L-carnitine enhances oocyte maturation and development of parthenogenetic embryos in pigs

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Theriogenology 76 (2011) 785–793 www.theriojournal.com

L-carnitine enhances oocyte maturation and development of parthenogenetic embryos in pigs G.-Q. Wu,1 B.-Y. Jia,1 J.-J. Li, X.-W. Fu, G.-B. Zhou, Y.-P. Hou, S.-E. Zhu* Key Laboratory of Animal Genetics, Breeding and Reproduction, Ministry of Agriculture and National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing, People’s Republic of China Received 24 November 2010; received in revised form 7 April 2011; accepted 7 April 2011

Abstract The objective was to determine whether adding L-carnitine in IVM/IVC medium enhanced maturation and developmental competence of porcine oocytes in vitro. Oocyte maturation rates did not differ significantly among groups supplemented with 0, 0.25, 0.5, or 1 mg/mL of L-carnitine added during IVM (although 2 mg/mL of L-carnitine reduced maturation rate). Compared with control oocytes, those treated with 0.5 mg/mL of L-carnitine during IVM had greater (P ⬍ 0.05) rates of blastocyst formation after parthenogenetic activation, and these blastocysts had less (P ⬍ 0.05) apoptosis. Adding 0.5 mg/mL of L-carnitine during IVM also significantly reduced intracellular reactive oxygen species (ROS), and increased glutathione (GSH) concentrations. With or without glucose supplementation, 0.5 mg/mL of L-carnitine in the IVM medium significantly hastened nuclear maturation of oocytes. Moreover, supplementing the IVM medium with either glucose or L-carnitine increased (P ⬍ 0.05) percentages of oocytes that reached the metaphase II (MII) stage, relative to a control group. Final maturation rates in IVM medium containing either glucose or L-carnitine were not significantly different. Adding L-carnitine (0 to 2 mg/mL) to IVC medium for activated porcine oocytes did not significantly affect development. However, 0.5 mg/mL of L-carnitine in IVC medium significantly reduced reactive oxygen species levels and apoptosis in activated blastocysts, although glutathione concentrations were not significantly altered. In conclusion, adding L-carnitine during IVM/IVC improved developmental potential of porcine oocytes, and also the quality of parthenogenetic embryos, probably by accelerating nuclear maturation, and preventing oxidative damage and apoptosis. © 2011 Elsevier Inc. All rights reserved. Keywords: Porcine oocyte; L-carnitine; Maturation; ROS; GSH; Apoptosis

1. Introduction The pig is important not only for agricultural production, but also for biomedical research [1]. In vitroproduced (IVP) porcine embryos are important for studies of the physiology of embryonic development

1

The first two authors contributed equally to this work.

* Corresponding author. Tel.: ⫹86 10 62731979; fax: ⫹86 10 62731767. E-mail address: [email protected] (S.-E. Zhu). 0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.04.011

and animal reproductive technologies, including embryo transfer, cloning, and transgenesis [2]. During oocyte/embryo culture, in vitro conditions are inevitably different from those in vivo; moreover, oocyte and embryo qualities are influenced by various factors, including time, temperature, composition of culture media, etc. [3]. Therefore, investigating the physiology, metabolism, and culture requirements of oocytes and embryos is critical to optimize IVP procedures. Several research groups have developed a range of culture media for IVP of porcine embryos [4 –7]. Regardless,

