or resveratrol to maturation medium before vitrification on in vitro-matured calf oocytes

Accepted Manuscript Assessment of the Effect of Adding L-Carnitine and/or Resveratrol to Maturation Medium Prior to Vitrification on in Vitro Matured Calf Oocytes José Felipe Sprícigo, Roser Morató, Núria Arcarons, Marc Yeste, Margot Alves Dode, Manuel López-Bejar, Teresa Mogas PII:

S0093-691X(16)30454-X

DOI:

10.1016/j.theriogenology.2016.09.035

Reference:

THE 13834

To appear in:

Theriogenology

Received Date: 8 June 2016 Revised Date:

13 September 2016

Accepted Date: 17 September 2016

Please cite this article as: Sprícigo JF, Morató R, Arcarons N, Yeste M, Dode MA, López-Bejar M, Mogas T, Assessment of the Effect of Adding L-Carnitine and/or Resveratrol to Maturation Medium Prior to Vitrification on in Vitro Matured Calf Oocytes, Theriogenology (2016), doi: 10.1016/ j.theriogenology.2016.09.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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REVISED

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ASSESSMENT OF THE EFFECT OF ADDING L-CARNITINE AND/OR

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RESVERATROL TO MATURATION MEDIUM PRIOR TO VITRIFICATION ON IN

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VITRO MATURED CALF OOCYTES.

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José Felipe Sprícigo a, b [email protected]

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Roser Morató a, [email protected]

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Núria Arcarons a, [email protected]

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Marc Yeste c, [email protected]

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Margot Alves Dode.b, [email protected]

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Manuel López-Bejar a, [email protected]

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Teresa Mogasa, *, [email protected]

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a

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Vallès, Spain.

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b

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Brazil.

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c

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Oxford, United Kingdom

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d

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Reproduction, Brasília-DF, Brazil.

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Facultat de Veterinària, Universitat Autònoma de Barcelona, Cerdanyola del

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School of Agriculture and Veterinary Medicine, University of Brasilia, Brasília-DF,

Nuffield Department of Obstetrics and Gynaecology, University of Oxford,

Embrapa Genetic Resources and Biotechnology, Laboratory of Animal

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*Corresponding author's address: Departament de Medicina i Cirurgia Animals,

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Universitat Autònoma de Barcelona, Cerdanyola del Vallès E-08193, Spain. E-

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mail: [email protected]; Tel: +34 93 581 1044; Fax: +34 93 581 20 06.

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ACCEPTED MANUSCRIPT Abstract

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Cryopreservation may lead bovine oocytes to undergo morphological changes and

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functional damage, due to the high lipid content in the cytoplasm and the formation

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of reactive oxygen species. Against this background, the present study aimed to

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improve the cryotolerance and developmental competence of prepubertal calf

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oocytes by adding L-carnitine (LC) and/or resveratrol (R) to the in vitro maturation

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(IVM) medium, as the former is involved in lipid metabolism and both are able to

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scavenge reactive oxygen species. With this purpose, different quality and

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functional oocyte parameters, such as spindle and chromosome configuration,

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DNA integrity, caspase activity and the profile of genes involved in lipid

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metabolism and oxidative stress were evaluated in IVM bovine oocytes before or

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after vitrification/warming. Oocytes were matured in the absence (control) or

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presence of LC (3.03 mM) and/or R (1 µM) and then vitrified/warmed prior to IVF

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and embryo culture. All treatment groups (control, LC, R and LC+R) of non-vitrified

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IVM oocytes showed similar rates (P>0.05) of a normal spindle and chromosome

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configuration to oocytes vitrified/warmed after maturation in the presence of LC+R.

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When oocytes in all treatment groups were compared before and after vitrification,

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no significant differences were detected in DNA fragmentation as measured using

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the TUNEL method. However, the proportion of early apoptotic oocytes increased

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following vitrification/warming, except when previously matured with R.

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Vitrified/warmed oocytes matured in the presence of LC did not differ with non-

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vitrified oocytes in terms of the expression of ACACA, SLC2A1, PLIN2, HSPA1A,

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GPX1 and SOD1 genes. Similarly, expression of ACACA, SLC2A1, PLIN2,

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HSPA1A and SOD1 genes in vitrified/warmed oocytes was similar to that of their

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fresh counterparts when matured in the presence of R. Finally, while the addition

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ACCEPTED MANUSCRIPT of LC and/or R to IVM medium had no effect on both cleavage and blastocyst

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rates either in fresh or vitrified oocytes. To conclude, the results of the present

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study demonstrate that the addition of LC and/or R to the in vitro maturation

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medium used for prepubertal bovine oocytes did not increase the embryo

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development potential of both fresh and vitrified oocytes. However, LC+R

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supplementation prior to vitrification decreased spindle damage, R addition

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modulated apoptosis and LC or R addition before vitrification positively affected

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the gene expression of vitrified/warmed oocytes.

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KEYWORDS:, cryopreservation, prepubertal, spindle configuration, DNA

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fragmentation, gene expression, apoptosis.

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1. Introduction

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There is mounting interest in the cryopreservation of mammalian oocytes and

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this is related to the introduction of procedures such as in vitro embryo production,

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nuclear transfer or gene banking. [1]. Much research in this field has been focused on improving the efficiency of

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oocyte cryopreservation. However, the practical use of vitrification to preserve

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bovine oocytes is still limited since vitrified oocytes seem to exhibit impaired in

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vitro development. Thus, blastocyst yields from vitrified/warmed IVM bovine

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oocytes have not been much improved (reviewed in [2]). Among the explanations

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provided for the inefficient cryopreservation of IVM bovine oocytes are the large

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size of oocytes, their low volume-to-surface ratio, high lipid content, and plasma

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membrane composition [3]. While lipids play an essential role in energy

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metabolism during oocyte maturation, fertilization and early embryonic

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development, high lipid content increases the cell susceptibility to cryoinjury [4].

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Lipid droplets have been localized at the plasma membrane or close to organelles

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such as mitochondria and endoplasmic reticulum [5], which seem to be main

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targets for cryoinjury [6, 7].

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The small water-soluble molecule L-carnitine (LC; β-hydroxy-γtrimethylammonium-butyric acid) plays a vital role for cell metabolism, as shuttles

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fatty acids into mitochondria to generate ATP and consequently affects

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intracellular ATP levels (reviewed in [8]). L-carnitine also has antioxidant

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properties, as reduces levels of reactive oxygen species (ROS) and protects cells

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against DNA damage [9] and apoptosis [10]. A study conducted in cattle showed

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that LC treatment during IVM of oocytes induced the translocation of active

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mitochondria to the inner oocyte cytoplasm driving embryonic development [11]. In

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maturation, increase the number of active mitochondria, diminish intracellular ROS

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levels and lipid droplets, and improve in vitro embryo development [12-14].

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Similarly, L-carnitine supplementation during in vitro maturation of sheep oocytes

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improves embryo developmental potential by reducing ROS and increasing GSH

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and alters the expression of antioxidant enzyme genes throughout the embryo

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development [15]. While this dual antioxidant/lipid metabolism role of LC makes it

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a potential candidate to improve the efficiency of oocyte cryopreservation,

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inconsistent results in terms of embryo development have been reported when

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using vitrified/warmed bovine oocytes previously matured in the presence of LC.

