BIOLOGY OF REPRODUCTION 62, 847–856 (2000)
Energy Metabolism in Preimplantation Bovine Embryos Derived In Vitro or In Vivo1
Narinder K. Khurana3 and Heiner Niemann2
Department of Biotechnology, Institute for Animal Husbandry and Behaviour, Mariensee, 31535 Neustadt, Germany
ABSTRACT
pregnancy after transfer [1–3]. In vitro-produced embryos
can differ in a number of properties from their in vivo counterparts. Differences include those related to gross morphology, ultrastructure, physiology, sensitivity to cryoinjury, and genomic activity, as well as fetal and postnatal
development [4–8]. Finally, IVP embryos differ from in
vivo-generated embryos with respect to their metabolism
[9–11]. A relationship between the metabolism of preimplantation embryos and their developmental competence
has been observed [12].
One of the possible causes for the various differences
observed between embryos generated in vitro and in vivo
could be suboptimal in vitro culture conditions. Energy
substrates are among the important ingredients of any culture medium. A substantial body of information indicates
the changing energy substrate requirements during early development of mammalian embryos with important differences among species [11–14]. An increased knowledge of
the metabolic fate of various energy substrates and the nutrient preferences of preimplantation embryos is crucial for
improvements of culture media. Although several reports
have dealt with the metabolism of bovine embryos produced in vitro or in vivo [15–20], a direct comparison of
the metabolic activity of embryos derived either in vitro or
in vivo at similar stages of development within the same
experiment has not yet been carried out. The purpose of
this study was to determine the metabolic needs of the bovine oocyte as it undergoes in vitro maturation (IVM), fertilization (IVF), and development to the hatched blastocyst
stage, as well as to compare these requirements with those
of their in vivo counterparts to evaluate the biochemical
normality of in vitro-produced embryos. The feasibility of
using metabolic activity as an index of developmental potential was also examined.
This study was an investigation of metabolism during bovine
preimplantation development from the oocyte up to the hatched
blastocyst derived in vitro or in vivo. Metabolism was determined by estimating the consumption of radiolabeled glucose,
pyruvate, or lactate during a 4-h incubation period in a closed
noninvasive system with NaOH as trap for the continuous collection of CO2. The postincubation medium was analyzed for
the presence of lactate. Embryonic metabolism from the matured oocyte to the 12-cell stage was more or less constant, with
pyruvate being the preferred substrate. The first marked increase
in oxidation of glucose occurred between the 12- and 16-cell
stage. Compaction of morula and blastocyst expansion was accompanied by significant increases in oxidation of all three energy substrates. The incorporation of glucose increased steadily
15-fold from the 1-cell to the blastocyst stage. In general, the
pattern of metabolism was similar between the embryos derived
in vitro and in vivo but with some distinct differences. The most
apparent feature of glucose metabolism by in vitro-produced
embryos was a 2-fold higher rate of aerobic glycolysis as compared to that in their in vivo counterparts. In vitro-matured oocytes produced measurable amounts of lactate, whereas in vivomatured oocytes exhibited a significantly lower metabolic activity and did not produce any lactate. When in vivo-collected embryos were preexposed to culture conditions, lactate production
increased significantly and at the hatched blastocyst stage
matched that of their in vitro counterparts. In vitro-produced
embryos up to the 8-cell stage oxidized significantly higher
amounts of lactate and had a lower ratio of pyruvate-to-lactate
oxidation than the in vivo-obtained embryos. The results of this
study show that under our culture conditions, important differences exist at the biochemical level between bovine embryos
produced in vitro and those generated in vivo that may well
affect the developmental capacity.
INTRODUCTION
MATERIALS AND METHODS
In vitro production of bovine embryos (IVP) from immature oocytes offers a commercially viable alternative to
breeding programs based on multiple ovulation and embryo
transfer techniques for the genetic improvement of livestock. Furthermore, IVP makes an abundant supply of oocytes and embryos of predefined stages available for basic
studies on preimplantation bovine development. However,
in spite of considerable progress in recent years in the development of IVP technology, the success rates in terms of
embryo yields remain modest and range between 30% and
40%, with approximately 50% of these being able to initiate
In Vitro Generation of Embryos
All chemicals, reagents, hormones, media, and antibiotics were purchased from Sigma Chemical (St. Louis, MO)
unless otherwise indicated. Ovaries, primarily from Holstein cows and heifers, were collected from a local abattoir
and transported to the laboratory within 2–3 h of collection
in PBS at ambient temperature (;258C). Upon arrival in
the laboratory, the ovaries were washed twice with fresh
PBS. Cumulus-oocyte complexes (COC) aspirated from
follicles 2–8 mm in diameter were allowed to settle for 8–
10 min. COC were collected into fresh PBS and classified
into one of four morphological categories mainly based
upon the characteristics of their cellular investments [21].
Oocytes having a homogenous, evenly granulated cytoplasm surrounded by a compact cumulus oophorus with
more than three layers were classified as category I, and
oocytes with fewer than 3 layers of cumulus cells or those
partially denuded but having a homogenous evenly granulated cytoplasm were classified as category II. Oocytes surrounded by corona radiata cells only were put in category
III, and denuded oocytes were grouped under category IV.
N.K.K. was financially supported by the Alexander von Humboldt Foundation, Bonn, Germany, in the form of a postdoctoral fellowship.
2
Correspondence. FAX: 49 05034 871 101; e-mail: [email protected]
3
Current address: Sector-I, Govt. Livestock Farm, Hisar-125001, Haryana,
India.
1
Received: 9 September 1999.
First decision: 5 October 1999.
Accepted: 8 November 1999.
Q 2000 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
847
848
KHURANA AND NIEMANN
Only oocytes belonging to categories I and II yielded morulae/blastocysts in vitro and were used in these experiments. However, in some metabolic studies, oocytes of category III were also included for comparison.
The basic medium for in vitro maturation and culture
was TCM-199 (no. M2520) containing glutamine and 25
mM Hepes and supplemented with 22 mg/ml pyruvate, 2.2
mg/ml sodium bicarbonate, and either penicillin G sodium
and streptomycin sulfate (0.5 mg/ml each) or gentamycin
sulfate (50 mg/ml). The medium was prepared fresh every
week and stored at 48C. For IVM, this medium was supplemented with 1 mg/ml estradiol-17b (Serva, Heidelberg,
Germany), 0.5 mg/ml FSH (Beckers, Faculte de Medicine
Veterinaire, Universite de l’Etat a Liegé, Belgium), 0.06 IU
hCG (Ekluton; Vemie, Kempen, Germany), and 20% heatinactivated (30 min at 568C) estrous cow serum (ECS) (i.e.,
maturation medium) [22].
