Download Investigation of respiration of individual bovine embryos produced in

Human Reproduction Vol.22, No.2 pp. 558–566, 2007
doi:10.1093/humrep/del404
Advance Access publication November 24, 2006.
Investigation of respiration of individual bovine embryos
produced in vivo and in vitro and correlation with viability
following transfer
A.S.Lopes1,2,5, S.E.Madsen3, N.B.Ramsing4, P.Løvendahl1, T.Greve2 and H.Callesen1
1
Department of Genetics and Biotechnology, Danish Institute of Agricultural Sciences, Tjele, 2Department of Large Animal Sciences,
The Royal Veterinary and Agricultural University, Frederiksberg C, 3Trans-Embryo Genetics, Brædstrup and 4Unisense FertiliTech A/S,
Aarhus C, Denmark
5
To whom correspondence should be addressed at: Research Centre for Reproductive Health, Department of Obstetrics and Gynaecology,
Faculty of Health Sciences, The University of Adelaide, 2nd Floor Medical School South, Frome Road, SA 5005 Adelaide, Australia.
E-mail: [email protected]
BACKGROUND: Quantification of oxygen consumption by individual preimplantation embryos has the potential to
improve embryo selection. This study investigated whether respiration rates of individual embryos are useful indicators of embryo viability. The effect of the Nanorespirometer on embryo viability was also evaluated. METHODS: The
respiration rates of individual day 7 bovine in vivo- (n = 44) and in vitro-produced (n = 156) embryos were measured
using the Nanorespirometer. In vivo-produced embryos were individually transferred to recipients. RESULTS: The
respiration rates of in vivo-produced embryos increased with increasing morphological quality and stage of development (P < 0.05). Pregnancy rates on days 35 and 60 were 65 and 60%, respectively. The mean respiration rate did not
differ significantly between embryos producing and not producing a pregnancy, but the transfer of embryos with respiration rates <0.78 nl/h, between 0.78 and 1.10 nl/h, and >1.10 nl/h resulted in 48, 100 and 25% pregnancy rate,
respectively. The mean respiration rate of in vitro-produced embryos was higher than that of in vivo-produced
embryos because of differences in the morphological quality and stage of development. CONCLUSION: The
Nanorespirometer does not adversely influence embryo viability, but the sample size was too small to confirm the
significance of the correlation observed between respiration rates and viability.
Key words: bovine embryo/embryo quality/Nanorespirometer/oxygen/respiration
Introduction
The ability to accurately and rapidly assess the quality and
viability of individual preimplantation embryos has been considered highly important for many years. In animal embryo
transfer units, the selection of those embryos that are most
likely to implant successfully will obviously assist in reducing
time and financial losses associated with keeping recipients for
long periods. Furthermore, the commercial value of the
selected embryos would be increased (Donnay, 2002). In
human medicine, an increased chance of pregnancy by transfer
of high-quality embryos would improve the success rates of
IVF treatments. High success rates will likely cause a reduction in the number of transferred embryos by treatment cycle,
thereby reducing the risk of multiple births, which are often
associated with obstetric and neonatal complications (Neuber
et al., 2003; Bergh, 2005).
Morphological evaluation using a standard stereomicroscope is still the most widely used technique for rapid routine
assessment of embryo quality and viability before transfer
(Boiso et al., 2002; Merton, 2002). Although a direct relationship between morphological quality [using the International
Embryo Transfer Society (IETS) evaluation system] and
embryo viability measured in terms of pregnancy rates has
been demonstrated previously (Hasler, 1998), the subjectivity
of such evaluation is widely recognized (Farin et al., 1995;
Overström, 1996; Farin et al., 1999). A combination of several
quality parameters will most likely result in improved embryo
selection criteria and in a better distinction between viable and
less- or non-viable embryos.
Oxygen consumption is a potential quality parameter in
itself, as it provides a valuable indication of the overall metabolic activity of a single embryo (Leese, 2003). Consequently,
the quantification of oxygen consumption by single embryos
combined with a morphological assessment is likely to
improve the selection of embryos by facilitating the evaluation
of their developmental competence. In fact, correlation
between oxygen consumption and morphological quality has
previously been demonstrated for in vitro-produced embryos
558 © The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Respiration and viability of in vivo and in vitro embryos
(Shiku et al., 2001; Lopes et al., 2005a) but not for in vivoproduced bovine embryos (Overström, 1992).
