Supplementation with exogenous coenzyme Q10
to media for in vitro maturation and embryo
culture fails to promote the developmental
competence of porcine embryos
Carolina Maside, Cristina Martinez-Serrano, Josep M. Cambra, Xiomara Lucas, Emilio A. Martinez, Maria Antonia Gil, Heriberto Rodriguez-Martinez, Inmaculada Parrilla and Cristina Cuello
The self-archived postprint version of this journal article is available at Linköping University Institutional Repository (DiVA):
http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-161619
N.B.: When citing this work, cite the original publication.
Maside, C., Martinez-Serrano, C., Cambra, J. M., Lucas, X., Martinez, E. A., Antonia Gil, M., Rodriguez-Martinez, H., Parrilla, I., Cuello, C., (2019), Supplementation with exogenous coenzyme Q10 to media for in vitro maturation and embryo culture fails to promote the developmental competence of porcine embryos, Reproduction in domestic animals, 54, 72-77.
https://doi.org/10.1111/rda.13486
Original publication available at:
https://doi.org/10.1111/rda.13486
Copyright: Wiley (12 months)
Supplementation with exogenous Coenzyme Q10 to media for in vitro maturation and embryo culture fails to promote the developmental competence of porcine embryos
C Maside1,2, CA Martinez1,3, JM Cambra1, X Lucas, EA Martinez1, MA Gil1, H Rodriguez-Martinez3, I Parrilla1*, C Cuello1
1Department of Animal Medicine and Surgery, University of Murcia, Murcia, Spain. 2SaBio IREC (CSIC –UCLM-JCCM), Albacete, Spain
3Department of Clinical & Experimental Medicine (IKE), BHK/Obstetrics & Gynaecology, Linköping University, Linköping, Sweden.
Corresponding author: Inmaculada Parrilla E-mail address: parrilla@um.es
Running title: Coenzyme Q10 and in vitro porcine embryo production Contents
The coenzyme Q10 (CoQ10) is a potent antioxidant with critical protection role against cell oxidative stress, caused by the mitochondrial dysfunction. This study evaluated the effects of CoQ10 supplementation to in vitro maturation or embryo culture media on the maturation, fertilization and subsequent embryonic development of pig oocytes and embryos. Maturation (Experiment 1) or embryo culture (Experiment 2) media were supplemented with 0 (control), 10, 25, 50 and 100 µM CoQ10. The addition of 10 to 50 µM CoQ10 to the in vitro maturation medium did not affect the percentage of MII oocytes nor the fertilization or the parameters of subsequent embryonic development. Exogenous CoQ10 in the culture medium neither did affect the development to the 2 to 4-cell stage nor rates of blastocyst formation. Moreover, the highest concentration of CoQ10 (100 µM) in maturation medium negatively affected blastocyst rates. In
conclusion, exogenous CoQ10 supplementation of maturation or embryo culture media failed to improve the outcomes of our in vitro embryo production system and its use as an exogenous antioxidant should not be encouraged.
porcine.
1. INTRODUCTION
In vitro production (IVP) of porcine embryos is an essential instrument for other reproductive biotechnologies, such as cloning or gene editing in a species which serves a suitable model for human research and even for xenotransplantation. Despite some remarkable achievements obtained for the production in vitro of pig embryos, the overall efficiency of porcine IVP is still low. Such low efficiency is due not only to the high incidence of polyspermy (Gil et al., 2010), but also to embryo culture conditions that are still sub-optimal or simply inadequate (Almiñana et al., 2010; Macháty, Day, & Prather, 1998). Although numerous studies have been conducted to increase the number and quality of porcine blastocysts produced in vitro, by the addition of different
supplements to the media base for in vitro maturation (IVM) or culture (IVC) (Gil et al., 2010), the progress achieved to date has been limited. A recurring concern of most researchers refers to the high oxidative stress exerted by the IVP culture conditions, which results in increased production of reactive oxygen species (ROS) by both the gametes, particularly the spermatozoa and further by the embryonic cells (Takahashi, Nagai, Okamura, Takahashi & Okano, 2002) with the subsequent damage to gametes and embryos (Kim, Jeon, Kim, Lee, & Hyun, 2015; Silva, Gadella, Colenbrander, & Roelen, 2007).