COC were washed twice in maturation medium without
estradiol, FSH, and hCG and transferred in groups of 10
into 100-ml drops of maturation medium under silicone oil
(Serva). They were incubated for 24 h at 398C in an atmosphere of high humidity and 5% CO2 in air. The IVF
medium consisted of Fert-TALP (TALP: Tyrode’s, albumin,
lactate, pyruvate) [23] containing 1 mM epinephrine, 10
mM hypotaurine, 20 mM penicillamine, 0.56 mg/ml heparin,
and 6 mg/ml BSA. After maturation, COC were inseminated for 18 h in 100-ml droplets of fertilization medium
under silicone oil with frozen/thawed semen from one Holstein bull with a history of proven fertility in our IVF program. After semen was thawed in a water bath at 378C for
1 min, motile spermatozoa were separated by the swim-up
procedure [24]. Sperm concentration was determined using
a Coulter counter (Coulter Electronics Ltd., Krefeld, Germany) or a hemocytometer, and the final concentration in
fertilization droplets was adjusted to one million sperm per
milliliter. After fertilization, presumptive zygotes with cumulus cells were washed twice and then transferred into
0.5 ml of development medium (TCM-199120%ECS)
placed under silicone oil in a 4-well Nunclon dish (Nunc,
Roskilde, Denmark). Cumulus cells attached to the bottom
of the dish and formed a monolayer within 2–3 days of
culture. When early cleavage-stage embryos were required,
cumulus cell remnants were removed by slight pipetting
without disturbing the cells attached to the bottom of the
dish. According to the requirements of the individual experiments, culture was either maintained undisturbed for 8–
10 days when the embryos should have reached the blastocyst stage or was terminated at appropriate intervals to
obtain embryos of predefined developmental stages for use
in the metabolic studies. The development medium was not
replaced during the 8- to 10-day culture period. All other
culture conditions were the same as for IVM/IVF. The viability of IVP embryos was examined by transferring blastocysts to 14 synchronized recipients, of which 6 become
pregnant. A total of 2281 embryos generated in 60 IVP
runs over a period of 18 mo were used for the present study.
Collection of In Vivo Embryos
Holstein cows and heifers were superovulated using either eCG or pFSH followed by prostaglandin F2a injection.
Uterine embryos were collected nonsurgically according to
standardized procedures. Oviductal stages were obtained after slaughter in the institute’s own slaughterhouse, using
PBS supplemented with 1% newborn calf serum as the
flushing medium. In vivo-matured oocytes were obtained
by aspirating follicles . 12 mm either from abattoir ovaries
or from the ovaries of superovulated animals slaughtered
72–74 h after the prostaglandin injection. Follicles measuring . 20 mm in diameter were considered cystic and were
not included in the study. In total, 373 in vivo-produced
embryos recovered from 42 animals in 15 sessions over a
period of 18 months were used for these experiments.
Metabolic Studies
Energy substrate-free medium. Studies on the metabolic
competence of bovine embryos generated in vitro or in vivo
were undertaken using medium based on Dulbecco’s PBS
without any additional energy substrates. It consisted of
NaCl, 130 mM; KCl, 2.38 mM; KH2PO4, 1.47 mM; Na2PO4,
8.10 mM; CaCl2, 0.71 mM; MgSO4, 0.92 mM; penicillin
G sodium, 60 mg/ml; streptomycin sulfate, 50 mg/ml; and
BSA (fraction V), 1.0 mg/ml. The medium was prepared
fresh every week and stored at 48C. This medium was used
for initial washing and after being enriched with the respective radiolabeled substrate was employed for the final
washing and incubation of the embryos for estimation of
metabolism.
Isotopes
All radioactive substrates including [U-14C]glucose, [1and [1-14C]lactate were obtained from Amersham Buchler GmbH & Co. KG (Braunschweig, Germany)
and stored at 2208C upon arrival. In preliminary experiments, an aliquot of each supply was tested for potential
toxic or inhibitory effects on embryonic development in
vitro. The stock isotopic solutions were dried under nitrogen and reconstituted by dilution with the respective nonradioactive substrate to give the required specific activity
and concentration. Calculated aliquots were dried in sterile
tubes and stored at 2208C until use. Prior to use, the dried
radioactive substrate was reconstituted by addition of a
known quantity of sterile culture medium. To increase the
accuracy of the measurements, particularly in studies with
single embryos, it was necessary to use the isotopic marker
at a high specific activity. In the present experiments, [U14C]glucose was used at a concentration of 0.28 mM (0.56
mM in a few preliminary experiments for comparison) at
a specific activity of 30 mCi/mmol (1.11 Mbq/mmol). Similarly, pyruvate was used at a concentration of 0.5 mM and
a specific activity of 15 mCi/mmol (0.55 MBq/mmol), while
lactate had a concentration of 2.5 mM at a specific activity
similar to that of pyruvate.
14C]pyruvate,
Estimation of CO2 Production
Basically, the methodology used for measuring oxidative
metabolism of embryos was similar to that used previously
[16, 25] with slight modifications. Embryos at a particular
stage of development, either single or in groups of 2–5,
were incubated for 4 h in a closed chamber in the presence
of a radioactive substrate, and the CO2 released was continuously collected in a NaOH trap. A washed (3 times)
and heat-sterilized 20-ml scintillation vial served as the incubation apparatus. Embryos were washed twice in energy
substrate-free medium and placed in 50-ml drops of radioactive medium for a final wash before being transferred in
a minimum volume (, 1 ml) into a sterile 600-ml microcentrifuge tube (Eppendorf-Netheler-Hinz, GmbH, Hamburg, Germany) containing 50 ml of radioactive incubation
medium. This tube was then placed in the scintillation vial
METABOLISM IN PREIMPLANTATION BOVINE EMBRYOS
with the lid of the tube acting as a support. Simultaneously,
0.5 ml of 2.5 M sodium hydroxide was added to the bottom
of the vial, which was then closed and incubated at 398C
for 4 h. At the end of the incubation period, the tube with
embryos was removed, and the radioactivity in the trapped
CO2 was determined after addition of Hionic Fluor (Packard Instruments, Dreieich, Germany), a pseudocumenebased scintillation cocktail. In all experiments, at least two
sham incubations were run in parallel to correct for background counts. The rate of CO2 production was estimated
from the disintegrations per minute detected in the sample
and the specific activity of the parent substrate. It was converted into picomoles per embryo per hour. This technique
was preferred in the present study as it required no transfer
of NaOH to the scintillation vial and provided sufficient
postincubation medium for the analysis of lactate production. Similarly, the viability of the embryos subjected to
metabolic measurements was examined by returning a total
of 25 embryos at the 2-cell stage to coculture with cumulus
cells after measuring their CO2 production. While 16 of
these embryos underwent two or more further divisions, 6
advanced to the morula/blastocyst stage. The rate of development was similar to that of control embryos.
Incorporation of Substrate Carbon
After the 4-h incubation period for measuring CO2 production, the embryos were recovered in a minimum volume
of incubation medium, washed three times in medium containing nonradioactive substrate at the same concentration,
and transferred to a scintillation vial containing 0.5 ml of
distilled water and processed for assay of radioactivity. A
sample of the last wash before radioassay was always included to provide background levels. The incorporation was
expressed as picograms atoms per embryo per hour as described in relation to CO2 production.
849
Metabolism of Glucose
Four experiments were conducted to study metabolism
of glucose by bovine embryos.
Experiment 1. The first experiment addressed utilization
of radiolabeled glucose as the sole energy substrate at a
concentration of 0.28 mM from the bovine immature oocyte
up to the hatched blastocyst stage derived in vitro or in
vivo. In preliminary trials, a 2-fold higher concentration
(0.56 mM) was also included to ensure a sufficient availability of the substrate.
Experiment 2. Based on the finding of the first experiment indicating large variations in repetitions from sizes
and individual blastocysts, the relationship between blastocyst size and its metabolic activity was evaluated. The
blastocysts were divided into three groups according to
their diameter (150–175, 176–200, and . 200 mm).
Experiment 3. The data from experiment 1 suggested
that preexposure of in vivo embryos to in vitro culture
(IVC) conditions affected their lactate production. To confirm this suggestion, this experiment addressed the metabolism of glucose by mid- and expanded blastocyst stage
embryos obtained after IVC of fresh in vivo-collected early
blastocysts. Utilizing data from experiment 1, a comparative study of glucose metabolism in embryos from three
different sources (in vitro produced, collected in vivo and
used immediately [fresh], and fresh embryos preexposed to
culture conditions) was accomplished.
