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Supplementation of culture medium with L-carnitine
improves the development and cryotolerance of
in vitro-produced porcine embryos
J. L. Lowe
A
,L. K. Bartolac
A
,R. Bathgate
B
and C. G. Grupen
A
,
C
A
Faculty of Veterinary Science, The University of Sydney, 425 Werombi Road, Camden,
NSW 2570, Australia.
B
Faculty of Veterinary Science, The University of Sydney, Regimental Drive,
NSW 2006, Australia.
C
Corresponding author. Email: christopher.grupen@sydney.edu.au
Abstract. Porcine oocytes and embryos contain substantial amounts of lipid, with little known regarding its metabolic
role during development. This study investigated the role of lipid metabolism and the interaction between carbohydrate
and lipid substrates in porcine embryos. Following in vitro fertilisation, presumptive zygotes were transferred to culture
medium supplemented with L-carnitine, a co-factor required for the metabolism of fatty acids. In porcine zygote medium-
3 (PZM-3), which contains pyruvate and lactate, 3 mM L-carnitine was the only dose that improved cleavage rates
compared with the control. In the absence of carbohydrates, all doses of L-carnitine from 1.5 to 12 mM increased cleavage
rates compared with the control. Culture in a PZM-3-based sequential media system (Days 0–3: pyruvate and lactate; Days
4–7: glucose) significantly increased blastocyst cell numbers compared with culture in standard PZM-3. Supplementing
PZM-3 with 3 mM L-carnitine produced blastocysts with cell numbers equivalent to those obtained in the sequential media
system. After vitrification, the post-warming survival rates of blastocysts obtained in media supplemented with 3 mM
L-carnitine were significantly greater than those of blastocysts obtained in standard PZM-3. In conclusion, L-carnitine
supplementation improved embryo development when the medium contained pyruvate and lactate or was lacking
carbohydrates completely, indicating a role for fatty-acid metabolism when the embryo’s requirements for carbohydrates
are not adequately met.
Additional keywords: blastocyst, carbohydrate, lipid, vitrification.
Received 4 November 2016, accepted 14 March 2017, published online 10 April 2017
Introduction
The in vitro production (IVP) of porcine embryos is an important
technology for biomedical research purposes, as well as the
development of advanced reproductive technologies (ARTs),
including vitrification and embryo transfer, to support pig-
breeding programs. However, in vitro-produced porcine embryos
display low developmental potential and are generally of poorer
quality compared with in vivo-derived embryos (Kikuchi 2004;
Grupen 2014), limiting these applications. Major differences seen
between in vitro-andin vivo-derived embryos include altered
metabolic processes (Thompson 1997;Durkin et al. 2001)and
changes in lipid-droplet morphology, size and number (Plante
and King 1994;Kikuchi et al. 2002a). These differences are likely
to contribute to variations in embryo developmental potential.
While common media preparations include exogenous car-
bohydrate substrates to support energy requirements, there is
evidence that embryos are capable of utilising endogenous lipid
reserves for ATP production. There is a decrease in fatty-acid
content (Romek et al. 2009,2011), with a corresponding
increase in O
2
consumption (Sturmey and Leese 2003), during
development of porcine embryos to the blastocyst stage. Fur-
ther, the addition of L-carnitine, a stimulant of lipid metabolism,
to murine (Dunning et al. 2010) and bovine (Sutton-McDowall
et al. 2012) embryo culture media improved embryo develop-
ment. Porcine embryos have a much greater intracellular lipid
content compared with those of other production animal species
(McEvoy et al. 2000), with in vitro-produced embryos having a
greater lipid content than in vivo-derived embryos (Kikuchi
et al. 2002a). Cytoplasmic lipid content is negatively correlated
with cryotolerance (Nagashima et al. 1995;Abe et al. 2002),
limiting the application of vitrification in porcine embryos.
It is well established that glucose, pyruvate and lactate are the
key carbohydrate substrates supporting preimplantation embryo
development and that the embryos’ preference for carbohydrate
substrates changes from the zygote to the blastocyst stages
(Gardner 1998;Leese 2012). However, many in vitro culture
(IVC) systems rely on single medium formulations, with energy
substrates unchanged for the duration of embryo culture.
CSIRO PUBLISHING
Reproduction, Fertility and Development
http://dx.doi.org/10.1071/RD16442
Journal compilation CSIRO 2017 www.publish.csiro.au/journals/rfd
In porcine embryos, glucose inhibits development when included
in culture medium during the earlycleavage stages, while glucose
consumption increases at the blastocyst stage (Flood and Wiebold
1988;Sturmey and Leese 2003). Consequently, sequential media
systems have been developed to better meet the energy require-
ments of the embryo, with altered concentrations of carbohydrate
substrates provided for different stages of development. Modified
porcine IVC media have led to increased blastocyst yields and
improved blastocyst quality by including pyruvate and lactate for
the first 48h following fertilisation and glucose for the remainder
of the culture period (Kikuchi et al. 2002b;Kim et al. 2004;Beebe
et al. 2007).
