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thawed in-vitro produced embryos
Research in Veterinary Science 102 (2015) 238–241
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/yrvsc
Amniotic fluid L-ergothioneine concentrations in pregnant sheep after natural mating and transfer of vitrified/thawed in-vitro produced embryos Salvatore Sotgia a,⁎, Angelo Zinellu a, Dionigia Arru a, Stefano Nieddu b, Alessandro Strina b, Federica Ariu b, Gianfranco Pintus a, Ciriaco Carru a,c, Luisa Bogliolo b, Sergio Ledda b a b c
Department of Biomedical Sciences, University of Sassari, Sassari, Italy Department of Veterinary Medicine, University of Sassari, Sassari, Italy Quality Control Unit, University Hospital Sassari (AOU), Sassari, Italy
a r t i c l e
i n f o
Article history: Received 18 May 2015 Received in revised form 26 August 2015 Accepted 1 September 2015 Available online xxxx Keywords: Antioxidant Pregnancy Vitrified/thawed IVP Sheep ART
a b s t r a c t L-ergothioneine levels were measured in amniotic fluid of pregnant sheep after natural mating and transfer of vitrified/thawed in-vitro produced embryos. Amniotic fluids were collected between 60 and 65 and 80–85 days of gestation and analysed by an ultra-performance liquid chromatographic (UPLC) method with fluorescence detection. L-Ergothioneine concentrations ranged between 0.23 and 9.36 μmol/L and were significantly higher in pregnancy obtained by the transfer of vitrified/thawed in-vitro produced embryos. Conversely, no significant changes in amniotic fluid L-ergothioneine concentrations were observed according to the stages of pregnancy considered in this study. These findings suggest that L-ergothioneine concentrations, are not affected as much by the gestational age, but rather by the method used to induce the pregnancy. On the whole, the measurement of L-ergothioneine in amniotic fluid could serve as a useful biomarker of oxidative stress and/or inflammatory state in pregnancy. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction In-vitro production of embryos (IVP) represents the third generation of high-tech procedures developed to achieve pregnancy by artificial or partially artificial means (Thibier, 2011). With other assisted reproductive technologies (ARTs), e.g. artificial insemination, multiple ovulation, and embryo transfer, IVP plays a key role in ensuring economic sustainability of animal husbandry (Cognié et al., 2004). However, in spite of efforts to improve its efficiency, IVP still shows shortcomings limiting its large-scale commercial use (Lonergan and Fair, 2008; Morton et al., 2005). A number of issues appear to influence both the production of transferable embryos and the success rates of pregnancies (Hasler, 2001). Growing evidence indicates that alteration of placental development in early pregnancy may play an essential part in the aetiology of pregnancy complications (Novakovic et al., 2010). Functional capacity of the placenta, central for optimal foetal growth and neonatal outcome, can be adversely affected by increased oxidative stress, as reported by several authors (Mutinati et al., 2013; Agarwal and Allamaneni, 2004; Agarwal et al., 2005). Due to its high-energy demand and increased oxygen requirement, pregnancy per se favours a state of oxidative stress ⁎ Corresponding author at: Department of Biomedical Sciences, Chair of Clinical Biochemistry, University of Sassari, Viale San Pietro 43/B, I-07100 Sassari, Italy. E-mail address: [email protected] (S. Sotgia).
http://dx.doi.org/10.1016/j.rvsc.2015.09.003 0034-5288/© 2015 Elsevier Ltd. All rights reserved.
(Mutinati et al., 2013). Once triggered, oxidative stress is directly and indirectly involved in many gestational diseases in humans and animals such as pre-eclampsia, eclampsia, abortion, premature birth or hypertension (Mutinati et al., 2013; Agarwal and Allamaneni, 2004; Agarwal et al., 2005). Recently, in pre-eclamptic women, an increased concentration of L-ergothioneine (ERT; 2-mercaptohistidine trimethylbetaine), a molecule with ROS-scavenging properties, has been observed in maternal erythrocytes (Turner et al., 2009). ERT, a natural non-toxic aminothiol, is biosynthesized exclusively by some non-yeast-like fungi (Paul and Snyder, 2010) and bacteria such as mycobacteria (Ey et al., 2007) and cyanobacteria (Pfeiffer et al., 2001). In mammals, ERT is acquired solely by dietary means and accumulates especially in mitochondria, cells and tissues normally exposed to oxidative stress and involved in the inflammatory response process (Paul and Snyder, 2010; Gründemann, 2012). ERT uptake, however, does not appear to be related to dietary sources but rather to the expression of the specific organic cation transporter protein (ETT) (Schauss et al., 2010). ETT-specific mRNA transcripts can be found in numerous tissues, including kidney, muscle, heart, placenta, and others (Tamai et al., 2004). In rat placenta, ETT expression is detectable throughout the labyrinthine zone, where maternaL-foetal nutrient exchange occurs (Wu et al., 2000). It is therefore conceivable that placental disorders associated with oxidative stress may also affect ERT concentrations. In this context, by virtue of its intimate contact with the foetus, placenta, and
S. Sotgia et al. / Research in Veterinary Science 102 (2015) 238–241
other structures, the amniotic fluid (AF) can be an important source of information. A large number of biochemical, cytological, biophysical and immunological parameters can be, in fact, assessed in AF to obtain information on the environment in the womb, development status, genetic anomalies and other pathologies in the foetus. Thus, the aim of this study was to evaluate if a) ERT is present in AF of pregnant sheep and b) its concentrations depend on whether pregnancy was derived by the transfer of vitrified/thawed IVP embryos (VT) or by natural mating (NM). For this purpose, AF of 38 pregnant sheep after VT or NM were collected between 60 and 65 and 80–85 days of gestation and analysed by an ultra-performance liquid chromatographic (UPLC) method with fluorescence detection (Sotgia et al., 2014a). 2. Materials and methods 2.1. Ethical procedures All animal experiments were performed in accordance with DPR 27/ 1/1992 (Animal Protection regulations of Italy) in conformity with European Community regulation 86/609. 2.2. Chemicals Acetonitrile (ACN) HPLC grade, sodium phosphate tribasic dodecahydrate, ammonium acetate, DMSO, and 7-diethylamino-3-[4(iodoacetamido)phenyl]-4-methylcoumarin (DCIA) were obtained from Sigma Aldrich Italia (Milan, Italy) whereas L-ergothioneine (ERT) was obtained from Vinci-Biochem (Florence, Italy). High-purity water, used throughout the experiments, was obtained by a Millipore Milli-Q system. 