Experiment 4. The fourth experiment examined glucose
metabolism in the presence of additional substrates (as
would be normally expected in vivo or under IVC conditions) in the form of pyruvate (0.5 mM) and lactate (2.5
mM) by embryos produced in vitro or in vivo at different
stages of preimplantation development.
Estimation of Lactate Production
Metabolism of Pyruvate and Lactate
Lactate production was estimated by separating lactate
from glucose in the postincubation media with descending
paper chromatography [26]. A total of 20 ml medium was
spotted on each strip of Whatman chromatography paper
(no. 3030917; Whatman Scientific Ltd., Kent, England) and
run for 16 h using butanol:water:acetic acid (4:5:1, v/v) as
solvent. Dried chromatograms were serially cut into 1-cm
lengths and processed for assay of radioactivity. The lactate
peak was identified by the Rf value and its comigration with
authentic lactate. Disintegrations per minute were converted
to picomoles per embryo per hour as described for CO2
production.
Three experiments were conducted to evaluate the metabolism of radiolabeled pyruvate and lactate by various
stages of preimplantation bovine embryos derived in vitro
or in vivo.
Experiment 5. This experiment evaluated the relationship
between the morphological quality of oocytes (categories I,
II, III) and their ability to metabolize pyruvate and lactate
immediately after aspiration (immature) or after IVM for
24 h. In vivo-matured (follicular) oocytes were also included. Nude oocytes (category IV) were not used, since preliminary trials showed that these oocytes had very low and
inconsistent metabolic activity.
Experiment 6. The sixth experiment was an investigation
of the utilization of [1-14C]pyruvate and [1-14C]lactate present as sole energy substrates by bovine embryos produced
in vitro or in vivo from the immature oocyte to the hatched
blastocyst stage.
Experiment 7. As pyruvate and lactate are known to have
interactions in embryos of various species [28], the effects
of both substrates on each other’s metabolism from the immature oocyte to the 16-cell stage using IVP embryos were
tested. In the first part of this experiment, the metabolism
of radiolabeled pyruvate (0.5 mM) was assessed in the presence of unlabeled lactate at increasing concentrations of 0,
0.5, 2.5, 5.0, and 25 mM; in the second part, the metabolism of radiolabeled lactate (2.5 mM) was evaluated in the
presence of 0 or 0.5 mM unlabeled pyruvate.
Experimental Design
The metabolism of three radiolabeled energy substrates
including glucose, pyruvate, and lactate during in vitro development from the immature oocyte to the hatched blastocyst was investigated during a 4-h incubation period. The
indicators of metabolic activity included production of carbon dioxide and incorporation of substrate carbon for all
three energy substrates, and accumulation of lactate from
glucose only. The cumulus cells surrounding early-stage
embryos were carefully removed prior to use for the metabolic studies. Complete absence of cumulus was verified
by microscopic examination (3100). In the present study,
only very good quality embryos were used for metabolic
investigations [27].
850
KHURANA AND NIEMANN
TABLE 1. Metabolism of [U-14C]glucose present as sole energy substrate by bovine embryos produced in vitro (IVP) versus in vivo at different stages
of development.
Biochemical parameter/source of embryos*
Stage of
embryonic
development
CO2 production (pmoles)
IVP
Immature oocyte
Matured oocyte
1-Cell
2-Cell
8-Cell
12-Cell
16-Cell
Early morula
Morula
Early blastocyst
Mid-blastocyst
Blastocyst
Hatching blastocyst†
Hatched blastocyst†
0.3
0.5
0.2
0.4
0.6
1.3
3.8
7.3
8.8
8.1
9.9
9.7
11.9
Lactate accumulation (pmoles)
In vivo
—
60
6 0.1a
6 0.0b
6 0.1a
6 0.1a
6 0.1c
6 0.5d
6 1.5eA
6 2.0eA
6 0.7e
6 1.1e
6 1.5e
6 1.4e
0.1
0.3
0.2
0.3
0.5
2.2
4.4
3.2
4.7
7.5
8.7
10.8
11.5
0.0
6 0.0aB
6 0.0b
6 0.0b
6 0.1b
6 0.1b
6 0.4c
6 0.3d
6 0.6dB
6 0.3dB
6 0.5e
6 1.0e
6 0.7e
6 0.8e
IVP
1.1
2.8
9.2
8.7
25.9
27.4
48.0
70.2
79.0
70.1
—
6 0.1a
0.0
0.0
0.0
6 1.3b
6 0.8cA
6 2.4c
6 5.6dA
6 6.4dA
6 7.6deA
6 4.6eA
6 4.4e
6 10.6e
Carbon uptake (pg atoms)
In vivo
0.0
0.0
0.0
0.0
0.0
0.0
5.4 6 0.9aB
6.0 6 1.1a
11.9 6 1.4bB
11.4 6 1.8bB
24.6 6 6.0cB
40.6 6 8.7dB
57.6 6 7.0d
58.1 6 23.0d
IVP
0.6
1.7
0.8
3.2
4.8
7.4
11.0
18.7
19.0
20.0
24.7
23.4
25.9
—
6 0.0aA
6 0.3ba
6 0.1a
6 0.8c
6 0.4c
6 1.1cA
6 0.8dA
6 4.1e
6 5.1e
6 2.1e
6 4.5e
6 2.7e
6 3.0e
In vivo
0.1
0.2
0.6
0.7
4.6
5.4
11.6
15.3
14.7
17.5
22.0
31.0
28.5
34.2
6
6
6
6
6
6
6
6
6
6
6
6
6
6
0.0a
0.1aB
0.1bB
0.1b
0.3c
0.7c
1.3dB
0.9eB
1.8e
0.8e
0.6f
3.7fg
1.6g
11.0g
* Values are means 6 SEM per embryo per hour pooled from 5–15 replicates.
† Values for in vivo hatching/hatched blastocysts are for those embryos which were obtained after 48–72 h in vitro culture of freshly obtained in vivo
embryos.
a–g Values with different superscripts within same column differ significantly (P , 0.05 at least).
A,B IVP vs. in vivo within same biochemical parameter and stage of embryonic development (P , 0.05 at least).
Statistical Analysis
Statistical significance of differences was tested by Student’s t-test, chi-square test, and protected least significant
difference (LSD) test where appropriate [29]. Differences
in the frequencies of maturation, fertilization, and cleavage,
as well as in the proportion of zygotes reaching the morula/
blastocyst stage in the two groups, were compared by chisquare test. The statistical significance of the differences of
the various indicators of metabolism between the two
sources of embryos and stages of development was calculated by Student’s t-test. Multiple means were compared by
protected LSD test. The data are presented as mean 6
SEM. Values at P , 0.05 were considered to be significant.
RESULTS
Metabolism of Glucose
The results of experiment 1 are presented in Table 1. In
initial trials, a 2-fold higher concentration (0.56 mM) of
glucose was also used. Since the pattern of glucose metabolism was not altered by this higher concentration of substrate, the data pertaining to the 0.56 mM concentration are
not presented. While immature oocytes did not oxidize any
glucose, there was a 38-fold increase in the rate of CO2
production as the matured oocyte progressed to the hatched
blastocyst stage in vitro. The first significant increase in
oxidation of glucose was noticed at the 16-cell stage when
TABLE 2. Effect of the size of the bovine blastocyst derived in vitro on
metabolism of [U-14C]glucose.
