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1 1 2 3Ocaña Quero, J.M.*, M. Moreno Millán , M. Pinedo Merlin , M.A. Ortega Mariscal y
1A. Rodero Franganillo
1Departamento de Genética. Facultad de Veterinaria. 14005 Córdoba.España.
2Departamento de Patología Animal. Facultad de Veterinaria. 14005 Córdoba. España.
3Departamento de Bromatología y Tecnología de los Alimentos. Campus de Rabanales. Córdoba. España.
Bovine oocyte. In vitro maturation. In vitro ferti Ovocito bovino. Maduración in vitro. Fecunda
lization. Serum. Gonadotropin hormones. ción in vitro. Suero. Hormonas gonadotrópicas.
On study the effect of different serum and monales, cuando se adicionan al medio de madu
hormonal supplements, when added to the ración, mantienen los procesos de maduración,
maturation medium, on the in vitro maturation and fecundación y división de ovocitos de bovino in
subsequent fertilization and cleavage process vitro. Los índices de maduración ovocitaria, fe
of bovine oocytes. The oocyte maturation, cundación y división, en presencia de los suple
fertilization and cleavage rates in the presence mentos ECS, FCS y hormonas fueron superiores
of ECS, FCS and hormonal supplements were significativamente (p<0,001) que aquellos ob
significantly higher (p<0.001) than that obtained servados en el grupo control. No se apreciaron
in the control group. No significant differences diferencias significativas para los estadíos de
for maturation, fertilization and development were maduración, fecundación y división entre los
observed between the different supplements. diferentes suplementos estudiados. Concluimos
The addition of serum and hormonal supplements que la adición de suplementos séricos y hormo
to the maturation medium improved the in vitro nales al medio de maduración mejora los índices
maturation rates and subsequent de maduración ovocitaria y subsiguientes índi
fertilization and cleavage rates of bovine oocytes. ces de fecundación y división in vitro de ovocitos
de bovino.
El objetivo del presente estudio fue investi
gar si diferentes suplementos séricos y hor
Techniques for producing pre
*Correspondencia implantation embryos by in vitro
Arch. Zootec. 48: 71 74. 1999.OCAÑA QUERO ET AL.
maturation/in vitro fertilization (IVM/ according to the following scheme:
IVF) are being used in many labo (1) a group of oocytes was cultivated
ratories worldwide. Viable embryos in basic maturation medium supple
can be produced from ovarian oocytes mented with 20 percent ECS; (2) other
collected hours after death of animals group of oocytes was matured in the
at the slaughterhouse. It was reported presence of 20 percent FCS; (3) a group
that hormonal and/or follicular factors of oocytes was matured in TCM 199
are needed to promote cytoplasmic medium supplemented with 4 IU/ml
maturation. Unfortunately, in vitro PMSG+2 IU/ml hCG (Laboratorios
matured bovine oocytes have lower Ovejero, León, España); (4) the last
fertilizability and developmental
capacity to the blastocyst stage than in basic maturation medium with no
vivo matured oocytes, although several supplements, serving as control group.
investigators have produced offspring After maturing period, some oocyte
from oocytes cultured in vitro (Fukuda were cytogenetically evaluated by
et al., 1990). These results suggest studying the meiotic maturation stage,
that failure in previous attempts of the remaining oocytes were fertilized
fertilization and further development and subsequently cultured for deve
of oocytes matured in vitro were due loping.
to inadequate oocyte culture systems. After 72 h in culture, presumptive
Several attempts have been made to zygotes were cytogenetically eva
modify the culture conditions, inclu luated. Data were statistically analysed
ding the addition of hormones, sera by Chi square test.
and granulosa cells to the maturation
medium for in vitro oocyte maturation,
RESULTS AND DISCUSSIONbut the percentage of oocytes reaching
the blastocyst stage with in vitro culture
As is shown in table I, whensystems is still low (Gliedt et al. , 1996).
follicular oocytes were matured inThe objetive of this study was to
TCM 199 medium supplemented withevaluate the effects of two different
ECS, FCS or hormonal supplementssera and two hormonal supplements
the maturation rates were significantlyon the in vitro maturation and
higher (80.4 percent, 78.3 percent andsubsequent fertilization and cleavage
85.1 percent respectively, p<0.001)of bovine oocytes.
than those obtained in control group
(51.9 percent). There were no sig
nificant differences between theMATERIALS AND METHODS
maturation percentages obtained in
The maturation, fertilization and supplemented oocyte groups.
culture procedures were made as In addition, the percentages of
described by Kim et al. (1990). oocytes matured in the presence of
ECS, FCS or hormonal supplements
EXPERIMENTAL DESIGN which reached the two pronuclei stage
The oocytes were matured for 24 h (fertilized oocytes) were significantly
Archivos de zootecnia vol. 48, núm. 181, p. 72.IN VITRO BOVINE EMBRYOS PRODUCTION
Table I. The influence of different sera and hormonal supplements added to the maturation
medium on in vitro maturation and subsequent fertilization and cleavage of bovine oocytes.
(Influencia de diferentes suplementos séricos y hormonales añadidos al medio de maduración sobre la
maduración in vitro y posterior fecundación y división de ovocitos de bovino).
