USE OF KETONE BODIES AS ENERGY SUBSTRATES AT DIFFERENT DEVELOPMENTAL STAGES AND UNDER DIFFERENT OXYGEN TENSIONS BY IN VITRO-PRODUCED BOVINE EMBRYOS (UTILIZACIÓN DE CUERPOS CETÓNICOS COMO SUSTRATO ENERGÉTICO EN DIFERENTES ESTADIOS Y BAJO DIFERENTES CONDICIONES ATMOSFÉRICAS DURANTE EL DESARROLLO EMBRIONARIO BOVINO IN VITRO)

Gómez E.

Description

Abstract
In vitro-produced bovine embryos can develop into hatched blastocysts in the presence of acetoacetate and b-hydroxybutyrate, which may be derived from lipid breakdown. The effects of these ketone bodies, added during different periods of early bovine embryo development and under different atmospheric conditions, were examined. In vitro produced bovine zygotes were cultured in modified-Synthetic Oviduct Fluid medium. Suplementation with 3.6 mM acetoacetate or b-hydroxybutyrate for 3 periods of culture gave blastocyst and expanded blastocyst rates comparable to controls with lactate/pyruvate. However, lactate/pyruvate produced higher blastocyst formation and expansion rates than ketone bodies when different proportions (10:1 and 1:10) of 3.6 mM acetoacetate/b-hydroxybutyrate and 3.6 mM lactate/pyruvate were used with either 20 percent O2 or 5 percent O2. However, no differences in development were attributable to redox status. When ketone bodies were added together in culture, lower blastocyst formation rates were obtained than using the same molecules as single substrates. It is concluded that bovine embryos can utilize ketone bodies at any period of early development and can develop withstanding different redox status.
Resumen
Los embriones bovinos producidos in vitro pueden desarrollarse hasta la eclosión del blastocisto en presencia de acetoacetato y b-Dhidroxibutirato, los cuales pueden proceder de la degradación de lípidos. Se estudiaron los efectos de estos cuerpos cetónicos, añadidos durante diferentes períodos del desarrollo embrionario preimplantatorio bajo diferentes condiciones atmosféricas. Zigotos bovinos producidos in vitro se cultivaron en medio modified-Synthetic Oviduct Fluid. La suplementación con acetoacetato o b-D-hidroxibutirato 3,6 mM en cada uno de los 3 períodos de cultivo considerados resultó en proporciones de blastocistos y de blastocistos expandidos comparables a los controles con lactato/piruvato. Sin embargo, la mezcla lactato/piruvato 3,6 mM dió lugar a índices de formación de blastocistos y de expansión más altos que los de los acetoacetato/ b-D-hidroxibutirato 3,6 mM cuando los elementos de cada par se utilizarón en proporciones 10:1 ó 1:10, tanto con una concentración de O2 del 20% como del 5%. El estado redox no dió lugar a diferencias en el desarrollo embrionario. Cuando los cuerpos cetónicos se añadieron juntos en cultivo, los porcentajes de blastocistos fueron inferiores a los obtenidos utilizando cada molécula por separado. En conclusión, los embriones bovinos pueden utilizar cuerpos cetónicos durante cualquier período del desarrollo embrionario preimplantatorio y pueden desarrollarse bajo diferentes estados redox.

