The use of mineral oil during in vitro maturation, fertilization, and embryo culture does not impair the developmental competence of pig oocytes

Accepted Manuscript The use of mineral oil during in vitro maturation, fertilization and embryo culture does not impair the developmental competence of pig oocytes Cristina Alicia Martinez, Alicia Nohalez, Cristina Cuello, Juan Maria Vazquez, Jordi Roca, Emilio A. Martinez, Maria Antonia Gil PII:

S0093-691X(14)00604-9

DOI:

10.1016/j.theriogenology.2014.11.001

Reference:

THE 12986

To appear in:

Theriogenology

Received Date: 26 September 2014 Revised Date:

29 October 2014

Accepted Date: 1 November 2014

Please cite this article as: Martinez CA, Nohalez A, Cuello C, Vazquez JM, Roca J, Martinez EA, Gil MA, The use of mineral oil during in vitro maturation, fertilization and embryo culture does not impair the developmental competence of pig oocytes, Theriogenology (2014), doi: 10.1016/ j.theriogenology.2014.11.001. 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

2 The use of mineral oil during in vitro maturation, fertilization and embryo culture

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does not impair the developmental competence of pig oocytes

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Cristina Alicia Martinez, Alicia Nohalez, Cristina Cuello, Juan Maria Vazquez,

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Jordi Roca, Emilio A. Martinez, Maria Antonia Gil*

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Department of Animal Medicine and Surgery. University of Murcia. Murcia, Spain.

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Corresponding author. Tel.: +34 868884734; fax: +34 868887069.

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E-mail address: [email protected] (Maria Antonia Gil).

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ABSTRACT

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This study evaluated the effects of mineral oil overlay during maturation, fertilization

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and embryo culture on the timing of nuclear maturation, the progesterone concentrations

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in the maturation medium and the subsequent developmental competence of the oocyte.

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The results from experiment 1 showed that under the typical humidity of laboratory

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incubators (95–97%), the culture media osmolality increased in the absence of oil

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overlay. For this reason, in experiment 2, maturation, fertilization and embryo culture

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media were incubated with either an oil cover (MO group) or a microenvironment

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system for maximum humidity (HM group). Under these conditions, the media

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osmolality was maintained below 300 mOsm/kg. A portion of oocytes (n = 1,414; four

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ACCEPTED MANUSCRIPT replicates) was removed from the maturation medium at 4- to 6-h intervals to evaluate

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the nuclear maturation stage. The corresponding medium was used for progesterone

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measurement. The remaining oocytes were inseminated with frozen-thawed ejaculated

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sperm and cultured for 12 h (n = 305) or 7 days (n = 619) to assess fertilization and

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embryo development parameters, respectively. The progesterone concentration of the

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maturation medium of the MO group was lower than 1.5 ng/mL at each time-point

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evaluated. The values obtained at 12 h of maturation and at the end of maturation were

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20 and 55 times lower than those of the HM group, respectively. However, compared

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with the HM group, oil overlay did not delay oocyte progression to metaphase I and II

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and did not influence normal fertilization, cleavage, blastocyst formation and total cell

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number in blastocysts. In conclusion, despite its pronounced impact on progesterone

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concentration, the use of mineral oil did not affect the time-course of oocyte maturation

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or oocyte developmental competence.

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In vitro production of embryos; porcine; oil overlay; in vitro fertilization; in vitro

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maturation; progesterone; blastocyst; mineral oil; osmolality; culture medium.

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1. Introduction

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Porcine embryo in vitro production (IVP) has become an important tool for basic

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science research, pig production and many emerging reproductive biotechnologies [1].

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Because of their physiological similarities to humans, cloned and genetically modified

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pigs are effective biomodels for many human diseases [2] and regenerative medicine

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research [3] and serve as potential donors of tissues and organs for xenotransplantation

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[4]. These biotechnologies require mature oocytes and embryos of good quality [5].

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ACCEPTED MANUSCRIPT Although many investigators have attempted to improve porcine IVP systems to

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produce high numbers of high-quality embryos, the overall efficiency of IVP

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technology is still very low, with normal fertilization and blastocyst rates lower than

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45% and 30%, respectively [6–13].