Previous studies reported important differences at the morphological, ultrastructural, physiological, transcriptional and
metabolic levels between bovine embryos produced in vivo and
those produced in vitro (Holm et al., 1998; Khurana and
Niemann, 2000; Bertolini et al., 2002; Lazzari et al., 2002;
Lonergan et al., 2003). Morphological differences between
these two types of embryos are often attributed to the way
embryo age is determined. In vivo recoveries are performed 7
days after estrus (day 0) and thus ∼6 days after fertilization,
whereas the age of in vitro-produced embryos is determined
from the time of IVF (day 0). Therefore, in vitro-produced
embryos are at least 1 day older than the corresponding in vivo
embryos (Hasler et al., 1995). This difference is reflected in
the stage of development of the respective embryos, where
early blastocyst and expanded blastocyst are the most common
stages for day 7 in vivo- and in vitro-produced embryos,
respectively (Merton, 2002). Moreover, the distinct culture
conditions in which both embryo types develop and the presence of certain media components also influence the observed
differences (Yoshioka et al., 1997, 2000; Peippo et al., 2001;
Holm et al., 2002).
Previous studies have provided evidence that embryonic respiration rates are directly correlated with viability following
embryo transfer. Overström et al. (1989, 1992) measured the
oxygen consumption of single mouse and bovine in vivoproduced blastocysts before transfer, and the highest survival
rates were observed in the group with the highest respiration
rates. Trimarchi et al. (2000) reported the birth of Aguti
(B6C6F1) mouse pups, resulting from the transfer of day 4 in
vitro-cultured blastocysts previously measured with the selfreferencing microelectrode technique (Smith, 1995; Land et
al., 1999). Nevertheless, all the previous studies comprised a
pooling of embryos before transfer which makes it impossible
to identify which embryo (and corresponding respiration rate)
produces the observed pregnancy, making the interpretation of
these results difficult.
We have earlier described and validated the nanorespirometer system (Nanorespirometer), which is a non-invasive and
highly sensitive technology developed for the measurement of
individual embryo respiration rates (Lopes et al., 2005a). However, that study did not assess embryo viability directly, as
embryo transfer to recipients following the measurements was
not performed. Nor was the effect of the measurement procedure itself on the subsequent viability of the embryos properly
evaluated.
In the present study, we determined the respiration rates of
day 7 in vivo-produced bovine embryos under the conditions
normally used for embryo transfer. We have investigated
whether embryos with higher respiration rates yield higher
pregnancy rates and consequently whether the measurement of
respiration rate will be a useful indicator of subsequent embryo
viability. We have further evaluated the relationship between
respiration rates and morphological quality, the stage of development and the diameter of day 7 embryos produced in vivo.
Moreover, we have demonstrated that the oxygen measurements performed with the Nanorespirometer did not influence
the subsequent viability of the embryo. Finally, we compared the
respiration rates of day 7 bovine in vivo- and in vitro-produced
embryos. Preliminary results of this work have been presented
earlier (Lopes et al., 2005b).
Materials and methods
Day 7 in vivo-produced embryos
In vivo embryo production
A total of 11 Danish Holstein–Friesian heifers (∼14–16 months) and
cows (∼30 months) from a single herd (milk production of 12 500 kg
per cow) were monitored daily for detection of estrous signs
(estrus = day 0) and subsequently superovulated between days 9 and
13 of the estrous cycle. Superovulation was induced with a total of
760 IU of FSH + 760 IU of LH (Pluset®, Laboratorios Calier S.A.,
Barcelona, Spain), administered i.m. in decreasing doses over 4 days
at 12 h intervals. Estrus was induced by a single i.m. injection of 3 ml
(75 μg/ml) D-cloprostenol (Genestran Vet, Scanvet Animal Health A/
S, Fredensborg, Denmark), a prostaglandin analogue, administered in
the morning of the fourth day of superovulation. Estrus was detected
∼48 h later (estrus was monitored twice daily), and artificial insemination
(AI) was carried out 12 h after the beginning of standing estrus with
frozen–thawed semen from Holstein–Friesian bulls of proven fertility
(10 × 106 sperm cells/straw). AI was normally performed once in heifers but twice in cows, 12 h apart and with semen from the same bull.
On day 7 of the estrous cycle, bovine in vivo-produced embryos
(average of 4.55 embryos/donor; n = 50) were non-surgically recovered
by flushing the uterine horns of the donors with phosphate-buffered
saline (PBS; Danish Veterinary Laboratory, Frederiksberg, Denmark)
supplemented with 10% cattle serum (CS; Danish Veterinary Laboratory)
and maintained at room temperature as previously described (Greve,
1981). The recovered embryos were washed in PBS and classified
according to the stage of development (compact morula or blastocyst)
and morphological quality (quality I, II, III or IV) by an experienced
practitioner using a stereomicroscope with a ×50 magnification and
based on the definitions specified in the IETS Manual (Wright J,
1998). Only transferable in vivo embryos, i.e. embryos likely to produce
a pregnancy (quality I, II and III; n = 47), were used in this study, and
thus, quality IV embryos (dead, degenerating or 1-cell embryos; n = 3)
were excluded.