For that reason, a commonly used strategy is the addition of exogenous antioxidants to the maturation and culture media, sometimes using empirical approaches. One of the main damages caused by oxidative stress on any cell targets the mitochondria causing a dysfunction of these essential organelles (Vercesi & Kowaltowski, 1999). Mitochondria are important regulators of numerous cellular functions, being fundamental for oocyte maturation, sperm survival, fertilization and embryonic development (Chappel, 2013; Schatten, Sun, & Prather, 2014). The coenzyme Q10 (CoQ10) is an essential cofactor of the electron transport chain present especially within the inner mitochondrial membrane where it serves as a potent antioxidant playing a critical role in protecting the cell in question against mitochondrial dysfunction mediated by oxidative stress (Hernández-Camacho, Bernier, López-Lluch, & Navas, 2018). In addition, CoQ10 promotes the synthesis of adenosine triphosphate (ATP), thus playing a crucial role in the activation of the fertilized oocyte towards further embryo development (Abdulhasan et al., 2017) owing to the fact that the mitochondria of the zygote are all derived from the oocyte and
thus constitute the major source of ATP during the development of the pre-implantation embryos (Babayev & Sel, 2015).
Despite these fundamental functions, little is known about the eventual actions of exogenous CoQ10 on the in vitro development of oocytes and embryos, and basically few, if any, experimental evidence has been presented for its beneficial or deleterious effects. In bovine, addition of exogenous CoQ10 to the maturation medium increased nuclear maturation rates and viability of oocytes (Abdulhasan et al., 2017). The effects of exogenous CoQ10 under moderate heat stress, not only improved mitochondrial distribution, membrane polarization, and differential expression of genes involved in the mitochondrial electron transport chain, but also improved oocyte developmental
competence (Gendelman & Roth, 2012). Exogenous CoQ10 in the maturation medium also improved oocyte survival and reduced the premature exocytosis of the cortical granules following vitrification and warming of immature oocytes (Ruiz-Conca, Vendrell, Sabés-Alsina, Mogas, & Lopez-Bejar, 2017). Unfortunately, corresponding data on the effects of exogenous CoQ10 in pigs is more limited. To the best of our knowledge, the study by Liang et al. (Liang, Niu, Shin, & Cui, 2017) is the only report describing pre-implantation embryo viability and quality after CoQ10 supplementation of the in vitro culture medium. These authors showed that exogenous CoQ10 resulted in beneficial effects on the development of porcine parthenogenetic embryos by
preventing oxidative damage and blastomere apoptosis.
The above studies suggest that exogenous CoQ10 could be an interesting molecule to use during porcine embryo IVP systems. The aim of this study was to test the
hypothesis that exogenous CoQ10 supplementation of porcine IVM or IVC media would improve the outcomes of oocyte maturation, fertilization and embryo development.
2. MATERIALS AND METHODS
2.1. Chemicals, culture media and culture conditions
Unless otherwise indicated, all chemicals used in the present study were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Pig cumulus–oocyte complexes (COCs) were washed in Tyrode’s lactate supplemented with 10 mM HEPES and 0.1% (w:v) polyvinyl alcohol (TL-PVA) (Martinez et al., 2014). The maturation medium used was TCM-199 (Gibco Life Technologies S.A., Barcelona, Spain) supplemented with 0.57
mM cysteine, 0.1% (w:v) PVA, 10 ng/mL EGF, 75 µg/mL of penicillin-G potassium, and 50 µg/mL of streptomycin sulfate. The basic medium used for in vitro fertilization (IVF) was a modified Tris-buffered medium (Abeydeera & Day, 1997) enriched with 1.0 mM caffeine and 0.2% (w:v) bovine serum albumin (BSA). A Dulbecco’s PBS (Gibco, Grand Island, NY) containing 4 mg/mL of BSA was used for washing
spermatozoa after their thawing and before re-suspension in IVF medium. The embryo culture medium used was the North Carolina State University-23 (NCSU23) (Petter and Wells, 1993) supplemented with 0.4% BSA. Both IVM, IVF and IVC were
performed under paraffin oil at 39 °C in 5% CO2 in air and 95–97% relative humidity.
2.2. Preparation of Coenzyme Q10
The CoQ10 was dissolved with chloroform to a stock concentration of 58 mM (50 mg/mL) and kept at -20 ºC until use. The stock was used to make fresh CoQ10 dosages in final concentrations of 10, 25, 50 and 100 µM in IVM or IVC media.