Sujets

• Acetoacetic acid
• Acetoacetato
• Lípido

Informations

• Publié par: erevistas
• Publié le: 01 janvier 1999
• Nombre de lectures: 61

Extrait

USE OF KETONE BODIES AS ENERGY SUBSTRATES AT
DIFFERENT DEVELOPMENTAL STAGES AND UNDER
DIFFERENT OXYGEN TENSIONS BY IN VITRO PRODUCED
BOVINE EMBRYOS*
UTILIZACIÓN DE CUERPOS CETÓNICOS COMO SUSTRATO ENERGÉTICO EN DIFE
RENTES ESTADIOS Y BAJO DIFERENTES CONDICIONES ATMOSFÉRICAS DURANTE
EL DESARROLLO EMBRIONARIO BOVINO IN VITRO*
Gómez, E. and C. Díez
Centro de Investigación Aplicada y Tecnología Agroalimentaria. Centro de Selección y Reproducción
Animal. Camino de los Claveles s/n. Somió. 33203 Gijón. Asturias. España.
Tlfno: 34 985195300. Fax: [phone]. E mail: [e-mail]
ADDITIONAL KEYWORDS PALABRAS CLAVE ADICIONALES
Cattle embryology. Acetoacetate. b hydroxy Embriología del ganado vacuno. Acetoacetato.
butyrate. Lipid. b D hidroxibutirato. Lípido.
SUMMARY
In vitro produced bovine embryos can develop However, lactate/pyruvate produced higher
into hatched blastocysts in the presence of blastocyst formation and expansion rates than
acetoacetate and b hydroxybutyrate, which may ketone bodies when different proportions (10:1
be derived from lipid breakdown. The effects of and 1:10) of 3.6 mM acetoacetate/b hydroxy
these ketone bodies, added during different butyrate and 3.6 mM lactate/pyruvate were used
periods of early bovine embryo development and with either 20 percent O or 5 percent O . However,2 2
under different atmospheric conditions, were no differences in development were attributable
examined. In vitro produced bovine zygotes were to redox status. When ketone bodies were added
cultured in modified Synthetic Oviduct Fluid together in culture, lower blastocyst formation
medium. Suplementation with 3.6 mM aceto rates were obtained than using the same
acetate or b hydroxybutyrate for 3 periods of molecules as single substrates. It is concluded
culture gave blastocyst and expanded blastocyst that bovine embryos can utilize ketone bodies at
rates comparable to controls with lactate/pyruvate. any period of early development and can develop
withstanding different redox status.
*This work was supported by a grant from FICYT,
project PA AGR96 01. Part of this work was
presented to the European Association of Embryo RESUMEN
Transfer meeting in 1997 (table IV) and to the
International Embryo Transfer Association mee
ting in 1998 (table I). Los embriones bovinos producidos in vitro
Arch. Zootec. 48: 207 217. 1999.GÓMEZ AND DÍEZ
pueden desarrollarse hasta la eclosión del and Bavister, 1995) and reach the blas
blastocisto en presencia de acetoacetato y b D tocyst stage (Gómez, 1997). However,
hidroxibutirato, los cuales pueden proceder de la it has been suggested that bovine
degradación de lípidos. embryos might metabolize endogenous
Se estudiaron los efectos de estos cuerpos substrates (Gordon, 1995; Gómez,
cetónicos, añadidos durante diferentes períodos 1997; Gómez and Díez, 1997; Rieger et
del desarrollo embrionario preimplantatorio bajo al., 1997), although their potential
diferentes condiciones atmosféricas. Zigotos contribution as energy sources has ten
bovinos producidos in vitro se cultivaron en me ded to be ignored (Leese et al. , 1993).
dio modified Synthetic Oviduct Fluid. La suplemen
Moreover, endogenous lipids could be
tación con acetoacetato o b D hidroxibutirato
metabolically useful at different
3,6 mM en cada uno de los 3 períodos de cultivo
developmental stages, as evidenced by
considerados resultó en proporciones de
the lipid content in preblastocyst stage
blastocistos y de blastocistos expandidos com
pig embryos being higher than that in
parables a los controles con lactato/piruvato. Sin
the more advanced stages (Dobrinsky,
embargo, la mezcla lactato/piruvato 3,6 mM dió
1996). Bovine embryos cultured in vitro
lugar a índices de formación de blastocistos y de
can develop up to the hatched blastocyst
expansión más altos que los de los acetoacetato/
stage by using either acetoacetate or b-b D hidroxibutirato 3,6 mM cuando los elemen
hydroxybutyrate as single energytos de cada par se utilizarón en proporciones
substrates at concentrations of 3.6 mM10:1 ó 1:10, tanto con una concentración de O
2
(Gómez, 1997). The blastocyst formationdel 20% como del 5%. El estado redox no dió
rates and cell number were comparablelugar a diferencias en el desarrollo embrionario.
to those obtained in modified SyntheticCuando los cuerpos cetónicos se añadieron jun
tos en cultivo, los porcentajes de blastocistos Oviduct Fluid (mSOF). Concentrations