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The high incidence of polyspermy and the low development and quality of in vitro-

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produced embryos compared with in vivo-derived embryos are the major limitations of

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current IVP systems, indicating improper culture conditions for IVM, IVF and/or IVC.

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The culture conditions used for IVM, IVF and IVC are constantly being studied and

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reinvented to reduce polyspermy through the in vitro simulation of the follicular,

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oviductal and uterine microenvironments (reviewed in [14–16]). Although the results

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from these studies suggest significant improvements, the current culture conditions

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remain suboptimal for achieving adequate fertilization and embryo development.

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To standardize the culture conditions and avoid the use of several undefined products

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that are commonly added to culture media (e.g., follicular fluid, BSA and sera) and

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influence oocyte and embryo developmental competence [1,17,18], chemically defined

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media have been developed (reviewed in [19]). The final objective of the development

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of these media is to improve the reliability and reproducibility of results among

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laboratories and to allow for the precise evaluation of the effects of different

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components on oocyte maturation, fertilization and embryo development. In contrast,

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little attention has been paid to the use of other routine components of the IVP systems,

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such as the oil covering. Covering the culture medium with mineral oil (MO) is a

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routine procedure used in culturing mammalian gametes and embryos. However, MO is

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derived from crude oil and is thus an undefined product that can vary in quality [20].

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The primary function of an oil overlay in embryo culture is to prevent liquid

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evaporation, which facilitates the maintenance of pH and osmotic pressure in the culture

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ACCEPTED MANUSCRIPT medium [21–23]. Osmolality of culture media is a key factor that affects the success of

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embryo IVP [24]. There is some evidence that the osmolality of culture media must

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remain stable during all IVP processes [25] because osmotic stresses can damage DNA

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and affect DNA replication, DNA transcription and mRNA translation, leading to

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cellular damage [26]. The desirable effects of oil overlay also include protecting the

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media from microbial contamination [27] and the absorption of accumulated toxic

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components [28]. However, several studies have shown that oil negatively influences

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the outcome of embryo IVP [17,22]. Cultures under mineral oil may lead to detrimental

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substances that accumulate in the oil during the production process or during storage

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being absorbed into the medium. The possibility that toxic elements that affect embryo

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development may be introduced into the culture media by the oil overlay has been

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described in mouse [29], bovine [30,31] and human [32,33] embryo culture studies.

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Furthermore, the use of MO overlay has been associated with delayed nuclear

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maturation and reduced oocyte developmental capacity in porcine IVP [34] and with

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delayed meiosis progression in mouse oocytes after in vitro follicle culture [35]. These

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adverse effects of oil have been attributed in part to the fact that oil extracts steroid

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hormones and meiosis-activating sterols from the maturation medium, which are

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secreted by cumulus cells and oocytes [34].

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To avoid the use of oil overlay during cultures without significant changes in the

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osmolality of the medium, several alternatives have been considered. Although oocytes

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and embryos may be cultured in larger volumes of media, this option may negatively

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affect embryo development because of the loss of beneficial substances secreted by the

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oocytes and embryos due to dilution [36,37]. Another alternative is the production of a

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high-humidity microenvironment in the culture dishes. The placement of a reasonable

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ACCEPTED MANUSCRIPT amount of water into the central hole of four-well dishes creates an appropriate humid

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microenvironment, preventing the increase of media osmolality during culture [38].

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The aim of this study was to evaluate the effects of MO overlay during maturation,

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fertilization and embryo culture on porcine IVP outcomes. We evaluated the media

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osmolality, time of nuclear maturation, progesterone (P4) concentration in maturation

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medium, and maturation, fertilization and embryo development parameters in the

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presence and absence of an oil overlay.

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2. Materials and methods

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All experimental procedures used in this study were performed in accordance with the

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2010/63/EU EEC Directive for animal experiments and were reviewed and approved by

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the Ethical Committee for Experimentation with Animals of the University of Murcia,

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Spain (research code: 1002/2012).

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2.1. Reagents and culture media

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All chemicals used in this study were purchased from Sigma–Aldrich Co. (Alcobendas,

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Madrid, Spain) unless otherwise indicated.