Previous studies in mouse and cattle embryos have suggested
a role for lipid metabolism during embryo IVC. In the absence of
exogenous glucose, pyruvate, lactate or an extracellular protein
source, the addition of 1 mM L-carnitine to culture medium
increased the proportion of IVP mouse embryos developing to
the blastocyst stage (Dunning et al. 2010). In cattle embryos,
supplementing complete culture medium (containing 0.5 mM
glucose, 0.35 mM pyruvate and 10.5 mM lactate) with 5 mM
L-carnitine improved development to the morula stage (Sutton-
McDowall et al. 2012). Porcine embryos contain a much larger
complement of endogenous lipids compared with embryos of
other domestic livestock species, making them an ideal model
for studying lipid metabolism. Supplementation of IVC medium
with 3.10 mM L-carnitine decreased the levels of reactive
oxygen species (ROS) and reduced the incidence of apoptosis
in porcine parthenogenetic blastocysts (Wu et al. 2011). Given
the changing needs of the developing embryo, it was hypothe-
sised that embryos may preferentially utilise lipids for energy
production at specific stages of development.
The aim of this study was to examine the role of lipid
metabolism during porcine embryo IVC. Therefore, the effect
of supplementing embryo culture medium with L-carnitine, a
known modulator of lipid metabolism, on embryo development
and cryotolerance was assessed.
Materials and methods
Chemicals and media
All chemicals were supplied by Sigma-Aldrich unless otherwise
stated. Washing and preparation of oocytes were carried out
using 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid
(HEPES)-buffered porcine X medium (PXM; 108 mM NaCl,
10 mM KCl, 0.35 mM KH
2
PO
4
, 0.4 mM MgSO
4
, 5.0 mM
NaHCO
3
, 25 mM HEPES, 0.2 mM sodium pyruvate, 2.0 mM
calcium lactate, 3.0mg mL
1
polyvinyl alcohol (PVA); Yoshioka
et al. 2008). In vitro maturation (IVM) was performed using
porcine oocyte medium (POM; 108 mM NaCl, 10 mM KCl,
0.35 mM KH
2
PO
4
,0.4mMMgSO
4
,25mMNaHCO
3
,4.0mM
glucose, 0.2 mM sodium pyruvate, 2.0 mM calcium lactate,
2.0 mM glutamine, 5.0 mM hypotaurine, 0.1 mM cysteamine,
minimum essential medium (MEM) amino acids (Gibco), MEM
non-essential amino acids (Gibco), 65 mgmL
1
penicillin G,
50 mgmL
1
streptomycin sulfate; Yoshioka et al. 2008) sup-
plemented with 3.0 mg mL
1
fatty acid-free bovine serum
albumin (BSA; IVP grade gamma irradiated; MP Biomedicals),
10 ng mL
1
epidermal growth factor (EGF), 10 IU mL
1
equine
chorionic gonadotrophin (eCG; Pregnecol; Bioniche Animal
Health Pty Ltd) and 10 IU mL
1
human chorionic gonadotro-
phin (hCG; Chorulon; Intervet Australia Pty Ltd). Tyrode’s
albumin lactate pyruvate–polyvinyl alcohol (TALP-PVA)
medium (Bavister 1989) supplemented with 3.0 mM calcium
lactate, 2.0 mM caffeine-sodium benzoate and 3.0 mg mL
1
BSA (Grupen and Armstrong 2010) was used for in vitro ferti-
lisation (IVF). The sperm preparation medium consisted of
TALP-PVA medium supplemented with 0.5 mg mL
1
BSA
(SpermTALP). Porcine zygote medium-3 (PZM-3; Yoshioka
et al. 2002) was used for embryo IVC. All droplets and wells of
media were covered with embryo-tested mineral oil and equil-
ibrated in a humidified atmosphere of 6% CO
2
in air at 38.58C
for at least 3 h before use.
In vitro maturation of oocytes
Ovaries from prepubertal gilts were collected immediately after
slaughter and transported to the laboratory at 34–388C in 0.9%
NaCl (Baxter) supplemented with an antibiotic–antimycotic
solution (100 IU mL
1
penicillin G, 0.25 mgmL
1
streptomycin
sulfate and 0.85% amphotericin B; Gibco) within 1 h. Cumulus–
oocyte complexes (COCs) were aspirated from antral follicles
3–6 mm in diameter using a 21-gauge needle, through which
constant suction (1 L min
1
) was applied, and collected in a
vacutainer tube. Collected COCs were washed twice in PXM.
Oocytes with an evenly granulated cytoplasm and at least three
complete layers of compact cumulus cells were selected and
washed in POM. Washed COCs were transferred to four-well
dishes (,50 COCs per well; Nunc) containing POM (500 mL per
well) and cultured at 38.58C in a humidified atmosphere of 6%
CO
2
in air.
In vitro fertilisation
After 44 h of IVM, oocytes were partially denuded by gentle
pipetting in PXM after brief exposure (,1 min) to 0.5 mg mL
1
hyaluronidase. Oocytes were washed and transferred to four-
well dishes containing TALP-PVA medium (500 mL per well).
Meanwhile, boar semen frozen in a 0.25-mL straw was thawed
immediately upon retrieval from liquid nitrogen storage by
agitating the straw in a water bath at 428C for 20 s. Spermatozoa
were purified by density-gradient centrifugation at 720gfor
10 min at 388C using a two-layer (45% and 90%) PureSperm
(Nidacon Laboratories AB) discontinuous gradient prepared
with SpermTALP medium. Following this, the pellet was gently
aspirated, made up to 1 mL in volume with SpermTALP
medium and centrifuged at 310gfor 5 min at 388C. The
supernatant was removed and the pellet was gently resus-
pended in 400 mL SpermTALP medium. Sperm motility and
concentration were then assessed. Spermatozoa were added to
the insemination wells (,50 oocytes per well) at a concen-
tration of 200 motile spermatozoa per oocyte. Gametes were
co-incubated for 30 min at 38.58Cin6%CO
2
in air, after which
the oocytes and zona-bound spermatozoa were carefully
transferred to a second well containing fresh TALP-PVA
medium (500 mL) and incubated for a further 5 h, resulting in a
total IVF co-incubation of 5.5 h (Grupen and Nottle 2000;Gil
et al. 2004;Bartolac et al. 2015).