2.3. Oocyte collection and in vitro maturation
Offal ovaries were obtained from pre-pubertal gilts at a local slaughterhouse (El Pozo S.A., Murcia, Spain) and transported to the laboratory at the University of Murcia in 0.9% (w/v) NaCl supplemented with 70 µg/mL kanamycin at 35 °C, within 1 h. The COCs were collected by slicing medium-sized follicles (3–6 mm in diameter) with a scalpel blade into TL-PVA. Only those COCs whose oocytes were surrounded by an complete, compact cumulus mass and having evenly granulated cytoplasm were selected and washed three times in maturation media. Groups of 45–50 COCs were transferred into each well of a four-well multi-dish (Nunc, Roskilde, Denmark) containing 500 µL of pre-equilibrated maturation mediasupplemented with 10 IU/mL eCG (Folligon, Intervet International B.V., Boxxmeer, The Netherlands) and 10 IU/mL hCG (Veterin Corion, Divasa Farmavic S.A., Barcelona, Spain) for 20–22 h. The oocytes were then incubated for another 20–22 h in maturation medium without hormones.
2.4. In vitro fertilization and in vitro culture
After maturation, the oocytes were denuded using 0.1% hyaluronidase to be placed into 50 µL drops in IVF medium until the addition of spermatozoa. Semen from a
mature, highly fertile boar, cryopreserved in 0.5 mL straws was thawed (two straws per replicate) in a circulating water bath at 37 °C for 20 s and washed three times in Dulbecco’s PBS by centrifugation at × 1,900 xg for 3 min. The pellet resulting from the final spinning was then resuspended in IVF media at a final sperm concentration of 3 × 105 spermatozoa/mL. The oocytes were co-incubated with spermatozoa for 5 h. Thereafter, the presumptive zygotes were washed and transferred (30 zygotes per well) into a four-well multi-dish containing 500 µL of glucose-free NCSU23
supplemented with 0.3-mM pyruvate and 4.5 mM lactate for 2 days and then changed to fresh embryo culture medium containing 5.5 mM glucose for an additional 5 day period. 2.5. Assessment of maturation, fertilization and embryo development
To evaluate maturation and fertilization parameters, matured oocytes and presumptive zygotes were fixed at 44 h of in vitro maturation and 18 h after in vitro fertilization, respectively, in 0.5% glutaraldehyde in PBS for 20 min at room temperature and placed on glass slides. After addition of 2 µL Vectashield (Vector, Burlingame, CA, USA) and 10 µg/mL Hoechst 33342 they were covered with a coverslip. The samples were then examined under a fluorescence microscope (Nikon, Eclipse 80i, Tokyo, Japan) using an excitation filter of 330 to 380 nm wave-length. The oocytes were considered mature when their chromosomes appeared organized at metaphase, as well as showing an extruded first polar body (Metaphase II, MII). The presumptive zygotes were
considered penetrated when they contained one or more male pronuclei and two polar bodies. The evaluated fertilization parameters were the rate of penetration (number of oocytes penetrated/total matured), the monospermic rate (number of oocytes containing only one male pronucleus/total penetrated), the number of spermatozoa/oocyte (mean number of spermatozoa per oocyte), and the efficiency of fertilization (number of monospermic oocytes/total inseminated).
Both cleavage rate (number of embryos cleaved to 2 to 4 cells of the total number of oocytes inseminated) and the rate of blastocyst formation (number of blastocysts of the total number of cleaved embryos) were morphologically evaluated using a stereomicroscope at 48 h and 168 h post-insemination, respectively. The total efficiency was finally assumed as the percentage of the total number of blastocysts/total oocytes inseminated.
2.6. Experimental design
To evaluate the dose-related effects of exogenous CoQ10 on maturation, fertilization and subsequent embryonic development, various concentrations of CoQ10 (0, 10, 25, 50 and 100 µM final concentration in the media) were added to the IVM (experiment 1) and IVC (experiment 2) media. A total number of 2,000 and 1,284 COCs were
respectively used in each experiment. 2.7. Statistical analyses
Statistical analysis was performed using the IBM SPSS 19 statistical software package (SPSS, Chicago, IL, USA). Binary variables (maturation, penetration, monospermy, cleavage, blastocyst formation, and efficiency rates) were obtained by calculating the percentage in every well of each experimental group and in each of the replicates. Data are expressed as the mean ± standard deviation (SD). Variables were analyzed to evaluate normality by the Kolmogorov–Smirnov test. Groups were compared using an unpaired Student´s t-test corrected for inequality of variances (Levene test) or a mixed-model ANOVA followed by the Bonferroni post-hoc test, as appropriate. Differences were considered significant at P<0.05.
