VII. SummaryBuffaloes represent vital part of theagricultural economy in Egypt and some other developing countries. However,buffaloes have low reproductive efficiency which could be related to the lowtotal number of follicles in the ovary, poor superovulatory response and highpercentage of atretic follicles. Moreover, recently efforts have been initiatedto improve the reproductive potential of these animals using biotechnologies asin vitro fertilization (IVF) and embryo transfer (ET).
Various studies have been shown that the in vitro culture (IVC)environment which the embryos are exposed after fertilization is the keydeterminant of the blastocyst quality. The assessment ofembryo quality is one of the most important factors, which determine asuccessful embryo transfer. Its significance is related to the essentialcorrelation between embryo quality and the stage of its development.The present study aimed to: 1. Assess the developmental competence of the in vitro producedembryos under different culture conditions.
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2. Qualify the invitro produced embryos under different culture conditions (addition ofgranulosa and oviductal cell monolayers, epidermal growth factor (EGF),lactoferrin (LF) and vitamin B12 on culture medium) using differentqualification methods including:v Morphometricevaluation without staining using stereomicroscope.v Stainingof embryos using different stains to assess cell count by Hoechst stain andviability by trypan blue stain.v Assessment ofmitochondrial function using 3-(4.5-Dimethylthiazol-2-yl)-2.5-diphenyltetrazoliumbromide (MTT) assay.
This study was carried out in the IVF unit,Department of Artificial Insemination and Embryo Transfer, Animal ReproductionResearch Institute, Haram, Giza-Egypt. This study was applied during the periodfrom June 2016 till June 2017. Ovaries and oviducts were collectedafter the slaughter of the animals from Bahteem slaughter house. Oocytes were aspirated and classifiedaccording to their quality into grades 1, 2, 3. Oocytes were grouped intocompact and denuded oocytes. The numberof oocytes used in Exp 1 (173), EXP 2 (99), EXP 3 (266), EXP4 (309) and EXP 5(201). Oocytes were cultured in TCM 199for 24 hrs at 38.
5°C in a CO2 incubator. Matured oocytes were fertilizedin vitro by frozen thawed semen. Spermatozoa were capacitated in vitro bycaffeine at final concentration 2×106 sperm/ml.
The sperm-oocytes were incubatedin TALP medium for 24 hrs. Fertilized oocytes werecultured under different culture conditions (Experimental design):EXP1. Cell free media or co cultured with granulosacell or oviductal cell monolayers.EXP2. Compact and denuded oocytes were cultured in CR1aa to evaluate the effect of cumulus cell on development ofembryos. EXP3. CR1aa with addition of EGF with different concentrations of 10, 20,50 and 100 ng /ml.EXP4.
CR1aawith addition of lactoferrin with different concentrations of10, 20, 50 and 100 µg /ml.EXP5. CR1aawith addition of vitamin B12 with concentrations of 10, 20 and50 µg /ml. Embryo culture was doneinside CO2 incubator with 5% CO2 at38°C. Observation of the cleavage rate was doneapproximately 24 hrs after the initiation of in vitro culture. Observation onthe development of pre-implantation embryo is done up to 5- 7 (morula) to 7- 9(blastocyst) days of in vitro culture.The assessment of embryo quality wascarried out under a stereomicroscope on grounds of morphological criteria.
Embryos were stained using different stains toassess cell count by Hoechst stain and viability by trypan blue stain. Embryoswere also assessed for mitochondrial function using MTT assay.The present studyrevealed the following results:1. Additionof oviductal cells (monolayer) to culture medium significantly improved thequality of in vitro produced buffalo embryos (developmental competence, morphology, cell number and mitochondrialfunction) compared with granulosa cell monolayer and cell free medium.
2. Usingof compact oocytes also improved development, morphology, cell number and mitochondrialfunction of in vitro produced buffalo embryos compared with denuded oocytes.3. Addition of EGF by concentration 20 or50ng/ml to culture media resulted in improvement ofthe quality of in vitro produced buffalo embryos (developmentalcompetence, morphology, cell number and mitochondrial function) when compared with other concentrations andcontrol group.4.
Addition of lactoferrinby concentration 50µg/ml to culture media improved the quality of in vitro produced buffaloembryos (developmentalcompetence, morphology, cell number and mitochondrial function) compared withother concentrations and control group. 5. Addition of vitamin B12by concentration 20 µg/ml to culture media resulted in improvement of the quality of in vitro producedbuffalo embryos (developmental competence, morphology, cell number and mitochondrial function)compared with other concentrations and control group .