BReproduction, Fertility and Development J. L. Lowe et al.
In vitro culture and embryo assessment
Presumptive zygotes were denuded of remaining cumulus cells
and loosely bound spermatozoa, washed and placed in 50-mL
droplets (maximum 15 zygotes per droplet) of culture medium
and incubated in 6% CO
2
,5%O
2
and 89% N
2
at 38.58C.
Cleavage was assessed and embryos were transferred to dif-
ferent media formulations as per the experimental design at 72 h
of IVC. On Day 4 of IVC, the culture media were supplemented
with 10% (v/v) fetal calf serum (FCS; heat inactivated,
Australian origin; Gibco) by adding 5 mL of equilibrated FCS to
each 50-mL droplet. Addition of FCS to culture media, which has
previously been shown to improve blastocyst development
(Pollard et al. 1995;Dobrinsky et al. 1996;Koo et al. 1997), was
done to maximise the blastocyst yield of all treatment groups.
The blastocyst formation rate, calculated as a percentage of the
embryos that cleaved, and the total number of cells per blasto-
cyst were assessed on Day 7 of IVC. Blastocysts were washed in
PXM and transferred to absolute ethanol containing 0.3 mgmL
1
Hoechst 33342. After staining for 30 min the blastocysts were
transferred to absolute ethanol, fixed overnight in the dark at 48C
and then mounted on slides. The stained nuclei were visualised
using fluorescence microscopy (Olympus BX61; Olympus) and
counted using ImageJ software (Version 1.46r; National Institutes
of Health).
Blastocyst vitrification and warming
On Day 7 of IVC blastocysts were vitrified as described previ-
ously (Vajta et al. 1998;Bartolac et al. 2015). Blastocysts were
classified morphologically as either A, B or C grade, according
to the criteria of the Society for Assisted Reproductive Tech-
nology (SART) grading system (A ¼good, B ¼fair, C ¼poor;
Racowsky et al. 2010). Only blastocysts of Grades A and B were
vitrified. At Day 7 of IVC, all A and B grade blastocysts had a
well-expanded blastocoele. Briefly, groups of up to 10 blas-
tocysts were washed in HEPES-buffered Medium 199 (Gibco)
supplemented with 20% FCS (H199-FCS) for 5 min. Blas-
tocysts were then transferred to equilibration medium (H199-
FCS supplemented with 7.5% ethylene glycol and 7.5%
dimethyl sulfoxide (DMSO)) and held for 3 min before being
transferred to vitrification medium (H199-FCS supplemented
with 17% ethylene glycol, 17% DMSO and 0.4 M sucrose) for
45 s. Embryos were loaded into a super-fine open-pulled straw
(SOPS; Minitube) within the 45-s period and immediately
plunged into liquid nitrogen. The equilibration and vitrification
media were used at room temperature. To warm, the open end of
the SOPS was placed directly into thaw medium 1 (H199-FCS
supplemented with 0.14 M sucrose) immediately upon retrieval
from liquid nitrogen. Blastocysts were held in this medium for
6 min before a 5-min hold in thaw medium 2 (H199-FCS sup-
plemented with 0.075 M sucrose) and a final 5-min hold in
H199-FCS. The warming media were used at 38.58C. The
blastocysts were then washed in PZM-3 supplemented with 20%
FCS, transferred to 50-mL droplets of PZM-3 supplemented with
20% FCS and cultured in 6% CO
2
,5%O
2
and 89% N
2
at 38.58C.
Survival was assessed after 24 h of post-warming culture, with
surviving blastocysts classified as those in which blastocoele
re-expansion had clearly occurred.
Experimental design
Experiment 1: dose-response effect of L-carnitine
supplementation in PZM-3
Presumptive zygotes were cultured for 7 days in PZM-3,
which contains 0 mM glucose, 0.2 mM pyruvate and 2.0 mM
lactate (PZM-PL), supplemented with either 0, 1.5, 3, 6 or
12 mM L-carnitine to determine the optimum dose. Cleavage
and blastocyst formation were assessed. The experiment was
replicated four times with 35–45 presumptive zygotes per
treatment group in each replicate.
Experiment 2: dose-response effect of L-carnitine
supplementation in carbohydrate-deficient medium
Presumptive zygotes were cultured for 7 days in either
control medium (PZM-PL; 0 mM glucose, 0.2 mM pyruvate
and 2.0 mM lactate) or in carbohydrate-deficient medium
(PZM–; 0 mM glucose, 0 mM pyruvate and 0 mM lactate)
supplemented with either 0, 1.5, 3, 6 or 12 mM L-carnitine.
Cleavage and blastocyst formation were assessed. The experi-
ment was replicated three times with 30–50 presumptive
zygotes per treatment group in each replicate.
Experiment 3: temporal effect of L-carnitine
supplementation in PZM-3
Presumptive zygotes were cultured for 7 days in PZM-PL
either:
(1) without (LC) or with 3 mM L-carnitine (þLC) for the
entire period,
(2) without 3 mM L-carnitine for the first 72 h and then without
(–LC/–LC) or with 3 mM L-carnitine (–LC/þLC),
(3) with 3 mM L-carnitine for the first 72 h and then without
(þLC/–LC) or with 3 mM L-carnitine (þLC/þLC).