3. RESULTS
Addition of the chloroform dilution vehicle for CoQ10 to either IVM or IVC media did not affect the developmental competence of oocytes (Figure 1) nor the ability of the presumptive zygotes to develop to the blastocyst stage (Figure 2), respectively. 3.1. Experiment 1: Exogenous CoQ10 in the medium of oocyte maturation.
There were no differences in the percentage of MII oocytes at 44 h of oocyte maturation between treatments (range: from 70.8 ± 1.3% to 76.4 ± 12.2%) and control (66.0 ± 10.6%) (Figure 3). The data from the in vitro fertilization (Table 1) clearly showed that exogenous CoQ10 neither had any effects on the fertilization parameters. The overall in vitro fertilization efficiency ranged from 24.5 ± 7.8% to 32.2 ± 7.9% without any differences between groups. In relation to the in vitro culture parameters, the addition of exogenous CoQ10 did not improve any parameter of embryonic development (Table 2). Moreover, the dose of 100 µM CoQ10 significantly reduced blastocyst formation and total efficiency of blastocyst production (P<0.05), as compared with the other groups.
3.2. Experiment 2: Exogenous CoQ10 in the medium of embryo culture. Regardless of the concentration used, addition of CoQ10 did not improve cleavage blastocyst formation rates (Table 3) by the end of the period of in vitro culture. The total efficiency of blastocyst production ranged from 24.6 ± 4.0% to 30.4 ± 7.3% with no differences between groups, i.e. not deviating from the controls.
4. DISCUSSION
The present study showed neither improvement of the different steps nor the overall efficiency of our porcine IVP system when adding CoQ10 as supplement for the different media involved.
The supplementation with CoQ10 to the IVM medium failed to increase not only maturation but also fertilization rates and the subsequent embryo development. These results are in disagreement with those reported in bovine, where CoQ10 treatment for 24 h of maturation increased oocyte survival and the percentage of oocytes achieving the MII-stage compared with control oocytes (Abdulhasan et al., 2017). These authors indicated that the improved conditions of maturation in vitro counteracted oxidative stress and that the CoQ10 increased ATP production and decreased AMPK activity, which improved maturation outcomes. The reasons for the discrepancies between the works are unclear. However, we could speculate that the effect of the CoQ10 added to the IVM medium depends on the stress level to which the oocytes are exposed. The following arguments support this statement; firstly, a higher proportion of bovine oocytes (30% – 50%), compared with porcine oocytes (10% -15%), die at the end of maturation (El-Raey et al., 2011; Nabenishi et al., 2012; Gil et al., 2017), which indicates that the stress during IVM is greater in bovine than in porcine IVM cultures and that CoQ10 could improve some functions in the bovine oocytes leading to an increased percentage of alive and MII oocytes. Secondly, the proportion of bovine oocytes developing to the blastocyst stage is highest in winter, intermediate in fall, and lowest in summer (Gendelman & Roth, 2012). These results have been related to alterations of the oocytes produced by seasonal heat stress (summer). While maturation with CoQ10 of bovine oocytes collected in winter (non-heat stress) or in summer (high-heat stress) did not affect the proportion of oocytes that developed to the blastocyst stage, in fall (moderate-heat stress), CoQ10 in the maturation medium increased the blastocyst formation rates to a level similar to that achieved during winter. Such data
suggests that CoQ10 positively supports oocyte developmental competence only under moderate damage (fall) but has not effect when the damage before culture is
insignificant (winter) or extreme (summer). Lastly, some studies reported positive effects of CoQ10 during IVM on survival post-warming following vitrification (Ruiz-Conca et al., 2017). In addition, supplementation of CoQ10 during the recovery culture (post-warming medium) has led to higher blastocyst yields among vitrified-warmed pig oocytes, obeying to increased survival rates and the presence of regulating mRNA expression (Hwang et al., 2016). The fact that vitrification and warming procedures importantly increase ROS levels (Castillo-Martín, Bonet, Morató, & Yeste, 2014) would confirm the suggestion that the addition of CoQ10 would only have beneficial consequences under oxidant circumstances in which the harmful effects of ROS are intensified.