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Indeed, in a study by Chankitisakul et al. [16], LC added to the bovine oocyte IVM

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medium led to higher blastocyst rates recorded 8 days after vitrification/warming

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and in vitro fertilization (IVF). In contrast, and despite an improved nuclear

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maturation rate, Phongnimitr et al. [17] found no increase in blastocyst rates using

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vitrified/warmed oocytes matured in a LC-supplemented medium. This indicates

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that research is warranted to address the effects of LC. On the other hand, although mature oocytes likely have efficient intracellular

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antioxidant systems, their redox potential could be affected by the harsh conditions

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of cryopreservation. As indirect evidence of impaired redox status, a significant

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rate of DNA damage was detected by the Comet assay in warmed cow oocytes

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[18]. Related to this, resveratrol (R; 3,4′,5-trihydroxystilbene), a natural type of

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phenol produced by several plants in response to injury [19], is a powerful

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antioxidant that could improve the cryotolerance of IVM bovine oocytes. In fact,

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adding the IVM media with R has been reported to improve fertilization outcomes

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and developmental capacity in both cow and pig oocytes, most likely through the

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of gene expression during oocyte maturation [20-22]. . While in the pig, R

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supplementation at various stages of IVM and vitrification/warming has been found

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to make the oocytes less susceptible to cryopreservation-induced damage [23],

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the effects upon the cryotolerance of IVM bovine oocytes are yet to be addressed.

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Therefore, and given the role of LC and R in lipid metabolism and oxidative stress, the present study examined whether the single or combined addition of LC

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and R to IVM medium of in vitro-matured bovine oocytes prior to

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vitrification/warming improved their cryotolerance and developmental competence

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following fertilization. In order to better understand the mechanisms underlying the

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effects mediated by LC and R as well as to fully address the inconsistencies found

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in the literature in the case of LC (reviewed in [2]), spindle and chromosome

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configuration, DNA integrity, caspase activity, embryonic cleavage, blastocyst

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formation and the expression levels of separate genes involved in lipid (ACACA,

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PLIN2) and glucose metabolism (SCL2A1), and in heat- (HSPA1A) and oxidative

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stress pathways (GPX1 and SOD1) were evaluated.

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2. Materials and Methods

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Unless otherwise specified, all reagents were purchased from Sigma-Aldrich

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(St. Louis, MO, USA). 2.1. Oocyte collection and in vitro maturation

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The methods used for the in vitro maturation of the oocytes have been

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described elsewhere [24]. Briefly, ovaries from slaughtered prepubertal calves (9

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months old) were transported from a local slaughterhouse to the laboratory in

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phosphate buffered saline (PBS) at 35-37ºC. Cumulus oocyte complexes (COCs)

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were obtained by aspiration from 2–8 mm follicles and only COCs with more than

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three cumulus cells layers and a homogeneous cytoplasm were selected. Once

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selected, groups of up to 40-50 COCs were transferred to 500 µL of maturation

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medium in a four-well plate and cultured for 24 h at 38.5°C in a 5% CO 2 humidified

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air atmosphere. The maturation medium was comprised of TCM-199

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supplemented with 10% (v/v) fetal calf serum (FCS), 10 ng/mL epidermal growth

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factor and 50 mg/mL gentamicin.

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2.2. Oocyte vitrification and warming

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2.2.1. Vitrification protocol

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Oocytes were vitrified using the Cryotop device (Dibimed-Biomedical Supply,

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S.L., Valencia, Spain) and vitrification and warming solutions described by

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Kuwayama et al. [25]. The holding medium (HM) for formulating all vitrification–

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warming solutions was HEPES-buffered TCM-199 containing 20% (v/v) FCS.

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Partially denuded oocytes were transferred to an equilibration solution (ES)

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consisting of 7.5% (v/v) ethylene glycol (EG) and 7.5% (v/v) dimethylsulfoxide

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(DMSO) in HM for 10 min and then transferred to the vitrification solution (VS)

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containing 15% (v/v) DMSO, 15% (v/v) EG and 0.5M sucrose dissolved in HM.

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After incubation for 30–40 s, the oocytes (up to five) were loaded onto the Cryotop

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device, almost all the solution removed to leave only a thin layer covering the

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oocytes, and the device plunged in liquid nitrogen. The entire process from

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exposure in VS to plunging in liquid nitrogen was completed within 90 s. 2.2.2. Warming protocol

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All warming steps were performed at 38.5°C. Vitrifi ed oocytes were warmed by

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directly immersing the Cryotop end into the warming solution consisting of 1M

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sucrose dissolved in HM for 1 min. The recovered oocytes were then transferred

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to HM solution containing 0.5M sucrose for 3 min and then incubated in HM for 5

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min. After two subsequent washes in HM for 5 min each, the oocytes were

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transferred back into maturation dishes and matured for approximately 2 h.

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2.3. In vitro fertilization and embryo culture

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Twenty four hours after the onset of in vitro maturation, in vitro matured oocytes from all treatment groups were in vitro fertilized. Briefly, the oocytes were washed

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once in fertilization medium before being transferred, in groups of 20-25, to four-

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well plates containing 250 µL of fertilization medium per well (Tyrode's medium

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supplemented with 25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6

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mg/mL fatty acid-free BSA and 10 µg/mL heparin–sodium salt (Calbiochem,

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Darmstadt, Germany). Motile spermatozoa were obtained by centrifuging frozen–

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thawed sperm from Asturian bulls of proven fertility (ASEAVA, Llanera, Asturias,

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Spain) on a discontinuous gradient (Bovipure, Nidacon International, Gothenburg,

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Sweden) for 10 min at 100 x g at room temperature. Viable spermatozoa collected

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from the bottom were washed (Boviwash, Nidacon International, Gothenburg,

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Sweden) and pelleted by centrifugation at 100 x g for 5 min. Spermatozoa were

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counted in a Neubauer chamber and diluted in fertilization medium to give a final

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concentration of 2 x 106 spermatozoa/mL. A 250 µL aliquot of this suspension was

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added to each fertilization well to obtain a final concentration of 1 x 106

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spermatozoa/mL. Plates were incubated at 38.5°C in a 5% CO2 humidified air

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atmosphere. At approximately 18 h post-insemination (pi), presumptive zygotes were

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denuded by gentle pipetting and transferred to 25-µL culture droplets of synthetic

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oviductal fluid (SOF) [26] (1 embryo/µL) containing 5% FCS (v/v) under mineral oil.

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Embryos were incubated at 38.5°C in a 5% CO 2, 5% O2, and 90% N2 humidified

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air atmosphere. Cleavage rates were recorded at 48 h post insemination (hpi) and

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blastocyst and hatching rates were also determined on days 7 (D7) and 8 (D8).