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because suboptimal metabolism, oxidative stress, and apoptosis are also important for oocytes and embryos cultured in vitro [8], we inferred that modifications in the culture system could enhance the efficiency of IVP. Free carnitine (3-hydroxy-4-N-trimethylammoniobutanoate) was first isolated from bovine muscle in 1905; however, only the L-isomer is bioactive [9]. It is well known that L-carnitine is a small, hydrophilic molecule which can promote fatty acid and energy utilization by transporting long chain fatty acids across the inner mitochondria membrane for ␤ oxidation, and thereby increase adenosine triphosphate (ATP) concentrations [10]. Furthermore, it also affected activities of several key enzymes involved in protein and lipid metabolism [11]. In addition, L-carnitine has antioxidant activity which may protect mitochondrial membranes and DNA against damage induced by reactive oxygen species (ROS) [12], and it strongly inhibited mitochondrial-dependent apoptosis [13]. L-carnitine has wide physiological functions; its clinical applications include cardiovascular diseases, urinary diseases, nervous system diseases, and male reproductive disorders [14 –17]. L-carnitine may be beneficial in improving pregnancy outcomes in assisted reproductive technologies, especially for patients with polycystic ovary syndrome and endometriosis [18 –20]. Furthermore, L-carnitine may protect cumulus cells from apoptosis and improve the meiotic competence and mitochondrial activity of porcine oocytes during in vitro culture [21]. However, the effects of L-carnitine during maturation and development of porcine oocytes are not well understood. The objectives of the present study were to determine whether adding L-carnitine during IVM/IVC could promote meiotic maturation of porcine oocytes, and ameliorate developmental competence and quality of parthenogenetic embryos in vitro. Furthermore, intracellular concentrations of glutathione (GSH) and ROS in oocytes and embryos were also examined. 2. Materials and methods All chemicals used in this study were purchased from Sigma Chemicals Co (St. Louis, MO, USA), unless otherwise indicated. 2.1. Oocyte collection and maturation Porcine ovaries were collected from prepubertal gilts at a local abattoir, transported to the laboratory at 37 °C, and then washed three times with 0.9% NaCl containing 75 mg/L potassium penicillin G and 50

mg/L streptomycin sulphate. Cumulus-oocyte complexes (COCs) were aspirated from superficial follicles (3 to 8 mm in diameter) using a 10 mL syringe fixed with an 18-gauge needle; those with uniform ooplasm and compact cumulus cells were washed three times in Tyrode’s lactate (TL)-HEPES-PVA (polyvinyl alcohol, 0.1%) [22]. Approximately 50 cumulus-oocyte complexes were placed into each well of a four-well multidish (Nunc, Roskilde, Denmark) containing 500 ␮L IVM medium and incubated for 44 h at 38.5 °C in an atmosphere of 5% CO2 in air with maximum humidity. The IVM medium was a modified North Carolina State University 37 medium (mNCSU37) [7], containing 0.6 mM cysteine, 10 ng/mL epidermal growth factor, 10 IU/mL equine chorionic gonadotropin, 10 IU/mL human chorionic gonadotropin, and 0.1% (wt/vol) PVA. 2.2. Assessment of nuclear status To examine nuclear status, oocytes were mechanically denuded from cumulus cells in Tyrode’s lactateHEPES-PVA medium containing 0.1% hyaluronidase. Oocytes with abnormal cytoplasm and chromatin morphology, broken membrane, and zona pellucida were recorded. The nuclear status of oocytes was determined by 4’,6-diamidino-2-phenylindole (DAPI) staining as previously described [23]. Briefly, oocytes were fixed in 4% paraformaldehyde overnight at 4 °C, stained in Dulbecco’s Phosphate Buffered Saline (D-PBS) containing 2.5% (wt/vol) DAPI for 10 min, then washed in D-PBS three times, and nuclear status assessed under an epifluorescence microscope (Olympus, Tokyo, Japan). Oocytes with a metaphase plate, but no polar body, were classified as being at the metaphase I (MI) stage, whereas oocytes with a polar body were classified as being at the metaphase II (MII) stage. 2.3. Parthenogenetic activation and in vitro culture of porcine embryos Oocytes were denuded as described above; those with intact cytoplasm and the first polar body were transferred into HEPES-buffered tissue culture medium 199 (H199) supplemented with 0.5% fetal bovine serum for the following experiments. Oocytes were first equilibrated in activation medium (0.3 mol/L mannitol, 0.1 mmol/L MgCl2, 0.05 mmol/L CaCl2, and 0.1% BSA) for 10 sec, then stimulated with a direct currentpulse of 1.2 kV/cm for 80 ␮sec, using a cell fusion machine (Fujihira Industry, Tokyo, Japan). Subsequently, they were initially transferred into porcine zygote medium-3 (PZM-3) [6] supplemented with 5 ␮g/mL cytochalasin B and 10 ␮g/mL cycloheximide