2.3. Vitrified/thawed IVP (VT) and natural mating (NM) groups A total of 38 Sarda breed sheep (3–5 years old) were enrolled from the experimental facilities of the Department of Veterinary Medicine, University of Sassari (Italy), and from a commercial farm in the province of Sassari (Italy). Animals were fed a live-weight maintenance ration throughout the experiments, and treated to synchronize estrous by using intravaginal progestagen-impregnated sponges (Cronogest, Intervet Srl, Italy) for 14 days and a single administration of 333 IU/ ewe of eCG (Folligon, Intervet Srl, Italy) on the day of the sponge removal. For VT group, ovaries of Sarda ewes were collected from a local slaughterhouse, oocytes were recovered, and embryos were produced in vitro as described by Bogliolo et al. (2011). Expanded blastocysts were recovered on the seventh day of culture and vitrified in Cryotop devices (Kitazato Ltd., Tokyo, Japan) using the minimum essential volume method (Nieddu et al., 2015). Embryos were thawed, cultured for 1 h before transfer in the recipient sheep. Twenty-four hours after sponge removal, a teaser ram was introduced in the flock to detect estrous. Seven days after the onset of estrous, three vitrified/thawed blastocysts were surgically transferred into each recipient via median laparotomy. For NM group, after the synchronization of the estrous, fertile rams were introduced in the flock for the breeding season and marked ewes were identified as mated. The pregnant ewes with a single foetus were identified by a transabdominal ultrasonography performed 15–20 days after natural mating or transfer of IVP embryos (Nieddu et al., 2014). Pregnancy by VT or NM was induced in 13 and 25 animals, respectively. Amniocentesis was subsequently performed in all animals between 60 and 65 and 80–85 days of gestation. Because sires contribute to embryonic/foetal development, as well as to placenta and foetal membranes (Anchamparuthy et al., 2009), in order to minimize this effect, different sires were used to produce embryos in vitro and for NM. 2.4. Collection, processing and chromatography of amniotic fluid A trans-abdominal ultrasound-guided amniocentesis, using a 9- to 4-MHz micro-convex probe, was performed to collect 10 mL of amniotic
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fluid at 60–65 (VT N = 8, NM N = 11) and 80–85 (VT N = 5, NM N = 14) days of gestation in 38 pregnant sheep (for details, see ref. Nieddu et al., 2015). The timing of amniocentesis was selected to ensure maximal safety during the procedure. In sheep, placental development is completed approximately within 60 days of gestation. After the third month, foetal movements may increase the risk of foetal/membrane damage. AFs were centrifuged at 4000 ×g for 10 min, then supernatants were stored at −80 °C until analysis by UPLC according to the method described by Sotgia et al. (2014a). Briefly, 50 μL of a 30 mmol/L sodium phosphate tribasic dodecahydrate solution and 50 μL of a 430 μmol/L DCIA fresh working solution were added to 150 μL of AF deproteinizated by ACN. After vortex mixing, the reaction mixture was allowed to stand in a light-protected area for 10 min at 90 °C. Then, samples were diluted two times with ACN and, after vortex mixing and centrifugation at 17,000 × g for 5 min at room temperature, analysed by UPLC. The apparatus employed for the analysis of ERT was a Waters system model Acquity UPLC equipped with a Waters Acquity fluorescence detector (Milford, MA, USA). Separation was achieved on a 100 × 2.1 mm Waters Cortecs UPLC HILIC 1.6-μm column by using a mixture of 30 mmol/L ammonium acetate/ACN (10:90, v/v) as a mobile phase, delivered to the column isocratically at a flow rate of 0.9 mL min−1. Separation was carried out at 45 °C, amount injected was 2 μL and column eluates were detected by a fluorescence detector at an excitation wavelength of 389 nm and emission wavelength of 467 nm with the signal gain set to 10. 2.5. Statistical analysis Statistical analyses were performed by using MedCalc for Windows, version 15.4 64 bit (MedCalc Software, Ostend, Belgium). Normality of distribution was checked by the Shapiro–Wilk test and data were presented as either mean ± SD or median and interquartile range (IQR), as appropriate. Homogeneity of variance was checked by F-test and differences between groups were compared by non-parametric Mann–Whitney U Test or by parametric independent t-test, with or without Welch's correction for unequal variances, as appropriate. Non-normally distributed variables were log10-transformed prior to being used with parametric tests, and the normal distribution of the residuals was checked to assess the goodness of fit of the transformations. A 2-sided P value of 0.05 was chosen as the cut-off for statistical significance. 3. Results All of the gestations were uncomplicated and, at term, ewes gave birth to a single and healthy lamb weighting 3–4 Kg, within range of the Sarda breed. ERT concentrations in AFs were normally distributed in NM (N = 14) and VT (N = 5) groups sampled at 80–85 days of gestation (NM8 and VT8, respectively). By contrast, they were skewed in NM (N = 11) and VT (N = 8) groups sampled at 60–65 days of gestation (NM6 and VT6, respectively). As shown in Table 1, the values of ERT ranged from a maximum of 9.36 μmol/L (VT group) to a minimum of 0.23 μmol/L (NM group). VT group showed the highest mean concentrations (1.81 ± 2.59 μmol/L) whereas the NM group showed the lowest (0.43 ± 0.31 μmol/L). As displayed in Table 1, after splitting the groups according to the days of gestation at which AFs were collected, NM8 group was the cluster with the lowest mean concentration (0.35 ± 0.10 μmol/L), while VT6 group showed the highest mean values (2.38 ± 3.38 μmol/L). Average concentrations in NM6 and VT8 groups were 0.53 ± 0.45 and 0.90 ± 0.34 μmol/L, respectively. Because log-10 transformation was unable to normalize the distribution of ERT values, further statistical analysis was conducted using non-parametric Mann–Whitney U Test. As shown in Fig. 1, the comparison of ERT concentrations between NM6 vs. NM8 and VT6 vs. VT8, showed a non-significant trend to decrease (p N 0.05). Conversely, as displayed in Fig. 2, a significant trend to increase was observed by
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Table 1 ERT concentrations between the groups. ERT concentrations are expressed as μmol per litre (μmol/L). NM
Lowest Value Highest value Mean ± SD Median (IQR)
VT
NM6 (N = 11)
NM8 (N = 14)
VT6 (N = 8)
VT8 (N = 5)
0.23 1.78 0.53 ± 0.45 0.37 (0.30–0.60)
0.24 0.60 0.35 ± 0.10 0.35 (0.27–0.41)
0.44 9.36 2.38 ± 3.38 0.68 (0.52–3.42)
0.47 1.31 0.90 ± 0.34 1.03 (0.58–1.12)