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2.4. Spindle and chromosome configuration

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According to a previously described protocol [27], after 24 h of in vitro maturation, oocytes were totally denuded of cumulus cells by gentle pipetting in

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PBS. Fresh and vitrified oocytes were fixed in a solution of 4% (w/v) formaldehyde

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in PBS for 30 min at 38.5°C, and permeabilized in T riton X-100 (2.5% (v/v) in PBS)

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for 15 min. For immunostaining, fixed oocytes were incubated with a monoclonal

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anti-α-tubulin antibody (Molecular Probes, Paisley, UK) (1:250) overnight, followed

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by incubation with an antimouse IgG antibody-Alexa Fluor 488 (Molecular Probes,

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Paisley, UK) (1:5000) for 1 h at 37ºC. Between incubations, the oocytes were

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washed three times in pre-warmed PBS for 5 min. Groups of five oocytes were

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mounted on poly-L-lysine treated coverslips fitted with a self-adhesive

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reinforcement ring and then stained with 4,6-diamidino-2-phenylindole

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hydrochloride (DAPI (Vysis Inc., Downers Grove, USA); 125 ng/mL). Preparations

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were sealed with nail varnish. An epifluorescence microscope (Axioscop 40FL,

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Carl Zeiss, Germany) was used to examine tubulin (Alexa fluor) and chromatin

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ACCEPTED MANUSCRIPT (DAPI). The criteria used to classify chromosome and microtubule distributions

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have been described elsewhere [27]. Oocytes were assigned a quality score

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based on the morphological normality of the meiotic spindle and chromatin. The

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meiotic spindle was classified as normal if it showed the classic symmetrical barrel

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shape, with chromosomes aligned regularly in a compact group along the

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equatorial plane. In contrast, abnormal spindles showed disorganized, clumped,

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dispersed, or unidentifiable spindle elements and chromatin that was clumped or

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dispersed from the spindle centre.

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2.5. DNA fragmentation by TUNEL

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Oocytes from fresh and vitrified/warmed treatment groups were completely

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denuded of cumulus cells by gentle pipetting in PBS and DNA fragmentation was

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assessed by the TUNEL method as described elsewhere [28]. Briefly, oocytes

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were fixed in 4% (w/v) paraformaldehyde in PBS for 1 h at 37°C. After fixation,

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they were washed at least three times in PBS containing polyvinylpyrrolidone

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(0.3% PVP in PBS; PBS-PVP) and permeabilized in 0.5% Triton X-100 for 2 min.

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The oocytes were then washed three times in PBS–PVP and incubated in the

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TUNEL reaction cocktail (In-situ Cell Death Detection System; Roche Diagnostic,

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Indianapolis, IN, USA) at 37°C for 1 h in the dark. Oocytes exposed to DNase I for

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15 min at room temperature (50 µL of RQ1 RNase-free Dnase (50 U/mL) served

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as positive controls and oocytes not exposed to the terminal TdT enzyme served

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as negative controls. Oocytes were washed thoroughly in PBS-PVP and finally

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mounted on poly-lysine-treated slides and covered with a 3-µl drop of Vectashield

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containing 125 ng/mL of DAPI to stain all nuclei and then a coverslip, sealing the

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edges with nail polish. The slides were then examined in an epifluorescence

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microscope (Axioscop 40FL, Carl Zeiss, Germany). Nuclei were scored as having

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either intact (TUNEL(+); blue stain) or fragmented (TUNEL(-); green stain) DNA.

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The apoptotic index was calculated as the ratio between TUNEL(-) cells and total

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number of nuclei. 2.6. Caspase labeling

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To detect active caspases in the oocytes, a FLICA Apoptosis Kit (FAM FLICA

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Poly Caspase Assay Kit, Immunochemistry Technologies, Bloomington, MN, USA)

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was used. Following the protocol described by Wasielak and Bogacki [29] and the

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manufacturer's instructions, oocytes from all groups were completely denuded of

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cumulus cells by gentle pipetting in PBS, and caspase activity was determined. As

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a caspase-positive control, fresh oocytes was incubated in maturation medium

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containing 1 µM staurosporine at 38.5°C overnight to activate cas pases.

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Immediately after washing in PVP-PBS, all oocytes were incubated in FLICA

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solution for 1 h at 38.5°C and 5% CO 2. A negative control (fresh oocytes incubated

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only in PBS) was included in each replicate. Next, all samples were washed in

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PVP-PBS and then stained in a mixture of propidium iodide (PI) (1.5 µg/mL in

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PBS) and Hoechst 33342 (1 µg/mL in PBS) for 5 min at 37°C to visualize cell

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nuclei and dead cell nuclei, respectively. Immediately before assessment, oocytes

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were mounted on slides and flattened with a coverslip. Slides were examined in an

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epifluorescence microscope (Axioscop 40FL, Carl Zeiss, Germany). Stained

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oocytes were classified as: viable oocytes without active caspases (FLICA−/PI−),

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viable oocytes with activated caspases (FLICA+/PI−) and dead oocytes (PI+).

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2.7. Gene expression

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Oocytes in the different experimental groups were denuded by pipetting,

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washed three times in Dulbecco’s PBS supplemented with 1 mg/ml PVA at

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38.5°C, snap-frozen in liquid nitrogen and stored a t -80°C for mRNA extraction

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ACCEPTED MANUSCRIPT and reverse transcription. Molecular biology procedures were carried out as

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previously described [30]. In brief, poly(A)-RNA was extracted from pools of 20

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oocytes per experimental group, following the manufacturer’s instructions using

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the Dynabeads mRNA Direct Extraction KIT (Dynal Biotech, Oslo, Norway) with

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minor modifications. Immediately after extraction, reverse transcription (RT) was

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performed in a thermocycler (Quanta Biosciences; Gaithersburg, MD, USA), at the

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following conditions: a first step of 5 min at 25°C , followed by 1 h at 42°C to allow

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the RT of mRNA and 10 min at 70°C to inactivate the RT enzyme. After RT, cDNA

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was diluted with 25 µL of the elution solution provided with the kit and stored at -

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20°C until use.

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The relative abundance of mRNA transcripts was quantified by real-time,

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quantitative RT-PCR (qRT-PCR) using the QuantStudio™ 7 Flex quantitative

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Real-Time PCR System (Applied Biosystems, Foster City, CA,USA) and SYBR®

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Select Master Mix (Life Technologies, Madrid, Spain). Six separate genes,

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ACACA, SLC2A1, PLIN2, HSPA1A, GPX1 and SOD1 were amplified. In each

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sample, cDNA was analyzed in triplicate to determine relative levels of each

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transcript of interest. Gene expression levels were normalized to levels of two

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separate housekeeping genes: glyceraldehyde-3-phosphate dehydrogenase

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(GAPDH) and peptidylprolyl isomerase A (PPIA). The qRT-PCR reaction mix

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contained 10 µL Fast SYBR Green Master Mix (Applied Biosystems), 0.5 µL

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forward and reverse primers (Life Technologies, Madrid, Spain) specific for the

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genes of interest and 2 µL of cDNA template. The final volume was made up to 20

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µL using nuclease-free water. Reactions were run at 95°C for 30 s followed by 40

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cycles and a standard dissociation curve.

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for each gene are provided in Table 1. The reactions were run in triplicate for each

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gene. The efficiency of primer amplification was 90 to 110%. Non-template

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controls were not amplified or returned a Ct value 10 points higher than the

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average Ct value for the genes. Expression levels of the target genes were

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normalized to average expression levels of GAPDH and PPIA, which were

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expressed at similar levels (Ct values) in all oocyte samples and were stable under

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the conditions used. The relative expression for each gene was calculated using

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the ∆∆Ct method with efficiency correction [31].