Cleavage and blastocyst formation were assessed and blas-
tocyst total cell numbers were determined. The experiment was
replicated three times with 30–45 presumptive zygotes per
treatment group in each replicate.
Experiment 4: effect of L-carnitine supplementation in a
sequential media system
The effect of L-carnitine supplementation during the first 72 h
of IVC was further examined using a sequential media system that
consisted of PZM-PL and a modified formulation containing
glucose but no pyruvate or lactate (PZM-G; 5.55 mM glucose,
0 mM pyruvate and 0 mM calcium lactate). The glucose concen-
tration was chosen based on that used in North Carolina State
University-23 (NCSU-23) medium (Petters and Wells 1993), a
commonly used porcine embryo culture medium, and previously
described refinements to sequential media used to culture porcine
embryos (Kikuchi et al. 2002b;Kim et al. 2004;Beebe et al. 2007).
Presumptive zygotes were cultured for 7 days in either:
(1) PZM-PL without or with 3 mM L-carnitine for the first 72 h
and then in PZM-PL (PL–/PL and PLþ/PL),
(2) PZM-PL without or with 3 mM L-carnitine for the first 72 h
and then in PZM-G (PL–/G and PLþ/G),
(3) PZM-G without or with 3 mM L-carnitine for the first 72 h
and then in PZM-G (G–/G and Gþ/G).
Lipid metabolism during IVC of porcine embryos Reproduction, Fertility and Development C
Cleavage and blastocyst formation were assessed and blas-
tocyst total cell numbers were determined. The experiment was
replicated three times with 30–50 presumptive zygotes per
treatment group in each replicate.
Experiment 5: effect of L-carnitine supplementation on
blastocyst cryotolerance
Embryos were cultured for 7 days in either PZM-PL without
(PL–) or with 3 mM L-carnitine (PLþ) or PZM-PL with 3 mM
L-carnitine for the first 72 h and then in PZM-G (PLþ/G). These
treatments were selected to examine the effects of L-carnitine-
supplemented single-step and sequential media on blastocyst
cryotolerance. Blastocysts of each group that were classified as
being of Grade A or B morphology on Day 7 of IVC were
vitrified and their post-warming survival was assessed. The
experiment was replicated six times.
Statistical analysis
Analyses were performed using GENSTAT 16th Edition (VSN
International Ltd). Data expressed as proportions were arcsine
transformed before analysis. Embryo cleavage, blastocyst for-
mation, blastocyst cell number and post-warming survival data
were analysed by one-way analysis of variance (ANOVA),
blocking by replicate. When significant differences were detec-
ted, the Fisher’s least significant difference (l.s.d.) test was used
for post hoc pairwise comparisons. All data are expressed as the
mean the standard error of the mean (s.e.m.) and P,0.05 was
considered to indicate a statistically significant difference.
Results
Experiment 1: dose-response effect of L-carnitine
supplementation in PZM-3
The effect of supplementing PZM-3 with different doses of
L-carnitine on embryo development is shown in Fig. 1. The
cleavage rate of the 3 mM L-carnitine group was greater than
that of the control group (80.7 4.6% vs 66.2 4.0%; P,0.05)
but did not differ from those of the other L-carnitine groups
(P.0.05). The blastocyst formation rate of the control was
greater than that of the 6 mM L-carnitine group (25.22.3% vs
8.5 1.8%; P,0.05) but did not differ from those of the other
L-carnitine groups (P.0.05).
Experiment 2: dose-response effect of L-carnitine
supplementation in carbohydrate-deficient medium
The effect of supplementing carbohydrate-deficient medium
(PZM–) with L-carnitine on embryo development is shown in
Fig. 2. In the absence of carbohydrate substrates, supplemen-
tation with all doses of L-carnitine increased the cleavage rate
(ranging from 26.8 11.0% to 42.1 3.2%) compared with
the unsupplemented control group (2.8 1.5%; P,0.05). The
cleavage rate of embryos cultured in the standard carbohydrate-
containing medium, PZM-PL (78.2 4.5%), was significantly
greater than those of embryos in all other groups. Whereas
embryos cultured in PZM-PL formed blastocysts as expected,
development to the blastocyst stage was markedly reduced in the
absence of carbohydrate substrates, regardless of L-carnitine
supplementation (P,0.05).
Experiment 3: temporal effect of L-carnitine
supplementation in PZM-3
The effect of supplementing PZM-PL with 3 mM L-carnitine
during the first 72 h of IVC and/or the remaining IVC interval on
embryo development is shown in Fig. 3. The cleavage and
blastocyst formation rates of embryos for all the treatment
groups did not differ significantly. The mean numbers of cells
per blastocyst were also similar between the treatment groups
(ranging from 20.2 3.7 cells to 31.8 6.1 cells; P.0.05).
0
10
20
30
40
50
60
70
80
90
0 1.5 3 6 12
Embryos developed (%)
L-carnitine concentration (mM)
ab
aab
ab
a
ab
b
b
ab
ab
Cleaved Blastocysts
Fig. 1. The dose-response effect of supplementing pyruvate- and lactate-
containing medium with L-carnitine on the development of IVP porcine
embryos. Following IVF, presumptive zygotes were cultured for 7 days in
PZM-PL supplemented with 0, 1.5, 3, 6 or 12 mM L-carnitine. Cleavage
rates (solid bars) are expressed as a percentage of total presumptive zygotes
cultured and blastocyst formation rates (open bars) are expressed as a
percentage of embryos that cleaved. Within data categories, bars without a
common letter differ significantly (P,0.05).