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for 4 h at 38.5 °C in an atmosphere of 5% CO2 in air with maximum humidity. Presumed activated embryos were washed three times in PZM-3 before culture in the same medium, under conditions described above. Cleavage and blastocyst rates were detected on Days 2 and 7 respectively (Day 0 ⫽ day of activation). 2.4. Total cell numbers and the incidence of apoptosis in blastocysts Apoptotic cells of blastocysts were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using an in situ cell death detection kit (Roche, Mannheim, Germany). At the end of the culture period, blastocysts were fixed in 4% paraformaldehyde overnight at 4 °C and permeabilized in 0.5% Triton X-100 in D-PBS for 1 h. Blastocysts were incubated in TUNEL reaction medium that specifically labeled the broken DNA ends of apoptotic cells with the fluorochrome fluorescein isothiocyanate for 1 h at 38.5 °C, and then were stained with 10 ␮g/mL Hoechst 33342 for 10 min. After being washed three times in D-PBS, blastocysts were mounted on microscope slides and examined using an epifluorescence microscope. Nuclei with green fluorescence were counted as apoptotic; cell numbers of blastocysts were determined based on Hoechst 33342 staining. Two-cell embryos collected after 2 d of culture were stained as negative controls; for a positive control, blastocysts treated with deoxyribonuclease I were used. 2.5. Measurement of intracellular ROS and GSH levels in oocytes and embryos The intracellular ROS levels of oocytes at the MII stage and embryos at the 3- to 4-cell stage were measured by 2=,7=-dichlorofluorescein (DCF) fluorescence assay, as previously described [24]. Briefly, oocytes and embryos from each treatment group were incubated (in the dark) in H199 supplemented with 1 mmol/L 2=,7=-dichlorodihydrofluorescein diacetate (DCHFDA) for 20 min at 38.5 °C, washed three times with D-PBS containing 0.1% (wt/vol) PVA, and then placed into 50 ␮L droplets. Fluorescence was measured under the epifluorescence microscope (with filters at 460 nm excitation), and fluorescence images were recorded as tagged image file format files. The fluorescence intensities were quantified using EZ-C1 Free Viewer software (Nikon, Tokyo, Japan). The experiment was replicated three times with a group of 15 to 20 oocytes/ embryos in each replicate. To minimize environmental influence, oocytes and embryos were manipulated un-

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der low light, and fluorescence intensities were recorded precisely 30 sec after exposure to light. Intracellular GSH concentrations of oocytes and embryos were measured by the 4-chloromethyl6.8-difluoro-7-hydroxycoumarin (CellTracker Blue CMF2HC) method, as previously described [24], using 10 ␮mol/L CellTracker and 370 nm excitation light. The procedure of experimental handling was the same as measurement of ROS levels described in this section. 2.6. Experimental design In Experiment 1, effects of various concentrations of L-carnitine added to IVM medium on maturation of porcine oocytes and subsequent parthenogenetic development were examined. Oocytes were randomly allocated to each treatment, and cultured in IVM medium supplemented in 0, 0.25, 0.5, 1, and 2 mg/mL L-carnitine. After 44 h of maturation, some oocytes were randomly selected to examine nuclear maturation by DAPI staining, whereas others with the first polar body were subjected to parthenogenetic activation. Cleavage rates, blastocyst rates, total number of nuclei, and incidence of apoptosis in blastocysts were recorded. The IVM medium was a standard mNCSU37 medium containing glucose. In Experiment 2, the effects of L-carnitine added to IVM medium, with or without glucose, on the progression of nuclear maturation, were assessed in a 2 ⫻ 2 factorial design. The concentration of L-carnitine was chosen based on the results of Experiment 1. Oocytes were cultured in IVM medium supplemented, with or without 0.5 mg/mL L-carnitine, in the presence or absence of 5.5 mmol/L glucose, and the progression of nuclear maturation was determined at specific intervals (after 36 and 44 h of maturation). In Experiment 3, the effect of various concentrations of L-carnitine supplemented in IVC medium on the developmental competence of parthenogenetic embryos was examined. All oocytes were cultured in the standard IVM medium containing 5.5 mmol/L glucose. After 44 h of maturation culture, oocytes with intact cytoplasm and first polar body were selected for parthenogenetic activation, and activated oocytes were cultured in PZM-3 medium supplemented with various concentrations of L-carnitine (0, 0.25, 0.5, 1, and 2 mg/mL). Cleavage rates, blastocyst rates, total number of nuclei, and incidence of apoptosis in blastocysts were recorded. In Experiment 4, the effects of L-carnitine during IVM/ IVC were determined on the levels of ROS and GSH in