comparing ERT concentrations between NM vs. VT (p = 0.0001), NM6 vs. VT6 (p = 0.021) and NM8 vs. VT8 (p = 0.002). 4. Discussion A literature search (Medline 1960–2015) failed to identify any publications on assessment of ERT in AF of pregnant sheep. We demonstrated that ERT is detectable and measurable in this fluid in the order of micromolar range. This is consistent with the concentrations reported in other body fluids (Sotgia et al., 2014a, 2014b, 2015), and testifies for the almost exclusive intracellular distribution of ERT. In this sense, an exception is the seminal plasma of some animals, where ERT is found at millimolar levels as an extracellular constituent (Nikodemus et al., 2011). However, once accumulated in cells, ERT is strongly retained by the organs. No significant changes in AF ERT concentrations were observed in the stages of pregnancy considered in this study, both in NM (NM6 vs. NM8, p N 0.05) and VT (VT6 vs. VT8, p N 0.05) groups. Interestingly, the differences were significant when comparing NM and VT as a whole (NM vs. VT, p = 0.0001) as well as according to the days of gestation during which AFs were collected (NM6 vs. VT6, p = 0.021; NM8 vs. VT8, p = 0.002). Although fluctuations of ERT concentrations after the pregnancy period assessed cannot be excluded, these findings suggest that the concentrations of ERT are not affected as much by gestational age. Rather, they seem affected by the method used to induce pregnancy, NM or VT. Several differences between NM and VT may account for the differences in the AF content of ERT. However, ERT-associated factors could also be involved. ERT has been directly associated with oxidative stress and is recognized to have ROS-scavenging functions (Brummel, 1985; Franzoni et al., 2006). During physiological pregnancy, all tissues and, mostly, placenta and
Fig. 1. Comparison of ERT concentrations according to the days of gestation at which AFs were collected both in ewes that were naturally mated (NM) and embryo transferred with in vitro–produced vitrified/thawed embryos (VT). To take account of the skewness towards large values, concentrations were plotted as log10 [ERT]. *p b 0.05; **p b 0.01; ***p b 0.001.
foetus require high amounts of oxygen (Mutinati et al., 2013). Therefore, pregnancy is a process that lends itself naturally to an increase in ROS production (Erisir et al., 2009). In this light, the higher ERT concentrations observed in the AF of VT vs. NM suggest an increased response against free radicals. In this sense, there is evidence of increased production of free radicals in pregnancies induced by ARTs (Gupta et al., 2010). Moreover, given the low tissue turnover of ERT (Kato et al., 2010; Kawano et al., 1982), an increase in AF concentrations might reflect an increase of apoptotic processes resulting from cell damage induced by free radicals. Oxidative stress is also causally linked to inflammatory processes, creating a vicious circle where, on one hand, oxidative stress triggers inflammation and, on the other, inflammatory cells produce high levels of ROS. Notably, the gene for ETT lies in close proximity to genes involved in inflammatory responses, and it is abundantly expressed in inflammatory cells such as CD14+ macrophages and monocytes (Paul and Snyder, 2010). ETT expression is upregulated by inflammatory cytokines and, in humans, its promoter shows binding sites for the transcription factor nuclear factor-kappa B, which regulates inflammatory genes (Paul and Snyder, 2010). In VT pregnancies, therefore, the higher ERT concentrations may be an indicator of a more severe inflammatory state compared to pregnancies induced by NM. Unfortunately, has not been possible to monitoring the levels of markers of oxidative stress, such as malondialdehyde (MDA), 8-isoprostane, or protein-bound carbonyl groups, to evaluate how they correlate with ERT levels. Moreover, only few gestations were induced after transfer of vitrified IVP embryos and all of NM and VT pregnancies were uncomplicated. It is, therefore, difficult to ascertain the real impact of ERT concentrations in either NM or VT pregnancies. However, taking into account the dependence of ERT concentration by the levels of the ETT-specific mRNA, as well as the modulating effect of oxidative stress/inflammation on the expression of ETT, it is plausible
Fig. 2. Differences between ERT concentrations according to the days of gestation and the method used to induce pregnancy. To take account of the skewness towards large values, concentrations were plotted as log10 [ERT]. *p b 0.05; **p b 0.01; ***p b 0.001.
S. Sotgia et al. / Research in Veterinary Science 102 (2015) 238–241
that the measurement of AF ERT might serve as a useful biomarker of oxidative stress and/or inflammatory state in pregnancy. 5. Conclusion For the first time, to our knowledge, ERT concentrations were measured in the amniotic fluid of pregnant sheep after natural mating and transfer of vitrified/thawed IVP embryos. ERT concentrations were significantly different between the groups, and were overall higher in the IVP group. On the whole, findings provide new insights, both in the monitoring and in understanding of the mechanisms accountable for the complications in pregnancy. Conflict of interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper. Acknowledgements Funding was provided by the Fondazione Banco di Sardegna, Sassari, Italy, and by the Ministero dell'Università e della Ricerca, Italy. It is a pleasure to thank Professor Arduino A. Mangoni from Flinders University (Australia) for his critical reading of the manuscript. References Agarwal, A., Allamaneni, S.S., 2004. Role of free radicals in female reproductive diseases and assisted reproduction. Reprod. BioMed. Online 9, 338–347. Agarwal, A., Gupta, S., Sharma, R.K., 2005. Role of oxidative stress in female reproduction. Reprod. Biol. Endocrinol. 3, 28. Anchamparuthy, V.M., Dhali, A., Lott, W.M., Pearson, R.E., Gwazdauskas, F.C., 2009. Vitrification of bovine oocytes: implications of follicular size and sire on the rates of embryonic development. J. Assist. Reprod. Genet. 26, 613–619. Bogliolo, L., Ariu, F., Leoni, G., Uccheddu, S., Bebbere, D., 2011. High hydrostatic pressure treatment improves the quality of in vitro-produced ovine blastocysts. Reprod. Fertil. Dev. 23, 809–817. Brummel, M.C., 1985. In search of a physiological function for L-ergothioneine. Med. Hypotheses 18, 351–370. Cognié, Y., Poulin, N., Locatelli, Y., Mermillod, P., 2004. State-of-the-art production, conservation and transfer of in-vitro-produced embryos in small ruminants. Reproduction Fertility and Development 16, 437–445. Erisir, M., Benzer, F., Kandemir, F.M., 2009. Changes in the rate of lipid peroxidation in plasma and selected blood antioxidants before and during pregnancy in ewes. Acta Vet. Brno 78, 237–242. Ey, J., Schömig, E., Taubert, D., 2007. Dietary sources and antioxidant effects of ergothioneine. J. Agric. Food Chem. 55, 6466–6474. Franzoni, F., Colognato, R., Galetta, F., Laurenza, I., Barsotti, M., Di Stefano, R., Bocchetti, R., Regoli, F., Carpi, A., Balbarini, A., Migliore, L., Santoro, G., 2006. An in vitro study on the free radical scavenging capacity of ergothioneine: comparison with reduced glutathione, uric acid and trolox. Biomed. Pharmacother. 60, 453–457. Gründemann, D., 2012. The ergothioneine transporter controls and indicates ergothioneine activity—a review. Prev. Med. 54, S71–S74. Gupta, S., Sekhon, L., Kim, Y., Agarwal, A., 2010. The role of oxidative stress and antioxidants in assisted reproduction. Curr. Womens Health Rev. 6, 227–238.