Subsequently, embryos in PBS plus 10% CS were individually
loaded into 0.25-ml sterilized straws. The straws containing the
embryos were placed in a portable incubator (37.5°C) and transported
to the laboratory, 1 h away from the embryo transfer facilities.
Digital records and diameter assessment
Upon arrival to the laboratory, the in vivo-produced embryos were
unloaded from the straws into a 4-well dish, and their outer diameter
was measured under a stereomicroscope (SMZ800; Nikon, Tokyo,
Japan) using an ocular micrometer eyepiece. Digital images of each
embryo were acquired using a digital still camera (GC-X3E; JVL,
Yokohama, Japan) mounted on an inverted optical microscope (TDM,
Nikon), with a thermal control microscope stage (CO 102; Linkam
Scientific Instruments Ltd, Tadworth, Surrey, UK).
Measurement of oxygen consumption
The Nanorespirometer (Unisense A/S, Aarhus, Denmark) used to
measure the oxygen consumption of single embryos has previously
been described (Lopes et al., 2005a). Briefly, the rosette with the
embryos was submerged into a beaker (80 ml) containing the culture
medium, which was composed of synthetic oviduct fluid (SOF)
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A.S.Lopes et al.
medium including amino acids, citrate and inositol (SOFaaci; Holm et al.
1999) supplemented with antibiotics (gentamycin sulphate, 50 μg/ml)
and 5% CS. The medium was maintained in a semi-closed system at
38.5°C and under a constant flow of humidified 5% CO2 in 19.5% O2.
Two rounds of measurements were performed to determine the oxygen
consumption of each embryo twice. Two empty glass capillaries
served as reference measurement without respiratory activity (= control).
Due to technical problems, accurate respiration rates were only determined in 44 of the 47 embryos. Respiration rates, determined twice
with a 43 ± 6 min interval between measurements, were similar, as the
regression line between the first and second determination was close
to unity [y2 = 0.93 (±0.06) y1 + 0.09 (±0.05); R2 = 0.89, P < 0.001; Proc
Reg, SAS]. Furthermore, the background noise of the nanorespirometer system was negligible (P > 0.05, two-tailed Student’s t-test), with
the empty control capillaries showing an apparent oxygen consumption of 0.007 ± 0.005 nl/h (n = 18), which was not significantly different from zero (P > 0.1). These results confirm the accuracy and
consistency of the measurements performed by the Nanorespirometer,
and hence, the background noise on each day of measurement was not
subtracted from the calculated embryonic respiration rates.
Embryo processing after the measurements
After the measurements, the embryos were individually transferred to
single wells of a 4-well dish (Nunc, Roskilde, Denmark) containing
SOFaaci culture medium supplemented with antibiotics and 5% CS.
After this, embryos were moved to a 4-well dish containing PBS plus
10% CS and individually loaded into 0.25-ml straws, which were transported to the embryo transfer facilities in a portable incubator maintained at 37.5°C.
Non-surgical embryo transfer
Forty-three Danish Holstein–Friesian heifers (∼14–18 months) were
used as recipients. Recipients were pre-selected based on normal
estrous activities and synchronized by i.m. injection of 2 ml (75 μg/
ml) D-cloprostenol ∼12 h before the corresponding injection in the
donor animals. Only recipients with detected signs of estrus and with
a palpable corpus luteum at the time of embryo transfer were used.
Single fresh day 7 bovine embryos produced in vivo (n = 43) were
non-surgically transferred to randomly assigned synchronized recipient
heifers on day 7 (±12 h) after estrus. Pregnancy was diagnosed by palpation per rectum on day 35 and confirmed by ultrasonography on day
60 after estrus. The annual pregnancy rate observed in the herd where
this study was carried out is ∼65%, following the transfer of an average
of 600 fresh embryos/year (Madsen SE, personal communication).
The present study was considered to be part of the routine work performed in relation to embryo transfer in practice. Embryos were
flushed in the morning, loaded into straws, transported to and from the
laboratory and transferred back to recipients ∼8 h later. As oxygen
measurements were non-invasive and expected not to interfere with
the subsequent viability of the embryos, permission from the Danish
experimental committee was not required for this study.
