الفهرس 
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Abstract
The current research was conducted to explore: a) the different variables factors influencing the rate of maturation, b) the different factors influencing the rate of cleavage and the growth of buffalo embryos in vitro and c) different methods used for evaluation of oocyte quality. Three experiments have been performed for this purpose.
Experiment I
It was conducted to explore the effect on maturation, cleavage rates and buffalo embryo growth by adding EGF, L-carnitine and dbcAMP to the maturation medium by using aspiration technique, oocytes were gathered. All COCs have been chosen for IVM with more than 3 layers of cumulus cells. Five groups of COCs were divided into:
A-First group: Matured in TCM (control group).
B-Second group Matured in TCM + 10 mg/ml EFG.
C-Third group: Matured in TCM + 0.2% w/v L-carnitine.
D-Fourth group: Matured in TCM + 0.1 mM dbcAMP (high dose).
E-Fifth group: Matured in TCM + 0.01 dbcAMP (low dose).COCs for 22-24 hours and elevated moisture were incubated at 38.5oC and 5% CO2. The number of M II oocytes was calculated after 22-24 hours, calculating the rate of M II oocytes as follows:
Rate of M II oocytes = no. M II oocytes / no. oocytes matured × 100.
The effect of EGF, L-carnitine and dbcAMP adding to the maturation medium on cleavage rates and embryo development has been investigated by using sperm from the same batch and the same buffalo bull for IVF. Using Percoll density gradient method, motile spermatozoa were isolated after thawing of frozen buffalo semen. The oocytes were then separated several times from the mass of the cumulus by gentile pipetting. Oocytes and spermatozoa were co-incubated in humified air at 5% CO2 at temperature of 38.5oC for 18-20 hrs. The presumptive zygotes were incubated in mSOF medium for 8 days under the environment of 5% CO2 and temperature of 38.5oC.
Development of cleavage and embryos was verified on days 2, 5, 7 and 8. It counted the amount of cleaved oocytes and calculated the rate of cleavage as follows:
Rate of cleavage = No. cleaved buffalo oocytes / No. fertilized buffalo oocytes × 100.
Experiment II.
Quality of buffalo oocytes was determined using morphology and Brilliant cresyle blue (BCB). There is high significant difference between morphological selection of buffalo oocytes using grades and biochemical selection using BCB.
Experiment III.
It was carried out to study the effect of buffalo oocytes staining with BCB before maturation on the cleavage rate and embryo development.
Oocytes were collected using aspiration method. All COCs with more than 3 layers of cumulus-cells were selected for IVM. In a humidified atmosphere, the oocytes were exposed to BCB diluted in mDPBS for 90 min at 38.5oC. with mDPBS the oocytes were rinsed twice , examined under a stereo microscope, and split into two groups depending on their cytoplasm coloration; those with or without blue cytoplasm coloration were referred to as BCB+ and BCB-..Oocytes were matured at 38.5oC below 5% CO2 and elevated moisture in the same BMM as earlier stated in Experiment I for 22-24 hrs. Sperm from the same batch and the same buffalo bull were acquired. By using Percoll density gradient method, motile spermatozoa were isolated after thawing of frozen buffalo semen. Oocytes were separated from the cumulus mass after 22-24 hours of IVM by gentile pipetting several times. In the same technique as earlier outlined in experiment I, oocytes and spermatozoa were co-incubated. Development of cleavage and embryos was verified on days 2, 5, 7 and 8.
The findings achieved were as follows:
Effect of EGF, L-carnitine and dbcAMP addition to the maturation medium on the maturation rate of buffalo oocytes.
1- In EGF, L-carnitine and low-dbcAMP, the maturation rate at the M II phase was significantly higher (P<0.01) than the control group.
2-Maturation rate to M II stage was significantly higher (P< 0.01) in EGF and L-carnitin than high and low-dbcAMP groups.
3-In low-dbcAMP, the M II phase maturation rate was considerably higher (P<0.05) than in the high-dbcAMP group.
Effect of EGF, L-Carnitine and dbcAMP addition to the maturation medium on buffalo oocyte cleavage rate and embryo development.
1-Addition of EGF, L-carnitine and low dose of dbcAMP into invitro maturation medium of buffalo oocytes significantly (P> 0.05) increase cleavage rate of buffalo zygotes.
2-Morula and blastocyst rate were higher (p> 0.05) in dbcAMP, EGF and L-carnitine where compared with control group
3- Development of Embryo to morula stage in low-dbcAMP was considerably greater than high dbcAMP group (P<0.01).
Assessment of buffalo oocytes quality using morphology and brilliant cresyle blue (BCB) in buffalo.
This study showed that brilliant cresyle blue (BCB) stain was a significant method in assessment of oocytes quality.
Effect of buffalo oocytes staining with BCB before maturation on cleavage rate and embryo development.
In the BCB+ group, the cleavage frequency and the growth of embryos at 8-16 cell, morula and blastocyst phases was considerably higher than in the BCB- group.
Conclusion
In conclusion, this study’s findings demonstrated that:
1- Adding EGF, L-carnitine and dbcAMP to maturation medium enhanced maturation rate to stage M II and also increase cleavage rate and embryo development of blastocyst phase buffalo oocytes.
2- Addition of low dose dbcAMP to maturation medium enhanced rates of maturation to stage M II and development of buffalo oocytes to stage blastocyst embryos than high dose.
3- Using biochemical method staining of oocyte with BCB for assessment of oocyte quality is a significant method.
4- Before maturation, oocyte staining with BCB stain is helpful for identifying buffalo oocytes with greater cleavage rates and embryo development.