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Hasler, J.F., 2001. Factors affecting frozen and fresh embryo transfer pregnancy rates in cattle. Theriogenology 56, 1401–1415. Kato, Y., Kubo, Y., Iwata, D., Kato, S., Sudo, T., Sugiura, T., Kagaya, T., Wakayama, T., Hirayama, A., Sugimoto, M., Sugihara, K., Kaneko, S., Soga, T., Asano, M., Tomita, M., Matsui, T., Wada, M., Tsuji, A., 2010. Gene knockout and metabolome analysis of carnitine/organic cation transporter OCTN1. Pharm. Res. 27, 832–840. Kawano, H., Otani, M., Takeyama, K., Kawai, Y., Mayumi, T., Hama, T., 1982. Studies on ergothioneine. VI. Distribution and fluctuations of ergothioneine in rats. Chem. Pharm. Bull. 30, 1760–1765. Lonergan, P., Fair, T., 2008. In vitro-produced bovine embryos-dealing with the warts. Theriogenology 69, 17–22. Morton, K.M., Catt, S.L., Maxwell, W.M., Evans, G., 2005. In vitro and in vivo developmental capabilities and kinetics of in vitro development of in vitro matured oocytes from adult, unstimulated and hormone-stimulated prepubertal ewes. Theriogenology 64, 1320–1332. Mutinati, M., Piccinno, M., Roncetti, M., Campanile, D., Rizzo, A., Sciorsci, R.L., 2013. Oxidative stress during pregnancy in the sheep. Reprod. Domest. Anim. 48, 353–357. Nieddu, S.M., Strina, A., Corda, A., Furcas, G., Ledda, M., Pau, S., Ledda, S., 2014. Comparation of foetal growth in natural mated and vitrified/warmed ovine embryos by ultrasonographic measurements. Global Vet. 12, 91–97. Nieddu, S.M., Mossa, F., Strina, A., Ariu, F., Pau, S., Ledda, M., Sotgia, S., Carru, C., Ledda, S., 2015. Differences in amniotic amino acid concentrations between pregnancies obtained with transfer of vitrified thawed in vitro-produced embryos and with natural mating in sheep. Theriogenology 83, 687–692. Nikodemus, D., Lazic, D., Bach, M., Bauer, T., Pfeiffer, C., Wiltzer, L., Lain, E., Schömig, E., Gründemann, D., 2011. Paramount levels of ergothioneine transporter SLC22A4mRNA in boar seminal vesicles and cross-species analysis of ergothioneine and glutathione in seminal plasma. J. Physiol. Pharmacol. 62, 411–419. Novakovic, B., Wong, N.C., Sibson, M., Ng, H.K., Morley, R., Manuelpillai, U., Down, T., Rakyan, V.K., Beck, S., Hiendleder, S., Roberts, C.T., Craig, J.M., Saffery, R., 2010. DNA methylation-mediated down-regulation of DNA methyltransferase-1 (DNMT1) is coincident with, but not essential for, global hypomethylation in human placenta. J. Biol. Chem. 285 (13), 9583–9593. Paul, B.D., Snyder, S.H., 2010. The unusual amino acid L-ergothioneine is a physiologic cytoprotectant. Cell Death Differ. 17, 1134–1140. Pfeiffer, C., Bauer, T., Surek, B., Schömig, E., Gründemann, D., 2001. Cyanobacteria produce high levels of ergothioneine. Food Chem. 129, 1766–1769. Schauss, A.G., Vértesi, A., Endres, J.R., Hirka, G., Clewell, A., Qureshi, I., Pasics, I., 2010. Evaluation of the safety of the dietary antioxidant ergothioneine using the bacterial reverse mutation assay. Toxicology 278, 39–45. Sotgia, S., Pisanu, E., Cambedda, D., Pintus, G., Carru, C., Zinellu, A., 2014a. Ultraperformance liquid chromatographic determination of lergothioneine in commercially available classes of cow milk. J. Food Sci. 79, 1683–1687. Sotgia, S., Zinellu, A., Mangoni, A.A., Pintus, G., Attia, J., Carru, C., McEvoy, M., 2014b. Clinical and biochemical correlates of serum L-ergothioneine concentrations in community-dwelling middle-aged and older adults. PLoS One 9, e84918. Sotgia, S., Zinellu, A., Pisanu, E., Vlahopoulou, G., Ariu, F., Ledda, S., Pintus, G., Carru, C., Bogliolo, L., 2015. Concentrations of L-ergothioneine in follicular fluids of farm animals. Comp. Clin. Pathol. http://dx.doi.org/10.1007/s00580-015-2098-8. Tamai, I., Nakanishi, T., Kobayashi, D., China, K., Kosugi, Y., Nezu, J., Sai, Y., Tsuji, A., 2004. Involvement of OCTN1 (SLC22A4) in pH-dependent transport of organic cations. Mol. Pharm. 1, 57–66. Thibier, M., 2011. Embryo transfer: a comparative biosecurity advantage in international movements of germplasm. Rev. Sci. Tech. 30, 177–188. Turner, E., Brewster, J.A., Simpson, N.A., Walker, J.J., Fisher, J., 2009. Imidazole-based erythrocyte markers of oxidative stress in preeclampsia—an NMR investigation. Reprod. Sci. 16, 1040–1051. Wu, X., George, R.L., Huang, W., Wang, H., Conway, S.J., Leibach, F.H., Ganapathy, V., 2000. Structural and functional characteristics and tissue distribution pattern of rat OCTN1, an organic cation transporter, cloned from placenta. Biochim. Biophys. Acta 1466, 315–327.