Day 7 in vitro-produced embryos
In vitro embryo production
The method used for in vitro production has been reported elsewhere
(Holm et al., 1999). In brief, bovine immature cumulus–oocyte complexes were aspirated from slaughterhouse-derived ovaries, selected
and matured for 24 h in 4-well dishes. Each well contained 400 μl of
bicarbonate-buffered TCM-199 medium (Gibco BRL, Paisley, UK)
supplemented with 15% CS, 10 IU/ml of equine CG and 5 IU/ml of
HCG (Suigonan Vet; Intervet Scandinavia, Skovlunde, Denmark).
The oocytes were matured under mineral oil at 38.5°C in 5% CO2 in
560
humidified air. Fertilization was performed in modified Tyrode’s
medium (Parrish et al., 1986) using frozen–thawed, Percoll-selected
sperm, which was pre-tested for in vitro production and different from
the semen used for AI of the superovulated donors. After 22 h, cumulus cells were removed by vortexing, and presumptive zygotes were
transferred to 400 μl of culture medium and incubated at 38.5°C in 5%
CO2, 5% O2, 90% N2 atmosphere with 100% humidity. The embryos
were produced in vitro over 18 independent replicates by using ∼50
oocytes/replicate. Mean cleavage and blastocyst rates were 78.4 and
41.5%, respectively.
In vitro embryo selection, evaluation and oxygen measurements
A total of 156 in vitro-produced embryos were measured during 18
experimental days. On each of the 18 experimental days, 5–10
embryos at the blastocyst stage (day 7 of culture) were randomly
selected to represent all morphological qualities and individually
transferred to one well of a 4-well dish containing culture medium.
Embryos were classified according to the stage of development:
blastocysts (n = 17), expanded blastocysts (n = 123; 27 early
expanded, 68 expanded or 28 late expanded), collapsed blastocysts
(n = 10) or hatching blastocysts (n = 6). Classification into four morphological qualities (quality I, II, III or IV), digital records, diameter
assessment as well as oxygen measurements were performed as previously described for in vivo-produced embryos.
Statistical analysis
Analysis of in vivo-produced embryos
Pregnancy status on day 35 was analysed using a linear mixed model
(Mixed Procedure, SAS). The models included the stage of embryonic
development and morphological quality as fixed effects and donor as
random effect. The random effect of donor was tested by a chi-square
analysis, before being introduced into the random part of the model.
Respiration rates were tested for the distributional properties, and
deviation from normal distribution was not significant (Univariate
Procedure, SAS). Respiration rates of day 7 embryos were analysed
using a linear mixed model (Mixed Procedure, SAS), including the
stage of embryonic development, diameter and morphological quality
as fixed effects and donor as random effect. Least squares means
(LSMs) for treatment factors were produced by this model.
The effect of embryonic respiration rate on pregnancy was tested
using a linear mixed model (Mixed Procedure, SAS) and included
pregnancy status on day 35 or 60 as fixed effect and donor as random
effect. Using an extended model, we subsequently introduced the factors morphological quality, stage of embryonic development and
embryo diameter as fixed effects. Mean embryo respiration rate for
cows subsequently diagnosed pregnant or non-pregnant were analysed
by the two-tailed Student’s t-test.
The embryos were divided into two and three even-sized categories
according to respiration rates, and differences in rates according to the
subsequent pregnancy status were estimated by chi-square analysis
(Freq Procedure, SAS). Embryos were subsequently divided into three
categories where the threshold values (0.78 and 1.10 nl/h) were
defined based on the pregnancy status on day 35. The degree of significance of these three categories was also tested using the chi-square
analysis (Freq Procedure, SAS).
Analysis of in vivo- and in vitro-produced embryos (combined)
Respiration rates of in vivo- and in vitro-produced embryos were
tested jointly for the distributional properties (Univariate Procedure,
SAS). Data were subsequently transformed using a square-root transformation, as this was effective in stabilizing variance and obtaining
approximate normal distribution of data and residuals.
Respiration and viability of in vivo and in vitro embryos
Following transformation, the respiration rates of in vivo- and
in vitro-produced embryos were evaluated by a linear mixed model
(Mixed Procedure, SAS), including the following fixed factors:
embryo morphological quality, the stage of development, type (in
vivo versus in vitro) and all interactions between the type, morphological quality and stage of development. Effects regarded as nonsignificant were subsequently removed from the analysis. The significance of the results was assessed on the transformed data, but
final results (LSM ± SEM) were generated by back transforming
(√x) to the original scale.