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The medium used to collect cumulus-oocyte complexes (COCs) was a modified

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Dulbecco’s phosphate-buffered saline (mDPBS) medium composed of 136.89 mM

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NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4 and 1.46 mM CaCl2·2H2O supplemented with 4

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mg/mL BSA, 0.34 mM sodium pyruvate, 5.4 mM D-glucose and 70 µg/mL kanamycin.

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The oocyte maturation medium was TCM-199 (Gibco Life Technologies S.A.,

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Barcelona, Spain) supplemented with 0.57 mM cysteine, 0.1% (w/v) polyvinylalcohol,

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10 ng/mL EGF, 75 µg/mL penicillin G potassium, and 50 µg/mL streptomycin sulfate.

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The basic medium used for IVF was a modified Tris-buffered medium [39] and

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(Trizma Base), 11.0 mM D-glucose and 5.0 mM sodium pyruvate supplemented with

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2.0 mM caffeine and 0.2% BSA. The embryo culture medium was North Carolina State

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University (NCSU-23) [40] supplemented with 0.4% BSA.

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2.2. Oocyte collection and in vitro maturation

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Ovaries were obtained from prepubertal gilts at a local slaughterhouse (El Pozo S.A.,

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Murcia, Spain) and transported to the laboratory at 35 °C within 1 h of collection in

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0.9% (w:v) NaCl containing 70 µg/mL kanamycin. The COCs were collected with a

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surgical blade from the surfaces of medium-sized follicles (3–6 mm in diameter).

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Follicular fluids containing the COCs were pooled in 15-mL conical tubes and were

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allowed to settle for 10 min. The sediment was collected in a 60-mm petri dish and

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COCs were located using a stereomicroscope (200×) and placed in a 35-mm petri dish

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with 2 mL of mDPBS. Oocytes surrounded by a compact cumulus mass and having

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evenly granulated cytoplasm were washed three times in maturation medium; 45–50

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COCs were transferred into each well of a 4-well multidish (Nunc, Roskilde, Denmark)

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containing 500 µL of pre-equilibrated maturation medium supplemented with 10 IU/mL

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eCG (Folligon, Intervet International B.V., Boxxmeer, The Netherlands) and 10 IU/mL

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hCG (Veterin Corion, Divasa Farmavic S.A., Barcelona, Spain) for 20–22 h (first IVM

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period). The oocytes were then incubated for another 20–22 h in maturation medium

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without hormones (second IVM period). Maturation was performed in an incubator

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(Labotect GmbH Labor-Technik-Göttingen, Göttingen, Germany) at 39 °C in 5% CO2

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in air and 95–97% relative humidity.

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After maturation, cumulus cells were removed with 0.1% hyaluronidase in maturation

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medium by vortexing for 2 min at 1660 rounds/min. The denuded oocytes were washed

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three times in maturation medium and fertilized as previously described [41]. Briefly,

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groups of 30 oocytes were placed in 50 µL drops of pre-equilibrated IVF medium in a

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35 mm x 10 mm Petri dish (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ,

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USA). The dishes containing oocytes were kept in the incubator at 39 °C in an

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atmosphere of 5% CO2 in air and 95–97% relative humidity for 30 min, at which point

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spermatozoa were added for fertilization.

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Semen from a mature Pietrain boar tested by his previous in vitro fertilizing potential

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was processed and cryopreserved as previously described [42]. Briefly, extended semen

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(1:1 in Beltsville thawing solution [43]) was cooled to 17 °C and held at this

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temperature for 3 h. After centrifugation at 2400 × g for 3 min, the pellets were diluted

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in LEY extender, which was composed of 80% (v:v) 310 mM ß-lactose solution, 20%

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(v:v) egg yolk and 100 µg/ml kanamycin sulfate, to a concentration of 1.5 × 10 9

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spermatozoa/mL. After further cooling to 5 °C over 120 min, the extended spermatozoa

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were suspended with LEY-Glycerol-Orvus Es Paste extender [92.5% LEY, 1.5% Equex

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STM (Nova Chemical Sales Inc., Scituate, MA, USA) and 6% glycerol] to a final

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concentration of 1 × 109 spermatozoa/mL. The cooled spermatozoa were packed into

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0.5-mL straws, which were frozen using a controlled-rate freezer (IceCube 1810;

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Minitüb, Tiefenbach, Germany) from 5 to -80 °C at 40 °C/min, held for 30 s at -80 °C,

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cooled at 70 °C/min to -150 °C and finally immersed in liquid nitrogen. One pool of

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two straws was thawed in a circulating water-bath at 37 °C for 20 s for each replicate.

