INFLUENCE OF HUMAN AND BOVINE INSULIN ON IN VITRO MATURATION, FERTILIZATION AND CLEAVAGE RATES OF BOVINE OOCYTES (INFLUENCIA DE LA INSULINA BOVINA Y HUMANA SOBRE LOS ÍNDICES DE MADURACIÓN, FECUNDACIÓN Y DIVISIÓN IN VITRO DE OVOCITOS DE BOVINO)

Ocaña-Quero J.M.

Description

Abstract
The present study was carried out to determine the effects of supplementation of the maturation media with human or bovine insulin on in vitro maturation, fertilization and cleavage rates of bovine oocytes. Cumulus-intact bovine oocytes were cultured in a maturation medium (TCM-199 containing 10 percent fetal calf serum) with or without human or bovine insulin supplementation (10 mg/ml). For the bovine insulin supplement, the maturation (80.3 percent), fertilization (61.3 percent) and cleavage (55.3 percent) rates were significantly higher (p<0.05) than those obtained in the control group (70.1
50.1 and 42.5 percent respectively). Thus, the percentages of cleavaged ova obtained in presence of human or bovine insulin (54.8 and 55.3 percent respectively) were significantly higher (p<0.05) than those observed in control group (42.5 percent). No difference was found among human and bovine insulin treatments. These results demonstrate that the addition of human or bovine insulin to the maturation medium increased the percentages of matured oocyte, increasing the subsequent fertilization and cleavage rates of bovine oocytes in vitro.
Resumen
Se realizó un estudio para determinar los efectos de la suplementación del medio de maduración con insulina humana o bovina sobre los índices de maduración, fecundación y división in vitro de ovocitos de bovino. Ovocitos de bovino se cultivaron en medio de maduración (TCM-199 conteniendo 10 p.100 de suero de ternero fetal) con o sin insulina humana o bovina (10 mg/ml). Para la insulina bovina, los índices de maduración (80,3 p.100), fecundación (61,3 p.100) y división (55,3 p.100) fueron superiores significativamente (p<0,05) que aquellos obtenidos por el grupo control (70,1
50,1 y 42,5 p.100 respectivamente). Además, los porcentajes de óvulos divididos obtenidos en presencia de insulina humana o bovina (54,8 y 55,3 p.100 respectivamente), fueron superiores significativamente (p<0,05) en comparación con los obtenidos por el grupo control (42,5 p.100). No se apreciaron diferencias significativas entre los tratamientos con insulina humana y bovina. Estos resultados ponen de manifiesto que la adición de insulina humana o bovina al medio de maduración incrementa los porcentajes de ovocitos maduros, mejorando posteriormente los índices de fecundación y división de ovocitos de bovino in vitro.