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Table 1 Effects of various concentrations of L-carnitine on maturation rates of porcine oocytes. L-carnitine (mg/mL)

No. oocytes

Maturation rates (%)

Degenerated (%)

0 0.25 0.5 1 2

155 137 146 156 152

56.4 ⫾ 1.7* 59.7 ⫾ 3.6* 60.7 ⫾ 5.3* 53.9 ⫾ 1.3*,† 44.9 ⫾ 2.7†

4.7 ⫾ 0.8 5.7 ⫾ 0.8 4.9 ⫾ 0.2 5.0 ⫾ 0.7 6.6 ⫾ 0.8

Values are mean ⫾ SEM of five replicates. ⴱ,† Within a column, means without a common superscript differed (P ⬍ 0.05).

oocytes/embryos. Oocytes were matured in the presence of 0 or 0.5 mg/mL of L-carnitine for 44 h, and embryos from normal activated oocytes not treated by L-carnitine were cultured in the presence of 0 or 0.5 mg/mL of L-carnitine for 2 days. Levels of ROS and GSH in oocytes and embryos, respectively, were determined. 2.7. Statistical analysis Oocytes and embryos were randomly distributed in each experimental group and at least three replicates were conducted for each experiment. Statistical analyses were performed using SPSS 17.0 (SPSS Inc, Chicago, IL, USA). Percentage data (e.g., rates of maturation, cleavage, and blastocyst formation) were arcsine-transformed before analysis to ensure homogeneity of variance. The normal distribution of the data was verified with a Shapiro-Wilk test. Data with a nonnormal distribution were analyzed with a Kruskal-Wallis test, whereas data with a normal distribution were analyzed with a generalized linear model procedure. Differences among treatments in each experiment were located with a Duncan test. For all analyses, P ⬍ 0.05 was considered significant. Results were expressed as mean ⫾ SEM.

3. Results 3.1. Effects of L-carnitine on maturation rates of porcine oocytes and subsequent parthenogenetic development There was no significant difference in maturation rates of porcine oocytes among groups treated with 0, 0.25, 0.5, or 1 mg/mL of L-carnitine. However, the maturation rate was decreased (P ⬍ 0.05) when oocytes were exposed to 2 mg/mL. There was also no significant difference in the percentages of degenerated oocytes, when they were cultured in various concentrations of L-carnitine (Table 1). When matured oocytes were subsequently cultured in IVC medium following parthenogenetic activation, blastocyst development rates of oocytes matured in 0.5 mg/mL of L-carnitine were increased (P ⬍ 0.05) compared with the control, and these blastocysts had a lower (P ⬍ 0.05) incidence of apoptosis. However, addition of 2 mg/mL L-carnitine to IVM medium decreased blastocyst rates (P ⬍ 0.05) compared with the control and other L-carnitine treated groups. Cleavage rates and cell numbers of blastocysts were not significantly different in IVM medium supplemented with various concentrations of L-carnitine (Table 2). 3.2. Effects of L-carnitine during IVM on progression of nuclear maturation of porcine oocytes Nuclear maturation of oocytes cultured in vitro was determined at 36 h. The maturation rates of oocytes cultured in L-carnitine-containing IVM medium with or without glucose was increased (P ⬍ 0.05) compared with the control. Moreover, in oocytes matured in glucose-free IVM medium with 0.5 mg/mL of L-carnitine, the percentages reaching MII did not differ significantly from those cultured in

Table 2 Effects of various concentrations of L-carnitine during IVM for porcine oocytes on developmental competence and embryonic apoptosis following parthenogenetic activation. L-carnitine (mg/mL)

No. oocytes*

No. cleaved (%)

No. blastocysts (%)

No. cells in blastocyst

No. apoptotic cells in blastocyst (%)

0 0.25 0.5 1 2

190 182 178 174 180

85.6 ⫾ 2.9 83.9 ⫾ 2.4 85.0 ⫾ 1.5 86.7 ⫾ 1.9 82.0 ⫾ 1.2

37.4 ⫾ 1.8† 40.0 ⫾ 2.0†,‡ 44.5 ⫾ 1.9‡ 39.7 ⫾ 1.2†,‡ 28.2 ⫾ 1.3§

46.9 ⫾ 3.6 44.7 ⫾ 2.9 53.7 ⫾ 4.1 44.3 ⫾ 3.0 43.4 ⫾ 3.0

9.2 ⫾ 0.7† 6.7 ⫾ 0.5‡ 5.6 ⫾ 0.7‡ 8.9 ⫾ 0.4† 9.6 ⫾ 0.3†

Values are mean ⫾ SEM of three replicates. * Total surviving numbers of oocytes transferred into culture after activation. †,‡,§ Within a column, means without a common superscript differed (P ⬍ 0.05).