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Just after thawing, 100 µL of sperm was washed three times by centrifugation at 1900 ×

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g for 3 min in mDPBS. Following the washing procedure, the sperm pellet was

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ACCEPTED MANUSCRIPT suspended in IVF medium. After appropriate dilution, 50 µL of this sperm suspension

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was added to the oocyte-containing medium to yield a final concentration of 3 × 105

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spermatozoa/mL. The spermatozoa:oocyte ratio was 1000:1. Oocytes were incubated

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with spermatozoa at 39 °C in an atmosphere of 5% CO2 in air and 95–97% relative

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humidity for 5 h.

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180 2.4. In vitro embryo culture

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After gamete co-incubation, presumed zygotes were washed by mechanical pipetting

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three times in pre-equilibrated embryo culture medium to remove spermatozoa that

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were not bound to the zona pellucida. The presumed zygotes were then transferred (40

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zygotes per well) to a 4-well multidish (Nunc) containing 500 µL of glucose-free

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embryo culture medium that was supplemented with 0.3 mM pyruvate and 4.5 mM

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lactate for 2 days and then changed to fresh embryo culture medium containing 5.5 mM

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glucose for an additional 5 days. Cultures were performed at 39 °C, 5% CO2 in air and

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95–97% relative humidity.

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2.5. Assessment of maturation, fertilization and embryo development parameters

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To evaluate the maturation and fertilization parameters, the oocytes and presumed

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zygotes were mounted on slides, fixed for 48–72 h in 25% (v:v) acetic acid in ethanol at

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room temperature, stained with 1% lacmoid in 45% (v:v) acetic acid, and examined

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under a phase-contrast microscope at 400 × magnification. Oocytes that possessed a

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nucleolus and filamentous chromatin located throughout the whole area or had a distinct

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nuclear membrane, a nucleolus, and chromatin stained in a ring-horseshoe shape were

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classified as being in the germinal vesicle stage. Oocytes with a plate but no polar body

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were classified as being in metaphase I (MI). Oocytes were considered mature when

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ACCEPTED MANUSCRIPT their chromosomes were at metaphase and they had an extruded first polar body (MII).

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The maturation rate was expressed as the number of MII-stage oocytes divided by all

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cultured oocytes. Oocytes were considered penetrated when they contained one or more

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swollen sperm heads and/or male pronuclei with their corresponding sperm tails and

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two polar bodies. The evaluated fertilization parameters were penetration (percentage of

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the number of penetrated oocytes/total mature oocytes inseminated), monospermy

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(percentage of the number of monospermic oocytes/total mature oocytes penetrated),

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number of spermatozoa per mature oocyte penetrated and efficiency of fertilization

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(number of monospermic oocytes/total mature oocytes inseminated). Degenerated

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oocytes (oocytes with a broken oolemma or abnormal appearance of the cytoplasm)

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were excluded from the study.

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In vitro embryo development was evaluated under a stereomicroscope at 2 and 7 days

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after insemination. An embryo that had cleaved to the 2- to 4-cell stage was defined as

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cleaved, and an embryo with a well-defined blastocoel and an inner cell mass and

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trophoblast totally discernible was defined as a blastocyst. The cleavage rate was

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determined at day 2 of culture and was defined as the percentage of oocytes that had

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divided to the 2- to 4-cell stage. Blastocyst formation was the percentage of 2- to 4-cell

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cell embryos that developed to the blastocyst stage. The total efficiency was described

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as the percentage of the total number of cultured oocytes that reached the blastocyst

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stage. All blastocysts were fixed in 4% paraformaldehyde in phosphate-buffered saline

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(PBS) for 30 min at room temperature (22–24 °C) to assess the total cell number, as an

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indicator of embryo quality. Embryos were washed with PBS containing 3 mg/mL

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BSA, placed on a slide in 4 µL of Vectashield (Vector, Burlingame, CA, USA)

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containing 10 µg/mL Hoechst 33342, and covered with a coverslip. Fixed embryos were

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examined with fluorescence microscopy using an excitation filter of 330 to 380 nm. The

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total number of nuclei that displayed blue fluorescence was counted.

