IJERD – International Journal of Environmental and Rural Development (2012) 3-2

Research article
erd

Effect of Culture Media on Fertilization Rate in Bovine andCaprine Embryos Production
SARUTTIWONG BOONKONG
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

CHAINARONG NAVANUKRAW*
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
Agricultural Biotechnology Research Center for Sustainable Economy (ABRCSE),
Khon Kaen, University, Khon Kaen, Thailand
Email: chanav@kku.ac.th

JIRATTI THAMMASIRI
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

WINAI JAIKAN
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

SUTHIPONG URIYAPONGSON
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
Received 19 December 2011

Accepted 7 February 2012(*: Corresponding Author)

Abstract The objective of this experiment was to determine the effect of culture media(Tyrode Albumin Lactate Pyruvate-In Vitro Fertilization-Synthetic Oviduct Fluid; TALPIVF-SOF and SAGE mediaTM) on fertilization rate in bovine and caprine embryos production. Thai crossbred bovine ovaries (n=40) were collected at a local abattoir located in Khon
Kaen municipality. Caprine ovaries (n=22) were collected by the surgery method at the Department of Animal Science, Faculty of Agriculture, Khon Kaen University. In order tocompare the culture media, oocytes were recovered and randomly cultured in TALP-IVFSOF and SAGE mediaTM during in vitro oocytes maturation and fertilization as described by
the standard protocols for in vitro maturation, in vitro fertilization and in vitro culture, respectively. Oocytes, obtained from ovaries with corpus luteum (CL) and without CL, wererecovered and determined as recovery rate prior in vitro culture. The results revealed that therecovery rates of bovine and caprine oocytes were not significantly different between ovaries
with CL (58.5 and 27.6%) without CL (43.5 and 29.4%) respectively; bovine oocytes had
significantly more layers of cumulus than caprine oocytes. IVF and IVC, developed fromtwo cells to blastocyst stage, derived from SAGE mediaTM were better than that of TALPIVF-SOF.
Keywords culture media, in vitro fertilization, embryo, beef, goat

INTRODUCTION
In vitro production techniques of bovine and caprine embryos become widely used to geneticimprovement. The improvement of bovine and caprine embryo culture systems is highly desirable
in term of the production of preimplantation stage embryos for biotechnological studies and for theembryo transfer. A standard medium for in vitro maturation is tissue culture  (Lonergan et al., 1994).
TALP-IVF-SOF is the medium commonly use for embryo fertilization and embryo culture in vitro.
SAGE MediaTM is also available as  a commercial product. This culture media has been modified byaddition of amino acids. The effect that the culture media may have on the developing embryos
between different media is unknown.
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Thus, the objective of this study was to determine the effect of culture media on thefertilization rate in bovine and caprine embryos production.
MATERIAL AND METHODSThe experimental procedures were approved by the Animal Ethic committee of Khon Kaen University(Reference No. 0514.1.12.2/88). Bovine ovaries were collected at a local abattoir located in Khon Kaenmunicipality and caprine ovaries were collected by surgery method at the Department of Animal
Science, Faculty of Agriculture, Khon Kaen University. In order to compare the culture media,oocytes were recovered and randomly cultured in two media: TALP-IVF-SOF (Table 1) and SAGE
mediaTM (CooperSurgical Inc., Trumbull, CT) as shown in Table 2.
Oocyte collection in bovine and caprine: Bovine oocyte collections were recovered fromslaughtered bovines at Khon Kaen municipality and transfered in 0.9% saline to the laboratory
within 1h (Yang et al., 1990). Follicles were aspirated with an 18 gauge needle using vacuumsuction.
Caprine oocyte collections were recovered from live goats by surgery method at the Department of Animal Science, Faculty of Agriculture, Khon Kaen University and transferred in 0.9%
saline  to the laboratory within 1 h  (Yang et al., 1990). Follicles were aspirated with a 21 gauge needle using vacuum suction.
Table 1 Chemical composition of culture media (TALP-IVF-SOF)
Composition
NaCl (mM)
KCl  (mM)
NaH2PO4 (mM)
CaCl26H2O (mM)
HEPES (mM)
Glucose (mM)
PVA  (mg/mL)
Lactate (sodium salt) (mM)
Pyruvate (mM)
Penicillamine (mM)
Gentamicin (µg/mL)
Sodium pyruvate (g)NaHCO3 (g)Glutamine (g)Glucose (g)Serum  (mg/ml)
EDTA (g)Streptomycin (g)TALP-IVF
114.00
3.20
0.40
2.00
0.50
5.00
1.00
13.00
10.40
3.35
50.00
-

SOF-IVC
0.33
1.00
1.50
10.00
0.10
0.10
0.10

Table 2  Chemical composition of culture media SAGE MediaTM ProductCatalog Number
SAGE MediaTMProductBasic Media Components
Non-Essential Amino Acids
Essential Amino Acids
Vitamins
Magnesium Sulfate Heptahydrate
Magnesium Chloride
Potassium Phosphate, Monobasic
Anhydrous
Calcium Lactate (L+)
TaurineCitric Acid
EDTA