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Five experiments were designed to determine whether the addition of LC and/or R

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to a conventional IVM medium improved oocyte survival after vitrification and

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warming. Based on previous reports in bovine oocytes, the concentration of L-

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carnitine used was 3.03 mM (0.6 mg/mL) [16] and that of R was 1 µM (0.23 µg/mL)

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[22]. For all experiments, bovine oocytes were in vitro maturated in conventional

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IVM medium (control), or in the same medium supplemented with 3.03 mM LC, 1

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µM R or both agents (LC+R). Twenty-two hours after the onset of IVM, half the

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oocytes in each treatment group were vitrified and warmed. After allowing the

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recovery of the vitrified/warmed oocytes for an additional 2-h period in their

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respective IVM medium, oocytes from each treatment group were collected to

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assess spindle and chromosome configuration (Experiment 1, n=5 replicates),

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DNA fragmentation (Experiment 2, n=5 replicates), caspase activity (Experiment 3,

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n=4 replicates) and gene expression (Experiment 4, n=4 replicates). For

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experiment 5, oocytes from each treatment group were in vitro fertilized and

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cultured to assess the effects of treatment during IVM on the embryo culture of

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non-vitrified and vitrified/warmed oocytes (n=7 replicates).

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2.9. Statistical analysis

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All statistical analyses were conducted with IBM SPSS Version 21.0 for Windows (IBM Corp.; Chicago, Illinois, USA). Data were first checked for normality

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and homogeneity of variances (homocedasticity) using Shapiro-Wilk and Levene

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tests, respectively. When required data transformed through arcsin √x.

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The effects of treatment (i.e. LC, R, LC+ R) and those of vitrification (i.e. fresh

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vs. vitrified-warmed) upon meiotic spindle configuration, cleavage rates, blastocyst

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yield, DNA fragmentation (TUNEL), caspase activity and mRNA expression level

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analyses were determined through a two-way analysis of variance (ANOVA)

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followed by Sidak’s test for pair-wise comparisons. Chi-square test was also used

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in the case of cleavage rates, DNA fragmentation and caspase activity. When

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even transformed, data did not fit with normal distribution a non-parametric

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Scheirer-Ray-Hare ANOVA for ranked data (approach) was performed. After

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calculating the ‘H’ statistic, the Mann-Whitney test was used for pair-wise

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comparisons.

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Data are expressed as means ± standard error of the mean (SEM). In all cases, significant level was set at P≤0.05.

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3. Results

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3.1. Experiment 1. Effects of LC, R or LC+R supplementation during the IVM of

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bovine oocytes on spindle configurations before and after vitrification The effects of supplementing the IVM medium with LC and/or R on chromosome and microtubule configurations before and after vitrification were

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established by staining with Alexa-fluor 488 and DAPI. According to the data in

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Table 2, the percentage of non-vitrified oocytes reaching the MII stage after IVM

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was significantly higher (P<0.05) than that observed in vitrified/warmed oocytes,

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regardless of the treatment. No significant effects of any of the IVM supplements

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were observed in both fresh and vitrified oocytes. Oocytes vitrified/warmed after

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IVM in the presence of LC+R showed similar normal spindle configuration rates

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(P>0.05) compared with their fresh counterparts, while those vitrified/warmed after

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LC, R or no additives showed significantly higher percentages of abnormal

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spindles and decondensed chromosomes compared to their non-vitrified

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counterparts.

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3.2. Experiment 2. Effects of LC, R or LC+R supplementation during the IVM of bovine oocytes on DNA fragmentation before and after vitrification

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Figure 1 shows the effects of LC and/or R treatment during IVM on DNA

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fragmentation measured by TUNEL assay before and after oocyte vitrification.

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Following IVM, DNA fragmentation rates were similar in all treatment groups

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though oocytes treated with LC+R showed the lowest rate (1.6%). Oocytes that

343

had been vitrified/warmed showed slightly higher percentages of DNA

344

fragmentation though differences were not significant when compared to their

345

fresh counterparts. Again, LC+R vitrified group showed the lowest rate of DNA

346

fragmentation (4.9%).

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ACCEPTED MANUSCRIPT 347 348 349

3.3. Experiment 3. Effects of LC, R or LC+R supplementation during the IVM of bovine oocytes on caspase activity before and after vitrification As shown in Table 3, early apoptosis, measured by active caspase labeling, was detected in 1.0-2.8% of the fresh oocytes after IVM without significant

351

differences between them (P>0.05), which could be due to the low number of

352

samples analyzed for each group. When these oocytes were vitrified and warmed,

353

rates of early apoptosis in control, LC and LC+R groups were significantly higher

354

than those observed in their fresh counterparts. In contrast, no significant

355

differences were observed for R groups. A significant increase in the percentage

356

of dead oocytes was observed between fresh and vitrified/warmed oocytes.

358 359

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3.4. Experiment 4. Effects of LC, R or LC+R supplementation during the in vitro maturation of bovine oocytes on gene expression before and after vitrification Data on the relative abundances of mRNA transcripts produced in response to LC, R or LC+R treatments before vitrification/warming of the oocytes are provided

361

in Figure 2. Supplementation of IVM medium with LC, R or LC+R for 24 h had no

362

effect on the expression profiles of target genes (ACACA, SLC2A1, PLIN2,

363

HSPA1A, GPX1 and SOD1). In contrast, vitrification and warming significantly

364

(P < 0.05) modified the expression of ACACA, SLC2A1, GPX1 and SOD1 genes,

365

though no effects were detected on PLIN2 and HSPA1A mRNA levels. A higher

366

expression of ACACA was observed after vitrification in all treatment groups, but

367

the difference was only significant for those oocytes matured in the presence of

368

LC+R. Whilst the expression of SOD1 was significantly up-regulated after

369

vitrification/warming in control and LC+R IVM-oocytes, no significant differences

370

were observed for vitrified/warmed oocytes that were in vitro matured in the

371

presence of LC or R. Contrarily, SLC2A1 expression was significantly

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ACCEPTED MANUSCRIPT 372

downregulated after vitrifying/warming control or LC+R oocytes but no differences

373

were noted for the L or R treatments. The expression of GPX1 was also

374

downregulated after vitrification/warming, but only control and R IVM-oocytes

375

showed significant differences from their fresh counterparts.

377

3.5. Experiment 5. Effects of LC, R or LC+R supplementation during the in vitro

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376

maturation of bovine oocytes on embryo development before and after vitrification Table 4 shows the effects produced on the survival and developmental

379

competence of calf oocytes after their Cryotop vitrification/warming following

380

maturation in an IVM medium supplemented with LC and/or R. No significant

381

effects of any of the IVM supplements on cleavage and blastocyst rates were

382

observed in fresh oocytes. However both parameters were significantly affected by

383

cryopreservation. Vitrified oocytes showed lower cleavage rates than non-vitrified

384

oocytes, regardless of IVM treatment. Similar effects of vitrification were observed

385

on blastocyst yields at Day 7 and 8 and, again, the previous IVM treatment had no

386

effect on this variable. In spite of this, the percentages of hatched blastocysts at

387

Day 8 were significantly higher in LC-treated fresh oocytes than in control and

388

LC+R fresh groups but similar to R-treated fresh oocytes. Although a similar effect

389

on hatching rates was observed for vitrified/warmed oocytes matured in the

390

presence of LC and LC+R, all vitrified oocytes showed significantly lower

391

percentages of hatched blastocysts when compared to their fresh counterparts.