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Table 3 Effects of L-carnitine during IVM on the progression of nuclear maturation of porcine oocytes. Glucose (mmol/L) 0 5.5

L-carnitine (mg/mL) 0 0.5 0 0.5

36 h

44 h

No. oocytes

MI (%)

MII (%)

No. oocytes

MI (%)

MII (%)

181 187 200 196

24.8 ⫾ 2.8 18.4 ⫾ 3.1 22.4 ⫾ 2.4 20.1 ⫾ 3.6

28.9 ⫾ 3.3* 41.8 ⫾ 2.3†,‡ 38.9 ⫾ 3.3† 48.0 ⫾ 1.9‡

188 183 207 208

15.8 ⫾ 2.3 14.8 ⫾ 1.9 12.7 ⫾ 2.2 10.9 ⫾ 2.2

46.4 ⫾ 2.2* 51.0 ⫾ 2.0*,† 56.9 ⫾ 1.9†,‡ 60.9 ⫾ 2.5‡

Values are mean ⫾ SEM of five replicates. ⴱ,†,‡ Within a column, means without a common superscript differed (P ⬍ 0.05).

glucose-containing IVM medium, with or without L-carnitine. Nuclear maturation was also examined when oocytes were cultured for 44 h; the proportions of oocytes developing to the MII stage were lower (P ⬍ 0.05) in glucose-free IVM medium without L-carnitine than in glucose-containing IVM medium with or without L-carnitine, however, there were no significant differences in maturation rates of oocytes between glucose-free IVM medium with L-carnitine and glucose-containing IVM medium without Lcarnitine (Table 3). 3.3. Effects of L-carnitine during IVC on developmental competence and apoptosis of porcine parthenogenetic embryos

3.4. Effects of L-carnitine during IVM/IVC on the intracellular levels of ROS and GSH in oocytes/ embryos Supplementing 0.5 mg/mL of L-carnitine to the IVM or IVC media decreased (P ⬍ 0.05) ROS levels in oocytes or embryos compared with the control. Adding 0.5 mg/mL of L-carnitine in IVM medium significantly increased GSH concentrations of porcine oocytes. When 0.5 mg/mL L-carnitine was added to IVC medium, the GSH concentrations in embryos did not differ between the control and 0.5 mg/mL L-carnitine treatment (Fig. 1 and Fig. 2). 4. Discussion

There were no significant effects of L-carnitine added to the IVC medium on cleavage rates and cell numbers of blastocysts, and blastocyst rates were not different among groups given 0, 0.25, 0.5, or 1 mg/mL of L-carnitine. However, 2 mg/mL decreased blastocyst rates (P ⬍ 0.05) compared with 0.25 and 0.5 mg/mL. Based on the TUNEL assay, the incidence of apoptosis in blastocysts was significantly decreased when embryos were cultured in IVC medium supplemented with 0.5 mg/mL of L-carnitine compared with the control (Table 4).

The progression of nuclear maturation of oocytes is a primary marker to predict subsequent developmental competence; fast progression of nuclear maturation was closely associated with high developmental competence of oocytes [25]. In pigs and cattle, if glucose, Mg2⫹, or follicular fluid from large follicles were added to the maturation medium, progression of nuclear maturation of oocytes and their subsequent development were enhanced [25–27]. In the current study, adding L-carnitine to the IVM medium accelerated

Table 4 Effects of various concentrations of L-carnitine during IVC on the developmental competence and apopotosis of parthenogenetic porcine embryos. L-carnitine (mg/mL)

No. oocytes*

No. cleaved (%)

No. blastocysts (%)

No. cells in blastocyst

No. apoptotic cells in blastocyst (%)

