The present study was
undertaken with the objective to produce in vitro sheep embryos using
different culture media. 572 presumptive zygotes  were cultured in three different culture media
viz. G1G2 sequential media, synthetic oviduct fluid amino acid (SOFaa), potassium
simplex optimizing media (KSOM) of which 162 (84.47 ± 3.07 per cent), 147
(75.93 ± 4.23 per cent) and 140 (72.27 ± 3.50) cleaved, respectively. The
cleavage rate was significantly higher in G1G2 sequential media than in SOFaa
and KSOM. In G1G2 sequential culture media, the mean percentage of 4 to 8
cell, 16 to 32 cell and morula were 148 (77.15 ± 3.04), 129 (66.39 ± 5.61), 99
(51.77 ± 2.86) and 86 (45.23 ± 2.07) respectively at different days. In
conclusion, the proportions of embryos reaching the advanced stages were
significantly higher in G1G2 culture media than the SOFaa and KSOM.

            In
recent years, several achievements have been made in reproductive
biotechnologies in domestic animals. The development of in vitro embryo production has led to the next generation of assisted
reproductive techniques, including intracytoplasmic sperm injection (ICSI),
transgenic animal production and cloning. Embryo culture environment has the
biggest influence on blastocyst quality, regardless of oocyte origin (Rizos et al., 2003). The objective of this
study was study the influence of different culture media on the production of in
vitro embryos in sheep.

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The ovaries of local sheep collected
from slaughterhouse except ovaries of pregnant ewes.  The oocytes were retrieved by slicing method and
only grade A,B and C oocytes were used for used for maturation. After washing,
the oocytes were transferred to the 50µl of in vitro maturation media droplets
and kept for incubation in CO2 incubator for about 24 h. The oocyte maturation
rate was assessed by the expansion of the cumulus cells and expulsion of the
first polar body.

            Sperms
were flushed from the cauda epididymis and swim up method was used to separate
the motile fraction of sperm. After washing, the oocytes were transferred into
the pre incubated 75µl droplets of in vitro fertilization tyrode’s albumin
lactate pyruvate (IVF TALP) medium containing heparin and cultured for 18 to 20
h. The fertilized oocytes were denuded from their cumulus attachment by
vigorous pipetting and fertilization rate was assessed. The fertilized oocytes
were cultured in 3 different culture medium viz., G1/G2 sequential medium, SOF
and KSOM and incubated in CO2 incubator for 5-6 days. The culture
media was changed frequently with the interval of 48 h and developmental rate
was assessed.  From
slaughtered sheep, totally 170 ovaries were collected, a total of 1001 oocytes
were harvested using slicing method. Among the collected oocytes 1.42 ± 0.16,
1.72 ± 0.22 and 1.34 ±0.17 per cent were classified as A, B and C grades
respectively with an average yield of 5.94± 0.36. A,B and C grade of oocytes
were used for maturation. A higher yield of good quality oocytes in this study
might have been due to the presence of more number of developing follicles in
the ovaries at the time of collection. The less physical force associated with
slicing was considered to cause less damage to cumulus cell layers which leads
to get good quality oocytes.

The total matured oocytes with good
expansion of cumulus (degree 2 and degree 1) used for fertilization was 572
with the mean value of 79.44 ± 2.90. The better maturation rates obtained in A
and B grade oocytes when compared to the C grade oocytes could be attributed to
the presence of the cumulus cells surrounding the zona pellucida in the former
grades (Shioya et al., 1988). The high maturation rate obtained in this
study could be due to supplementation of culture medium with FCS (Totey et
al., 1992).

 

All the 572 oocytes
used for maturation were cultured in three different culture media viz. G1G2
sequential media, synthetic oviduct fluid amino acid (SOFaa), potassium simplex
optimizing media (KSOM) of which 162 (84.47 ± 3.07 per cent), 147 (75.93 ± 4.23
per cent) and 140 (72.27 ± 3.50) cleaved respectively. The cleavage rate was
significantly higher in G1G2 sequential media than in SOFaa and KSOM.

In G1G2 sequential
culture media, the mean percentage of 4 to 8 cell, 16 to 32 cell and morula
were 148 (77.15 ± 3.04), 129 (66.39 ± 5.61), 99 (51.77 ± 2.86) and 86 (45.23 ±
2.07) respectively at different days. In SOFaa media, the mean percentages of 4
to 8 cell, 16 to 32 cell and morula were 125 (65.04 ± 3.91), 94 (49.39 ± 4.33),
66 (35.02 ± 3.58) and 57 (32.47 ± 6.75) respectively. In KSOM, the mean percentages
of 4 to 8 cell, 16 to 32 cell and morula were 103 (53.79 ± 3.11), 77 (41.20 ±
4.18),54 (28.83 ± 3.03) and 35 (19.29 ± 2.73) respectively. Comparison of
various developmental rates in G1G2 media, SOFaa and KSOM, the proportion of
embryos reaching the advanced stages were significantly higher in G1G2 culture
media than the SOF medium at all stages. Heindryckx et al. (2001)
cleared that culture conditions that maintain the early embryo might not be
ideal for the post compaction embryo and vice versa. Therefore, in this study,
sequential media were introduced; during the first 3 days embryos were cultured
in a simple low-phosphate and low-glucose medium, which was then replaced by a
medium with higher glucose content, which promoted morula-to-blastocyst
transition.  G1.5 has nonessential amino
acids plus methionine while G2.5 harbours essential and nonessential amino
acids.  G1.5/G2.5 also contains vitamins.
The present experiment result also agrees that groups cultured in pyruvate /
lactate for 72 h with additional supplementation of glucose until day 6.

While
comparing SOFaa with KSOM, the proportion of embryos reaching the advanced
stages was significantly higher in SOF medium than in KSOM at all stages. In
the present study, the development rate to morula stage in SOF was
significantly higher than that in KSOM media. However, the ingredients of SOF
and KSOM media differed slightly. While KSOM have glutamine, SOF does not have
it. There were recent reports investigating the inhibitory and teratological
effects of glutamine on embryos (Summers et al., 2005). Also, the
concentration of BSA in SOF was higher than that in KSOM. These differences
could have an effect on the embryo developments data in our study. The
commercially available medium manufactured by Vitrolife, Sweden and used in
this study supported better development compared to two other media used (KSOM
or mSOF), which were prepared in laboratory. 
The presence of additional constituents in KSOM in comparison to mSOF
and the quality of water used for preparation of media might have been the
causative factor for poorer development of embryos in these media than in
G1.5/G2.5.

SUMMERY

The aim of this study
was to produce in vitro sheep embryos using different culture media. We
concluded that G1G2 culture media was better than the SOFaa and KSOM for embryos
reaching the advanced stages.