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ACCEPTED MANUSCRIPT 392

6. Discussion

393

Given the antioxidant and lipolytic actions of R and LC, we hypothesized that the addition of these two molecules to the IVM medium could improve the

395

cryotolerance of IVM bovine oocytes to vitrification/warming. Cryotolerance was

396

assessed in terms of effects on meiotic spindle structure, apoptosis and gene

397

expression of the oocyte and its potential for embryo development after in vitro

398

fertilization.

Our data indicate that neither LC, nor R or LC+R supplementation of the IVM

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399

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medium helped to improve nuclear maturation or had any effects on apoptosis in

401

the non-vitrified (fresh) oocytes. Several studies have shown beneficial effects of

402

LC on the maturation rates of pig [12, 13] and cow oocytes [17]. In contrast, our

403

results are similar to those reporting no improvement in maturation rates after

404

supplementing the IVM medium with LC (pig [14]; sheep [15]) or different

405

concentrations of R (0.1 µM to 2 µM) (pig [32]; goat [33]). In addition, in the

406

present study, cleavage rates and blastocyst yields on Days 7 or 8 after

407

insemination were also similar in the LC, R, LC+R and control groups. In fact, the

408

role of LC or R during IVM in embryo development has not been clearly

409

established. Some authors have reported improved blastocyst development after

410

IVF attributable to the supplementation of IVM medium with LC (cow [17]; pig [13];

411

sheep [15]) or R [22], while others have observed no such a benefit [12]. We did,

412

however, note that the hatching ability of Day 8 blastocyts was significantly better

413

for those fresh oocytes treated with LC. It is generally accepted that prepubertal

414

oocytes are less developmentally competent than oocytes from cows (reviewed by

415

[34]). Although rates of fertilization and cleavage of prepubertal calf oocytes are

416

similar to those of cow oocytes, their capacity to reach the blastocyst stage is

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20

ACCEPTED MANUSCRIPT reduced [35, 36]. In addition, pregnancy rates for in vivo or in vitro-produced

418

embryos derived from oocytes of prepubertal animals are low [36-38]. Several

419

explanations for the reduced developmental potential of prepubertal oocytes have

420

been proposed, including incomplete or deficient reorganization of organelles in

421

the cytoplasm, modified protein synthesis, reduced oocyte size, and impaired

422

metabolism [39-41]. The findings of several studies indicate improved embryo

423

development in response to LC through the modification of amounts and

424

distribution of mitochondria, reduced apoptosis and intracellular ROS levels along

425

with increased GSH levels. In vitro matured prepubertal lamb and calf oocytes

426

have lower volume density, volume, size, and number of mitochondria [39, 42],

427

which could be related to their diminished developmental potential. Thus, a critical

428

number and distribution pattern of mitochondria seems to exist for appropriate

429

oocyte and embryo development, with evenly distributed mitochondria reflecting

430

improved oocyte quality [43, 44]. In effect, there is an increase in the proportions

431

of in vitro matured cow and pig oocytes when mitochondria are evenly distributed

432

in response to LC supplementation [11, 12]. LC has also been found to decrease

433

percentages of apoptotic oocytes in pigs, and to decrease intracellular ROS and

434

increase GSH levels in pig and sheep oocytes [13, 15]. GSH is an indicator of a

435

mature oocyte cytoplasm with well-established beneficial effects on blastocyst

436

formation and quality [45]. We could thus hypothesize that the cumulative effects

437

of LC on cytoplasmic maturation may have helped to improve the embryo hatching

438

ability of prepubertal bovine oocytes matured in the presence of LC.

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439

As expected, we observed lower proportions of bovine MII–oocytes showing a

440

normal spindle morphology when in vitro matured oocytes were vitrified/warmed in

441

the basic medium. This finding is consistent with previous observations in our

21

ACCEPTED MANUSCRIPT laboratory, as MII–oocyte vitrification has adverse effects on MII-spindle assembly

443

and chromosome alignment [46, 47]. Although no significant differences were

444

observed between the vitrified/warmed LC+R group and their fresh un-vitrified

445

counterpart, it is difficult to establish a possible positive effect of LC+R

446

supplementation prior to vitrification on spindle morphology when the difference

447

between both groups was more than 20%. Only in the mouse, Moawad et al. [48]

448

reported the beneficial effects of LC supplementation during vitrification/warming

449

and IVM of GV–oocytes on MII-spindle assembly. As far as we know, the effects of

450

R supplementation or the combination of R and LC during IVM and

451

vitrification/warming of MII-oocytes on MII-spindle assembly have not been

452

previously reported.

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In the present study, no significant differences in percentages of apoptosis determined using the TUNEL method were seen among treatments after

455

vitrification. However, while effects of LC and LC+R treatment on caspase activity

456

before vitrification differed significantly between vitrified and control fresh oocytes,

457

R supplementation of IVM medium significantly reduced caspase activity after

458

vitrification/warming. Resveratrol has been attributed several dose-dependent

459

health benefits functioning both as a pro-apoptotic and anti-apoptotic agent

460

(reviewed by [49]). Hence, when given at a lower dose, R provided

461

cardioprotection by acting as an anti-apoptotic agent and decreasing the number

462

of apoptotic cardiomyocytes [50]. Furthermore, Giaretta et al. [23] reported

463

significantly higher proportions of viable MII pig oocytes with inactive caspases

464

after their vitrification/warming in an IVM medium supplemented with 2 µM R,

465

which matches with our results found in bovine IVM oocytes.

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22

ACCEPTED MANUSCRIPT Two recent studies have revealed contradictory results related to the effects on

467

embryo development of LC added to the IVM medium before vitrification/warming

468

of adult bovine oocytes. Chankitisakul et al. [16] reported a greater embryo

469

development rate up to the blastocyst stage 8 days after vitrification and IVF when

470

LC was added to the IVM medium. In contrast, Phongnimitr et al. [17] reported no

471

such beneficial effects of LC on blastocyst development. Here, we detected

472

significantly lower percentages of Day 8 blastocysts after oocytes were

473

vitrified/warmed, irrespective of the supplement used during in vitro maturation

474

when compared to fresh oocytes. However, while neither LC or R or both were

475

able to improve embryo development, the hatching capacity of these blastocysts

476

derived from vitrified/warmed oocytes was improved when these had been in vitro

477

matured in the presence of LC or LC+R. As mentioned earlier, LC could have

478

improved oocyte quality because of its antioxidant properties and beneficial effects

479

on mitochondrial lipid metabolism, ATP contents and GSH levels. But, it is worth

480

mentioning that, because of the low efficiency of vitrified/warmed calf oocytes to

481

reach the blastocyst stage, the number of hatched blastocysts reported in this

482

study does not allow us to reach any reliable conclusion on the effect of the

483

addition of LC and /or R prior to a vitrification on embryo development.

