This work was carried out to evaluate the quality and developmental competence of
the in vitro produced buffalo embryos under different culture conditions as co
culture with cumulus cell and buffalo oviductal cell monolayers (BOCM) or
supplementation of lactoferrin to the culture medium. Oocytes were aspirated
and classified according to their quality into grades 1, 2, 3. Oocytes were matured
in TCM 199 for 24 hrs at 38.5°C in a CO2 incubator. After
maturation, oocytes were in vitro fertilized by frozen thawed sperms capacitated in vitro by caffeine at final concentration 2
million sperm /ml. The sperm-oocytes were incubated in TALP
medium for 24 hrs. Fertilized oocytes were cultured in Cell free media or co cultured with cumulus
cell or oviductal cell monolayers (EXP1), CR1aa with addition of lactoferrin with different concentrations (EXP2) inside CO2 incubator
with 5% CO2 at
38°C. Addition of BOCM to culture media resulted in a significant
increase in morula and blastocyst
compared with cell free media and media with cumulus cell monolayer and also resulted
in a significant increase in number of cells
and mitochondrial function of in vitro produced buffalo embryos. Addition of lactoferrin by concentration 50µg/ml to culture media resulted in a significant increase in rates of cleavage, morula and blastocyst compared
with control or other concentrations and also resulted in an increase in number
of cell and mitochondrial function of in vitro produced buffalo embryos. It
could be concluded that, addition of oviductal cells (monolayer) or lactoferrin
to culture medium significantly improved the quality of in vitro produced
Key words: In vitro fertilization, Buffalo embryos,
BOCM, Lactoferrin, Quality.
a vital part of the agricultural
economy in Egypt. But, they have lower fertility rate which may be attributed to lower follicular
number in the ovary, low response to superovulation and higher number of atretic follicles.
Recently efforts are established to
enhance the reproductive efficiency of
these animals using reproductive biotechnology such as in vitro fertilization
(IVF) technology and embryo transfer
Reproductive biotechnologies as IVEP and ET may
enhance genetic improvement and are needed as a tool to obtain large number of
offspring of superior animals. So, it has been expected that there is a
positive relationship between the use of these technologies and production (2).
Buffalo embryos can be cultured under different conditions and it is indicated
that the culture system and the composition of the medium influence embryo
quality (3). Some studies have been demonstrated that
the quality of the oocyte is the most important factor that influences the
blastocyst rate and that the in vitro culture medium in which the embryos are
cultured is the most important determinant of the quality of blastocyst (4). Several authors favour
the co-culture of embryos with cumulus and oviductal epithelial cells or with cell lines as Buffalo rat liver (BRL) for production of buffalo embryos. It was demonstrated that cellular
co culture produces specific embryo trophic factors which improve the in
vitro embryo development (1).
evaluation of quality of embryo is one of the most critical factors, which
determine the output of embryo transfer. Its significance is attributed to the
essential relationship between embryo quality and its developmental stage (5). Thus, this study contributed to study the effect of cellular
co culture (cumulus or oviductal cells monolayers) and lactoferrin additives to
improve the quality of in vitro produced
Materials and methods
Ovaries were collected within a few minutes after the slaughter of the
animals from Bahteem slaughter house. They were put in a well-sealed container
containing physiological saline and traces of Streptomycin and Penicillin at a
temperature 28-33°C. Oviducts also were dissected from the mesosalpinix.
Transportation of the ovaries and oviducts to the laboratory occurred within
2 hrs from slaughtering of buffalo, then
washed in warm physiological saline
several times at 37°C until obtaining
clear saline and put in water bath at 37 °C during collection of oocyte (6). Before oocytes recovery, drying of the ovaries with sterile towel paper was necessary.
Aspiration of oocytes from follicles (2-8mm) carried out by using a needle of 18
gauge attached to a 10 ml plastic syringe containing few Millis of warmed
aspiration medium (7). The examination and
classification of recovered oocytes occurred according to their quality into:
grade 1 (good), 2 (fair), 3 (poor) (8).
