Production of cloned-caprine embryos in vitro obtained from interspecies nuclear transfer using bovine cytoplast and caprine karyoplast / Soh Hui Hui
Production of cloned-caprine embryos using the intraspecies somatic cell nuclear transfer is limited by low source of caprine oocytes as the recipient cytoplast in Malaysia. Therefore, using the bovine oocytes as recipient cytoplast in interspecies somatic cell nuclear transfer is an alternative...
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| Format: | Thesis |
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2012
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| Online Access: | http://studentsrepo.um.edu.my/4156/1/SHH_1%2D280pg.pdf http://studentsrepo.um.edu.my/4156/ |
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| Summary: | Production of cloned-caprine embryos using the intraspecies somatic cell nuclear
transfer is limited by low source of caprine oocytes as the recipient cytoplast in
Malaysia. Therefore, using the bovine oocytes as recipient cytoplast in interspecies
somatic cell nuclear transfer is an alternative approach to produce large number of
cloned-caprine embryos and subsequently offspring at a rapid rate. This study was
aimed to produce cloned-caprine embryos in vitro by specifically evaluating the
interspecies nuclear transfer technique such as enucleation methods, nuclear transfer
methods and in vitro culture systems. Briefly, the bovine ovaries were collected from
local abattoir and transported to the laboratory within 2 to 3 hours in saline solution
(0.9% NaCl). Bovine oocytes were recovered by checkerboard slicing the entire surface
of the ovary inside the culture dish by using the razor blade. Oocytes with several
compact layers of cumulus cells were selected and cultured in in vitro maturation (IVM)
medium for 20 to 22 hours. After maturation, cumulus oocyte complexes (COC) were
denuded in hyaluronidase (0.1%) to remove the cumulus cells. The matured oocytes
with extrusion of first polar body were selected for enucleation to remove the spindle.
Caprine-foetal fibroblast cells (donor karyoplasts) were harvested and transferred into
enucleated bovine oocytes. The reconstructed oocytes were activated and the
reconstructed couplets were cultured in KSOM medium for in vitro embryos
development in CO2 (5%) incubator at 38.5oC in humidified atmosphere for 8 days. The
medium was changed every 2 days of in vitro culture. Samples of embryos from each
stage were stained with Hoechst 33342 to examine the number of nuclei of the embryos.
The data were presented as mean±SEM and were analysed using one-way ANOVA.
The significant differences among treatments were further analysed by DMRT and
P<0.05 was considered significant.
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In Experiment 1, two enucleation methods were compared, i.e. squeezing and
aspiration. There was no significant difference in the percentages successfully
enucleated oocytes (P>0.05) for both enucleation methods (squeezing vs. aspiration:
88.01±3.00% vs. 91.68±1.92%, respectively). In terms of manipulation efficiency, the
duration needed to complete the enucleation together with injection, the speed for
aspiration with sub-zonal injection was significantly faster (P<0.05) than squeezing with
sub-zonal injection (41.18±2.77 and 83.82±3.16 minutes, respectively). In in vitro
embryo development, the percent of cloned-caprine embryos from 2 cell stage up to
blastocyst stage using squeezing (2 cell: 60.18±2.43%, 4 cell: 53.80±2.84%, 8 cell:
37.71±3.30%, morula: 24.45±2.71% and blastocyst: 12.08±2.95%) and aspiration (2
cell: 61.55±4.20%, 4 cell: 49.86±3.87%, 8 cell: 39.22±4.26%, morula: 21.07±3.94%
and blastocyst: 10.93±1.87%) methods did not differ significantly (P>0.05).
In Experiment 2, two nuclear transfer methods were compared, i.e. sub-zonal
injection with electro-fusion and intracytoplasm injection. There were no significant
differences (P>0.05) in the injection and reconstruction rates for both nuclear transfer
methods. The percent cloned-caprine embryos obtained from interspecies SCNT at 2-
and 8 cell using SUZI and ICI methods did not differ significantly (P>0.05). However,
the percentages of cloned-caprine embryos at 4 cell, morula and blastocyst derived from
SUZI method were significantly higher (P<0.05) compared to the ICI method
(53.80±2.84% vs. 38.60±2.25%, 24.45±2.71% vs. 16.05±1.43% and 12.08±2.95% vs.
4.51±1.45%, respectively).
In Experiment 3, three different in vitro culture system were compared, i.e.
Group 1 (KSOM A throughout duration of the culture), observation of the embryos
were recorded on days 3, 5, 7 and 8 without changing the medium; Group 2 (KSOM A
on days 1-3, changed with KSOM A on days 3 and 5), the embryos were observed and
recorded on days 3, 5, 7 and 8; and Group 3 (KSOM A on days 1-3, changed with
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KSOM B on days 3 and 5), the embryos were observed and recorded on days 3, 5, 7 and
8. Group 3 (2 cell: 60.18±2.43%, 4 cell: 53.80±2.84%, 8 cell: 37.71±3.30%, morula:
24.45±2.71% and blastocyst: 12.07±2.95%) showed significantly higher (P<0.05) in the
in vitro development competence from 2 cell up to blastocyst stages compared to
Groups 1 (2 cell: 49.01±2.02%, 4 cell: 36.92±3.02%, 8 cell: 26.46±1.74%, morula:
8.42±0.47% and blastocyst: 0.00±0.00%) and 2 (2 cell: 49.85±3.27%, 4 cell:
39.68±2.72%, 8 cell: 29.34±1.87%, morula: 10.22±1.49% and blastocyst: 0.00±0.00%).
In Experiment 4, an attempt to produce pregnancy after oviduct embryo transfer
of interspecies SCNT embryos and using ultrasound scanning for pregnancy diagnosis
were carried out. A total of 63 cloned-caprine embryos were obtained from interspecies
SCNT experiment. However, only 55 embryos of Grades 1 and 2 (4- and 8 cell stages)
were chosen and transferred into 9 recipients with at least 1 CL. Unfortunately, after
ultrasound scanning on day 30 of gestation age after embryo transfer, there was no
pregnancy observed in the recipient goats after embryo transfer experiment.
It can concluded from the present study that caprine embryos could be
successfully produced through interspecies SCNT using caprine foetal fibroblast cell as
donor karyoplast and bovine oocyte as recipient cytoplast under local setting in
Malaysia. It is believe this is the first report of producing cloned-caprine embryos with
satisfactory blastocyst rate in interspecies SCNT using KSOM supplementation
additional of glucose. With this encouraging findings and future refined research, using
caprine-bovine in interspecies SCNT to produce caprine embryos and offspring may
offer a new approach to increase genetically superior goat population in Malaysia at a
rapid rate to meet the goat meat and milk demand for the industry. |
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