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Because of the observed effects of LC and/or R supplementation during IVM, it

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485

was also our aim to gain further knowledge on the molecular mechanisms

486

underlying such effects. As mentioned previously, different studies have shown

487

that supplementation with exogenous LC during maturation increased intracellular

488

ATP levels and reduced intracellular lipid content in the oocyte, demonstrating that

489

LC increased utilization of intracellular fatty acid stores [12]. Similarly, LC and R

490

have been proven to diminish intracellular ROS levels [14, 15, 22]. In this context,

23

ACCEPTED MANUSCRIPT we hypothesized that transcript abundance of genes involved in fatty acid

492

metabolism and glucose transport would change after treatment with LC while IVM

493

in presence of LC and/or R would modify the expression of genes related to redox

494

balance. In these experiments and although a significant increase of embryo

495

hatchability was observed when LC was added during IVM, no significant changes

496

in relative mRNA abundance of the oocytes matured in presence of LC were found

497

for genes related to fatty acid metabolism (ACACA and PLIN2), glucose transport

498

(SLC2A1) and heat- (HSPA1A) and oxidative stress (GPX1 and SOD1) compared

499

with control fresh oocytes. This lack of variation in mRNA expression observed in

500

this study is consistent with the results observed by Paczkowski et al. [51], who

501

found no differences in the expression of genes related to fatty acid metabolism

502

when 1mM of LC was added to IVM medium of murine oocytes. Regarding genes

503

associated with oxidative stress, LC supplementation during IVM of sheep oocytes

504

significantly upregulates the expression of GPX1 whereas the expression of SOD1

505

is not altered. However, in our study, transcriptions of genes involved in redox

506

balance in vitrified/warmed oocytes were not altered when compared to control

507

oocytes. This could be due to the fact that redox balance was not much affected in

508

our in vitro culture conditions, which would explain why the impact upon gene

509

expression was not apparent, or that the ROS produced was neutralized by the

510

free radical scavenging effect of LC. Nonetheless, vitrification and warming

511

affected the transcription of most of the evaluated genes when compared to their

512

fresh counterparts. Strikingly, transcript abundance of SOD1 and GPX1 in vitrified

513

oocytes after treatment with LC or transcript abundance of SOD1 after treatment

514

with R did not differ from their fresh counterparts. Again, this suggests that both

515

molecules could prevent ROS formation produced by the vitrification/warming

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24

ACCEPTED MANUSCRIPT process. SOD1 takes part in the initial defense against the superoxide anion,

517

mediating the conversion of O2- into H2O2 (for a review, see [52]). Although no

518

previous works have been conducted in vitrified bovine oocytes, data published

519

from other species are not explicit. In effect, while studies conducted in murine

520

species have shown that vitrification upregulates SOD1 expression in oocytes [53],

521

Turathum et al. [54] observed no effects of vitrification on SOD1 expression in

522

canine oocytes. Although the effect on SOD1 expression of adding LC or R to the

523

IVM medium prior to vitrification/warming has not been determined previously in

524

the literature, it has been shown that treatment with LC or R during maturation

525

decreases oocyte ROS levels [12-14, 22]. Hence, LC or R treatment before

526

vitrification may have helped levels of SOD1 expression to resemble to those of

527

fresh oocytes. Expression levels of glutathione peroxidase 1 (GPX1) gene serve

528

as an indicator of GSH content. GPX1 transcripts are present in both oocytes and

529

embryos [55] and have been used as a marker of oocyte quality [56].

530

Vitrification/warming led to significant downregulation of the GPX1 gene, which is

531

consistent with the observation by Somfai et al. [57] of a significantly reduced GSH

532

level after vitrifying IVM porcine oocytes. Interestingly, when oocytes were vitrified

533

after IVM with LC, levels of GPX1 expression did not differ from LC-treated fresh

534

(non-vitrified) ones, probably due to an increase in oocyte GSH content after IVM

535

in the presence of LC as previously observed in pigs [12, 13]. Besides, we also

536

observed that oocytes vitrified after treatment with LC+R usually exhibited similar

537

levels of gene expression that those of control vitrified oocytes. Although there is

538

no explanation for the differences obtained between cellular and molecular tests, it

539

appears that reaching a firm conclusion about the effects of supplementation of

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25

ACCEPTED MANUSCRIPT 540

IVM media with LC and/or R on the fatty acid metabolism, glucose transport or

541

redox status of vitrified oocytes requires a more complex analysis.

542

In conclusion, the addition of LC and/or R to the IVM medium of prepubertal calf oocytes does not improve embryo development either of fresh or vitrified/warmed

544

oocytes. Exceptions were higher hatchability for the LC-treated fresh oocytes and

545

LC- and LC+R-treated vitrified oocytes. LC+R supplementation prior to vitrification

546

decreased spindle damage, R addition modulated apoptosis and LC or R addition

547

restored gene expression at the same levels than fresh controls. Despite the new

548

insights gained by the assessment of gene expression herein, it appears that more

549

research is warranted to fully address the effects of vitrification upon the metabolic

550

and redox status of oocytes, which would benefit from evaluating other genes and

551

the activity of other clue enzymes. Additional molecular information on the effects

552

of vitrification methods of bovine oocytes may unravel the genetic responses of

553

this cell to the cryopreservation process and consequently improve

554

cryopreservation strategies.

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ACCEPTED MANUSCRIPT 556

6. Acknowledgments This study was supported by the Spanish Ministry of Science and Innovation

558

(Project AGL2013–46769), Generalitat de Catalunya (Project No. 2014 SGR 547)

559

and CAPES Foundation, Ministry of Education of Brazil.

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Goncalves PB, et al. Mitochondrial distribution and adenosine triphosphate content

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morphological criteria and developmental capacity after in vitro fertilization and

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culture. Biology of reproduction. 2001;64:904-9.

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[44] Brevini TA, Cillo F, Antonini S, Gandolfi F. Cytoplasmic remodelling and the

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acquisition of developmental competence in pig oocytes. Animal reproduction

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beta-mercaptoethanol can increase the glutathione content of pig oocytes matured

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in vitro and the rate of blastocyst development after in vitro fertilization.

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Theriogenology. 1998;50:747-56.

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[46] Morato R, Izquierdo D, Paramio MT, Mogas T. Cryotops versus open-pulled

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straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on

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2008;57:137-41.

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707

effects of maturation stage and prematuration treatment on the nuclear and

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cytoskeletal components of oocytes and their subsequent development. Mol

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Reprod Dev. 2005;72:239-49.

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[48] Moawad AR, Xu B, Tan SL, Taketo T. l-carnitine supplementation during

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vitrification of mouse germinal vesicle stage-oocytes and their subsequent in vitro

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maturation improves meiotic spindle configuration and mitochondrial distribution in

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metaphase II oocytes. Human reproduction. 2014;29:2256-68.

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[49] Das S, Das DK. Resveratrol: a therapeutic promise for cardiovascular

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diseases. Recent Pat Cardiovasc Drug Discov. 2007;2:133-8.

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2001;496:181-90.

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[51] Paczkowski M, Schoolcraft WB, Krisher RL. Fatty acid metabolism during

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maturation affects glucose uptake and is essential to oocyte competence.

721

Reproduction. 2014;148:429-39.

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[52] Takahashi M. Oxidative stress and redox regulation on in vitro development of

723

mammalian embryos. J Reprod Dev. 2012;58:1-9.

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[53] Habibi A, Farrokhi N, Moreira da Silva F, Bettencourt BF, Bruges-Armas J,

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Amidi F, et al. The effects of vitrification on gene expression in mature mouse

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oocytes by nested quantitative PCR. J Assist Reprod Genet. 2010;27:599-604.