All statistical procedures were performed using the computational
software of SAS (SAS Institute, 1999). The level of significance was
P < 0.05, unless otherwise indicated.
Results
Respiration rates of in vivo-produced embryos
The overall mean respiration rate of day 7 in vivo-produced
embryos was 0.82 ± 0.05 nl/h (n = 44). The mean respiration
rate decreased with decreasing morphological quality, being
1.01 ± 0.06a (n = 18), 0.89 ± 0.07a (n = 14) and 0.73 ± 0.08b nl/h
(n = 12) for quality I, II and III embryos, respectively (a,b: P < 0.05;
values with different superscripts differ significantly; Figure 1).
Blastocysts had a 35% higher oxygen consumption than compact morulae (P < 0.01). On the contrary, the respiration rates
of in vivo-produced embryos were not related to embryo
diameter.
Embryo viability following transfer of in vivo-produced
embryos
Pregnancy rates following the transfer of pre-measured in vivoproduced embryos were 65% (28/43) and 60% (26/43), on
days 35 and 60 post-estrus, respectively. This is in accordance
with the rates routinely observed following embryo transfer in
the herd where this study was carried out. Of the blastocysts
transferred, 50% (3/6) produced a pregnancy on days 35 and
2.10
a
Respiration rate (nl/h)
1.80
a
1.50
b
1.20
0.90
0.60
60 as opposed to 68% (25/37) and 62% (23/37) pregnancy
rates observed on days 35 and 60 for the compact morula stage,
respectively. After the transfer of quality I, II and III embryos,
the associated pregnancy rates on day 35 were 75, 57 and 62%,
respectively. Neither the morphological quality nor the stage of
embryonic development influenced pregnancy rates on days 35
and 60. A total of 26 normal, viable calves were born following
the transfers.
The mean respiration rate for embryos producing a pregnancy was slightly higher but not significantly different from
the mean respiration rate of embryos that did not produce a
pregnancy (0.79 ± 0.04 versus 0.73 ± 0.07 nl/h; P > 0.05).
Neither was the variance of the measured respiration rates significantly different in embryos producing a pregnancy versus
those not producing a pregnancy on day 35. Pregnancy on days
35 and 60 was not influenced by respiration rates of day 7 in vivoproduced embryos (P < 0.05). When embryos were divided
into two even-sized groups according to their respiration rates,
the probability of producing a pregnancy was not significantly
different between embryos with high and low respiration rates
(P = 0.33; Table I). A similar result was found for three evensized groups.
The cumulative frequency curves for respiration rates of
embryos associated with pregnant and non-pregnant animals
are shown in Figure 2. The pink curve for respiration rate of
embryos associated with pregnancy closely followed the sigmoid pattern expected for a normal distribution. However,
the blue curve—showing respiration rates for embryos that
did not induce pregnancy in their recipient—was different.
Embryos subsequently associated with non-pregnant animals
seemed to show both very low and very high respiration rates.
However, all of the 13 embryos with a respiration rate
between 0.78 and 1.10 nl/h resulted in a pregnancy on day 35.
The blue curve in Figure 2 thus contains a conspicuous vertical line segment from 0.78 to 1.10 nl/h. This is in agreement
with the hypothesis that a normal range of respiration rates
exists and that embryos with respiration rates below or above
this range have a reduced probability of generating a pregnancy. Thus, three biologically meaningful categories are
likely to exist, and the selection of boundaries for the three
categories is therefore reasonable. We defined a low, a
medium and a high respiratory category, which included
embryos with respiration rates <0.78 nl/h, between 0.78 and
1.10 nl/h and >1.10 nl/h, respectively. The pregnancy status
of the recipients following transfer of embryos of distinct respiratory categories is summarized in Table II, and no statistically significant differences among the three categories were
detected.
0.30
0.00
I
II
III
(n = 18)
(n = 14)
(n = 12)
Morphological quality
Figure 1. Respiration rates (nl/h) of day 7 in vivo-produced embryos
according to the morphological quality (a,b: P < 0.05; n = 44). The
horizontal bars in the figure represent the mean respiration rates for
each morphological quality.
Table I. Pregnancy status according to respiratory category (high versus low)
of bovine in vivo-produced embryos
Respiratory category
Pregnant
Non-pregnant
High (>0.75 nl/h)
Low (<0.75 nl/h)
70% (n = 14)
45% (n = 11)
30% (n = 6)
55% (n = 9)
Threshold limit of 0.75 nl/h was chosen to generate two equal-sized groups.
561
A.S.Lopes et al.