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Osmolality (mOsm/kg) of the culture media was determined with a vapor pressure

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osmometer (Vapro® Model 5520; Wescor Inc, Logan, UT, USA) according to the

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manufacturer’s instructions. To measure osmolality, 10 µL of medium was taken from

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each dish and placed on the sample loading area in the osmometer. Measurements of

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each sample were made in duplicate, and the mean value was calculated. Prior to each

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experimental run, the osmometer was calibrated using 10 µL of osmolality standards.

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2.7. Progesterone measurement

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Concentrations of P4 in the maturation media were assayed utilizing an Immulite kit on

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an Immulite 1000 analyzer (Siemens Healthcare Diagnostics, Deerfield, IL, USA), a

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solid phase, competitive immunoassay using enzyme-labeled chemiluminescent

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technology. The assay sensitivity was 0.2 ng P4/mL, and the intraassay and interassay

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coefficients of variation were less than 10%.

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2.8.1. Experiment 1

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This experiment was performed to evaluate the effects of MO (cat. no. M8410) overlay

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of culture media on oocyte developmental competence. Oocyte maturation, fertilization

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and embryo culture were performed in the presence of MO cover (MO group) or

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without oil overlay under the typical humidity of laboratory incubators (OF group), in a

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ACCEPTED MANUSCRIPT total of two replicates. The volume of oil added as a cover was 300 µL, 2 mL and 300

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µL in IVM, IVF and IVC media, respectively. In each replicate, a random subset of

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oocytes and presumed zygotes was fixed and stained at 44 h of maturation (n = 154) and

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18 h after fertilization (n = 158) to evaluate the maturation and fertilization parameters,

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respectively. The remaining presumed zygotes (n = 314) were cultured to evaluate the

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in vitro embryo development at 2 and 7 days of culture. Media osmolality was measured

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before and at the end of the each incubation period.

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2.8.2. Experiment 2

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According to the results of experiment 1 (the media osmolality in the OF group was

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higher than those in MO group), maturation, fertilization and embryo culture media

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were incubated under MO cover (MO group) or without oil but surrounded by purified

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water, creating a humid microenvironment (HM group). The humid microenvironment

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was created by adding 5 mL of purified water to the middle hole of the 4-well dishes

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used for maturation and embryo culture and by situating two Petri dishes (60 × 15 mm),

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each containing 5 mL of purified water, next to each fertilization plate. The volume of

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oil used was the same as described in experiment 1. A total of 2,838 oocytes in four

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replicates were used in the experiment. A portion of the oocytes (n = 1,414) was

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removed from the maturation medium at 4–6 h intervals from the start until the end of

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maturation period (44 h); the oocytes were fixed and stained to evaluate their nuclear

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maturation stage, and the corresponding medium was frozen and stored (-20 °C) until

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the assay for P4 was performed. The concentration of P4 was also measured in

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maturation media with or without oil overlay not containing oocytes. At the end of the

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maturation period, the remaining oocytes were inseminated with thawed sperm, and a

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random subset of the presumed zygotes (n = 305) was fixed and stained at 18 h after

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fertilization to evaluate fertilization parameters. The remaining presumed zygotes (n =

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619) were cultured to evaluate the in vitro embryo development at 2 and 7 days of

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culture. Media osmolality was measured before and at the end of each incubation

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period.

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For statistical purposes, each drop was considered an experimental unit. Continuous

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variables (osmolality, P4 concentrations and total cell number per blastocyst) are

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expressed as the mean ± SD of two (Experiment 1) or four (Experiment 2) replicates.

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The mean ± SD of binary variables (MI and MII oocytes, fertilized and monospermic

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oocytes and cleaved embryos or embryos that developed to blastocyst stage) were

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obtained after calculating the percentage in every drop of each group and in each

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replicate. Significant differences between the MO and OF groups (Experiment 1) and

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between the MO and HM groups (Experiment 2) were determined by an unpaired

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Student's t-test corrected for inequality of variances (Levene’s test). Statistical analysis

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was performed using the IBM SPSS 19 Statistics package (SPSS, Chicago, IL, USA).

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Differences were considered significant when P < 0.05.

