Elsevier

Theriogenology

Volume 85, Issue 4, 1 March 2016, Pages 688-697
Theriogenology

Research article
L-ergothioneine supplementation during culture improves quality of bovine in vitro–produced embryos

https://doi.org/10.1016/j.theriogenology.2015.10.008Get rights and content

Abstract

The aim of this study was to evaluate whether supplementation of bovine culture medium with the natural antioxidant L-ergothioneine (LE), improves in vitro blastocyst development and quality, assessed as resistance to cryopreservation, total cells number, cellular differentiation, and apoptosis index. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid with 0, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM of LE (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On the basis of the results of this dose-response trial, the range of concentrations to test was reduced in experiment 2, in which presumptive zygotes were cultured with 0, 0.05 mM, and 0.1 mM of LE. On Day 7, embryo yields were assessed, and the blastocysts (BL) were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO and 0.5 M sucrose. Finally, BL produced on Day 8 in the absence (control) and presence of 0.1 mM LE were used for transferase-mediated dUTP nick end labeling and differential staining to evaluate, respectively the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm lineages (experiment 3). Despite similar blastocyst yields, supplementation of culture medium with 0.1 mM LE improved the cryotolerance of in vitro–produced (IVP) embryos compared to the control group, as indicated by higher (P < 0.05) hatching rates recorded after 48-hour post-warming culture (48.5%, 50.0%, and 63.8%, respectively with 0, 0.05, and 0.1 mM LE). Interestingly, when embryos were cultured in the presence of 0.1 mM LE, the percentage of BL with the most physiological ICM:total cells ratio (20%–40%) increased (85.1 vs. 66.0%, P < 0.05), confirming a beneficial effect on embryo quality. Furthermore, 0.1 mM LE decreased (P < 0.01) both the average number (4.3 ± 0.2 vs. 9.1 ± 0.3) and the proportion (3.6 ± 0.3 vs. 8.1 ± 0.5) of apoptotic cells in BL compared to the control. In conclusion, the enrichment of bovine culture medium with 0.1 mM LE improves embryo quality, as indicated by the improved cryotolerance, the lower apoptotic rate, and the higher percentage of BL with the most physiological ICM:total cells ratio.

Introduction

Assisted reproductive technologies involving the in vitro production of preimplantation stage embryos are imperative for the treatment of infertility and/or for fertility management in both human and veterinary reproductive medicine [1], [2]. The total number of in vitro–produced (IVP) bovine embryos transferred to recipients worldwide in 2011 alone was 373,836 [3]. Despite this widespread use, the fertilization and culture of early livestock embryos in suboptimal, foreign microenvironments lead to a high frequency (50%–70%) of early embryonic demise during about 5 to 7 days of in vitro development [4], [5]. In addition, numerous studies have shown that IVP bovine embryos differ from their in vivo–derived counterparts in timing of development, morphology [6], metabolism [7], intactness of zona pellucida (ZP) [8], blastocyst numbers [9], cryotolerance [10], relevant gene expression [11] and, ultimately, pregnancy rates [12].

One of the most important parameters for pregnancy establishment and maintenance after embryo transfer (ET) is embryo quality and hence cryotolerance. Pregnancy rates after ET of cryopreserved IVP embryos are lower than those recorded with in vivo–produced embryos, due to their poorer quality [13]. Even if the background of this difference is not completely understood, it is known that the culture medium has dramatic effect on developmental competence and resistance to cryopreservation of the resulting embryos [14]. Indeed, the high incidence of developmental failure has been attributed to a wide range of non-mutually exclusive dysfunctions including poor gamete quality, chromosomal abnormalities, telomere uncapping, and suboptimal culture conditions that induce oxidative stress [15], [16].

An increase of reactive oxygen species generation and subsequent oxidative stress is the major factor affecting in vitro mammalian embryo development; indeed, oxidative stress is implicated in many different types of cell injuries, including membrane lipids peroxidation, oxidation of amino acids and nucleic acids, apoptosis and necrosis, which may subsequently decrease the viability of IVP embryos [17], [18]. To overcome this, various antioxidants such as β-mercaptoethanol, selenium, and resveratrol have been used during in vitro culture to enhance the antioxidant capacity of IVP embryos and hence to improve the development of preimplantation embryos [14], [19], [20].

L-ergothioneine (LE), a naturally occurring amino acid, is a powerful scavenger of OH and an inhibitor of iron or copper ion–dependent generation of OH from H2O2 [21]. Ergothioneine is synthesized by some bacteria and fungi, but not by animals [22], [23]. Therefore, mammals acquire ergothioneine from their diet, including mushrooms, oats, corn, and meat [24]. Indeed, food-derived LE is found in brain, erythrocytes, liver, kidney, heart, seminal fluid, and ocular tissues of several species [25]. Ergothioneine has been shown to scavenge singlet oxygen, hydroxyl radicals, hypochlorous acid, and peroxyl radicals [26], [27] and inhibit peroxynitrite-dependent nitration of proteins and DNA [28]. Furthermore, a protective effect against diabetic embryopathy in pregnant rats has been reported [29]. Recently, it has been shown that antioxidant activity of LE is the highest, and it eliminates the most active free radicals compared to some classic well-known antioxidants such as glutathione (GSH), uric acid and trolox [30].

It has been reported that the addition of LE during IVM, IVF, and IVC in the sheep has a beneficial effect on in vitro maturation of oocytes and embryonic development, especially from cleavage to morulae stages [31]. To our current knowledge, there is no study available in literature on the effects of an antioxidant with strong and unique features, such as LE in cattle. Therefore, the aim of this work was to evaluate whether supplementation of culture medium with LE improves in vitro blastocyst development and embryo quality in cattle, the latter assessed in terms of cryotolerance, allocation of blastocyst cells into the inner cell mass (ICM) and trophectoderm (TE) lineages and apoptotic cells rate.

Section snippets

Experimental design

In experiment 1, presumptive zygotes were cultured in vitro with 0 (control, n = 232), 0.05 mM (n = 240), 0.1 mM (n = 236), 0.5 mM (n = 238), and 1 mM LE (n = 244), over four replicates. On the basis of the results of this dose-response trial, the range of concentrations to test was reduced in experiment 2, in which presumptive zygotes were cultured in vitro with 0 (control, n = 282), 0.05 mM (n = 286), and 0.1 mM LE (n = 285), over five replicates. The blastocysts (BL) produced (on average 94

Results

The dose-response trial in experiment 1 showed a remarkable decrease (P < 0.01) of both cleavage and blastocyst development with 0.5 mM LE and an evident toxic effect, with no embryo production, with 1 mM LE (Fig. 3).

In experiment 2, the supplementation of LE during culture did not affect cleavage and post-fertilization embryo development, both in terms of total embryo output (grades 1,2 tight morulae and blastocyst) and superior quality BL (grade 1,2 BL), as summarized in Table 1. The

Discussion

The results of the present study demonstrated that enrichment of culture medium with low doses of the natural antioxidant LE, improves bovine embryo quality, as indicated by increased cryotolerance, improved ICM:total cells ratio, and reduced incidence of apoptosis.

The preliminary dose-response trial (experiment 1) showed a negative effect on both cleavage and blastocyst rates with 0.5 mM LE and an evident toxic effect, with further reduction of cleavage and no blastocyst production at the

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