In the present study, the addition of CoQ10 to the IVC medium did neither affect the cleavage or blastocyst formation rates. Recently, a positive effect of CoQ10 was observed using porcine parthenogenetic embryos (Liang et al., 2017). These authors reported that CoQ10 in the culture medium significantly increased the rates of cleavage and blastocyst formation and further improved blastocyst quality by preventing
oxidative damage and blastomere apoptosis. Again, the beneficial effects of CoQ10 on parthenogenetically derived embryos could be due to the greater sensitivity of these embryos to oxidative stress than those embryos produced by IVF (Booth, Holm, & Callesen, 2005) because of their substantial biological differences (Gómez et al., 2009). Furthermore, whereas in the present study the addition of 100 μM of exogenous CoQ10 was inefficient when added to the IVC medium, such concentration was the one
considered optimal for the culture of porcine parthenogenetic embryos (Liang et al., 2017). At last, we showed the harmful effect of that concentration of CoQ10 on the developmental competence of oocytes when added to the IVM medium. This fact was not surprising since it is known that an excess of antioxidants can be detrimental for fertilization and embryonic development in bovine (Gonçalves, Barretto, Arruda, Perri, & Mingoti, 2010).
In conclusion, supplementation of IVM or IVC media with CoQ10 at final doses of 10 to 100 µM failed to improve the overall efficiency of our porcine IVP system. In addition, high concentrations of CoQ10 (100 µM) in the IVM medium negatively affected blastocyst formation rate.
ACKNOWLEDGEMENTS
The authors are grateful to El Pozo company (Murcia, Spain) for supplying the ovaries used in this study and to Moises Gonzalvez for his assistance throughout this work. We thank the Seneca Foundation, Murcia, Spain (Saavedra Fajardo Program; 20027/SF/16) and the Junta de Comunidades de Castilla-La Mancha, Spain (PRT program;
SBPLY/17/180501/000500) for co-funding support of C Maside and the Ministry of Economy and Competitiveness (Madrid, Spain) for its grant-based support of CA Martinez and JM Cambra (BES-2013-064087 and BES-2016-077869, respectively). This study was supported by MINECO-FEDER (AGL2015-69735-R), Madrid, Spain, Fundacion Seneca (19892/GERM/15), Murcia, Spain and the Research Council FORMAS, Stockholm (Project 2017-00946).
CONFLICT OF INTEREST
The authors have no conflict of interest to declare. AUTHOR CONTRIBUTIONS
CM conceived and designed the study. CM, CAM, JMC, XL, MAG, IP and CC performed the experiments. CM, HR-M and EAM analyzed and interpreted the data. CM wrote the primary manuscript. All authors revised and approved the manuscript for publication. EAM, MAG and HR-M obtained the funding to carry out the study.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
REFERENCES
Abdulhasan, M. K., Li, Q., Dai, J., Abu-Soud, H. M., Puscheck, E. E., & Rappolee, D. A. (2017). CoQ10 increases mitochondrial mass and polarization, ATP and Oct4 potency levels, and bovine oocyte MII during IVM while decreasing AMPK activity and oocyte death. Journal of Assisted Reproduction and Genetics, 34(12), 1595–1607. https://doi.org/10.1007/s10815-017-1027-y
Almiñana, C., Gil, M. A., Cuello, C., Parrilla, I., Caballero, I., Sanchez-Osorio, J., … Martinez, E. A. (2010). Capability of frozen-thawed boar spermatozoa to sustain pre-implantational embryo development. Animal Reproduction Science, 121(1–2),
145–151. https://doi.org/10.1016/j.anireprosci.2010.05.004
Booth, P. J., Holm, P., & Callesen, H. (2005). The effect of oxygen tension on porcine embryonic development is dependent on embryo type. Theriogenology, 63(7), 2040–2052. https://doi.org/10.1016/j.theriogenology.2004.10.001
Castillo-Martín, M., Bonet, S., Morató, R., & Yeste, M. (2014). Comparative effects of adding β-mercaptoethanol or L-ascorbic acid to culture or vitrification-warming media on IVF porcine embryos. Reproduction, Fertility and Development, 26(6), 875–882. https://doi.org/10.1071/RD13116
Chappel, S. (2013). The Role of Mitochondria from Mature Oocyte to Viable Blastocyst. Obstetrics and Gynecology International, 2013, 1–10. https://doi.org/10.1155/2013/183024
El-Raey, M., Geshi, M., Somfai, T., Kaneda, M., Hirako, M., Abdel-Ghaffar, A. E., … Nagai, T. (2011). Evidence of melatonin synthesis in the cumulus oocyte
complexes and its role in enhancing oocyte maturation in vitro in cattle. Molecular Reproduction and Development, 78(4), 250–262.