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[54] Turathum B, Saikhun K, Sangsuwan P, Kitiyanant Y. Effects of vitrification on

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nuclear maturation, ultrastructural changes and gene expression of canine

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ACCEPTED MANUSCRIPT [55] El Mouatassim S, Guerin P, Menezo Y. Expression of genes encoding

731

antioxidant enzymes in human and mouse oocytes during the final stages of

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maturation. Mol Hum Reprod. 1999;5:720-5.

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[56] Maedomari N, Kikuchi K, Ozawa M, Noguchi J, Kaneko H, Ohnuma K, et al.

734

Cytoplasmic glutathione regulated by cumulus cells during porcine oocyte

735

maturation

736

Theriogenology. 2007;67:983-93.

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[57] Somfai T, Ozawa M, Noguchi J, Kaneko H, Kuriani Karja NW, Farhudin M, et

738

al. Developmental competence of in vitro-fertilized porcine oocytes after in vitro

739

maturation and solid surface vitrification: effect of cryopreservation on oocyte

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antioxidative system and cell cycle stage. Cryobiology. 2007;55:115-26.

741

[58] Brisard D, Chesnel F, Elis S, Desmarchais A, Sanchez-Lazo L, Chasles M, et

742

al. Tribbles expression in cumulus cells is related to oocyte maturation and fatty

743

acid metabolism. J Ovarian Res. 2014;7:44.

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[59] Goovaerts IG, Leroy JL, Rizos D, Bermejo-Alvarez P, Gutierrez-Adan A,

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Jorssen EP, et al. Single in vitro bovine embryo production: coculture with

746

autologous cumulus cells, developmental competence, embryo quality and gene

747

expression profiles. Theriogenology. 2011;76:1293-303.

748

[60] Khalil WA, Marei WF, Khalid M. Protective effects of antioxidants on linoleic

749

acid-treated

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development. Theriogenology. 2013;80:161-8.

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[61] Dhali A, Anchamparuthy VM, Butler SP, Mullarky IK, Pearson RE,

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Gwazdauskas FC. Development and quality of bovine embryos produced in vitro

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using growth factor supplemented serum-free system. Open Journal of Animal

754

Sciences. 2011;01:97-105.

fertilization

and

embryonic

development

in

vitro.

AC C

EP

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SC

affects

RI PT

730

bovine

oocytes

during

maturation

and

subsequent

embryo

35

ACCEPTED MANUSCRIPT [62] Sastre D, da Costa NN, de Sa AL, Conceicao SD, Chiaratti MR, Adona PR, et

756

al. Expression of PLIN2 and PLIN3 during oocyte maturation and early embryo

757

development in cattle. Theriogenology. 2014;81:326-31.

758

[63] Bessa IR, Nishimura RC, Franco MM, Dode MA. Transcription profile of

759

candidate genes for the acquisition of competence during oocyte growth in cattle.

760

Reprod Domest Anim. 2013;48:781-9.

761

[64] Machado GM, Ferreira AR, Pivato I, Fidelis A, Spricigo JF, Paulini F, et al.

762

Post-hatching development of in vitro bovine embryos from day 7 to 14 in vivo

763

versus in vitro. Mol Reprod Dev. 2013;80:936-47.

764

[65] Valckx SD, Van Hoeck V, Arias-Alvarez M, Maillo V, Lopez-Cardona AP,

765

Gutierrez-Adan A, et al. Elevated non-esterified fatty acid concentrations during in

766

vitro murine follicle growth alter follicular physiology and reduce oocyte

767

developmental competence. Fertil Steril. 2014.

AC C

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SC

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ACCEPTED MANUSCRIPT

36 768

Table 1. Primers used in this study to amplify gene fragments for real time RT-qPCR

769 Annealing

Primer sequences

o

temp ( C)

188

60

R:CTGCCATCCTCACGACCT F:GGCGTGAACCACGAGAAGTATAA GAPDH F:GGGACTACACCCAGATGAATGA GPX 1

172 R:AGCATAAAGTTGGGCTCGAA F:CAAGATCACCATCACCAACG 219 R:AAATCACCTCCTGGCACTTG F:AGTGAACTTGCCAGGAAGAATG

PLIN2

TE D

HSPA1A

120

F:GCCATGGAGCGCTTTGG PPIA

EP

R:TTCATCTGTATCATCGTAGCCG

60

M AN U

119 R:CCCTCCACGATGCCAAAGT

65

60

56

772 773 774 [59]

775 NM_174076.3

776 [60]

777 NM_174550.1

778 [61]

779 60

NM_173980 [62]

60

NM_178320.2 [63]

60

NM_174602 [64]

787

309

Abbreviations: F, forward; R, reverse

784 785

F:GTGCAAGGCACCATCCACTTCG

R:CACCATCGTGCGGCCAATGATG

782 783

258

R:CACAAATAGCGACACGACAGT SOD1

780 781

F:CAGGAGATGAAGGAGGAGAGC SLC2A1

771

[58]

NM_001034034.2

R:CCACAGTCAGCAATGGTGATCT

AC C

Literature

NM_174224

SC

ACACA

770

GenBank accession

size(bp) F:TGCTTCCCATTTGCCATC

RI PT

Amplicon Gene

56

NM_174615 [65]

786

ACCEPTED MANUSCRIPT

37 788

Table 2. Effects of L-carnitine and/or resveratrol supplementation during the in vitro maturation of bovine oocytes on spindle and chromosome

789

configurations before and after vitrification/warming

RI PT

790 Spindle* Oocytes,

MII

Group

Normal

Abnormal

n (%±SEM)

n (%±SEM)

CTR

91

72 (79.1±2.9)

a

57 (79.2±2.1)

abc

LC

71

54 (76.1±2.6)

a

48 (88.9±2.3)

a

R

60

45 (75.0±2.6)

a

39 (86.7±4.3)

ab

LC+R

80

64 (80.0±4.1)

a

53 (82.8±2.7)

ab

CTR vit

101

57 (56.4±3.7)

b

25 (43.9±2.3)

d

LC vit

80

40 (50.0±2.3)

b

22 (55.0±1.8)

R vit

81

46 (56.7±4.0)

b

21 (45.7±4.0)

LC+R vit

78

44 (56.4±5.5)

b

27 (61.4±4.8)

n (%±SEM)

15 (20.8±2.1)

abc

M AN U

n (%±SEM)

n (%±SEM)

13 (18.1±1.7)

ab

793

2 (2.8±1.1)

a

794 795

6 (11.1±2.3)

a

5 (9.3±1.5)

a

1 (1.9±1.4)

a

6 (13.3±4.3)

ab

4( 8.9±2.0)

ab

2 (4.4±2.2)

ab

2 (3.1±3.1)

ab

ab

9 (14.1±1.1)

32 (56.1±2.3)

d

18 (31.6±3.6)

cd

18 (45.0±1.8)

cd

9 (22.5±3.3)

d

25 (54.3±4.0)

d

12 (26.1±2.4)

bcd

17 (38.6±4.8)

bcd

5 (11.4±3.8)

TE D

11 (17.2±2.7)

AC C

EP

792 Decondensed

Dispersed

SC

n

791

Chromosomes*

ab

b

ab

ab

ab

796 797 798 c

15 (26.3±2.3) 9 (22.5±2.2)

799

bc

800 c

10 (21.7±2.9) 8 (18.2±1.2)

801

b

802

803

abcd

804

*Percentages referred to the total number of oocytes at MII.