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/yrvsc
Amniotic fluid L-ergothioneine concentrations in pregnant sheep after natural mating and transfer of vitrified/thawed in-vitro produced embryos Salvatore Sotgia a,⁎, Angelo Zinellu a, Dionigia Arru a, Stefano Nieddu b, Alessandro Strina b, Federica Ariu b, Gianfranco Pintus a, Ciriaco Carru a,c, Luisa Bogliolo b, Sergio Ledda b a b c
Department of Biomedical Sciences, University of Sassari, Sassari, Italy Department of Veterinary Medicine, University of Sassari, Sassari, Italy Quality Control Unit, University Hospital Sassari (AOU), Sassari, Italy
a r t i c l e
i n f o
Article history: Received 18 May 2015 Received in revised form 26 August 2015 Accepted 1 September 2015 Available online xxxx Keywords: Antioxidant Pregnancy Vitrified/thawed IVP Sheep ART
a b s t r a c t L-ergothioneine levels were measured in amniotic fluid of pregnant sheep after natural mating and transfer of vitrified/thawed in-vitro produced embryos. Amniotic fluids were collected between 60 and 65 and 80–85 days of gestation and analysed by an ultra-performance liquid chromatographic (UPLC) method with fluorescence detection. L-Ergothioneine concentrations ranged between 0.23 and 9.36 μmol/L and were significantly higher in pregnancy obtained by the transfer of vitrified/thawed in-vitro produced embryos. Conversely, no significant changes in amniotic fluid L-ergothioneine concentrations were observed according to the stages of pregnancy considered in this study. These findings suggest that L-ergothioneine concentrations, are not affected as much by the gestational age, but rather by the method used to induce the pregnancy. On the whole, the measurement of L-ergothioneine in amniotic fluid could serve as a useful biomarker of oxidative stress and/or inflammatory state in pregnancy. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction In-vitro production of embryos (IVP) represents the third generation of high-tech procedures developed to achieve pregnancy by artificial or partially artificial means (Thibier, 2011). With other assisted reproductive technologies (ARTs), e.g. artificial insemination, multiple ovulation, and embryo transfer, IVP plays a key role in ensuring economic sustainability of animal husbandry (Cognié et al., 2004). However, in spite of efforts to improve its efficiency, IVP still shows shortcomings limiting its large-scale commercial use (Lonergan and Fair, 2008; Morton et al., 2005). A number of issues appear to influence both the production of transferable embryos and the success rates of pregnancies (Hasler, 2001). Growing evidence indicates that alteration of placental development in early pregnancy may play an essential part in the aetiology of pregnancy complications (Novakovic et al., 2010). Functional capacity of the placenta, central for optimal foetal growth and neonatal outcome, can be adversely affected by increased oxidative stress, as reported by several authors (Mutinati et al., 2013; Agarwal and Allamaneni, 2004; Agarwal et al., 2005). Due to its high-energy demand and increased oxygen requirement, pregnancy per se favours a state of oxidative stress ⁎ Corresponding author at: Department of Biomedical Sciences, Chair of Clinical Biochemistry, University of Sassari, Viale San Pietro 43/B, I-07100 Sassari, Italy. E-mail address: [email protected] (S. Sotgia).
http://dx.doi.org/10.1016/j.rvsc.2015.09.003 0034-5288/© 2015 Elsevier Ltd. All rights reserved.
(Mutinati et al., 2013). Once triggered, oxidative stress is directly and indirectly involved in many gestational diseases in humans and animals such as pre-eclampsia, eclampsia, abortion, premature birth or hypertension (Mutinati et al., 2013; Agarwal and Allamaneni, 2004; Agarwal et al., 2005). Recently, in pre-eclamptic women, an increased concentration of L-ergothioneine (ERT; 2-mercaptohistidine trimethylbetaine), a molecule with ROS-scavenging properties, has been observed in maternal erythrocytes (Turner et al., 2009). ERT, a natural non-toxic aminothiol, is biosynthesized exclusively by some non-yeast-like fungi (Paul and Snyder, 2010) and bacteria such as mycobacteria (Ey et al., 2007) and cyanobacteria (Pfeiffer et al., 2001). In mammals, ERT is acquired solely by dietary means and accumulates especially in mitochondria, cells and tissues normally exposed to oxidative stress and involved in the inflammatory response process (Paul and Snyder, 2010; Gründemann, 2012). ERT uptake, however, does not appear to be related to dietary sources but rather to the expression of the specific organic cation transporter protein (ETT) (Schauss et al., 2010). ETT-specific mRNA transcripts can be found in numerous tissues, including kidney, muscle, heart, placenta, and others (Tamai et al., 2004). In rat placenta, ETT expression is detectable throughout the labyrinthine zone, where maternaL-foetal nutrient exchange occurs (Wu et al., 2000). It is therefore conceivable that placental disorders associated with oxidative stress may also affect ERT concentrations. In this context, by virtue of its intimate contact with the foetus, placenta, and
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other structures, the amniotic fluid (AF) can be an important source of information. A large number of biochemical, cytological, biophysical and immunological parameters can be, in fact, assessed in AF to obtain information on the environment in the womb, development status, genetic anomalies and other pathologies in the foetus. Thus, the aim of this study was to evaluate if a) ERT is present in AF of pregnant sheep and b) its concentrations depend on whether pregnancy was derived by the transfer of vitrified/thawed IVP embryos (VT) or by natural mating (NM). For this purpose, AF of 38 pregnant sheep after VT or NM were collected between 60 and 65 and 80–85 days of gestation and analysed by an ultra-performance liquid chromatographic (UPLC) method with fluorescence detection (Sotgia et al., 2014a). 2. Materials and methods 2.1. Ethical procedures All animal experiments were performed in accordance with DPR 27/ 1/1992 (Animal Protection regulations of Italy) in conformity with European Community regulation 86/609. 2.2. Chemicals Acetonitrile (ACN) HPLC grade, sodium phosphate tribasic dodecahydrate, ammonium acetate, DMSO, and 7-diethylamino-3-[4(iodoacetamido)phenyl]-4-methylcoumarin (DCIA) were obtained from Sigma Aldrich Italia (Milan, Italy) whereas L-ergothioneine (ERT) was obtained from Vinci-Biochem (Florence, Italy). High-purity water, used throughout the experiments, was obtained by a Millipore Milli-Q system. 