0
10
20
30
40
50
60
70
80
90
PZM-PL 0 1.5 3 6 12
Embryos developed (%)
L-carnitine concentration (mM) in PZM−
Cleaved Blastocysts
b
a
b
c
c
a
bb
c
b
c
b
Fig. 2. The dose-response effect of supplementing carbohydrate-deficient
medium with L-carnitine on the development of IVP porcine embryos.
Following IVF, presumptive zygotes were cultured for 7 days in control
medium (PZM-PL; 0 mM glucose, 0.2 mM pyruvate and 2.0 mM lactate)
without L-carnitine or modified PZM-3 (PZM–; 0 mM glucose, 0 mM
pyruvate and 0 mM lactate) supplemented with 0, 1.5, 3, 6 or 12 mM
L-carnitine. Cleavage rates (solid bars) are expressed as a percentage of
total presumptive zygotes cultured and blastocyst formation rates (open
bars) are expressed as a percentage of embryos that cleaved. Within data
categories, bars without a common letter differ significantly (P,0.05).
DReproduction, Fertility and Development J. L. Lowe et al.
Experiment 4: effect of L-carnitine supplementation in a
sequential media system
The effect of supplementing sequential media containing
different carbohydrate substrates (PZM-PL and PZM-G) with
3 mM L-carnitine during the first 72 h of IVC on embryo
development is shown in Fig. 4. Regardless of L-carnitine
supplementation, embryos cultured in PZM-PL for the first 72 h
of IVC cleaved at a greater rate (ranging from 67.9 8.3% to
77.2 4.9%) than those cultured in PZM-G for the first 72 h
(44.3 5.4% and 44.4 10.8%; P,0.05). The incidence of
blastocyst formation was much lower when embryos were cul-
tured in PZM-G for the first 72 h (0.0 0.0% and 4.2 4.2%)
compared with those cultured in PZM-PL for the first 72 h
(ranging from 24.1 7.5% to 33.2 4.2%; P,0.05). The
presence of L-carnitine during the first 72 h did not significantly
affect the rates of cleavage or blastocyst formation. However,
when embryos were cultured in the PL/PL system, supplemen-
tation with L-carnitine during the first 72 h increased the mean
total number of cells per blastocyst (43.4 6.3 cells vs
24.0 3.0 cells; P,0.05). Culture in the PL/G sequential
media system (with or without L-carnitine) also produced
blastocysts with mean total cell numbers (40.1 6.5 cells and
40.3 6.7 cells respectively) greater than those of the PL/PL
(without L-carnitine) group (24.0 3.0 cells; P,0.05). Insuf-
ficient blastocysts were obtained from embryos cultured in the
G/G system (with or without L-carnitine) to effectively analyse
the blastocyst cell numbers of these groups.
Experiment 5: effect of L-carnitine supplementation on
blastocyst cryotolerance
The effect of L-carnitine supplementation of different culture
systems on the cryotolerance of Day-7 IVP porcine blastocysts
is shown in Fig. 5. Blastocysts produced in the presence of
L-carnitine in both the single-step medium (PLþ) and the
sequential media system (PLþ/G) survived vitrification and
warming at greater rates (48.3 5.0% and 48.1 4.5%
0
10
20
30
40
50
60
70
80
90
⫺LC ⫹LC ⫺LC/⫺LC ⫹LC/⫺LC ⫺LC/⫹LC ⫹LC/⫹LC
Embryos developed (%)
Culture medium group
Cleaved Blastocysts
Fig. 3. The effect of supplementing pyruvate- and lactate-containing
medium with L-carnitine during the pre- and/or post-compaction stages on
the development of IVP porcine embryos. Following IVF, presumptive
zygotes were cultured for 7 days in PZM-PL either without (–LC) or with
3 mM L-carnitine (þLC) without a medium change, without (–LC/–LC) or
with 3 mM L-carnitine (þLC/þLC) with a medium change at 72 h, with
3 mM L-carnitine for the first 72 h and then without supplementation ( þLC/
–LC) or without supplementation for the first 72 h and then with 3 mM L-
carnitine (–LC/þLC). Cleavage rates (solid bars) are expressed as a
percentage of total presumptive zygotes cultured and blastocyst formation
rates (open bars) are expressed as a percentage of embryos that cleaved.
0
10
20
30
40
50
60
70
80
90
PL/PL PL/PL PL/G PL/G G/G G/G
⫺⫹⫺⫹⫺⫹
Embryos developed (%)
Culture medium group
a
aaa
a
aa
a
b
b
b
b
Cleaved Blastocysts
Fig. 4. The effectof supplementingmedia containingdifferent carbohydrate
substrates with L-carnitine during the pre-compaction stages on the develop-
ment of IVP porcine embryos. Following IVF, presumptive zygotes were
cultured for 7 days in media containing either pyruvate and lactate with a
medium change at 72 h (PL/PL), pyruvate and lactate for the first72 h and then
glucose (PL/G) or glucose with a medium change at 72h (G/G). Additionally,
the medium used for the first 72h was either supplemented with 3 mM
L-carnitine (þ) or not (–). Cleavage rates (solid bars) are expressed as a
percentage of total presumptive zygotes cultured and blastocyst formation
rates (open bars) areexpressed as a percentage ofembryos that cleaved. Within
data categories, bars without a common letter differ significantly (P,0.05).