Biochemical parameter*
Size of
blastocyst
(mm)
150–175
176–200
.200
n
CO2 production
(pmoles)
Lactate
accumulation
(pmoles)
Carbon uptake
(pg atoms)
7
8
7
7.3 6 1.0
7.0 6 0.7
8.5 6 1.0
51.7 6 12.9
61.5 6 12.2
69.8 6 7.1
29.7 6 8.1
17.9 6 4.3
27.5 6 6.0
* Values are means 6 SEM per embryo per hour for 7–8 replicates.
the rate of CO2 production exceeded the level of 1 pmol/
embryo per hour. The in vitro-matured oocyte exhibited
aerobic glycolysis by accumulating small quantities of lactate in the incubation medium. Lactate production by IVP
embryos fell to undetectable levels until the 12-cell stage,
when lactate was again detected. In contrast, in vivo-generated embryos did not produce any lactate up to the 16cell stage. Thereafter, a gradual increase in the production
of lactate was recorded as the embryo progressed to the
blastocyst stage and underwent hatching. The bulk of glucose was utilized through aerobic glycosis by later preimplantation stages. In general, the in vivo-grown embryos
produced only half the quantity of lactate that their in vitro
counterparts did. However, no true comparisons could be
made at the hatching and hatched blastocyst stages between
embryos of differing origin, as these could not be collected
in vivo and were obtained by IVC of freshly collected early
blastocysts for 48–72 h. Interestingly, lactate production of
these in vivo-grown and in vitro-cultured blastocysts
reached levels almost equal to that of their IVP counterparts. While only a small quantity of glucose was incorporated by the immature oocyte, it increased steadily thereafter, showing a 15-fold increase between the 1-cell and the
blastocyst stages. In vivo-generated embryos followed a
similar pattern with a few minor differences.
The results of experiment 2 are summarized in Table 2.
There were no differences among the three groups of blastocysts based on their diameters in their metabolic activity
as measured by CO2 production, lactate accumulation, and
carbon uptake from glucose. CO2 production of 22 blastocysts employed in this experiment ranged from 3 to 12.7
pmol/embryo per hour. Lactate production and substrate
carbon uptake ranged from 27.3 to 133.8 pmol and 5.2 to
79.3 pg atoms per embryo per hour, respectively.
The results of experiment 3 are shown in Table 3. The
preexposure of in vivo-generated embryos to culture conditions significantly increased the rate of aerobic glycosis,
and the differences disappeared at the hatched blastocyst
stage obtained after 72 h of IVC.
The data on the metabolism of radiolabeled glucose in
851
METABOLISM IN PREIMPLANTATION BOVINE EMBRYOS
TABLE 3. Comparison of metabolism of [U-14C]glucose by bovine blastocysts produced in vitro (IVP), obtained in vivo, and used immediately (fresh)
or obtained after in vitro culture for 1–3 days of freshly collected early blastocysts (Fresh 1 IVC).
Stage of development*
Biochemical parameter/
source of embryo
Mid-blastocyst
Catabolic utilization
CO2 (pmoles)
IVP embryos
Fresh embryos
Fresh 1 IVC embryos
Lactate production (pmoles)
IVP embryos
Fresh embryos
Fresh 1 IVC embryos
Incorporation (pg atoms)
IVP embryos
Fresh embryos
Fresh 1 IVC embryos
Expanded blastocyst
Hatched blastocyst
8.1 6 0.7a
7.5 6 0.5a
10.4 6 0.4b
10.0 6 1.1
8.7 6 1.0
9.4 6 0.4
11.9 6 1.4
NP
11.5 6 0.8
48.0 6 7.6a
24.7 6 6.0b
42.6 6 1.6a
70.3 6 4.5a
20.8 6 5.0b
40.6 6 8.7c
70.2 6 10.6
NP
58.1 6 22.9
20.0 6 2.1a
22.0 6 0.6a
48.5 6 10.7b
24.7 6 4.5
26.5 6 4.0
31.0 6 3.7
25.9 6 3.0
NP
34.2 6 11.0
* Values are means 6 SEM (per embryo per hour) pooled from 5–11 replicates; NP, not performed.
a–c Values with different superscripts within biochemical parameter of a stage of development differ significantly (P , 0.05).
TABLE 4. Metabolism of [U-14C]glucose in the presence of unlabeled pyruvate (0.5 mm) and lactate (2.5 mM) by bovine embryos derived in vitro or
in vivo.
Biochemical parameter/source of embryos*
CO2 production (pmoles)
Stage of embryonic
development
Matured oocyte
2-Cell
8-Cell
16-Cell
Early blastocyst
Blastocyst
IVP
0.1 6
0.0
0.3 6 0.2b
0.5 6 0.1b
1.3 6 0.2c
3.4 6 0.4d
0.0a
Lactate accumulation (pmoles)
In vivo
IVP
0.0
0.0
0.3 6 0.1a
0.7 6 0.2b
0.9 6 0.2b
3.6 6 0.5c
2.2 6
0.0
0.0
1.7 6 0.3aA
11.7 6 1.5bA
28.7 6 2.2cA
0.7a
Incorporation (pg atoms)
In vivo
IVP
0.0
0.0
0.0
0.9 6 0.3aB
5.3 6 0.7bB
16.8 6 1.8cB
6
6
6
6
6
6
0.5
0.8
2.1
3.9
5.8
18.4
0.1aA
0.2a
0.4b
0.3c
0.6c
2.3d
In vivo
0.1
0.6
2.3
5.4
5.4
19.9
6
6
6
6
6
6
0.0aB
0.2b
0.4c
0.7d
0.6d
2.7e
* Values are means 6 SEM per embryo per hour for 5–6 replicates.
A,B Paired values in the same biochemical parameter differ significantly (P , 0.05).
a–e Values with different superscripts in the same column differ significantly (P , 0.05).
TABLE 5. Metabolism of [1-14C]pyruvate and [1-14C]lactate used as sole energy substrate in vitro by oocytes belonging to different morphological
categories before and after maturation.
Energy substrate*
Pyruvate
Stage/source/morphological
category of oocytes
Category-I
Category-II
Category-III
Matured oocyte
Category-I (IVM)
Category-II (IVM)
Category-III (IVM)
In vivo matured
Lactate
CO2
(pmoles)
Incorporation
(pg atoms)
CO2
(pmoles)
Incorporation
(pg atoms)
2.7 6 0.1a
1.9 6 0.3b
1.0 6 0.1c
0.5 6 0.1a
0.2 6 0.0b
0.1 6 0.0c
1.2 6 0.2
0.9 6 0.1
0.9 6 0.2
0.3 6 0.0
0.3 6 0.0
0.5 6 0.1
3.6
3.7
1.3
2.4
6
6
6
6
0.2a
0.2a
0.1b
0.2c
0.8
0.9
0.7
0.5
6
6
6
6
0.0
0.1
0.0
0.1
2.1
2.3
1.1
1.2
6
6
6
6
0.1a
0.2a
0.1b
0.1b
1.9
1.1
0.8
0.5
6
6
6
6
0.4a
0.1a
0.1b
0.1c
* Values are means 6 SEM for five replicates.
a–c Means with different superscripts in the same column and stage of development differ significantly (P , 0.05).
the presence of unlabeled pyruvate and lactate (experiment
4) are presented in Table 4. The overall pattern of glucose
metabolism and the production of lactate by the IVM oocyte remained unaltered in the presence of alternate energy
substrates. However, the total consumption of glucose was
reduced by 60–70% as compared to values obtained in experiment 1 when glucose was used as the sole energy substrate. Approximately 70–90% of it was metabolized
through aerobic glycolysis. The IVP embryos continued to
produce twice as much lactate as did their in vivo counterparts
but on a much lower scale in absolute terms. The incorporation of glucose carbon at the blastocyst stage was not
affected when compared with the data from experiment 1.