805

CTR: control group; LC: supplementation with 3.03 mM L-carnitine; R: supplementation with 1 µM resveratrol; LC+R: supplementation with 3.03 mM LC

806

plus 1 µM R; vit: oocytes in the respective treatment groups subjected to vitrification/warming.

807

Different superscripts within columns indicate significant differences between treatments (P<0.05). SEM: standard error of the mean.

ACCEPTED MANUSCRIPT

38 Table 3: Effects of L-carnitine and/or resveratrol supplementation during the IVM of bovine oocytes on caspase activation as determined by

809

FLICA/Hoechst 33342/PI staining before and after vitrification/warming

810 Total

FLICA−/PI-

812

n

N

(%± SEM)

FLICA+/PIN

(%± SEM)

PI+

N

(%± SEM)

0

(0.0±0.0)

a

68

(97.1±2.9)

1

(1.5±1.5)

a

LC

79

78

(97.8±2.3)

a

1

(1.1±1.1)

a

0

(0.0±0.0)

a

814

R

77

74

(92.2±2.8)

a

2

(2.8±1.7)

ab

1

(1.1±1.1)

a

815

LC+R

84

82

(95.8±4.2)

a

1

(1.0±1.0)

a

1

(1.0±1.0)

a

816

CTR vit

73

61

(72.0±3.2)

b

6

(7.0±1.1)

b

6

(7.1±1.3)

b

817

LC vit

74

66

(80.5±4.2)

b

3

(3.4±1.2)

b

5

(6.3±1.5)

b

818

R vit

80

73

(83.8±6.2)

ab

1

(1.3±1.3)

a

6

(6.9±2.3)

b

819

L+R vit

76

67

(77.7±5.9)

b

2

(1.9±1.9)

a

7

(9.2±3.5)

b

820

EP

821

SC

69

TE D

CTR

a

813

M AN U

811

RI PT

808

822

ab

823

CTR: control group; LC: supplementation with 3.03 mM L-carnitine; R: supplementation with 1 µM resveratrol; LC+R: supplementation with 3.03 mM LC

824

plus 1 µM R; vit: oocytes in the respective treatment groups subjected to vitrification/warming. FLICA-/PI-: viable oocyte/inactive caspase; FLICA+/PI-:

825

viable oocyte/active caspase; PI+: dead oocyte.

826

AC C

Different superscripts within columns indicate significant differences between treatments (P<0.05). SEM: standard error of the mean.

ACCEPTED MANUSCRIPT

39 827

Table 4. Developmental competence of bovine oocytes in vitro matured in the presence of 3.03 mM L-carnitine and/or 1 µM resveratrol before and after

828

vitrification/warming

RI PT

829 D7 Blastocyst Oocytes

D8 Blastocyst

D2 Cleaved*

Groups

Total*

Expanded**

n (%±SEM)

n (%±SEM)

n (%±SEM) CRT

138

110 (79.7±2.7)

a

28 (20.3±1.0)

LC

130

110 (84.6±1.7)

a

37 (28.5±1.3)

a

22 (59.6±4.4)

R

149

124 (83.2±1.1)

a

36 (24.2±2.4)

a

23 (63.81±5.2)

LC+R

128

103 (80.5±2.9)

a

31 (24.2±2.1)

a

16 (51.6±5.2)

CRT vit

123

34 (27.6±3.4)

b

3 (2.4±1.0)

LC vit

112

38 (33.9±3.2)

b

5 (4.5±1.5)

R vit

123

45 (36.6±4.1)

b

LC+R vit

115

40 (34.8±3.5)

b

M AN U

12 (42.8±5.2)

Total*

Hatched***

n (%±SEM)

n (%±SEM)

b

28 (20.3±1.0)

a

10 (35.7±5.4)

b

ab

37 (28.5±1.3)

a

25 (67.6±3.0)

a

36 (24.2±2.4)

a

19 (52.8±2.3)

ab

31 (24.2±2.1)

a

13 (41.9±5.7)

b

a

ab

3 (2.4±1.0)

b

0

b

0

5 (4.5±1.5)

b

1 (20.0±9.2)

4 (3.3±1.0)

b

0

4 (3.3±1.0)

b

0

3 (2.6±1.3)

b

0

3 (2.6±1.3)

b

1 (33.3±4.6)

EP

TE D

0

c

c

AC C

830

b

a

SC

n

831

abc

832

total number of oocytes.**Percentages of expanded blastocysts referred to the total number of D7 embryos.***Percentages of hatched blastocysts referred

833

to the total number of D8 embryos.

834

CTR: control group; LC: supplementation with 3.03 mM L-carnitine; R: supplementation with 1 µM resveratrol; LC+R: supplementation with 3.03 mM LC

835

plus 1 µM R; vit: oocytes in the respective treatment groups subjected to vitrification/warming; SEM: standard error of the mean.

Different superscripts within columns denote significant differences between treatments (P<0.05). *Percentages of D7 and D8 blastocysts referred to the

40

ACCEPTED MANUSCRIPT Figure legends

837

Figure 1. Rates of DNA fragmentation (TUNEL) recorded in bovine oocytes in vitro matured in the

838

presence of 3.03 mM L-carnitine (LC) and/or 1 µM resveratrol (R) followed by vitrification/warming.

839

Data are the mean ± s.e.m. Different letters indicate significant differences (P<0.05). CTR: control

840

group (n=59); LC: LC supplementation group (n=43); R: R supplementation group (n=58); LC+R:

841

LC+R supplementation group (n=61). CTR vit (n=67), LC vit (n=49), R vit (n= 52), LC+R vit (n=59):

842

oocytes in the respective treatment groups subjected to vitrification/warming.

AC C

EP

TE D

M AN U

SC

RI PT

836

41

ACCEPTED MANUSCRIPT Figure 2. Relative expression levels of the genes ACACA, PLIN2, SLC2A, HSPA1A, GXP1 and

844

SOD1 recorded in bovine oocytes in vitro matured in the presence of 3.03 mM L-carnitine (LC)

845

and/or 1 µM resveratrol (R) before and after vitrification/warming. Data are the mean ± s.e.m.

846

Different letters indicate significant differences (P<0.05) in the expression of a given gene.

AC C

EP

TE D

M AN U

SC

RI PT

843

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

A study designed to improve the cryotolerance and developmental competence of prepubertal calf oocytes by adding L-carnitine (LC) and/or resveratrol (R) to the in vitro maturation medium,

RI PT

The addition of LC and/or R to the IVM medium of prepubertal calf oocytes did not improve embryo development either of fresh or vitrified/warmed oocytes. LC and /R during maturation didn’t have any effect on the proportion of oocytes showing a normal spindle morphology and chromosome distribution but LC+R addition prior vitrification decreased spindle damage of the oocytes.

SC

The proportion of early apoptotic oocytes increased after vitrification/warming, except for those oocytes previously matured with R. LC or R addition before vitrification positively affected the expression of genes

AC C

EP

TE D

M AN U

involved in lipid metabolism and heat and oxidative stress,