0
10
20
30
40
50
60
PL⫺PL⫹PL⫹/G
Post-warming survival (%)
Culture medium
g
roup
a
b
b
Fig. 5. The effect of supplementing different media systems with
L-carnitine on the cryotolerance of Day-7 IVP porcine blastocysts. Blas-
tocysts were vitrified following culture in medium that contained either
pyruvate and lactate without L-carnitine for the entire period (PL–), pyruvate
and lactate with L-carnitine for the entire period (PLþ) or pyruvate and
lactate with L-carnitine for the first 72 h and then glucose for the remainder
of the period (PLþ/G). Survival rates are expressed as a percentage of
blastocysts that were vitrified and warmed. Bars without a common letter
differ significantly (P,0.05).
Lipid metabolism during IVC of porcine embryos Reproduction, Fertility and Development E
respectively) than those produced in the absence of L-carnitine
in the single-step medium (PL–; 31.0 3.9%; P,0.05).
Discussion
This study showed that in the absence of carbohydrate substrates,
L-carnitine supplementation of culture medium markedly
increased the incidence of cleavage in IVP porcine embryos. In
PZM-3, a commonly used porcine embryo culture medium that
contains pyruvate and lactate, the beneficial effect of L-carnitine
supplementation on embryo development was less evident, with
increasesin cleavage rate and improvements to blastocyst quality
observed under only some of the conditions tested. This finding
suggests that the carbohydrate substrates provided in PZM-3 do
not fully meet the metabolic requirements of IVP porcine
embryos.Furthermore,given that the magnitude of the L-carnitine
effect varied with the provision of carbohydrate substrates, any
inconsistencies in the findings of previous L-carnitine investiga-
tions may be attributed to differences in the culture media used.
When pyruvate, lactate and glucose were omitted from the
medium, all doses of L-carnitine (1.5–12 mM) increased the rate
of cleavage, although only to about half of that achieved in
carbohydrate-containing medium (0.2 mM pyruvate and 2 mM
lactate) without L-carnitine. Contrary to this, cattle embryos
showed similar cleavage rates regardless of carbohydrate or
L-carnitine supplementation (Sutton-McDowall et al. 2012). In
the present study, blastocyst formation rates were not increased
by L-carnitine supplementation in the absence of carbohydrates.
Similarly, cattle embryos were arrested in the early cleavage
stages of development when cultured without carbohydrates
(Ferguson and Leese 2006). However, the addition of 1 mM
L-carnitine to culture medium without carbohydrates supported
cattle morula development to the same level as that achieved in
carbohydrate-containing medium; moreover, the addition of
5 mM L-carnitine to carbohydrate-deficient medium promoted
a higher rate of morula development (Sutton-McDowall et al.
2012). Carbohydrate substrates in culture are not necessarily
used for energy generation, with certain metabolic pathways
utilising carbohydrate substrates to produce intermediaries
necessary for other cellular functions. For example, NADPH
is produced by the pentose phosphate pathway (PPP) for
biosynthetic reactions and as a reducing agent to protect against
toxic ROS accumulation. These alternate purposes may explain
why porcine embryos developed poorly in the absence of
carbohydrates despite stimulation of lipid metabolism.
Intermediaries of alternate pathways are also required for full
metabolic breakdown of lipid products. Carbohydrates are
needed to produce oxaloacetate, which is required to react with
acetyl-CoA to prime the tricarboxylic acid (TCA) cycle. The
addition of oxaloacetate to carbohydrate-free IVM medium
improved subsequent development to a level similar to that of
oocytes matured with glucose and decreased the triglyceride
content (Sturmey and Leese 2008). It is possible that in the
absence of carbohydrates the required intermediaries for com-
plete oxidation of lipid substrates are also lacking, preventing
lipid from being utilised effectively as an energy substrate.
Further, embryos from ruminant species exhibit a higher level of
glycolytic activity than porcine embryos (Thompson et al. 1991;
Rieger et al. 1992;Gardner et al. 1993;Swain et al. 2002), which
may account for some of the reported metabolic differences
between species.
In the single-step PZM-3, which contained pyruvate and
lactate, inclusion of 3 mM L-carnitine for the duration of IVC
significantly increased the incidence of cleavage. There is
evidence that embryos from rabbits (Khandoker and Tsujii
1998), mice (Hillman and Flynn 1980) and cattle (Ferguson
and Leese 1999,2006) are capable of metabolising fatty acids to
produce energy during the early cleavage stages. Further,
inhibition of b-oxidation during porcine embryo culture blocked
development at the zygote stage (Sturmey and Leese 2008),
suggesting a requirement for lipid metabolism at the initial
cleavage division. In contrast, Wu et al. (2011) found no
difference in the cleavage of porcine parthenotes when they
were cultured in standard PZM-3 supplemented with 0–12 mM
L-carnitine. In a comparison of bovine IVF embryos and
parthenotes, differences in the expression levels of genes relat-
ing to metabolism, including glucose transport and the PPP,
have been reported (Go´ mez et al. 2009). Therefore, the incon-
sistent findings on the effects of L-carnitine supplementation
during the early cleavage stages may be due to metabolic
differences between porcine IVF embryos and parthenotes.