In vitro maturation of oocytes:
of selected oocytes three times in warmed aspiration medium should be applied and finally washing by IVM medium before their transfer to IVM medium. Oocytes were cultured
in groups (5–15 oocytes) in 50 µl of IVM
medium under paraffin oil in a CO2 incubator (5% CO2 and
90–95% relative humidity) at 38.5°C for
24 hrs (7).
In vitro fertilization:
Partial denudation of the cumulus cells surrounding the oocytes should
be applied to facilitate penetration of the sperm cells
to the oocytes. Washing of oocytes two
times in warmed IVF medium was very necessary. Microdroplet (100µl) of F-TALP medium
containing BSA and caffeine with 2 million sperm/ ml was prepared to
accommodate 10 oocytes. Co-incubation of sperm with oocyte was carried out in a
humidified environment with 5% CO2 and 38.9°C temperature for 24 hrs
In vitro culture of fertilized oocytes:
Washing of oocytes four times in culture medium should be applied before their
transfer to the in vitro culture
medium. Oocytes were transferred to the in vitro culture medium. In vitro
embryo culture was applied inside
incubator with 5% CO2 at 38°C humidified air.
1- Influence of monolayer on the quality
and in vitro produced buffalo embryos development (EXP1):
Fertilized oocytes were cultured in:
a. Cell free media (CR1aa) and Cellular
media by co-cultured in (Granulosa cells monolayers (GCM) and Bovine oviductal cell monolayers
2- Influence of lactoferrin on the quality and in vitro produced
buffalo embryos development (EXP 2):
Fertilized oocytes were cultured in CR1aa with addition of lactoferrin
with different concentrations of 10, 20, 50 and 100 µg /ml. The cleavage rate was observed after the
begining of in vitro culture by 24 hrs. The development of in vitro produced
embryo was observed 7 to 9 days of in vitro culture.
Evaluation of in vitro produced embryos:
The evaluation of embryo quality is
applied under a stereomicroscope according to morphological criteria of in
vitro produced embryos. The most important criteria during the morphological
evaluation of embryos include damage and fracture of the zona pellucida, shape,
size, colour and the number of damaged blastomeres and the presence of granules
in the subvitelline space (5).
Staining with Hoechst 33342:
Preparation of Embryos:
Embryos were removed from culture medium and were
washed 2 times in 100 ?l of phosphate buffer saline containing 1 mg/ml
polyvinyl pyrrolidone. The embryos were
fixed in 100 ?l of paraformaldehyde solution for 1 h at room temperature. Embryos
were washed in 100 ?l phosphate buffer saline / polyvinyl pyrrolidone for 3
Staining the Embryo:
Staining should be carried out with dimmed lights. Embryos were transfered to a 50 ?l of working
solution. The cells were stained for 10
minutes. The embryos were washed two times by using 100 ?l of phosphate buffer
saline – polyvinyl pyrrolidone.
Mounting Embryos to Slides:
slides were prepared by putting them in a 1:10 poly-L-lysine solution for few
minutes. They were allowed to dry. Embryos
were transferred to a slide coated with poly-L-lysine and embryos were allowed
to dry for 20 minutes at room temperature.
Cover slip was put over the slide and was allowed to dry for 2 hrs in a
dark place. The cells were counted under
the fluorescent microscope with ultraviolet (UV) filter. Nuclei had blue color
3. Evaluation by MTT:
This test was carried out by
incubating embryos with 0.25 mg/ml MTT in the culture media for 3 hrs at 37 °C.
Insoluble formazan products were solubilized by overnight incubation in 50%
N,N-dimethyl formamide, 20% sodium dodecyl sulfate, pH 4.8 (11).
Quantitative measurement of the absorbance of this colored solution can be performed at wave length 575 nm by ELISA reader.
Each experiment was replicated at least three times. The data were
expressed as means ± SEM. The statistical significant difference was analyzed
by ANOVA using SPSS. Values with different letters across treatments differed
significantly. Differences of P