0 0.25 0.5 1 2

183 171 189 192 178

86.0 ⫾ 4.2 85.3 ⫾ 3.7 86.0 ⫾ 2.3 83.3 ⫾ 1.8 82.3 ⫾ 1.2

38.3 ⫾ 1.7†,‡ 40.0 ⫾ 1.2† 41.9 ⫾ 1.2† 37.2 ⫾ 1.9†,‡ 33.9 ⫾ 1.8‡

44.1 ⫾ 3.1 48.7 ⫾ 3.2 43.3 ⫾ 2.7 43.2 ⫾ 1.9 40.9 ⫾ 2.9

8.8 ⫾ 0.8† 8.4 ⫾ 0.9† 5.3 ⫾ 0.6‡ 7.5 ⫾ 0.6†,‡ 9.7 ⫾ 0.5†

Values are mean ⫾ SEM of three replicates. Within a column, means without a common superscript differed (P ⬍ 0.05).

ⴱ,†,‡

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Fig. 1. Fluorescent photomicrographs of (A) control and (B) L-carnitine treatment matured porcine oocytes, and (C) control and (D) L-carnitine treatment Day-3 cleaved porcine embryos after staining with 2=,7=-dichlorodihydrofluorescein diacetate (DCHFDA), observed under blue light, showing intracellular reactive oxygen species (ROS) levels. Fluorescent photomicrographs of (E) control and (F) L-carnitine treatment matured oocytes and (G) control and (H) L-carnitine treatment Day-3 cleaved embryos after staining with CellTracker were observed under ultraviolet light, showing intracellular glutathione (GSH) concentrations.

nuclear maturation of porcine oocytes, and increased the percentage of blastocyst development after parthenogenetic activation. We inferred that L-carnitine was involved in nuclear maturation, which is a factor in improving developmental competence of porcine oocytes. Glucose, an essential exogenous energy substrate, improved oocyte maturation and embryonic development, involving physiological mechanisms regulating the nuclear and cytoplasmic maturation of porcine oocytes [25,26,28]. Metabolic products derived from glucose have important roles in progression of nuclear maturation [26]. In the present study, adding glucose to

Fig. 2. Effects of L-carnitine during IVM/IVC on intracellular levels of reactive oxygen species (ROS) and glutathione (GSH) in porcine oocytes/embryos. Fluorescence intensities were correlated with intracellular levels of ROS and GSH. Values are mean ⫾ SEM. a and b, For adjacent pairs of columns, means without a common letter differed (P ⬍ 0.05).

the IVM medium accelerated the progression of nuclear maturation of porcine oocytes. In this study, L-carnitine significantly accelerated nuclear maturation, with or without supplemental glucose in the IVM medium; therefore, L-carnitine enhanced final maturation rates of porcine oocytes matured in glucose-free IVM medium. L-carnitine appeared to be involved in the transport of long chain fatty acids into the mitochondria for ␤ oxidation; therefore, L-carnitine supplementation could improve the utilization of fatty acid and energy in order to meet physiological requirements [29]. Porcine oocytes contain substantial endogenous lipid, most of which is triglyceride (TG) [30]. Endogenous triglycerides play an important role in energy metabolism during in vitro maturation of porcine oocytes, providing ATP for the protein synthesis that is necessary for meiosis and cytoplasmic maturation [31]. Sturmey and Leese [31] demonstrated that the triglyceride content of porcine oocytes decreased during in vitro maturation. When triglyceride metabolism was inhibited using methyl palmoxirate (a stereo-specific irreversible inhibitor of the carnitine palmitoyl transferase complex responsible for transporting fatty acyl-coenzyme A (acylCoA) across the mitochondrial membrane) during in vitro maturation of porcine oocytes, developmental competence of oocytes after fertilization was impaired, suggesting that endogenous fatty acid metabolism was important during in vitro maturation of porcine oocytes [32]. Therefore, we inferred that acceleration of nuclear maturation and enhancement of subsequent parthenogenic development may have been due to promotion of fatty acid metabolism through the addition of L-carnitine,