Metabolism of Pyruvate and Lactate
The results of experiment 5 indicate that the morphological
quality of the immature oocyte significantly affected oxi-
852
KHURANA AND NIEMANN
FIG. 1. CO2 production from [U-14C]glucose (diamonds), [1-14C]pyruvate (squares), and [1-14C]lactate (triangles) by immature oocytes and in vitroderived embryos at various stages of development.
from its rate of oxidation as compared to that of lactate or
glucose (Fig. 1). The rate of utilization of pyruvate was
relatively constant as the oocyte advanced to the morula
stage in comparison to few ups and downs noted in the
utilization of lactate. Only a small but consistent amount
of substrate carbon was incorporated throughout preimplantation development with no effects of source of embryos or the employed substrate.
dation and uptake of pyruvate but not that of lactate (Table
5). In general, oocytes from categories I and II had a similar
pattern of metabolic activity. In vivo-matured oocytes had
significantly lower rates of oxidation of pyruvate and lactate as compared to IVM oocytes in categories I and II.
The results of experiment 6 are presented in Table 6.
Irrespective of the origin of the embryos, pyruvate was the
preferred substrate until the 16-cell stage. This was evident
TABLE 6. Comparative utilization of [1-14C]pyruvate or [1-14C]lactate present as sole energy substrates by bovine IVP and in vivo embryos at various
stages of their development.
Energy substrate/biochemical parameter/source of embryos*
Pyruvate
CO2
Stage of embryonic
development
IVP
Immature oocyte
Matured occyte
1-Cell
2-Cell
8-Cell
12-Cell
16-Cell
Early morula
Morula
Early blastocyst
Blastocyst
Hatched blastocyst
—
6 0.2aA
6 0.2bA
6 0.2b
6 0.3b
6 0.2b
6 0.2b
6 0.7b
6 2.1c
6 1.4c
6 1.3d
6 1.4d
3.6
2.9
2.4
2.8
2.1
3.0
2.9
5.4
5.0
11.5
12.3
Lactate
Incorporation (pg atoms)
In vivo
2.7
2.4
2.2
2.3
2.6
2.6
3.3
3.2
5.4
7.8
9.8
6 0.1a
6 0.2aB
6 0.1aB
6 0.1a
6 0.5a
6 0.4a
6 0.2b
6 0.4b
6 1.0c
6 1.3d
6 1.1d
NP
IVP
0.8
0.6
1.2
1.4
1.2
1.1
0.7
1.6
1.2
2.6
0.6
—
6 0.0a
6 0.1a
6 0.1b
6 0.1b
6 0.4b
6 0.1b
6 0.1a
6 0.2b
6 0.3b
6 0.6c
6 0.1a
CO2 (pmoles)
In vivo
0.5
0.5
0.9
1.2
1.4
1.0
1.0
1.3
2.0
1.5
2.4
6 0.1a
6 0.1a
6 0.1b
6 0.1b
6 1.0b
6 0.2b
6 0.1b
6 0.4b
6 0.3c
6 0.1b
6 0.6c
NP
IVP
2.1
1.0
1.7
2.4
1.1
1.5
2.8
2.6
4.6
6.7
6.5
—
6 0.1aA
6 0.1bA
6 0.2aA
6 0.5aA
6 0.1b
6 0.1b
6 0.4a
6 0.3a
6 0.5c
6 0.7d
6 0.4d
* Values are means 6 SEM per embryo per hour for 5–15 replicates; NP, not performed.
A,B IVP vs. in vivo embryos (P , 0.05) within same biochemical parameter and stage of development.
a–d Values with different superscripts in the same column differ significantly (P , 0.05).
Incorporation (pg atoms)
In vivo
1.2
1.2
0.6
0.6
0.7
1.1
1.1
1.5
2.1
4.5
5.7
6 0.2a
6 0.1aB
6 0.1bB
6 0.2bB
6 0.1bB
6 0.2a
6 0.2a
6 0.4a
6 0.3c
6 0.8d
6 0.5d
NP
IVP
1.9
1.0
1.0
1.7
1.1
0.7
1.4
1.5
0.9
1.3
2.4
—
6 0.4aA
6 0.1aA
6 0.1a
6 0.4a
6 0.1a
6 1.1a
6 0.5a
6 0.3a
6 0.1a
6 0.1a
6 0.7b
In vivo
0.3
0.5
1.4
1.3
0.9
1.0
1.0
1.7
2.5
2.3
4.3
6 0.1a
6 0.1aB
6 0.1bB
6 0.2b
6 0.2b
6 0.1c
6 0.7c
6 0.5b
6 0.2d
6 0.5d
6 1.5d
NP
853
METABOLISM IN PREIMPLANTATION BOVINE EMBRYOS
TABLE 7. Production of CO2 from [(1-14C]pyruvate in the presence of unlabeled lactate and from [1-14C]lactate in the presence of unlabeled pyruvate
by immature oocytes and IVP embryos at different stages of development.
Substrate (conc. mM)
Stage of embryonic development*
Radiolabeled
Pyruvate (0.5) alone
Lactate (2.5) alone
Unlabeled
1
1
1
1
Lactate
Lactate
Lactate
Lactate
(0.5)
(2.5)
(5.0)
(25.0)
1 Pyruvate (0.5)
Immature
oocyte
2.7
2.1
1.1
0.6
0.3
1.2
0.5
6
6
6
6
6
6
6
0.1a
0.2b
0.1c
0.0d
0.0d
0.2A
0.1B
Matured
oocyte
3.6
3.2
1.9
1.0
0.2
2.1
1.3
6
6
6
6
6
6
6
0.2a
0.1a
0.2b
0.1c
0.1d
0.1A
0.1B
1-Cell
2-Cell
6
6
6
6
6
6
6
6
6
6
6
6
6
6
2.9
2.0
1.3
0.7
0.2
1.0
0.8
0.2a
0.2b
0.1c
0.1d
0.0e
0.1
0.1
2.4
2.5
1.4
0.8
0.2
1.7
1.2
0.2a
0.1a
0.1b
0.1c
0.1d
0.2
0.1
8-Cell
2.8
2.4
1.6
0.9
0.8
2.4
0.6
6
6
6
6
6
6
6
0.3a
0.3a
0.3b
0.1bc
0.2c
0.1A
0.1B
12-Cell
2.1
1.6
1.6
0.5
0.1
1.1
0.8
6
6
6
6
6
6
6
0.2a
0.5a
0.2a
0.1b
0.0c
0.1A
0.0B
16-Cell
3.0
2.0
1.8
0.8
0.4
1.5
1.1
6
6
6
6
6
6
6
0.2a
0.3b
0.4b
0.1c
0.1c
0.1A
0.1B
* Values are means 6 SEM per embryo per hour for 4–5 replicates.
a–e Means with different superscripts in the same column differ significantly (P , 0.05 at least).
A,B P , 0.05, paired values within same stage of development.
The data pertaining to both parts of experiment 7 are
summarized in Table 7. In general, the oxidation of pyruvate was significantly reduced as the concentration of lactate was increased. At 25 mM lactate, the highest level
tested, oxidative metabolism of pyruvate was drastically reduced to 4–15% of the control values in all stages of development. Oxidation of C-1 of lactate in the presence of
0.5 mM pyruvate was significantly decreased in all developmental stages except at the 1- and 2-cell stage.