Sujets

• Cattle
• In vitro fertilisation
• Fecundación in vitro

Informations

• Publié par: erevistas
• Publié le: 01 janvier 1998
• Nombre de lectures: 25

Extrait

INFLUENCE OF HUMAN AND BOVINE INSULIN ON IN VITRO
MATURATION, FERTILIZATION AND CLEAVAGE RATES OF
BOVINE OOCYTES
INFLUENCIA DE LA INSULINA BOVINA Y HUMANA SOBRE LOS ÍNDICES DE MADU
RACIÓN, FECUNDACIÓN Y DIVISIÓN IN VITRO DE OVOCITOS DE BOVINO
1* 2 3 1Ocaña Quero, J.M. , M. Pinedo Merlín , M. Ortega Mariscal y M. Moreno Millán
1Departamento de Genética. Laboratorio de Citogenética. Facultad de Veterinaria.14005 Córdoba.
España.
2Departamento de Patología Clínica Veterinaria. Facultad de Veterinaria. 14005 Córdoba. España.
3Departamento de Bromatología y Tecnología de los Alimentos. Campus Rabanales. Edificio C I anexo.
14071 Córdoba. España.
ADDITIONAL KEYWORDS PALABRAS CLAVE ADICIONALES
Hormonal supplementation. Cattle. In vitro Suplementación proteica. Vacuno. Fecundación
fertilization. In vitro bovine embryos. in vitro. Embriones de bovino in vitro.
SUMMARY
The present study was carried out to determi significantly higher (p<0.05) than those observed
ne the effects of supplementation of the maturation in control group (42.5 percent). No difference was
media with human or bovine insulin on in vitro found among human and bovine insulin
maturation, fertilization and cleavage rates of treatments. These results demonstrate that the
bovine oocytes. Cumulus intact bovine oocytes addition of human or bovine insulin to the
were cultured in a maturation medium (TCM 199 maturation medium increased the percentages
containing 10 percent fetal calf serum) with or of matured oocyte, increasing the subsequent
without human or bovine insulin supplementation fertilization and cleavage rates of bovine oocytes
(10 mg/ml). For the bovine insulin supplement, in vitro.
the maturation (80.3 percent), fertilization (61.3
percent) and cleavage (55.3 percent) rates were
significantly higher (p<0.05) than those obtained RESUMEN
in the control group (70.1; 50.1 and 42.5 percent
respectively). Thus, the percentages of cleavaged Se realizó un estudio para determinar los
ova obtained in presence of human or bovine efectos de la suplementación del medio de ma
insulin (54.8 and 55.3 percent respectively) were duración con insulina humana o bovina sobre los
índices de maduración, fecundación y división in
vitro de ovocitos de bovino. Ovocitos de bovino*Correspondencia.
E mail: [e-mail] se cultivaron en medio de maduración (TCM 199
Arch. Zootec. 47: 85 93. 1998.OCAÑA QUERO ET AL.
conteniendo 10 p.100 de suero de ternero fetal) al., 1993; Spicer et al., 1993).
con o sin insulina humana o bovina (10 mg/ml). Therefore, it is expected that insulin
Para la insulina bovina, los índices de madura and the growth factor IGF I have some
ción (80,3 p.100), fecundación (61,3 p.100) y beneficial effects on in vitro maturation
división (55,3 p.100) fueron superiores signi of bovine oocytes. Some studies
ficativamente (p<0,05) que aquellos obtenidos showed that supplementation of the
por el grupo control (70,1; 50,1 y 42,5 p.100 maturation medium with insulin
respectivamente). Además, los porcentajes de improved cumulus expansion and
óvulos divididos obtenidos en presencia de insu oocyte fertilizability in vitro (Lorenzo
lina humana o bovina (54,8 y 55,3 p.100 respec
et al., 1994; Webb et al., 1994), but
tivamente), fueron superiores significativamente
other reports showed that insulin had
(p<0,05) en comparación con los obtenidos por
no significant effect on the fertilization
el grupo control (42,5 p.100). No se apreciaron
rate or morula formation (Stubbings et
diferencias significativas entre los tratamientos
al., 1990). The role of insulin on in
con insulina humana y bovina. Estos resultados
vitro maturation, fertilization and
ponen de manifiesto que la adición de insulina
cleavage of bovine oocytes has not
humana o bovina al medio de maduración
been fully understood.
incrementa los porcentajes de ovocitos madu
The aim of the present study was toros, mejorando posteriormente los índices de
obtain information about the effects offecundación y división de ovocitos de bovino in
supplementation of the maturation me vitro.
dia with human or bovine insulin on in
vitro maturation, fertilization and
cleavage rates of bovine oocytes.INTRODUCTION
Embryos may be obtained from a
MATERIALS AND METHODSwide range of mammalian species by
flushing the reproductive tract or
COLLECTION AND PREPARATION OFproduced from the recovery of mature
OOCYTES FOR MATURATIONor immature oocytes from antral