100
Cumulative frequency (%)
80
60
40
Non-pregnant
Pregnant
20
0
0
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
Respiration rate (nl/h)
Figure 2. Cumulative frequency (%) curves of the respiration rates (nl/h) of day 7 in vivo-produced embryos retrospectively categorized by the
pregnancy status of the recipient. The pink and blue curves represent pregnant (n = 28) and non-pregnant animals (n = 15), respectively. The
curves indicate the percentage of the given pool of embryos that had a respiration rate less than or equal to a given value (e.g. from the blue curve,
it can be observed that 80% of the embryos that did not produce a pregnancy had a respiration rate <0.8 nl/h).
Table II. Pregnancy status according to respiratory category (high versus
medium versus low) of bovine in vivo-produced embryos
Respiratory category
Pregnant
Non-pregnant
High (>1.10 nl/h)
Medium (0.78–1.10 nl/h)
Low (<0.78 nl/h)
25% (n = 1)
100% (n = 13)
48% (n = 11)
75% (n = 3)
0% (n = 0)
52% (n = 12)
Table III. Respiration rates (nl/h; mean ± SEM, back-transformed from
square root) of in vivo- and in vitro-produced embryos according to the
morphological quality
Morphological quality
Threshold limits (0.78 and 1.1 nl/h) were chosen to reflect the apparent
pattern shown in the cumulative frequency curves from Figure 2.
Comparison of respiration rates of in vivo- and
in vitro-produced embryos
The overall mean respiration rate of day 7 in vitro-produced
embryos was 1.35 ± 0.06 nl/h (n = 156), which was considerably higher than the 0.82 ± 0.05 nl/h (n = 44) observed for
day 7 in vivo-produced embryos. After square-root transformation, the corresponding back-transformed values were
1.26 ± 0.05 and 0.79 ± 0.05 nl/h, respectively. Regardless
of the apparent difference in mean respiration rates between
in vivo- and in vitro-produced embryos, embryo type was
found not to influence embryonic oxygen consumption.
Instead, the stage of embryonic development and morphological quality within type significantly affected respiration
rates, with embryos of better morphological quality and at
more advanced stages of development showing higher respiration rates (Table III; Figure 3).
562
I
II
III
IV
Embryo type
In vivo
In vitro
n
Respiration rate
n
Respiration rate
18
14
12
–
1.26 ± 0.14a,x
1.24 ± 0.20a,x
0.96 ± 0.19b*,x
–
38
52
47
19
1.70 ± 0.11a,y
1.27 ± 0.08b,x
1.15 ± 0.08b,x
0.79 ± 0.11c
a,b,c,x,y
: P < 0.05, b*: P = 0.07; values with different superscripts within the
same column (a, b, b*, c) and within the same row (x, y) vary significantly.
Discussion
For the first time, the respiration rates of individual day 7
in vivo-produced embryos were accurately measured with the
Nanorespirometer and the measured embryos subsequently
individually transferred fresh to synchronized recipients.
Hence, this study allowed us to compare the respiration rate
and viability of a single embryo following transfer.
The Nanorespirometer was accurate and consistent in measuring individual embryonic respiration rates, as demonstrated
by the high correlation between the first and the second measurement of the same embryo as well as by the negligible background noise of the empty control capillaries (which is likely
Respiration and viability of in vivo and in vitro embryos
3.00
d
Respiration rate (nl/h)
2.50
2.00
c
b
1.50
a,b
a,b
a
1.00
a
0.50
0.00
CM
B
EXB
XB
LXB
(n = 38)
(n = 19)
(n = 29)
(n = 75)
(n = 31)
CB
(n = 10)
HB
(n = 7)
Stage of embryonic development
Figure 3. Respiration rates (mean ± SEM; nl/h) of individual embryos according to the stage of embryonic development (in vivo and in vitro
embryos analysed together; back-transformed data from square-root transformation; CM, compact morula; B, blastocyst; EXB, early expanded
blastocyst; XB, expanded blastocyst; LXB, late expanded blastocyst; CB, collapsed blastocyst; HB, hatching blastocyst; a,b,c,d: P < 0.05, values
with different superscripts vary significantly).
attributed to the extremely small oxygen consumption by the
electrochemical microsensor during the measurements). Furthermore, this technology did not interfere with embryo viability, as pregnancy rates resulting from the transfer of
embryos after measurements of respiration rate were in
agreement with those published by others working with similar systems (Hasler, 1998; Farin et al., 1999) and with the
results routinely achieved following embryo transfer in the
herd where this study was carried out. In fact, the pregnancy
rates obtained in this study following measurements of
embryo respiration rate were slightly higher than the rates
normally observed for each morphological quality. These
normally range between 65 and 70%, 50–70% and 30–50%
following the transfer of quality I, II and III embryos, respectively
(Madsen SE, personal communication).