2.3. Vitrified/thawed IVP (VT) and natural mating (NM) groups A total of 38 Sarda breed sheep (3–5 years old) were enrolled from the experimental facilities of the Department of Veterinary Medicine, University of Sassari (Italy), and from a commercial farm in the province of Sassari (Italy). Animals were fed a live-weight maintenance ration throughout the experiments, and treated to synchronize estrous by using intravaginal progestagen-impregnated sponges (Cronogest, Intervet Srl, Italy) for 14 days and a single administration of 333 IU/ ewe of eCG (Folligon, Intervet Srl, Italy) on the day of the sponge removal. For VT group, ovaries of Sarda ewes were collected from a local slaughterhouse, oocytes were recovered, and embryos were produced in vitro as described by Bogliolo et al. (2011). Expanded blastocysts were recovered on the seventh day of culture and vitrified in Cryotop devices (Kitazato Ltd., Tokyo, Japan) using the minimum essential volume method (Nieddu et al., 2015). Embryos were thawed, cultured for 1 h before transfer in the recipient sheep. Twenty-four hours after sponge removal, a teaser ram was introduced in the flock to detect estrous. Seven days after the onset of estrous, three vitrified/thawed blastocysts were surgically transferred into each recipient via median laparotomy. For NM group, after the synchronization of the estrous, fertile rams were introduced in the flock for the breeding season and marked ewes were identified as mated. The pregnant ewes with a single foetus were identified by a transabdominal ultrasonography performed 15–20 days after natural mating or transfer of IVP embryos (Nieddu et al., 2014). Pregnancy by VT or NM was induced in 13 and 25 animals, respectively. Amniocentesis was subsequently performed in all animals between 60 and 65 and 80–85 days of gestation. Because sires contribute to embryonic/foetal development, as well as to placenta and foetal membranes (Anchamparuthy et al., 2009), in order to minimize this effect, different sires were used to produce embryos in vitro and for NM. 2.4. Collection, processing and chromatography of amniotic fluid A trans-abdominal ultrasound-guided amniocentesis, using a 9- to 4-MHz micro-convex probe, was performed to collect 10 mL of amniotic
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fluid at 60–65 (VT N = 8, NM N = 11) and 80–85 (VT N = 5, NM N = 14) days of gestation in 38 pregnant sheep (for details, see ref. Nieddu et al., 2015). The timing of amniocentesis was selected to ensure maximal safety during the procedure. In sheep, placental development is completed approximately within 60 days of gestation. After the third month, foetal movements may increase the risk of foetal/membrane damage. AFs were centrifuged at 4000 ×g for 10 min, then supernatants were stored at −80 °C until analysis by UPLC according to the method described by Sotgia et al. (2014a). Briefly, 50 μL of a 30 mmol/L sodium phosphate tribasic dodecahydrate solution and 50 μL of a 430 μmol/L DCIA fresh working solution were added to 150 μL of AF deproteinizated by ACN. After vortex mixing, the reaction mixture was allowed to stand in a light-protected area for 10 min at 90 °C. Then, samples were diluted two times with ACN and, after vortex mixing and centrifugation at 17,000 × g for 5 min at room temperature, analysed by UPLC. The apparatus employed for the analysis of ERT was a Waters system model Acquity UPLC equipped with a Waters Acquity fluorescence detector (Milford, MA, USA). Separation was achieved on a 100 × 2.1 mm Waters Cortecs UPLC HILIC 1.6-μm column by using a mixture of 30 mmol/L ammonium acetate/ACN (10:90, v/v) as a mobile phase, delivered to the column isocratically at a flow rate of 0.9 mL min−1. Separation was carried out at 45 °C, amount injected was 2 μL and column eluates were detected by a fluorescence detector at an excitation wavelength of 389 nm and emission wavelength of 467 nm with the signal gain set to 10. 2.5. Statistical analysis Statistical analyses were performed by using MedCalc for Windows, version 15.4 64 bit (MedCalc Software, Ostend, Belgium). Normality of distribution was checked by the Shapiro–Wilk test and data were presented as either mean ± SD or median and interquartile range (IQR), as appropriate. Homogeneity of variance was checked by F-test and differences between groups were compared by non-parametric Mann–Whitney U Test or by parametric independent t-test, with or without Welch's correction for unequal variances, as appropriate. Non-normally distributed variables were log10-transformed prior to being used with parametric tests, and the normal distribution of the residuals was checked to assess the goodness of fit of the transformations. A 2-sided P value of 0.05 was chosen as the cut-off for statistical significance. 3. Results All of the gestations were uncomplicated and, at term, ewes gave birth to a single and healthy lamb weighting 3–4 Kg, within range of the Sarda breed. ERT concentrations in AFs were normally distributed in NM (N = 14) and VT (N = 5) groups sampled at 80–85 days of gestation (NM8 and VT8, respectively). By contrast, they were skewed in NM (N = 11) and VT (N = 8) groups sampled at 60–65 days of gestation (NM6 and VT6, respectively). As shown in Table 1, the values of ERT ranged from a maximum of 9.36 μmol/L (VT group) to a minimum of 0.23 μmol/L (NM group). VT group showed the highest mean concentrations (1.81 ± 2.59 μmol/L) whereas the NM group showed the lowest (0.43 ± 0.31 μmol/L). As displayed in Table 1, after splitting the groups according to the days of gestation at which AFs were collected, NM8 group was the cluster with the lowest mean concentration (0.35 ± 0.10 μmol/L), while VT6 group showed the highest mean values (2.38 ± 3.38 μmol/L). Average concentrations in NM6 and VT8 groups were 0.53 ± 0.45 and 0.90 ± 0.34 μmol/L, respectively. Because log-10 transformation was unable to normalize the distribution of ERT values, further statistical analysis was conducted using non-parametric Mann–Whitney U Test. As shown in Fig. 1, the comparison of ERT concentrations between NM6 vs. NM8 and VT6 vs. VT8, showed a non-significant trend to decrease (p N 0.05). Conversely, as displayed in Fig. 2, a significant trend to increase was observed by
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Table 1 ERT concentrations between the groups. ERT concentrations are expressed as μmol per litre (μmol/L). NM
Lowest Value Highest value Mean ± SD Median (IQR)
VT
NM6 (N = 11)
NM8 (N = 14)
VT6 (N = 8)
VT8 (N = 5)
0.23 1.78 0.53 ± 0.45 0.37 (0.30–0.60)
0.24 0.60 0.35 ± 0.10 0.35 (0.27–0.41)
0.44 9.36 2.38 ± 3.38 0.68 (0.52–3.42)
0.47 1.31 0.90 ± 0.34 1.03 (0.58–1.12)