ART-1020/1021
Fertilization (HTF)X
X

ART-1026/1027
Cleavage
X
X

X

X

X

ART-1029
Blastocyst
X
X
X
X
X
X

X
X
X
X

X
X
X
X

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X

IJERD – International Journal of Environmental and Rural Development (2012) 3-2

In vitro maturation (IVM): Oocytes were placed in maturation medium  (TCM-199) under mineral oil (Rose et  al.,  1992). Maturation proceeded for 22-24 h at 38.5 ºC in an environment of 5%
CO2 in air.
In vitro  fertilization (IVF): Frozen semen was thawed at 37 ºC for 30 second and washed by centrifuging at 700 g for 5 minutes to remove any remaining semen extender (Gandhi et al. 2000). Before transfer to fertilization drops, oocytes were transferred into drop of fertilization medium undermineral oil. Oocytes were coincubated with spermatozoa for 22-24 h at 38.5 ºC in an environmentof 5% CO2 in air.
In vitro  culture (IVC):  At 24 h post-insemination, the presumptive embryos were washed in culture medium and embryos were placed into culture medium under mineral oil at 38.5 ºC in an environment of 5% CO2 in air. Cleavage rate was recorded at 24, 48, 72, 96 and 120 h postinsemination.
Statistical analysis: Data were analyzed using Chi-square test  (Steel et al., 1997). Statistical analysis was performed in all tests at a 95% confidence interval.
RESULTS
Studies on the influence of ovaries with CL (ovaries within the development of CL) and without
CL showed that   ovaries with CL and without CL were not significantly different (P>0.05) (Table3).
Table 3 Recovery rate of oocyte obtained from bovine ovaries
Ovarywith CL
without CL

NumberFollicle
123
147% Recovery of oocyte
Oocyte7264

58.54 (72/123)
43.54  (64/147)

Table 4 The recovery rate of oocyte obtained from caprine ovaries
Ovarywith CLwithout CL

NumberFollicle
94
17% Recovery of oocyteOocyte
26527.66 (26/94)
29.41    (5/17)

Studies on the influence of ovaries with CL (ovaries within the development of CL) and without CL showed that   ovaries with CL and without CL were not significantly different (P>0.05) (Table 4).
Morphology of bovine oocytes and caprine oocytes
Study of the morphology of bovine and caprine cumulus cells have shown that bovine oocytes had
significantly more layers of cumulus than caprine oocytes and can effect fertilization of oocytes.

Fig. 1 Morphology of bovine oocytes (left) and caprine oocytes (right)ⒸISERD58

IJERD – International Journal of Environmental and Rural Development (2012) 3-2

Effect of culture media on in vitro fertillization in bovine embryo production
The results revealed that IVF and IVC, developed from two cells to the blastocyst stage of SAGE
mediaTM, were better than that of TALP-IVF-SOF as determined by the embryo image analysis.

AB

2 cell (SAGE mediaTM)

2 cell (TALP-IVF-SOF)CD

4 cell (SAGE mediaTM)

8 cell (SAGE mediaTM)

E

F

Morula (SAGE mediaTM)

Blastocyst  (SAGE mediaTM)

Fig. 2 (A-F) Morphology of bovine embryo during early development
CONCLUSION
The present study suggested that the number of cumulus cells is important for supporting embryo
development and culture media can increase the cleavage.
ACKNOWLEDGEMENTS
The authors would like to express the most appreciation to the Thailand Research Fund Master,
Research Grants: TRF-MAG Window II (MRG-WII535S012), and Research Group of Beef andBuffalo production, Department of Animal Science, Faculty of Agriculture, Khon Kaen University.

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REFERENCES
Gandhi, A.P., Lane, M.L., Gardner, D.K. and Krisher, R.L. 2000. A single medium supports development ofbovine embryos throughout maturation fertilization and culture. Human Reproduction, 15, 395-401.
Gordon. I. 1994. Laboratory production of cattle embryos. CAB International, Oxon, UK, 640.
Lonergan, P., Carolan, C. and Mermillod, P.  1994. Development of bovine embros in vitro following oocyte
maturation under defined conditions. Reproduction Nutrition Development, 34, 329-339.
Nagano, M., Katagiri, S. and Takahashi, Y. 2006. Relationship between bovine oocyte morphology and invitro development potential. Zygote, 14, 53-61.
Rose, T.A. and Bavister, B.D. 1992. Effect of oocyte maturation medium on in vitro development of in vitro
fertilized bovine embryos. Molecular Reproduction Development, 31(1), 72-7.
Steel, R., Torrie, J. and Dickey, D. 1997. Principles and procedures of statistics: A biometrical approach.
WCB/McGraw-Hill, a Division of the McGraw-Hill Companies, 666.
Yang, N.S., Lu, K.H. and Gordon, I. 1990. In vitro fertilization (IVF) and culture (IVC) bovine oocytes fromstored ovaries. Theriogenology, 33(1), 352-362.

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