The respiration rates obtained in this study for in vivoproduced embryos are considerably different from those documented by Thompson et al. (1995) and Overström (1996), as
they were 0.7 ± 0.1 and 1.47 ± 0.44 nl/h, respectively. The
observed difference might be attributed to differences in
methodology, techniques and approaches, and hence, direct comparisons may not be possible. The respiration rates of in vivoproduced embryos decreased with decreasing morphological
quality, indicating that morphological differences are at least
partly reflected at the level of oxygen consumption. Nevertheless, the respiration rates of quality I and II embryos were not
significantly different. A similar result was found by Overström (1992), who did not observe any differences in oxygen
consumption among embryos of different morphological qualities. The significant but limited correlation between embryo
morphological quality and respiration rate observed in this
study and in a previous study using in vitro-produced embryos
(Lopes et al., 2005a) suggests that combining measurements of
oxygen consumption with assessment of morphological quality
might improve embryo selection.
As opposed to our previous observations for in vitroproduced embryos (Lopes et al., 2005a), we could not detect a
significant correlation between embryo diameter and respiration rate for in vivo-produced embryos. Correlation between
embryo diameter and oxygen consumption has been reported
earlier (Shiku et al., 2001). The lack of correlation between
respiration and diameter observed in this study could be related
to the fact that in vivo-produced embryos are a more homogeneous population with a more even size distribution as compared with in vitro-produced embryos (Holm et al., 1998).
Another explanation could be that very few in vivo-produced
embryos were at the blastocyst stage (n = 9), where the diameter and associated respiration rate increase considerably.
The higher respiration rates observed for blastocysts as compared with morulae have previously been described (Thompson
et al., 1996; Harvey et al., 2002, 2004; Abe et al., 2005) and
seem to be associated with a higher energy demand for protein
synthesis and activity of the plasma membrane Na+-K+dependent ATPase during blastocoel formation. However, this
association can also reflect the difference in cell number
between these two stages of embryonic development, as the
number of cells of an embryo appears to be correlated with
oxygen consumption (Donnay, 2002).
No significant correlation between individual respiration
rates and subsequent viability of the embryos was demonstrated in our study, where embryo viability was assessed by
evaluating the pregnancy status of the recipients receiving the
pre-measured embryos. No single threshold value beyond
which more pregnancies could be observed was established.
563
A.S.Lopes et al.
However, such a simple interpretation is in most cases inadequate for crucial physiological parameters like respiration. For
such key parameters, we frequently observe an optimal range,
and adverse conditions as well as a poor prognosis are likely to
occur when the observed parameter is below or above this
range.
The cumulative frequency distribution of the pregnancies
according to respiration rate in Figure 2 (and notably the observation that three out of four embryos with the highest respiration were associated with non-pregnancy) led us to speculate
that both very high and very low levels of embryonic respiration rates could be associated with non-pregnancy, whereas the
best chance for pregnancy would be found among embryos
with middle values of respiration. This interpretation of the
data is reflected in the two chosen threshold limits that form
the basis for Table II. In this case, very low respiration rates
would correlate with adverse alterations in oxidative phosphorylation and in other oxygen-related processes, and consequently with reduced embryo viability and increased mortality.
Moreover, a very high respiration rate could represent a manifestation of metabolic stress, which is associated with reduced
embryo viability. The fact that two of the three embryos that
did not produce a pregnancy in the high respiratory category
(Table II) were at the blastocyst stage suggests that the lower
pregnancy rates obtained in this group could also be associated
with the reduced capacity of fast-cleaving embryos to produce
a pregnancy, as previously reported (Ziebe et al., 1997). Nevertheless, none of the previous hypotheses could be statistically
verified with the limited number of embryos investigated in
this study. Previous attempts to correlate respiration rate and
embryo viability suggested that higher survival rates of mouse
and bovine embryos were achieved with the highest respiration
rates (Overström et al., 1989, 1992). Yet, in these studies, the
survival rates were evaluated after the transfer of groups of,
and not individual, embryos.
Various factors can account for the observed difference in
respiration rates even for in vivo-produced embryos that subsequently produced a pregnancy. Among these factors is the precise time of fertilization, which cannot be accurately
determined, particularly when cows were used as donors where
insemination was performed twice. Indeed, embryos were collected either at the compact morula or at the blastocyst stage,
and 60% of the donors yielding compact morulae with respiration rates <0.78 nl/h and resulting in pregnancy were cows reinseminated 12 h after the first insemination. The possibility of
these embryos being only produced after the second insemination, thus being substantially younger, could partly explain the
successful implantation of embryos with low respiration rates.