comparing ERT concentrations between NM vs. VT (p = 0.0001), NM6 vs. VT6 (p = 0.021) and NM8 vs. VT8 (p = 0.002). 4. Discussion A literature search (Medline 1960–2015) failed to identify any publications on assessment of ERT in AF of pregnant sheep. We demonstrated that ERT is detectable and measurable in this fluid in the order of micromolar range. This is consistent with the concentrations reported in other body fluids (Sotgia et al., 2014a, 2014b, 2015), and testifies for the almost exclusive intracellular distribution of ERT. In this sense, an exception is the seminal plasma of some animals, where ERT is found at millimolar levels as an extracellular constituent (Nikodemus et al., 2011). However, once accumulated in cells, ERT is strongly retained by the organs. No significant changes in AF ERT concentrations were observed in the stages of pregnancy considered in this study, both in NM (NM6 vs. NM8, p N 0.05) and VT (VT6 vs. VT8, p N 0.05) groups. Interestingly, the differences were significant when comparing NM and VT as a whole (NM vs. VT, p = 0.0001) as well as according to the days of gestation during which AFs were collected (NM6 vs. VT6, p = 0.021; NM8 vs. VT8, p = 0.002). Although fluctuations of ERT concentrations after the pregnancy period assessed cannot be excluded, these findings suggest that the concentrations of ERT are not affected as much by gestational age. Rather, they seem affected by the method used to induce pregnancy, NM or VT. Several differences between NM and VT may account for the differences in the AF content of ERT. However, ERT-associated factors could also be involved. ERT has been directly associated with oxidative stress and is recognized to have ROS-scavenging functions (Brummel, 1985; Franzoni et al., 2006). During physiological pregnancy, all tissues and, mostly, placenta and
Fig. 1. Comparison of ERT concentrations according to the days of gestation at which AFs were collected both in ewes that were naturally mated (NM) and embryo transferred with in vitro–produced vitrified/thawed embryos (VT). To take account of the skewness towards large values, concentrations were plotted as log10 [ERT]. *p b 0.05; **p b 0.01; ***p b 0.001.
foetus require high amounts of oxygen (Mutinati et al., 2013). Therefore, pregnancy is a process that lends itself naturally to an increase in ROS production (Erisir et al., 2009). In this light, the higher ERT concentrations observed in the AF of VT vs. NM suggest an increased response against free radicals. In this sense, there is evidence of increased production of free radicals in pregnancies induced by ARTs (Gupta et al., 2010). Moreover, given the low tissue turnover of ERT (Kato et al., 2010; Kawano et al., 1982), an increase in AF concentrations might reflect an increase of apoptotic processes resulting from cell damage induced by free radicals. Oxidative stress is also causally linked to inflammatory processes, creating a vicious circle where, on one hand, oxidative stress triggers inflammation and, on the other, inflammatory cells produce high levels of ROS. Notably, the gene for ETT lies in close proximity to genes involved in inflammatory responses, and it is abundantly expressed in inflammatory cells such as CD14+ macrophages and monocytes (Paul and Snyder, 2010). ETT expression is upregulated by inflammatory cytokines and, in humans, its promoter shows binding sites for the transcription factor nuclear factor-kappa B, which regulates inflammatory genes (Paul and Snyder, 2010). In VT pregnancies, therefore, the higher ERT concentrations may be an indicator of a more severe inflammatory state compared to pregnancies induced by NM. Unfortunately, has not been possible to monitoring the levels of markers of oxidative stress, such as malondialdehyde (MDA), 8-isoprostane, or protein-bound carbonyl groups, to evaluate how they correlate with ERT levels. Moreover, only few gestations were induced after transfer of vitrified IVP embryos and all of NM and VT pregnancies were uncomplicated. It is, therefore, difficult to ascertain the real impact of ERT concentrations in either NM or VT pregnancies. However, taking into account the dependence of ERT concentration by the levels of the ETT-specific mRNA, as well as the modulating effect of oxidative stress/inflammation on the expression of ETT, it is plausible
Fig. 2. Differences between ERT concentrations according to the days of gestation and the method used to induce pregnancy. To take account of the skewness towards large values, concentrations were plotted as log10 [ERT]. *p b 0.05; **p b 0.01; ***p b 0.001.
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that the measurement of AF ERT might serve as a useful biomarker of oxidative stress and/or inflammatory state in pregnancy. 5. Conclusion For the first time, to our knowledge, ERT concentrations were measured in the amniotic fluid of pregnant sheep after natural mating and transfer of vitrified/thawed IVP embryos. ERT concentrations were significantly different between the groups, and were overall higher in the IVP group. On the whole, findings provide new insights, both in the monitoring and in understanding of the mechanisms accountable for the complications in pregnancy. Conflict of interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper. Acknowledgements Funding was provided by the Fondazione Banco di Sardegna, Sassari, Italy, and by the Ministero dell'Università e della Ricerca, Italy. It is a pleasure to thank Professor Arduino A. Mangoni from Flinders University (Australia) for his critical reading of the manuscript. References Agarwal, A., Allamaneni, S.S., 2004. Role of free radicals in female reproductive diseases and assisted reproduction. Reprod. BioMed. Online 9, 338–347. Agarwal, A., Gupta, S., Sharma, R.K., 2005. Role of oxidative stress in female reproduction. Reprod. Biol. Endocrinol. 3, 28. Anchamparuthy, V.M., Dhali, A., Lott, W.M., Pearson, R.E., Gwazdauskas, F.C., 2009. Vitrification of bovine oocytes: implications of follicular size and sire on the rates of embryonic development. J. Assist. Reprod. Genet. 26, 613–619. Bogliolo, L., Ariu, F., Leoni, G., Uccheddu, S., Bebbere, D., 2011. High hydrostatic pressure treatment improves the quality of in vitro-produced ovine blastocysts. Reprod. Fertil. Dev. 23, 809–817. Brummel, M.C., 1985. In search of a physiological function for L-ergothioneine. Med. Hypotheses 18, 351–370. Cognié, Y., Poulin, N., Locatelli, Y., Mermillod, P., 2004. State-of-the-art production, conservation and transfer of in-vitro-produced embryos in small ruminants. Reproduction Fertility and Development 16, 437–445. Erisir, M., Benzer, F., Kandemir, F.M., 2009. Changes in the rate of lipid peroxidation in plasma and selected blood antioxidants before and during pregnancy in ewes. Acta Vet. Brno 78, 237–242. Ey, J., Schömig, E., Taubert, D., 2007. Dietary sources and antioxidant effects of ergothioneine. J. Agric. Food Chem. 55, 6466–6474. Franzoni, F., Colognato, R., Galetta, F., Laurenza, I., Barsotti, M., Di Stefano, R., Bocchetti, R., Regoli, F., Carpi, A., Balbarini, A., Migliore, L., Santoro, G., 2006. An in vitro study on the free radical scavenging capacity of ergothioneine: comparison with reduced glutathione, uric acid and trolox. Biomed. Pharmacother. 60, 453–457. Gründemann, D., 2012. The ergothioneine transporter controls and indicates ergothioneine activity—a review. Prev. Med. 54, S71–S74. Gupta, S., Sekhon, L., Kim, Y., Agarwal, A., 2010. The role of oxidative stress and antioxidants in assisted reproduction. Curr. Womens Health Rev. 6, 227–238.