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3. Results

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3.1. Experiment 1

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The osmolality of the maturation, fertilization and embryo culture media before and at

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the end of the each incubation period is shown in Figure 1. There were no differences

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between the initial and final osmolalities when the media were overlaid with MO, with

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osmolality values of approximately 295 mOsml/kg in the maturation and fertilization

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ACCEPTED MANUSCRIPT media and 275 mOsml/kg in the embryo culture media. In contrast, when the media

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were not covered by oil overlay, an increase (P < 0.001) in osmolality was detected in

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all media, achieving values above 305 mOsm/kg in the maturation and embryo culture

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media and of 445 mOsml/kg in the fertilization medium. At 44 h of maturation, there

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was no difference in the percentage of MII-oocytes between the MO and OF groups

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(76.6 ± 7.8% and 70.4 ± 6.3%, respectively). Tables 1 and 2 show the fertilization

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parameters and embryo development in the presence or absence of an oil cover. While

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there were no differences between groups in the penetration, monospermic or efficiency

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rates, the cleavage and blastocyst rates were higher (P < 0.003) when the culture media

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were covered with MO. The blastocyst rates in relation to the number of cleaved

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embryos were almost twice as high (P < 0.003) in the MO group compared with those

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of the OF group. The total efficiency of blastocyst development (number of blastocysts

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obtained from the total number of presumed zygotes cultured) was increased four times

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(P < 0.001) when the IVM, IVF and IVC media were covered with MO.

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3.2. Experiment 2

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The osmolality of the media at the beginning and end of each incubation period did not

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vary between the oil overlay and humid microenvironment conditions, except when the

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fertilization medium was incubated without an oil overlay. In this case, the osmolality

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was slightly higher (P < 0.05) at the end of incubation period compared with those

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observed at the beginning of incubation or in the presence of oil, although was

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maintained to levels of below 300 mOsm/kg in all cases (Figure 2).

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The P4 concentrations in the media in which oocytes were matured are represented in

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Figure 3. The maturation medium from the MO group had a P4 concentration lower

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ACCEPTED MANUSCRIPT than 1.5 ng/mL at each time-point evaluated, with values 20 and 55 times lower at 12 h

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and at the end of maturation, respectively, than those in the HM group (P < 0.0001).

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The pattern of meiosis progression in oocytes matured in medium covered with or

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without oil was similar (Figure 4). At the beginning of maturation, all oocytes were at

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the germinal vesicle stage. At 22 h, a minor proportion of oocytes were in MI, whereas

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at 28 h and 34 h, the majority of oocytes were at that stage (MI). The first oocytes in

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MII stage were visualized at 34 h of maturation. At 40 h and 44 h of culture,

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approximately 60% and 75% of oocytes, respectively, had progressed to MII, regardless

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of the presence or absence of oil overlay.

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The fertilization and embryo development results are indicated in Tables 3 and 4. At 18

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h after IVF, there were no differences in penetration, monospermic or efficiency rates

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between MO and HM groups. The presence or absence of an oil cover during culture

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did also not influence the cleavage, blastocyst formation or total cell number in

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blastocysts.

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4. Discussion

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This study demonstrated that the use of MO to cover the culture media in porcine

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embryo IVP system profoundly decreased the P4 concentration during oocyte

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maturation but did not affect the time-course of oocyte maturation or the oocyte

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developmental competence.

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The results from experiment 1 show that culture media incubated without oil overlay

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resulted in a significant increase in osmolality compared with media covered with oil.

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This result indicates that the typical relative humidity used in IVP-laboratory incubators

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(95–97%) is not sufficient to prevent medium evaporation and avoid changes in

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osmolality. In contrast to this assertion, Shimada et al [34] found that the osmolality of

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However, these authors did not mention the type of incubator used or any specific

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conditions for humidity, which limit the potential comparisons. To the best of our

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knowledge, there are no studies comparing the osmolality of culture media in the

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presence or absence of MO incubated in IVF incubator units.

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The osmolality of the maturation and embryo culture media incubated in the absence of

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oil overlay were high (close to 310 mOsm/kg) compared with those before incubation

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and in oil-overlaid media (below 298 mOsm/kg). Moreover, the osmolality of

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fertilization medium underwent an extreme increase in the absence of oil overlay,

357

reaching values of 445.5 mOsmol/kg, compared with those of the oil-overlaid medium

358

(298.0 mOsm/kg) or the original medium before incubation (295.5 mOsm/kg).