Inconsistent results between experiments can also be attributed
to differences in the source of the pig ovaries collected, as
maternal age at slaughter influences oocyte quality (Grupen
et al. 2003;Bagg et al. 2007), or the time of the year in which
experiments are conducted, as oocyte quality is known to be
seasonally affected (Bertoldo et al. 2010,2012).
Compared with the single-step PZM-3 without L-carnitine,
culture in the sequential carbohydrate-containing media system
was beneficial to blastocyst quality. Modifying the carbohydrate
substratesin culture medium from pyruvate and lactate during the
pre-compaction stages to glucose during the post-compaction
stages has previously been demonstrated to be beneficial to
porcine embryo development (Kikuchi et al. 2002b;Beebe
et al. 2007). It is well established that preimplantation embryos
of numerous species meet their energy requirements by preferen-
tially consuming pyruvate before the morula stage and glucose
after the morula stage (Leese 2012). While porcine embryos are
known to consume pyruvate and glucose as energy substrates
(Swain et al. 2002), early studies showed that glucose alone can
support their development throughout preimplantation (Petters
and Wells 1993). In the present study, the quality of the embryos
cultured in medium containing pyruvate and lactate for the
duration of culture was improved by the inclusion of 3mM
L-carnitine for the first 72 h. This improvement to embryo quality
was similar to that achieved by culturing embryos in glucose-
containing medium after they had been cultured in pyruvate- and
lactate-containing medium for the first 72h (i.e. in the PL/G
system). This result suggests that the pyruvate- and lactate-
containing medium is suboptimal and that the subsequent provi-
sion of glucose compensates, at least to some degree, for the early
shortfall in ATP production. Stimulating lipolysis through
L-carnitine supplementation for the first 72 h of IVC appeared
to better meet the embryos’ energy needs when the carbo-
hydrate substrates were suboptimal (i.e. in the PL/PL system).
Further studies are needed to determine whether L-carnitine
FReproduction, Fertility and Development J. L. Lowe et al.
supplementation enhances ATP production in porcine embryos
at the early cleavage stages.
Carnitine is an important co-factor of the carnitine shuttle for
entry of free fatty acids into the mitochondrial matrix. Lipids
provide a dense energy source and contribute to ATP production
via metabolic breakdown through b-oxidation and oxidative
phosphorylation. Supplementation of IVM medium with
L-carnitine led to decreased lipid-droplet density in porcine
oocytes (Somfai et al. 2011), increased levels of b-oxidation in
murine oocytes (Dunning et al. 2010) and tended to increase the
ATP content in bovine oocytes (Chankitisakul et al. 2013). Also,
supplementation of IVC medium with L-carnitine decreased
lipid-droplet density and increased ATP levels in two-cell
bovine embryos (Takahashi et al. 2013). Interestingly, inhibi-
tion of oxidative phosphorylation at the peri-compaction stage
improved the development of porcine embryos (Macha´ty et al.
2001), highlighting the importance of the metabolic switch from
oxidative phosphorylation to glycolysis. L-carnitine may also
protect against oxidative stress, having been shown to alter the
redox state of somatic cells via mitochondrial pathways (Pillich
et al. 2005;Ye et al. 2010). In support of this, addition of
L-carnitine to embryo culture medium was found to reduce ROS
formation in mouse (Abdelrazik et al. 2009) and cattle (Sutton-
McDowall et al. 2012) embryos, with a concomitant improve-
ment in development to the blastocyst stage. Similarly, in
porcine parthenotes, L-carnitine supplementation of culture
medium reduced intracellular ROS levels and decreased the
incidence of apoptosis in cells of the resulting embryos (Wu
et al. 2011). It is unclear whether L-carnitine treatments provide
a dual effect of stimulating lipid metabolism and protecting
against oxidative stress or if one effect is more dominant than
the other.
In this study, culture in glucose-containing medium for the
entire period did not support high rates of cleavage and blasto-
cyst formation. This finding appears to conflict with those of
porcine embryo IVP studies conducted using NCSU-23 medium
(Petters and Wells 1993), a commonly used formulation that
contains 5.55 mM glucose and no pyruvate or lactate (Rath et al.
1995;Abeydeera et al. 1998a,1998b). However, direct compar-
isons of PZM-3 and NCSU-23 medium have consistently shown
that PZM-3 is superior, better supporting the development of
porcine embryos to the blastocyst stage (Yoshioka et al. 2002;
Im et al. 2004;Nana´ssy et al. 2008;Wang et al. 2009). Porcine
embryos are capable of utilising glucose for the duration of
culture (Swain et al. 2002), although there is limited glucose
metabolism before compaction (Biggers et al. 1967;Flood and
Wiebold 1988) and the majority of ATP produced is derived
from oxidative phosphorylation (Sturmey and Leese 2003).
Furthermore, Karja et al. (2006) found that hydrogen peroxide
levels were greater in Day-1 porcine embryos cultured in
medium containing glucose (1.5–20 mM) as the sole energy
substrate than in those cultured in medium containing pyruvate
and lactate without glucose. Given that the inclusion of
L-carnitine during the first 72 h of IVC increased blastocyst cell
numbers in the absence of glucose (i.e. in PZM-PL), it appears
that medium devoid of glucose, despite containing pyruvate and
lactate, does not fully meet the metabolic needs of porcine
embryos during the early cleavage stages. While the results of
the present study suggest that stimulation of lipid metabolism
better supports the needs of early cleavage-stage porcine embryos,
inclusion of glucose at a low concentration (e.g. 0.2 mM) may also
provide some benefit. This proposal is supported by the finding
that porcine oviduct fluid contains ,0.2 mM glucose at the time
the embryos are at the four-cell stage (Nichol et al. 1998). As
mentioned previously, provision of a higher concentration
(5.55mM) of glucose after compaction may compensate for a
deficiency in carbohydrate substrates before compaction and is
therefore recommended for porcine embryo sequential media
systems. It should be noted that components of fetal calf serum,
which was added on Day 4 of IVC, may have contributed to the
compensatory effect observed. However, even with the addition
of FCS, the PL/PL system did not support blastocyst development
to the same extent as the PL/G system, indicating that the
enhanced development can be largely attributed to the presence
of glucose.