Types of Number of oocytes Number of ova
supplements cultured matured* inseminated fertilized* cleaved*
a a aECS 250 80.4±4.7 250 53.7±4.4 57.7±3.7
a a aFCS 250 78.3±4.5 250 50.4±3.1 55.4±3.7
a a cPMSG+hCG 250 85.1±5.1 250 57.1±3.7 60.1±4.0
b b bControl 250 51.9±3.2 250 32.7±2.9 20.4±2.0
a,bDifferent superscripts in the same column denote significant differences when compared by Chi square
test (p<0.001). Data are showed as mean percentages±SEM from 5 replicates.
higher (53.7 percent, 50.4 percent, and The ECS supplement is widely used
57.1 percent respectively; p<0.001) in the maturation medium of preo
than those found in control group (32.7vulatory bovine oocytes (Sanbuissho
percent). No significant differences and Threlfall, 1990), in vitro ferti
between ECS, FCS and hormonal lization (Kim et al., 1990) and
treatments were found. subsequent embryonic development
Finally, the percentages of clea (Lambert et al. , 1986). Our results are
vaged ova in the presence of ECS (57.7 similar to those reported by Sche
percent), FCS (55.4 percent) or llander et al. (1990), who postulated
PMSG+hCG (60.1 percent) were that the beneficial effects of ECS
significantly higher (p<0.001) than supplement might be due to the
those observed in control group (20.4 relatively high LH content. This
percent). No significant differences hormone is known to be responsible
were observed between the supple for breaking the COC oocyte inte
mented oocytes groups. raction and activating maturation
The results obtained in our study process (Saeki et al., 1990).
demonstrated the important role that FCS is an important serum supple
serum and hormonal supplementation ment widely used in the oocyte
plays in the maturation and subsequent maturation and fertilization in vitro . It
fertilization and cleavage process was postulated that the beneficial effect
supporting our studies (Gliedt et al., of FCS might be due to maturation
1996). It is suggested that serum and stimulating components such as
hormonal supplements contain factors hormones, proteins and growth factors,
which stimulate in vitro maturation, similar to those found in ECS su
fertilization and subsequent embryonic pplement. These stimulating factors
development (Mattioli et al., 1991; are active on the germinal vesicle
Schroeder et al., 1991). breakdown inducing the meiotic
Archivos de zootecnia vol. 48, núm. 181, p. 73.OCAÑA QUERO ET AL.
resumption of the immature oocytes and embryonic development (Sanbuiis
(Mattioli et al., 1991). ho and Threlfall, 1990; Brackett and
It has been reported that FCS Zuelke, 1993). These hormones are
contains some undefined growth known to contain high LH hormone
promoting components that are absent levels.
in the serum of adult animals (Mochi In conclusion, the addition of serum
zuki et al., 1991). or gonadotropin hormones to the
Finally, It has been reported that maturation medium improved the
hormonal supplementation is nece meiotic maturation and subsequent
ssary for activating both cytoplasmic fertilization and cleavage rates of
and nuclear oocyte maturation mecha bovine oocytes matured and fertilized
nism, as well as in vitro fertilization in vitro.
In vitro fertilization of bovine oocytes maturedBrackett, B.G. and Z.A. Zuelke. 1993. Analysis of
factors involved in the in vitro production of in vivo and collected at laparoscopy.
Theriogenology, 25: 117 133.bovine embryos. Theriogenology, 39: 43 64.
Fukuda, Y., M. Ichikawa and K. Naito. 1990. Birth Mattioli, M., M.L. Bacci and G. Galeati. 1991. Effects
of normal calves resulting from bovine oocytes of LH and FSH on the maturation of pig oocytes
matured, fertilized and cultured with cumulus in vitro. Theriogenology, 36: 95 105.
Mochizuki, H., Y. Fukui and H. Ono. 1991. Effectcells in vitro up to the blastocyst stage. Biology
Reproduction, 42: 114 119. of the number of granulosa cells added to
culture medium for in vitro maturation,Gliedt, D.W., C.F. Rosenkraus, R.W. Rprie, A.L.
Munyon, J.N. Pierson, G.F. Miller and J.M. fertilization and development of bovine
Rakes. 1996. Effects of media, serum, oocytes. Theriogenology, 36: 973 985.
oviductal cells, and hormones during Sanbuiisho, A. and W.R. Threlfall. 1990. The
influence of serum and gonadotropins on inmaturation on bovine embry development in
vitro. J. Dairy Sci. , 79: 536 542. vitro maturation and fertilization of bovine
oocytes. Theriogenology, 34: 341 347.Kim, C.I., J.E. Ellington and R.H. Foote. 1990.
Maturation, fertilization and development of Schellander, K., F. Fuhrer and B.G. Brackett.
bovine oocytes in vitro using TCM 199 and a 1990. In vitro fertilization and cleavage of
simple defined medium with co culture. bovine oocyte matured in medium suplemen
ted with estrous cow serum. Theriogenology,Theriogenology, 33: 433 439.
Lambert, R.D., M.A. Sirard and C. Bernard. 1986. 33: 477 485.
Recibido: 6 2 97. Aceptado: 12 11 97.
Archivos de zootecnia vol. 48, núm. 181, p. 74.