Although most of the embryos were collected at the compact
morula stage, the degree of compaction possibly differed, and
this could also partly explain the surprisingly low respiration
rates of some of the embryos that were subsequently associated
with a pregnancy.
The recipients are obviously a potential confounding factor,
when the evaluation of embryo viability is based solely on the
subsequent pregnancy status. Pregnancy rates are highly influenced by the quality of the recipients. In fact, the ability of the
recipients to produce and maintain a pregnancy following
564
embryo transfer has been regarded as more important than the
morphological quality of the embryos transferred (McMillan,
1998).
Moreover, as oxygen consumption correlates with the
number of cells of an embryo (Kaidi et al., 2001) and the cell
number does not apparently reflect the viability of an embryo
after transfer (Donnay, 2002), it is likely that some bias has
been introduced. The limited number of embryos transferred in
this study has likewise limited the statistical significance of the
observations, and therefore, further investigations involving a
higher number of individual transfers are required to clarify the
nature of the relationship between embryonic respiration and
viability.
Overall, the respiration rates of in vivo-produced embryos
were not significantly lower than those of day 7 in vitroproduced embryos. The apparent difference in mean respiration rates between in vivo- and in vitro-produced embryos was
mostly explained by the different ratio of embryos of each
morphological quality within embryo type. Differences
between these two embryo types were only observed for quality
I embryos and are likely associated with differences in the
stage of development. These results are somewhat surprising,
as in vivo-produced embryos have previously been described
as being different from their in vitro counterparts also at the
metabolic level (Partridge and Leese, 1996; Khurana and
Niemann, 2000). Furthermore, it could be expected that the
culture conditions and media components such as glucose,
amino acids, pyruvate and serum, which are known to affect
embryo metabolism (Eckert et al., 1998; Lane and Gardner,
2000; Donnay, 2002; Abe et al., 2005), would have had quantifiable effects on the resulting embryos, which would have been
detected during the respiration measurements.
Consistent with previous observations for in vivo-produced
embryos, when both embryo types were jointly analysed, a
strong correlation between respiration rate and the stage of
embryonic development could be established. Such correlation
has previously been described (Thompson et al., 1996; Donnay
and Leese, 1999; Abe et al., 2005). Moreover, in line with previously reported data (Thompson et al., 1995; Sturmey and
Leese, 2003), reduced levels of embryo respiration were
observed after maximum expansion, i.e. at the collapsing stage
before hatching. The reduced respiration rate of the collapsed
embryos has earlier been explained by the reduced activity of
Na+-K+-dependent ATPase after complete formation of the
blastocoel. One could also speculate that embryos enter a stage
of ‘temporary quietness’ before the strong metabolic outburst
occurs, at the time of hatching.
The presented experiment allows us to conclude that measuring the respiration rates of individual bovine embryos using
the Nanorespirometer did not influence their subsequent viability, as embryos developed into viable fetuses at rates considered normal under farm conditions and all calves born were
normal and viable. As no statistically validated correlation
between respiration rate and pregnancy status could be demonstrated, it is possible that the use of this equipment will not
help in identifying the most viable in vivo-produced embryos
just by measuring the respiration rates, both because in vivoproduced embryos are known to be a more homogeneous
Respiration and viability of in vivo and in vitro embryos
population and because confounding factors such as the quality of the recipients seem to play a major role in the pregnancy
outcome. On the contrary, it is possible that the use of this
technology can bring new possibilities and prove beneficial in
improving selection of in vitro-produced embryos, especially
in the human field where the selection of proper recipients is
obviously not an issue.
Acknowledgements
The authors thank Anette Pedersen, Klaus Villemoes, Lars Hauer
Larsen, Lars Borregard Pedersen and Ruth Kristensen for their technical assistance. Dr P. Horn is appreciated for critically reviewing the
manuscript. Novo Nordisk Foundation financially supported the
acquisition of the nanorespirometer technology and the Danish Directorate for Food, Fisheries and Agri Business (TFH03-8) the embryo
work. Ana Sofia Lopes is supported by a grant from the Foundation
for Science and Technology (FCT), Ministry of Science and Technology, Portugal (SFRH/BD/8486/2002).
Conflict of interest
Niels Ramsing holds shares of Unisense FertiliTech A/S.
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Submitted on June 24, 2006; resubmitted on August 19, 2006; accepted on
September 19, 2006