Ovaries were collected from cowsfollicles in the ovary. Once that oocytes
at a local slaughterhouse, put intoare fully grown, they may be matured,
physiological saline (0.9 percent, w/v,fertilized and cultured in vitro, pro
NaCl) with antibiotics (100 mg/mlducing viable embryos with full
streptomycin, 100 IU/ml penicillin anddevelopmental potential.
0.25 mg/ml anphotericin) mantainedIt is known that follicular deve
at 30 to 37°C, and transported to thelopment is controlled by various factors
laboratory within 1 to 2 h of slaughter.(Gonadotrophins, steroids, growth
factors) of endocrine and paracrine The cumulus oocyte complexes were
origin (Ireland, 1987; Webb et al., aspirated from small antral follicles of
1994). Several studies have indicated 1 to 5 mm of diameter with an 18 ga
that insulin and insulin like growth needle attached to a 5 ml syringe
factor I (IGF I) stimulate the proli containing PBS (phosphate buffered
feration of granulosa cells and the saline supplemented with 5 percent (v/
production of progesterone (Gong et v) heat inactivated fetal calf serum
Archivos de zootecnia vol. 47, núm. 177, p. 86.IN VITRO PRODUCTION OF BOVINE EMBRYOS
(FCS) and antibiotics. After washing the capacitated sperm suspension were
once in Hank's salt solution and twice added to 1 ml of freshly prepared
in maturation medium, oocytes with fertilization medium (TCM 199
compact cumulus cells were placed in supplemented with 10 percent FCS),
a Falcon plastic culture dish (3.5 x 1.0containing 20 40 matured oocytes at a
6cm) containing 2 ml of maturation concentration of 1 2x10 total sperma
medium and cultured for 24 h at 39°Ctozoa/ml and cultured for 24 h at 39°C
under 5 percent CO in air. The basic under 5 percent CO in air.
2 2
maturation medium was TCM 199
with Earle's salts and Hepes (Sigma IN VITRO ZYGOTE CULTURE
Chemical Company, St. Louis, MO, Twenty four hours after gamete co
USA) plus 0.2 percent L glutamine, culture, the oocytes were gently
0.02 percent of an antibiotic antimyco washed twice in Hank's solution in a
tic solution (100 mg/ml streptomycin, 10 ml plastic tube in order to remove
100 IU/ml penicillin and 0.25 mg/ml the cumulus cells and free sperma
anphotericin; Sigma) and supple tozoa, and then transferred to a culture
mented with 10 percent fetal calf serumdish containing 1 ml of TCM 199
(FCS). supplemented with 10 percent FCS
and incubated for 96 h at 39°C under 5
SPERM CAPACITATION AND IN VITRO percent CO in air. The culture was
2
FERTILIZATION observed every 12 h by microscope in
For washing spermatozoa, 30 and order to evaluate the cleavage stage.
45 percent isotonic Percoll solutions
were prepared by diluting of 90 percentEXPERIMENTAL DESIGN
isotonic Percoll solution (Percoll, The influence of human and bovine
Pharmacia LBK Biotecnology AB, insulin added to the maturation
Uppsala, Sweden). Then, 2 ml of 30 medium on the in vitro nuclear
maturation rate and subsequentpercent Percoll solution was placed on
fertilization and cleavage rates of2 ml of 45 percent Percoll in a 10 ml
test tube. For the preparation of matured oocytes was evaluated. TCM
capacitated spermatozoa, two straws 199 containing 10 percent FCS was
of frozen semen were thawed for 5 used as maturation medium. COCs
seconds in 39°C water, and 2 ml of were matured in the maturation
semen were deposited on the upper medium with or without 10 mg/ml of
layer of the percoll gradient solution. human (Humulina NPH 40 IU, Eli Lilly
The tube was centrifuged for 15 min at and Company, Indianapolis, USA) or
700 x g. The sedimented spermatozoa bovine (Sigma) insulin supplemen
tation. After maturation period, somedisplaying good motility in the bottom
oocytes were cytogenetically proce of tube were resuspended in 1 ml of H
TALP medium containing 0.6 percent ssed to evaluate the nuclear maturation
BSA (Sigma), 5 mM caffeine and 100stage, the remaining oocytes were
mg/ml heparin (Sigma) and were fertilized and cultured for developing.
incubated for 15 min in 5 percent CO Finally, the presumptive zygotes were
2
incubator for capacitation. 300 ml of cultured for 96 h in TCM 199 medium
Archivos de zootecnia vol. 47, núm. 177, p. 87.OCAÑA QUERO ET AL.
Table I. Effect of supplementation of the maturation medium with human or bovine insulin on
in vitro maturation rates of bovine oocytes. (Efecto de la suplementación del medio de maduración
con insulina humana o bovina sobre los índices de maduración in vitro de ovocitos de bovino).
Nuclear maturation stage (percent)
Treatment Nº of trials Nº oocytes Germinal M