General Considerations
An overview of oxidative metabolism of IVP embryos
is depicted in Figure 1. The metabolic activity was more
or less constant until the 12-cell stage, with pyruvate being
the preferred energy substrate. The immature oocyte was
not able to utilize any glucose, but glucose utilization gradually increased from the mature oocyte to the hatched blastocyst stage with the first marked increase occurring between the 12- and 16-cell stages. In contrast, the oxidation
of pyruvate and lactate remained within a narrow range of
1–3 pmol until the morula stage, when there was a 2- to 4fold increase. Only a small amount of substrate carbon from
pyruvate and lactate was incorporated throughout preimplantation development. In comparison, incorporation of
glucose increased more than 150-fold from the immature
oocyte to the blastocyst stage. The bulk of glucose was
metabolized through aerobic glycolysis at the expanded and
hatched blastocyst stages. The addition of one or more alternate energy substrates significantly affected the utilization of glucose, pyruvate, or lactate.
In general, the pattern of metabolism was similar between the embryos from the two sources, with some important differences such as the higher metabolic activity of
IVM oocytes with characteristic aerobic glycolysis. Similarly, embryos at later stages of preimplantation development, either generated in vitro or preexposed to culture conditions for 1–3 days after being collected in vivo, produced
double the amounts of lactate from glucose as compared to
their in vivo counterparts.
DISCUSSION
For the first time the metabolism of in vivo- and in vitroderived bovine embryos was investigated in parallel, and
several distinct differences were detected under our experimental conditions. The glucose concentrations (0.28 mM)
employed in the present experiments are close to those
found in bovine oviductal fluid [30, 31], as concentrations
above the physiological levels may disturb embryonic glu-
cose metabolism [32]. Similarly, the concentrations of lactate (2.5 mM) and pyruvate (0.5 mM) seem to be within
the physiological range [33].
In general, the pattern of utilization of glucose, pyruvate,
and lactate was similar between the embryos derived in
vitro and in vivo. The overall pattern of glucose metabolism
from the 1-cell to the blastocyst stages was similar to that
reported for murine [34], human [35], ovine [36], porcine
[37], and bovine [15–17] embryos. The first marked increase in the metabolism of glucose was observed between
the 8- and 16-cell stage, the time of activation of the embryonic genome [38]. However, in bovine embryos collected from superovulated animals, the first marked increase in utilization of glucose was detected between the
16-cell and morula stage [16]. This significant increase in
glucose metabolism in early embryos may either be related
to the synthesis of one or more key glycolytic enzymes as
it coincides with the activation of the embryonic genome
or be due to changes in the allosteric regulation of enzyme
activity [17]. However, the present observations on the production of lactate by in vitro-matured oocytes would suggest that bovine embryos possess the full machinery of glycolytic enzymes from very early stages onward. This suggestion is supported by a study in which lactate production
by all stages of in vitro-derived bovine embryos between
the 1-cell and the blastocyst stage was detected [15]. In
contrast, lactate production was not detected from the 1cell to the 12-cell stage in the present study. However, these
two studies used different methodologies and may not be
directly comparable. It is possible that the enzymes were
present throughout the early period of development, but
were inhibited by intracellular regulations until the 12-cell
stage when the growth rate and energy demands of the embryo increased significantly.
The present data on incorporation of glucose carbon suggest that glucose metabolism plays a crucial role in the
synthetic activity, as incorporation increased more than six
times between the 12-cell and compact morula stages. This
suggestion is further supported by the observations made
in experiment 4, in which glucose carbon incorporation was
substantially reduced in the presence of alternate energy
substrates at all stages of development but not at the blastocyst stage when a great deal of synthetic activity is expected. Several studies using mouse morulae/early blastocysts have shown that a significant fraction of glucose is
incorporated into macromolecules [26, 39, 40]. In general,
the developmental pattern of changes in the incorporation
resembled that of human [35] and murine [41] embryos.
One of the striking findings of the present metabolic
854
KHURANA AND NIEMANN
studies was the production of lactate and the considerably
higher rates of oxidation of all three energy substrates by
IVM oocytes as compared to their in vivo counterparts.
Differences at the ultrastructural level between oocytes matured in vitro and in vivo [42, 43] are likely reflected at the
biochemical level. Alternately, follicular oocytes used in
the present study might not yet have acquired their full
metabolic competence, which occurs prior to the time of
ovulation. A comparison of the metabolism of oocytes collected from preovulatory follicles at different intervals prior
to ovulation and oocytes collected from the oviduct immediately after ovulation would be useful.
The most apparent characteristic of glucose metabolism
in the present study was the 2-fold higher production of
lactate by the in vitro-produced blastocysts as compared to
their in vivo counterparts. The rate of lactate production of
hatched blastocysts obtained after 72-h IVC of freshly collected early blastocysts matched that of their IVP counterparts. Others have also reported findings similar to ours,
that is, that the bulk of glucose is metabolized through aerobic glycolysis by mammalian morulae and blastocysts
[15–17, 36, 41, 44–46]. In contrast, earlier it was suggested
that glycolysis was blocked in bovine blastocysts because
of a lack or inhibition of pyruvate kinase [47]. The high
rate of aerobic glycolysis does not seem to be incompatible
with an active tricarboxylic acid (TCA) cycle. This is
shown by the 3- to 4-fold increase in the rates of oxidation
of pyruvate and lactate during development from the morula to the blastocyst stage in the present experiments as
well as in other studies [15, 48]. The results of experiment
4 also suggest the operation of an active TCA cycle in
embryos, as the total consumption of glucose was reduced
by more than 50% when pyruvate and lactate were added
to the incubation medium.
Several explanations for the high rate of lactate production by IVP embryos can be put forward. Glycolysis was
functional at the blastocyst stage (except in IVM oocytes),
when there is a high demand for energy to meet the needs
of compaction, expansion, and blastocoele formation [49].
Presumably, bovine embryos are able to enhance glycolysis
to a maximum capacity to gain additional energy when the
other pathways are already operating at maximum. This is
supported by the finding that the rate of oxidation of glucose did not increase significantly beyond the morula stage
although the energy demand is increased during blastulation. Why, in spite of the high demands of energy, the embryo chose this less efficient pathway of energy production
is not clear, particularly in light of the fact that the TCA
cycle was active. It is most likely a manifestation of metabolic stress [16] attributable either to separation from the
natural environment or to the extended period of suboptimal IVC conditions. A stress response could also explain
the lactate production shown by in vitro-matured oocytes
in the first stage of separation from their natural environment (follicle) and the high rates of lactate production at
the blastocyst stage after additional exposure to IVC conditions. Similarly, the low rate of lactate production by in
vivo-collected embryos could also be explained on this basis. An enhanced aerobic glycolysis is a characteristic feature of adult cells when they are isolated from their natural
environment and cultured in vitro. The same findings have
been obtained for tumor cells, which resemble early embryos in that they have pluri- or totipotent properties and
show rapid cell division [13].
Recently, it has been reported that culture medium used
for production of embryos can affect metabolism of the
resulting embryos. Embryos generated in a completely defined medium had lower rates of glycolysis than embryos
grown in medium supplemented with serum [50]. This
could be similarly true for the present study, since the IVP
embryos were produced in medium containing 20% ECS.
Whether this high rate of lactate accumulation that can
cross cell membranes and affect intracellular pH [51] has
any physiological significance or any effect on viability of
the bovine embryos raises questions yet to be resolved. The
production of lactate was suggested to be a physiological
requirement for the maintenance of cytoplasmic redox equilibrium of (NADH):(NAD1) [52]. Embryos that are likely
to have reduced viability such as those exhibiting a slow
rate of development and having fewer blastomeres displayed higher rates of glycolysis [32]. Similarly, increased
rates of glycolysis have been associated with reduced embryo viability in mice [53].
