group 42.11±4.44. Abnormal vitrified-thawed oocytes with cracked zona and leakage was higher in the melatonin treated group 24.44±4.97 and 30.93±2.14, respectively than control and vitamin C treated group.
Experiment 3.3: Effect of antioxidants on the fertilization rate on the vitrified-thawed in-vitro matured dromedary camel oocytes
There was a highly significant difference (P<0.001) between the control group and the treated groups in the mean of fertilization rate of vitrified-thawed in-vitro matured dromedary camel oocytes. The mean of fertilization rate in the control group 40% and increased to be 61% in the vitamin C treated group and 64% in the melatonin treated group. These results reflect the positive impacts of addition of vitamin C and melatonin on the in vitro maturation media on the fertilization rate of vitrified in vitro matured oocytes after thawing.
Experiment 4: Effect of antioxidants on the viability and mitochondrial intensity of vitrified-thawed in-vitro matured Dromedary Camel oocytes.
Excellent and good quality camel oocytes (n=195) were divided in two groups: the first group (n=107) for staining of fresh in vitro matured dromedary camel oocytes and the second (n=88) for staining of vitrified-thawed in-vitro matured dromedary camel oocytes. Our results showed that there was a significant difference (P<0.001) in the mitochondrial distribution (diffused, periphery and semi-periphery) between the control group and treated groups either in fresh or vitrified-thawed in-vitro matured dromedary camel oocytes. Also, the mitochondrial intensity of treated groups was significantly higher than the control group (P<0.001) either in fresh or vitrified-thawed in vitro matured dromedary camel oocytes.
It can be concluded from the study that the addition of vitamin C and melatonin as antioxidants significantly increases the rate of maturation and fertilization rate of in-vitro matured oocytes and in-vitro matured vitrified-thawed dromedary camel oocytes through improving the mitochondrial intensity and distribution of matured oocytes.