https://doi.org/10.1002/mrd.21295
Gendelman, M., & Roth, Z. (2012). Incorporation of coenzyme Q10 into bovine oocytes improves mitochondrial features and alleviates effects of summer thermal stress on developmental competence. Biology of Reproduction, 87(Suppl_1), 115–115. https://doi.org/10.1093/biolreprod/87.s1.115
Gil, M. A., Cuello, C., Parrilla, I., Vazquez, J. M., Roca, J., & Martinez, E. A. (2010). Advances in swine in vitro embryo production technologies. Reproduction in Domestic Animals, 45(SUPPL. 2), 40–48. https://doi.org/10.1111/j.1439-0531.2010.01623.x
Gil, M. A., Nohalez, A., Martinez, C. A., Ake-Villanueva, J. R., Centurion-Castro, F., Maside, C., … Martinez, E. A. (2017). Effects of meiotic inhibitors and
gonadotrophins on porcine oocytes in vitro maturation, fertilization and development. Reproduction in Domestic Animals, 52(5), 873–880. https://doi.org/10.1111/rda.12993
Gómez, E., Gutiérrez-Adán, A., Díez, C., Bermejo-Alvarez, P., Muñoz, M., Rodriguez, A., … Caamaño, J. N. (2009). Biological differences between in vitro produced bovine embryos and parthenotes. Reproduction, 137(2), 285–295.
https://doi.org/10.1530/REP-08-0220
(2010). Effect of antioxidants during bovine in vitro fertilization procedures on spermatozoa and embryo development. Reproduction in Domestic Animals, 45(1), 129–135. https://doi.org/10.1111/j.1439-0531.2008.01272.x
Hernández-Camacho, J. D., Bernier, M., López-Lluch, G., & Navas, P. (2018). Coenzyme Q10 supplementation in aging and disease. Frontiers in Physiology, 9(FEB), 1–11. https://doi.org/10.3389/fphys.2018.00044
Hwang, I. S., Kwon, D. J., Kwak, T. U., Lee, J. W., Im, G. S., & Hwang, S. (2016). Improved survival and developmental rates in vitrified-warmed pig oocytes after recovery culture with coenzyme Q10. Cryo-Letters, 37(1), 59–67.
Kim, E., Jeon, Y., Kim, D. Y., Lee, E., & Hyun, S. H. (2015). Antioxidative effect of carboxyethylgermanium sesquioxide (Ge-132) on IVM of porcine oocytes and subsequent embryonic development after parthenogenetic activation and IVF. Theriogenology, 84(2), 226–236.
https://doi.org/10.1016/j.theriogenology.2015.03.006
Liang, S., Niu, Y. J., Shin, K.-T., & Cui, X.-S. (2017). Protective effects of coenzyme q10 on developmental competence of porcine early embryos. Microscopy and Microanalysis, 23(04), 849–858. https://doi.org/10.1017/s1431927617000617 Macháty, Z., Day, B. N., & Prather, R. S. (1997). Development of early porcine
embryos in vitro and in vivo1. Biology of Reproduction, 59(2), 451–455. https://doi.org/10.1095/biolreprod59.2.451
Martinez, E. A., Angel, M. A., Cuello, C., Sanchez-Osorio, J., Gomis, J., Parrilla, I., … Gil, M. A. (2014). Successful non-surgical deep uterine transfer of porcine morulae after 24 hour culture in a chemically defined medium. PLoS ONE, 9(8).
https://doi.org/10.1371/journal.pone.0104696
Nabenishi, H., Takagi, S., Kamata, H., Nishimoto, T., Morita, T., Ashizawa, K., & Tsuzuki, Y. (2012). The role of mitochondrial transition pores on bovine oocyte competence after heat stress, as determined by effects of cyclosporin A. Molecular Reproduction and Development, 79(1), 31–40. https://doi.org/10.1002/mrd.21401 Ruiz-Conca, M., Vendrell, M., Sabés-Alsina, M., Mogas, T., & Lopez-Bejar, M. (2017).