fueron inferiores a los obtenidos utilizando cada of 3.3 mM lactate and 0.3 mM pyruvate,
molécula por separado. En conclusión, los em normally contained in mSOF, have also
briones bovinos pueden utilizar cuerpos cetónicos been reported to be optimal for ovine
durante cualquier período del desarrollo embrio embryos (Thompson et al., 1993) and
nario preimplantatorio y pueden desarrollarse allow for the reliable culture of bovine
bajo diferentes estados redox. embryos (Takahashi and First, 1992).
If acetoacetate and b hydroxy
butyrate were derived from endogenous
INTRODUCTION
lipids the survival after freezing and
thawing of embryos is likely to increase,
In vitro produced (IVP) bovine
as is the case following the mechanical
embryos can utilize different exogenous
delipidation of IVP bovine embryos
energy substrates during their develop
(Leibo et al., 1995; Díez et al., 1996;
ment (Peura, 1990; Rieger et al., 1992;
Ushijima et al., 1996). Attempts to
Takahashi et al., 1992; Rosenkrans et improve freezability by triggering lipid
al., 1993; Pinnyopummintr and Bavister, breakdown and subsequent fatty acid
1995; Thompson et al., 1996a). In the b oxidation in embryonic cells using
absence of exogenous energy sources, epinephrine have been carried out, so
few embryos are able to complete the far without positive results (Gómez
first cleavage stage (Pinnyopummintr and Díez, 1997).
Archivos de zootecnia vol. 48, núm. 182, p. 208.USE OF KETONE BODIES AS ENERGY SUBSTRATES
Intracellular b hydroxybutyrate/ were placed in an embryo filter (Em
acetoacetate and lactate/pyruvate Con, Eurofomento Pecuario, Madrid,
+ratios are related to the NADH/NAD Spain) and rinsed 3 times with holding
ratios in the mitochondria and cyto medium, consisting of PBS, pyruvate
1plasm, respectively. The relative 0.16 mM, BSA 3 mg ml and gentamycin
1concentrations of these substrates can solution 5 ml ml (Gibco, Barcelona,
alter via the respective redox couples Spain).
and may affect embryo development.
At the same time, oxygen concentra IN VITRO MATURATION
tion influences the redox status and, as Only oocytes enclosed in a compact
a consequence, preferential utilization cumulus with evenly granulated
of substrates might occur. cytoplasm were selected for matu
The aim of this work was to ration. The COCs were washed 3 ti
characterize bovine embryo develop mes in maturation medium, which
ment (in vitro 1) in the presence of consisted of Medium 199 (Sigma, Ma
acetoacetate and b hydroxybutyrate drid, Spain), fetal bovine serum (FBS,
1during three periods of culture (0 to 4810 percent v/v), FSHp (1 mg ml ,
1h, 48 to 120 h and 120 to 216 h); and 2) Sigma), LH (5 mg ml , Sigma) and
1in the presence of different proportions17b estradiol (1mg ml , Sigma).
(10:1 and 1:10) of acetoacetate/b- Maturation was performed by culturing
hydroxybutyrate (3.6 mM) and lactate/ approximately 50 COCs in 500 ml of
pyruvate (3.6 mM) under 5 percent maturation medium in four well dishes
CO in air and 5 percent CO , 5 percent at 39ºC in 5 percent CO under air and
2 2 2
O and 90 percent N . high humidity for 23 to 24 h.
2 2
IN VITRO FERTILIZATION
MATERIALS AND METHODS In vitro fertilization was carried
out using a swim up procedure similar
OOCYTE RECOVERY to that previously reported by Parrish
Bovine ovaries recovered from a et al. (1986). Briefly, semen from 1
slaughterhouse were placed in NaCl frozen straw of a single bull was thawed
1solution (9 mg ml ) containing in a water bath and added to a
1antibiotics (Penicillin, 100 u.i. ml and polystyrene tube containing 1 ml of
1streptomycin sulfate, 100 mg ml ) and pre equilibrated Sperm TALP. After 1
maintained at 30 35ºC until recovery of h of incubation, 700 ml of the upper layer
cumulus oocyte complexes (COCs). of supernatant containing the motile
Ovaries were washed twice in distilled sperm was removed. The sperm were
water and once in freshly prepared centrifuged for 7 min at 600 g and the
saline and antibiotics. The COCs were supernatant aspirated to leave a pellet
aspirated from follicles (2 7 mm) approximately 100 ml in volume. Sperm
through an 18 gauge needle connected concentration was determined with a
to a vacuum system and recovered intohaemocytometer. After maturation, the
a 50 ml plastic tube (Nunc, Roskilde, COCs were washed 3 times in holding
Denmark). Follicular fluid and COCs medium and placed in four well culture
Archivos de zootecnia vol. 48, núm. 182, p. 209.GÓMEZ AND DÍEZ
Table I. In vitro development up to the hatched blastocyst stage of bovine embry