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and the effect of glucose metabolism on porcine oocytes maturation can be compensated by lipid metabolism. In vitro environmental factors, e.g., exposure to light, greater oxygen stress, and culture medium composition, induced metabolic variations in oocytes and embryos, leading to an imbalance in the production and degradation of ROS [33]. Consequently, this could cause lipid peroxidation, enzyme inactivation, oxidative modification of proteins, and DNA fragmentation, known to have detrimental effects, such as mitochondrial alterations, apoptosis, and embryo cell block, on oocytes and embryos [34,35]. Supplementing a number of antioxidants such as catalase, superoxide dismutase, or glutathione peroxidase to culture medium could protect oocytes and embryos from oxidative damage [36]. L-carnitine also exhibited strong antioxidant effects, as it was able to antagonize a very high concentration of H2O2 (up to 500 ␮mol/L) in culture medium of mouse embryos [18]. In the present study, adding 0.5 mg/mL L-carnitine into IVM/IVC medium reduced ROS levels in oocytes/embryos. The mechanism of the antioxidation of L-carnitine may be via a beneficial effect on 1,1-diphenyl2-picryl-hydrazyl free radical (DPPH · ) scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, total reducing power, and metal chelating on ferrous ion activities [12]. Intracellular GSH concentrations increased during maturation of porcine oocytes [37]; GSH concentrations at completion of oocyte maturation may be a maker for cytoplasmic maturation [38]. Increased GSH concentrations in matured oocytes stimulated male pronuclear (MPN) formation and improved developmental competence by protecting oocytes or embryos from oxidative stress [37]. Furthermore, adding cysteine and ␤-mercaptoethanol to IVM medium of porcine oocytes increased GSH and decreased ROS levels [39]. Anthocyanin (an antioxidant) treatment during IVM improved developmental competence of somatic cell nuclear transfer (SCNT) embryos by increasing intracellular GSH synthesis and reducing ROS levels in pigs [40]. In the present study, adding 0.5 mg/mL L-carnitine to IVM medium significantly increased intracellular GSH concentrations and improved development of parthenogenetic embryos; this was attributed to L-carnitine counteracting ROS and thus sustaining stores of GSH in matured porcine oocyte. Therefore, we inferred that L-carnitine was involved in cytoplasmic maturation through increasing intracellular GSH concentrations and nuclear maturation through accelerating nuclear progression, which improved parthenogenetic development of porcine oocytes.

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In this study, adding L-carnitine to the IVC medium containing significantly decreased ROS levels in oocytes compared with the control, although the L-carnitine failed to improve the blastocyst percentage or total cell numbers. Furthermore, the presence or absence of L-carnitine had no relationship with intracellular GSH concentrations in embryos. Early embryos have only a limited ability to synthesize GSH, and the intracellular GSH concentrations gradually increased from the four-cell to the blastocyst stage in porcine embryos [41]. Conversely, other antioxidants, namely glutamine and hypotaurine were included in the IVC medium. It has been reported that the presence of glutamine and hypotaurine in the IVC medium promoted development of porcine embryos and decreased intracellular ROS levels [42]. Therefore, glutamine and hypotaurine might mask the action of L-carnitine to development of porcine embryos during IVC. However, L-carnitine supplementation during IVC might act as a defense mechanism against ROS, and maintain the GSH system. Apoptosis was much more pronounced in embryos produced in vitro compared with those produced in vivo [43]; in that regard, little or no apoptotic DNA fragmentation was found in porcine blastocysts produced in vivo [44]. Therefore, suboptimal culture conditions in vitro can cause apoptosis in embryos, and the rate of apoptotic cells in embryos can be used to evaluate embryo quality [45]. L-carnitine strongly inhibits mitochondrial-dependent apoptosis both in vivo and in vitro [46]. L-carnitine is able to stabilize mitochondrial membranes and increase the supply of energy to the organelle and protect the cell from apoptosis [13]. It has been reported that L-carnitine reduces apoptosis through the mitochondrial pathway in mouse fibroblasts [13]. In murine embryos, L-carnitine at 0.3 and 0.6 mg/mL significantly decreased the incidence of apoptosis compared with the group treated with H2O2 alone (500 ␮mol/L) [18]. In the present study, supplementation of 0.5 mg/mL of L-carnitine in IVM/IVC medium significantly inhibited the incidence of apoptosis in porcine parthenogenetic blastocysts. In conclusion, adding L-carnitine to IVM medium accelerated the progression of nuclear maturation, increased blastocyst rates of porcine oocytes following parthenogenetic activation, and reduced the incidence of apoptosis in parthenogenic embryos. Meanwhile, L-carnitine treatment during IVM reduced intracellular ROS and increased GSH concentrations. The addition of L-carnitine to IVC medium also inhibited generation of ROS, and reduced the incidence of apoptosis in

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