VII. Summary Buffaloes represent vital part of the

VII. Summary

Buffaloes represent vital part of the
agricultural economy in Egypt and some other developing countries. However,
buffaloes have low reproductive efficiency which could be related to the low
total number of follicles in the ovary, poor superovulatory response and high
percentage of atretic follicles. Moreover, recently efforts have been initiated
to improve the reproductive potential of these animals using biotechnologies as
in vitro fertilization (IVF) and embryo transfer (ET).

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Various studies have been shown that the in vitro culture (IVC)
environment which the embryos are exposed after fertilization is the key
determinant of the blastocyst quality. The assessment of
embryo quality is one of the most important factors, which determine a
successful embryo transfer. Its significance is related to the essential
correlation between embryo quality and the stage of its development.

The present study aimed to:

     1. Assess the developmental competence of the in vitro produced
embryos under different culture conditions.

    2. Qualify the in
vitro produced embryos under different culture conditions (addition of
granulosa and oviductal cell monolayers, epidermal growth factor (EGF),
lactoferrin (LF) and vitamin B12 on culture medium) using different
qualification methods including:

v Morphometric
evaluation without staining using stereomicroscope.

v Staining
of embryos using different stains to assess cell count by Hoechst stain and
viability by trypan blue stain.

v Assessment of
mitochondrial function using 3-(4.5-Dimethylthiazol-2-yl)-2.5-diphenyltetrazolium
bromide (MTT) assay.

      This study was carried out in the IVF unit,
Department of Artificial Insemination and Embryo Transfer, Animal Reproduction
Research Institute, Haram, Giza-Egypt. This study was applied during the period
from June 2016 till June 2017.  Ovaries and oviducts were collected
after the slaughter of the animals from Bahteem slaughter house.  Oocytes were aspirated and classified
according to their quality into grades 1, 2, 3. Oocytes were grouped into
compact and denuded oocytes.  The number
of oocytes used in Exp 1 (173), EXP 2 (99), EXP 3 (266), EXP4 (309) and EXP 5
(201).  Oocytes were cultured in TCM 199
for 24 hrs at 38.5°C in a CO2 incubator.  Matured oocytes were fertilized
in vitro by frozen thawed semen. Spermatozoa were capacitated in vitro by
caffeine at final concentration 2×106 sperm/ml.  The sperm-oocytes were incubated
in TALP medium for 24 hrs.

 Fertilized oocytes were
cultured under different culture conditions (Experimental design):

EXP1.  Cell free media or co cultured with granulosa
cell or oviductal cell monolayers.

EXP2. 
Compact and denuded oocytes were cultured in CR1aa to evaluate the effect of cumulus cell on development of
embryos.

 EXP3. CR1aa with addition of  EGF with different concentrations of 10, 20,
50 and 100 ng /ml.

EXP4. CR1aa
with addition of   lactoferrin with different concentrations of
10, 20, 50 and 100 µg /ml.

EXP5. CR1aa
with addition of   vitamin B12 with concentrations of 10, 20 and
50 µg /ml.

      Embryo culture was done
inside CO2  incubator  with 5% CO2  at
38°C.  Observation of the cleavage rate was done
approximately 24 hrs after the initiation of in vitro culture. Observation on
the 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 was
carried out under a stereomicroscope on grounds of morphological criteria.  Embryos were stained using different stains to
assess cell count by Hoechst stain and viability by trypan blue stain. Embryos
were also assessed for mitochondrial function using MTT assay.

The present study
revealed the following results:

1.     Addition
of oviductal cells (monolayer) to culture medium significantly improved the
quality of in vitro produced buffalo embryos 
(developmental competence, morphology, cell number and mitochondrial
function) compared with granulosa cell monolayer and  cell free medium.

2.     Using
of compact oocytes also improved development,  morphology, cell number and mitochondrial
function of in vitro produced buffalo embryos compared with denuded oocytes.

3.    
Addition of EGF by concentration 20 or
50ng/ml to culture media resulted in improvement of
the quality of in vitro produced buffalo embryos (developmental
competence, morphology, cell number and mitochondrial function)  when compared with other concentrations and
control group.

4.    
Addition of lactoferrin
by concentration 50µg/ml to culture media improved the quality of in vitro produced buffalo
embryos  (developmental
competence, morphology, cell number and mitochondrial function) compared with
other concentrations and control group.

5.    
Addition of vitamin B12
by concentration 20 µg/ml to culture media resulted in improvement of the quality of in vitro produced
buffalo embryos (developmental competence,  morphology,  cell number and mitochondrial function)
compared with other concentrations and control group .

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