241
Hasler, J.F., 2001. Factors affecting frozen and fresh embryo transfer pregnancy rates in cattle. Theriogenology 56, 1401–1415. Kato, Y., Kubo, Y., Iwata, D., Kato, S., Sudo, T., Sugiura, T., Kagaya, T., Wakayama, T., Hirayama, A., Sugimoto, M., Sugihara, K., Kaneko, S., Soga, T., Asano, M., Tomita, M., Matsui, T., Wada, M., Tsuji, A., 2010. Gene knockout and metabolome analysis of carnitine/organic cation transporter OCTN1. Pharm. Res. 27, 832–840. Kawano, H., Otani, M., Takeyama, K., Kawai, Y., Mayumi, T., Hama, T., 1982. Studies on ergothioneine. VI. Distribution and fluctuations of ergothioneine in rats. Chem. Pharm. Bull. 30, 1760–1765. Lonergan, P., Fair, T., 2008. In vitro-produced bovine embryos-dealing with the warts. Theriogenology 69, 17–22. Morton, K.M., Catt, S.L., Maxwell, W.M., Evans, G., 2005. In vitro and in vivo developmental capabilities and kinetics of in vitro development of in vitro matured oocytes from adult, unstimulated and hormone-stimulated prepubertal ewes. Theriogenology 64, 1320–1332. Mutinati, M., Piccinno, M., Roncetti, M., Campanile, D., Rizzo, A., Sciorsci, R.L., 2013. Oxidative stress during pregnancy in the sheep. Reprod. Domest. Anim. 48, 353–357. Nieddu, S.M., Strina, A., Corda, A., Furcas, G., Ledda, M., Pau, S., Ledda, S., 2014. Comparation of foetal growth in natural mated and vitrified/warmed ovine embryos by ultrasonographic measurements. Global Vet. 12, 91–97. Nieddu, S.M., Mossa, F., Strina, A., Ariu, F., Pau, S., Ledda, M., Sotgia, S., Carru, C., Ledda, S., 2015. Differences in amniotic amino acid concentrations between pregnancies obtained with transfer of vitrified thawed in vitro-produced embryos and with natural mating in sheep. Theriogenology 83, 687–692. Nikodemus, D., Lazic, D., Bach, M., Bauer, T., Pfeiffer, C., Wiltzer, L., Lain, E., Schömig, E., Gründemann, D., 2011. Paramount levels of ergothioneine transporter SLC22A4mRNA in boar seminal vesicles and cross-species analysis of ergothioneine and glutathione in seminal plasma. J. Physiol. Pharmacol. 62, 411–419. Novakovic, B., Wong, N.C., Sibson, M., Ng, H.K., Morley, R., Manuelpillai, U., Down, T., Rakyan, V.K., Beck, S., Hiendleder, S., Roberts, C.T., Craig, J.M., Saffery, R., 2010. DNA methylation-mediated down-regulation of DNA methyltransferase-1 (DNMT1) is coincident with, but not essential for, global hypomethylation in human placenta. J. Biol. Chem. 285 (13), 9583–9593. Paul, B.D., Snyder, S.H., 2010. The unusual amino acid L-ergothioneine is a physiologic cytoprotectant. Cell Death Differ. 17, 1134–1140. Pfeiffer, C., Bauer, T., Surek, B., Schömig, E., Gründemann, D., 2001. Cyanobacteria produce high levels of ergothioneine. Food Chem. 129, 1766–1769. Schauss, A.G., Vértesi, A., Endres, J.R., Hirka, G., Clewell, A., Qureshi, I., Pasics, I., 2010. Evaluation of the safety of the dietary antioxidant ergothioneine using the bacterial reverse mutation assay. Toxicology 278, 39–45. Sotgia, S., Pisanu, E., Cambedda, D., Pintus, G., Carru, C., Zinellu, A., 2014a. Ultraperformance liquid chromatographic determination of lergothioneine in commercially available classes of cow milk. J. Food Sci. 79, 1683–1687. Sotgia, S., Zinellu, A., Mangoni, A.A., Pintus, G., Attia, J., Carru, C., McEvoy, M., 2014b. Clinical and biochemical correlates of serum L-ergothioneine concentrations in community-dwelling middle-aged and older adults. PLoS One 9, e84918. Sotgia, S., Zinellu, A., Pisanu, E., Vlahopoulou, G., Ariu, F., Ledda, S., Pintus, G., Carru, C., Bogliolo, L., 2015. Concentrations of L-ergothioneine in follicular fluids of farm animals. Comp. Clin. Pathol. http://dx.doi.org/10.1007/s00580-015-2098-8. Tamai, I., Nakanishi, T., Kobayashi, D., China, K., Kosugi, Y., Nezu, J., Sai, Y., Tsuji, A., 2004. Involvement of OCTN1 (SLC22A4) in pH-dependent transport of organic cations. Mol. Pharm. 1, 57–66. Thibier, M., 2011. Embryo transfer: a comparative biosecurity advantage in international movements of germplasm. Rev. Sci. Tech. 30, 177–188. Turner, E., Brewster, J.A., Simpson, N.A., Walker, J.J., Fisher, J., 2009. Imidazole-based erythrocyte markers of oxidative stress in preeclampsia—an NMR investigation. Reprod. Sci. 16, 1040–1051. Wu, X., George, R.L., Huang, W., Wang, H., Conway, S.J., Leibach, F.H., Ganapathy, V., 2000. Structural and functional characteristics and tissue distribution pattern of rat OCTN1, an organic cation transporter, cloned from placenta. Biochim. Biophys. Acta 1466, 315–327.