The inclusion of L-carnitine, either for the entirety of IVC in
the single-step medium (PLþ) or for the first 72 h in the
sequential media system (PLþ/G), significantly improved the
cryotolerance of porcine blastocysts. The lipid content of
embryos has a major influence on their cryosensitivity
(Dobrinsky and Johnson 1994;Nagashima et al. 1995;Abe
et al. 2002;Sudano et al. 2011), with lipids having been shown
to cause an increase in oxidative and mechanical damage during
ice-crystal formation (Isachenko et al. 1998). Stimulation of
lipolysis during embryo culture has previously been shown to be
an effective approach to reduce cytoplasmic lipid content and
enhance cryotolerance. The addition of 10 mM forskolin to
culture medium for 24 h on Day 5 of IVC increased lipolytic
activity and markedly improved the cryosurvival of vitrified
Day-6 porcine blastocysts (Men et al. 2006). L-carnitine sup-
plementation of IVC medium also improved the cryotolerance
of vitrified buffalo blastocysts (Boccia et al. 2013) and bovine
blastocysts subjected to conventional slow freezing (Takahashi
et al. 2013). In the present study, the cytoplasmic lipid content
was not measured. Therefore, it is unclear whether the
L-carnitine treatment reduced the lipid content of the blastocyst
cells or, via the antioxidant activity of L-carnitine, provided
protection against ROS formed during the vitrification–
warming process. Alternatively, the cryotolerance of the blas-
tocysts may have been related to the total cell numbers of
blastocysts in each treatment group. The results of Experiment
4 revealed that the PL/PL system without L-carnitine produced
blastocysts with fewer cells than the PL/PL system with
L-carnitine and the PL/G system with L-carnitine. However,
all the vitrified blastocysts were classified as being of Grade A or
B morphology, which we have found display similar rates of
post-warming survival (L. K. Bartolac, C. Sjo¨ blom and C. G.
Grupen, unpubl. data).
Accumulation of ROS is accelerated when embryos are
exposed to environmental stressors, such as those experienced
during vitrification and warming. Oxidative stress can lead to
mitochondrial damage, ATP depletion, apoptosis and develop-
mental blocks (Gue´rin et al. 2001;Gupta et al. 2009). Also, due
to the large amount of cytoplasmic lipid, porcine embryos are
particularly susceptible to lipid peroxidation, as evidenced by a
reduction in the intracellular accumulation of H
2
O
2
when
Lipid metabolism during IVC of porcine embryos Reproduction, Fertility and Development G
porcine embryos were delipated (Yoneda et al. 2004). The
addition of 1.25 mg mL
1
(7.8 mM) L-carnitine to IVM medium
reduced the density of lipid droplets and the levels of intracellular
H
2
O
2
in metaphase II-stage porcine oocytes (Somfai et al. 2011).
Similarly, culture of bovine embryos in medium containing 1.5 or
3.0 mM L-carnitine reduced the density of lipid droplets and
improved cryotolerance (Takahashi et al. 2013). In porcine
parthenotes, the addition of 0.5 mg mL
1
(3.1 mM) L-carnitine
to PZM-3 decreased intracellular ROS levels at the three- to four-
cell stage and reduced the incidence of apoptosis at the blastocyst
stage (Wu et al. 2011). As the addition of L-ascorbic acid, a
powerful antioxidant, to NCSU-23 medium was also found to
reduce ROS levels and improve the cryotolerance of porcine IVP
embryos (Castillo-Martı´n et al. 2014), the improved cryotoler-
ance observed in the present study appears to be most likely dueto
the antioxidant property of L-carnitine.
In conclusion, the addition of L-carnitine, a co-factor of
b-oxidation, to porcine embryo culture medium increased the
rate of cleavage, enhanced blastocyst quality and improved the
cryotolerance of Day-7 blastocysts. In the complete absence of
carbohydrate substrates, the ability of fertilised oocytes to
undergo the initial cleavage division was vastly improved by
supplementing the medium with L-carnitine. Even when the
medium contained pyruvate and lactate, L-carnitine supplemen-
tation significantly increased the rate of cleavage, suggesting
that the PZM-3 formulation does not provide adequate energy
substrates at the early stages of embryo development. This
finding suggests that lipid metabolism plays an important role
in energy generation during early porcine embryo development.
The observed increase in blastocyst total cell number and
improvement in blastocyst cryotolerance may alternatively be
attributed to the antioxidant properties of L-carnitine. Regardless
of the mode of action, supplementing medium with L-carnitine
during the first 72 h of IVC was found to be aneffective strategy to
improve the viability of IVP porcine embryos.
Acknowledgements
The authors thank the staff of Wollondilly Abattoir Pty Ltd (Picton, NSW,
Australia) for supplying the ovaries used in this study.
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