All three measures of glucose metabolism displayed
wide individual variations at the blastocyst stage. The data
presented in Table 2 suggest that these variations are not
related to size or morphological quality of the embryo, as
only very good quality embryos were employed for these
metabolic studies. However, glucose uptake and lactate production have been employed to select viable bovine blastocysts upon freezing and thawing [54]. Perhaps cryopreservation is particularly detrimental to embryos with a metabolism operating at its maximum capacity and only those
embryos can survive that have a metabolism within the
physiological range.
The data on the metabolism of pyruvate by immature
oocytes belonging to different morphological categories indicated that the morphological differences were reflected at
the biochemical level. However, these metabolic indicators
cannot be used as selection criteria for the developmental
potential of oocytes in vitro, as the overall metabolic activity of immature oocytes was relatively low and the differences in metabolic activity between category I and II oocytes disappeared after maturation. The pattern of utilization of pyruvate and lactate suggested that pyruvate was
the preferred substrate up to the 12-cell stage, and its oxidation further increased during compaction and blastulation, similarly to that of glucose. A similar increased uptake
and oxidation of pyruvate has been reported for bovine [13,
47, 48] and ovine [36, 52] embryos. This is in contrast to
murine embryos, which show a dramatic decrease in pyruvate uptake at the blastocyst stage [55, 56]. Our results
show that only a small fraction of pyruvate and lactate carbon was incorporated by embryos at all stages of development, which is in agreement with the findings for murine
embryos [57]. We observed that early embryos up the to
8-cell stage oxidized significantly higher amounts of lactate
and had a lower ratio of pyruvate-to-lactate oxidation than
their in vivo counterparts. These observations indicate differences in ATP/ADP ratio and oxidation/reduction status
of the embryos grown in vitro versus in vivo. A proper
lactate-to-pyruvate ratio is essential for balancing the oxidation/reduction potential in embryos [58]. However, it has
been reported that lactate alone can support development
of early bovine [59] and ovine [60] embryos.
An interesting finding of our study was that lactate and
pyruvate each affected metabolism of the other. In the presence of 25 mM lactate, utilization of pyruvate was drastically reduced, raising doubts about the need for high
amounts of lactate in culture media. Most culture media
have a higher pyruvate-to-lactate ratio than the maximum
(1:50) used in the present study. It is possible that the pres-
METABOLISM IN PREIMPLANTATION BOVINE EMBRYOS
ence of somatic cells and glucose during IVC of bovine
embryos may change this ratio, as in the mouse, cumulus
cells are capable of producing pyruvate [61]. Additionally,
pyruvate was required for meiotic maturation in denuded
but not in cumulus-enclosed bovine oocytes [62]. The present results suggest that a low pyruvate-to-lactate ratio
should be beneficial for culture of bovine oocytes/embryos
in chemically defined medium. A low pyruvate-to-lactate
ratio for culture of bovine embryos in semidefined media
was compatible with rates of development similar to those
obtained with complex culture conditions and a high lactate-to-pyruvate ratio [59].
In conclusion, the results of the present experiments
demonstrate that in vivo- and in vitro-derived bovine embryos possess to a large extent similar metabolic activities.
However, several distinct metabolic differences were identified for in vitro-derived oocytes and embryos as compared
with their in vivo counterparts. The most prominent features were the production of lactate and high rates of oxidation of energy substrates by in vitro-matured oocytes.
Similarly, IVP blastocysts produced twice as much lactate
as did embryos generated in vivo. A stress response to suboptimal culture conditions is the most probable explanation
for this high rate of lactate production. The overall low
metabolic activity of immature oocytes and the high individual variability of the metabolic rates of blastocysts may
not allow employment of metabolic indicators as an index
of development potential.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
ACKNOWLEDGMENTS
The authors thank Mr. K.-G. Hadeler and Dr. L. Bungartz for their help
in collection of embryos.
26.
REFERENCES
27.
1. Trounson AO, Pushett D, MacLellon LJ, Lewis J. Current status of
IVM/IVF and embryo culture in humans and farm animals. Theriogenology 1994; 41:57–66.
2. Hasler JF. The current status of oocyte recovery, in vitro embryo production, and embryo transfer in domestic animals with an emphasis
on the bovine. J Anim Sci 1998; 76(suppl 3):52–74.
3. Krisher RL, Bavister BD. Response of oocytes and embryos to the
culture medium. Theriogenology 1998; 49:103–114.
4. Holm P, Callesen H. In vivo versus in vitro produced bovine ova:
similarities and differences relevant for practical application. Reprod
Nutr Dev 1998; 38:579–594.
5. Wrenzycki C, Herrmann D, Carnwath JW, Niemann H. Expression of
the gap junction gene connexin 43 (Cx43) in preimplantation bovine
embryos derived in vitro or in vivo. J Reprod Fertil 1996; 108:17–24.
6. Wrenzycki C, Herrmann D, Carnwath JW, Niemann H. Expression of
RNA from developmentally important genes in preimplantation bovine embryos produced in TCM supplemented with BSA. J Reprod
Fertil 1998; 112:387–398.
7. Farin PW, Farin CE. Transfer of bovine embryos produced in vivo or
in vitro: survival and fetal development. Biol Reprod 1995; 52:676–
682.
8. Kruip TAM, DenDaas JHG. In vitro produced and cloned embryos:
effects on pregnancy, parturition and offspring. Theriogenology 1997;
47:43–52.
9. Rieger D. Relationship between energy metabolism and development
of early mammalian embryos. Theriogenology 1992; 37:75–93.
10. Thompson JG. Comparison between in vivo-derived and in-vitro-produced pre-elongation embryos from domestic ruminants. Reprod Fertil
Dev 1997; 9:341–354.
11. Gardner DK. Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture. Theriogenology 1998; 49:83–102.
12. Barnett DK, Bavister BD. What is the relationship between the metabolism of preimplantation embryos and their developmental competence? Mol Reprod Dev 1996; 43:105–133.
13. Leese HJ. Metabolism of the preimplantation mammalian embryo. In:
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
855
Milligan SR (ed.), Oxford Reviews of Reproductive Biology. Oxford:
Oxford University Press; 1992: 13:35–72.
Bavister BD. Culture of preimplantation embryos: facts and artifacts.
Hum Reprod Update 1995; 1:91–148.
Thompson JG, Partridge RG, Houghton FD, Cox CI, Leese HJ. Oxygen uptake and carbohydrate metabolism by in vitro derived bovine
embryos. J Reprod Fertil 1996; 106:299–306.
Javed MH, Wright RW Jr. Determination of pentose phosphate and
Embden-Meyerhof pathway activities in bovine embryos. Theriogenology 1991; 35:1029–1037.
Rieger D, Loskutoff NM, Betteridge KJ. Developmentally related
changes in the uptake and metabolism of glucose, glutamine and pyruvate by cattle embryos produced in vitro. Reprod Fertil Dev 1992;
4:547–557.
Rose-Hellekant TA, Libersky-Williamson EA, Bavister BD. Energy
substrates and amino acids provided during in vitro maturation of
bovine oocytes alter acquisition of developmental competence. Zygote
1998; 6:285–294.
Partridge RJ, Pullar D, Wrathball AE, Leese HJ. Consumption of amino acids by in vivo and in vitro-derived bovine embryos. Theriogenology 1996; 45:181 (abstract).
Ferguson EM, Leese HJ. Triglyceride content of bovine oocytes and
early embryos. J Reprod Fertil 1999; 116:373–378.