Coenzyme Q10 supplementation during in vitro maturation of bovine oocytes (Bos taurus) helps to preserve oocyte integrity after vitrification. Reproduction in
Domestic Animals, 52, 52–54. https://doi.org/10.1111/rda.13056 Schatten, H., Sun, Q. Y., & Prather, R. (2014). The impact of mitochondrial
Reproductive Biology and Endocrinology, 12(1), 1–11. https://doi.org/10.1186/1477-7827-12-111
Silva, P. F. N., Gadella, B. M., Colenbrander, B., & Roelen, B. A. J. (2007). Exposure of bovine sperm to pro-oxidants impairs the developmental competence of the embryo after the first cleavage. Theriogenology, 67(3), 609–619.
https://doi.org/10.1016/j.theriogenology.2006.09.032
Vercesi, A. E., & Kowaltowski, A. J. (1999). Mitochondrial damage induced by conditions of oxidative stress. Free Radical Biology and Medicine, 26(3–4), 463– 471. https://doi.org/10.1016/S0891-5849(98)00216-0
Figure legends
Figure 1. Developmental ability of porcine oocytes matured in the absence (control; n=414) or presence of 0.002% chloroform dissolving vehicle (n=423). Data are presented as the mean ± standard deviation of four replicates.
Figure 2. Developmental ability of presumptive porcine zygotes cultured in the absence (control; n=218) or presence of 0.002% chloroform dissolving vehicle (n=232). Data are presented as the mean ± standard deviation of four replicates.
Figure 3. Effects of coenzyme Q10 (CoQ10) treatment at various concentrations during IVM on the percentage of oocytes achieving the metaphase II stage after 40-44 h of
maturation. Data are presented as the mean ± standard deviation of four replicates. Numbers in parentheses show the number of oocytes used.
Table 1. Effect of exogenous CoQ10 treatment at various concentrations during oocyte maturation on fertilization parameters after 18 h of in vitro fertilization.
Oocytes CoQ10
(µM) N Penetrated (%) Monospermic (%) Efficiency (%) Sperm/oocyte
0 59 89.3 ± 3.6 32.6 ± 11.3 26.8 ± 9.6 2.1 ± 0.2
10 118 94.6 ± 3.2 35.3 ± 11.4 29.3 ± 10.6 1.8 ± 0.7
25 88 97.2 ± 4.8 35.4 ± 7.2 32.2 ± 7.9 1.9 ± 0.1
100 114 96.1 ± 3.7 37.6 ± 9.1 31.5 ± 9.9 1.9 ± 0.4 Data are expressed as the mean ± SD (four replicates).
Table 2. Effect of exogenous CoQ10 treatment at various concentrations during oocyte maturation on embryonic development after 48 h and 168 h
post-insemination.
Embryo development to CoQ10
(µM) N 2-4-cell % Blastocyst % Efficiency %
0 203 67.9 ± 9.8 43.5 ± 7.1a 28.9 ± 4.8ª
10 200 60.6 ± 6.0 49.9 ± 6.0ª 30.1 ± 3.4ª
25 199 66.5 ± 9.7 43.3 ± 6.3a 28.3 ± 7.0ª
50 199 74.4 ± 9.2 45.1 ± 7.4 a 33.3 ± 4.8ª
100 202 67.8 ± 5.7 26.8 ± 12.6b 17.8 ± 7.6b
Data are expressed as the mean ± SD (four replicates). Different letters within the same column indicate statistical differences (P<0.05).
Table 3. Effect of exogenous CoQ10 treatment at various concentrations during embryo culture on embryonic development after 48 h and 168 h post-insemination.
Embryo development to CoQ10
(µM) N 2-4-cell % Blastocyst % Efficiency %
0 299 61.5 ± 7.6 48.9 ± 15.5 29.7 ± 8.8
10 245 58.7 ± 8.6 40.2 ± 5.7 23.6 ± 4.9
25 249 65.3 ± 5.6 46.6 ± 10.6 30.4 ± 7.3
100 254 64.7 ± 9.9 39.2 ± 11.3 24.6 ± 4.0 Data are expressed as the mean ± SD (four replicates).