Leibfried L, First NL. Characterization of bovine follicular oocytes
and their ability to mature in vitro. J Anim Sci 1979; 48:76–86.
Eckert J, Niemann H. Effects of platelet-derived growth factor
(PDGF) on the in vitro production of bovine embryos in protein free
media. Theriogenology 1996; 46:307–320.
Parrish JJ, Susko-Parrish JL, Winer MA, First NL. Capacitation of
bovine sperm by heparin. Biol Reprod 1988; 38:1171–1180.
Parrish JJ, Susko-Parrish JL, Leibfried-Rutledge ML, Critser ES, Eyestone WH, First NL. Bovine in vitro fertilization with frozen-thawed
semen. Theriogenology 1986; 25:591–600.
O’Fallon JV, Wright RW Jr. Quantitative determination of the pentose
phosphate pathway in preimplantation mouse embryos. Biol Reprod
1986; 34:58–64.
Khurana NK, Wales RG. Effects of oestradiol and progesterone on
the metabolism of (U-14C)glucose by mouse morulae and early blastocysts in vitro. J Reprod Fertil 1987; 79:267–273.
Lindner GM, Wright RW Jr. Bovine embryo morphology and evaluation. Theriogenology 1983; 20:407–416.
Wales RG. Biochemistry of the developing embryo. J Reprod Fertil
Suppl 1973; 18:117–125.
Snedecor GW, Cochran WG. Statistical Methods. Ames, IA: Iowa
State University Press; 1980: 234–235.
Carlson D, Black DL, Howe GR. Oviduct secretion in the cow. J
Reprod Fertil 1970; 22:549–552.
Parrish JJ, Susko-Parrish JL, Handrow RR, Sims MM, First NL. Capacitation of bovine spermatozoa by oviduct fluid. Biol Reprod 1989;
40:1020–1025.
Rieger D, Grisart B, Semple E, Van Langendonckt A, Betteridge KJ,
Dessy F. Comparison of the effects of oviductal cell co-culture and
oviductal cell-conditioned medium on the development and metabolic
activity of cattle embryos. J Reprod Fertil 1995; 105:91–98.
Leese HJ. The formation and function of oviduct fluid. J Reprod Fertil
1988; 82:843–856.
Wales RG. Measurement of metabolic turnover in single mouse embryos. J Reprod Fertil 1986; 76:717–725.
Wales RG, Whittingham DG, Hardy K, Craft IL. Metabolism of glucose by human embryos. J Reprod Fertil 1987; 79:289–297.
Gardner DK, Lane M, Batt P. Uptake and metabolism of pyruvate and
glucose by individual sheep preattachment embryos developed in
vivo. Mol Reprod Dev 1993; 36:313–319.
Flood MR, Wiebold JL. Glucose metabolism by preimplantation pig
embryos. J Reprod Fertil 1988; 84:7–12.
Telford NA, Watson AJ, Schultz GA. Transition from maternal to
embryonic control in early mammalian development: a comparison of
several species. Mol Reprod Dev 1990; 26:90–100.
Edirisinghe WR, Wales RG, Pike IL. Synthesis and degradation of
labelled glycogen pools in preimplantation mouse embryos during
short periods of in vitro culture. Aust J Biol Sci 1984; 37:137–146.
Wales RG, Hunter J. Participation of glucose in the synthesis of glycoproteins in preimplantation mouse embryos. Reprod Fertil Dev
1990; 2:35–50.
Gardner DK, Leese HJ. The role of glucose and pyruvate transport in
regulating nutrient utilization by preimplantation mouse embryos. Development 1988; 104:423–429.
856
KHURANA AND NIEMANN
42. Hyttel P, Callesen H, Greve T. A comparative ultrastructural study of
in vivo versus in vitro fertilization of bovine oocytes. Anat Embryol
1989; 179:435–442.
43. DeLoos F, Van Maurik P, Van Beneden T, Kruip TAM. Structural
aspects of bovine oocyte maturation in vitro. Mol Reprod Dev 1989;
31:208–214.
44. Rieger D, Loskutoff NM, Betteridge KJ. Developmentally related
changes in the metabolism of glucose and glutamine by cattle embryos
produced and cocultured in vitro. J Reprod Fertil 1992; 195:585–595.
45. Waugh EE, Wales RG. Oxidative utilization of glucose, acetate and
lactate by early preimplantation sheep, mouse and cattle embryos.
Reprod Fertil Dev 1993; 5:123–133.
46. Eckert J, Pugh PA, Thompson JG, Niemann H, Tervit HR. Exogenous
protein affects developmental competence and metabolic activity of
bovine preimplantation embryos in vitro. Reprod Fertil Dev 1998; 10:
327–332.
47. Rieger D, Guay P. Measurements of the metabolism of energy substrates in individual bovine blastocysts. J Reprod Fertil 1988; 83:585–
591.
48. Donnay I, Leese HJ. Embryo metabolism during the expansion of the
bovine blastocyst. Mol Reprod Dev 1999; 53:171–178.
49. Benos DJ, Balaban RS. Energy requirements of the developing mammalian blastocyst for active ion transport. Biol Reprod 1980; 23:941–
947.
50. Krisher RL, Lane M, Bavister BD. Developmental competence and
metabolism of bovine embryos cultured in semi-defined and defined
culture media. Biol Reprod 1999; 60:1345–1352.
51. Edwards LJ, Williams DA, Gardner DK. Intracellular pH of the preimplantation mouse embryo: effects of extracellular pH and weak acids. Mol Reprod Dev 1998; 50:434–442.
52. Thompson JG, Bell ACS, Pugh PA, Tervit HR. Metabolism of pyruvate by pre-elongation sheep embryos and effect of pyruvate and lactate concentrations during in vitro culture. Reprod Fertil Dev 1993; 5:
417–423.
53. Lane M, Gardner DK. Prospective selection of viable mouse embryos
prior to transfer using metabolic rate. Hum Reprod 1996; 11:1975–
1978.
54. Gardner DK, Pawelczynski M, Trounson AO. Nutrient uptake and
utilization can be used to select viable Day 7 bovine blastocysts after
cryopreservation. Mol Reprod Dev 1996; 44:472–475.
55. Leese HJ, Barton AM. Pyruvate and glucose uptake by mouse ova
and preimplantation embryos. J Reprod Fertil 1984; 72:9–13.
56. Martin KL, Leese HJ. Role of glucose in mouse preimplantation embryo development. Mol Reprod Dev 1995; 40:436–443.
57. Wales RG, Whittingham DG. The metabolism of specifically labelled
lactate and pyruvate by two cell mouse embryos. J Reprod Fertil 1973;
33:207–222.
58. Cross PC, Brinster RL. The sensitivity of one-cell mouse embryos to
pyruvate and lactate. Exp Cell Res 1973; 77:57–62.
59. Takahashi Y, First NL. In vitro development of bovine one-cell embryos: influence of glucose, lactate, pyruvate, amino acids and vitamins. Theriogenology 1992; 37:963–978.
60. Boone WR, Dickey JF, Luszcz LJ, Dantzler JR, Hill JR Jr. Culture of
ovine and bovine ova. J Anim Sci 1978; 47:908–913.
61. Leese HJ, Barton AM. Production of pyruvate by isolated mouse cumulus cells. J Exp Zool 1985; 234:231–236.
62. Susko-Parrish J, Aktas H, Leibfried-Rutledge ML. The effect of energy substrates during maturation on the fertilization and development
of bovine oocytes. Theriogenology 1992; 37:305 (abstract).