359

Although it seems clear that the small IVF drop volume (100 µL), compared with those

360

of the maturation and embryo culture drops (500 µL), had a large influence on the

361

osmolality of medium in the absence of MO, the type of the dish used for IVF (Petri

362

dish) should also be considered.

363

It was proposed that the osmolality of IVM/IVF/IVC media should remain stable during

364

incubation [25] because it plays an important role in the success of embryo IVP

365

programs [44]. Treating porcine [45] and bovine [46] oocytes with hyperosmotic

366

solutions caused approximately one-half of oocytes to lose the ability to develop into

367

blastocysts. Other studies have also indicated that high osmolality (>300 mOsm/kg) is

368

detrimental to mouse embryo development [47–49] because the increased intracellular

369

salt concentration destabilizes proteins and disrupts biochemical reactions and

370

metabolism [50]. In our study, although there were no differences in nuclear maturation

371

and fertilization parameters between the MO and OF groups, the cleavage and

372

blastocyst formation rates were lower (P < 0.003) when using culture media without oil

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348

15

ACCEPTED MANUSCRIPT overlay (high osmolality), thus confirming the negative effect of inadequate osmolality

374

on the embryo development. We can speculate that the high osmolality of the oil-free

375

fertilization medium was primarily responsible for the low embryo development

376

obtained in this group. Two facts support this speculation: first, the osmolality in the

377

maturation and embryo culture media in the absence of oil overlay was comparable with

378

the physiological osmolality reported for porcine follicular, oviductal and uterine fluids

379

(304.5 ± 0.5, 318 ± 8.0 and 293 ± 8.0 mOsm/kg, respectively) [24,51]; second, the

380

maturation and embryo culture media contain glycine, glutamine and hypotaurine,

381

among other amino acids, which are considered effective osmoprotectant organic

382

osmolytes [49]. In the presence of these osmolytes, oocytes and embryos develop in

383

culture at higher osmolalities that are similar to those they experience under in vivo

384

conditions. In their absence, however, as in the case of the fertilization medium used in

385

this study, the oocytes and embryos develop only at lower osmolalities in the medium

386

[52].

387

To prevent water evaporation and minimize osmolality changes in oil-free cultures, we

388

investigated the effects of a humid microenvironment by depositing purified water near

389

the culture dishes. As reported for in vitro bovine embryo production [38], the creation

390

of such a humid microenvironment was able to maintain osmolality in the maturation,

391

fertilization and embryo culture media at similar levels (< 300 mOsm/kg) to those of the

392

cultures covered with MO. The possibility of performing IVP of porcine embryos while

393

maintaining an adequate osmolality, in either the presence or absence of MO, allows for

394

the evaluation of the effect of oil overlay on oocyte development. Our results clearly

395

demonstrated that under similar osmotic conditions, oocytes can be matured, fertilized

396

and cultured in media covered with or without MO with similar success rates, indicating

397

that an oil overlay did not have a negative effect on the developmental ability of porcine

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16

ACCEPTED MANUSCRIPT oocytes. These results differ from those reported by Shimada et al [34], who found a

399

delay of nuclear maturation and a reduction in the developmental competence of pig

400

oocytes cultured in medium covered with MO. These authors found that the P4

401

concentrations in the maturation medium were very low when the oocytes were matured

402

under MO, indicating that P4 was absorbed by the oil during incubation. In addition,

403

they reported that at 28 and 40 h of maturation, the proportion of oocytes that

404

underwent germinal vesicle breakdown (GVBD), and those that reached the MII stage,

405

were lower in oocytes cultured in oil-covered medium. However, at 32 and 44 h of

406

maturation, the percentage of oocytes at the GVBD and MII stages, respectively, was

407

similar in each cultivation system. These authors suggested that the adverse effects of

408

oil on the developmental capacity of the oocyte might be explained by absorption by the

409

oil of cumulus secreted P4. In our study, as previously reported [34,53], we observed an

410

extreme decrease in the P4 concentration in the maturation medium covered by MO.

411

Moreover, the P4 concentrations were low at all maturation times evaluated, indicating

412

that the oocytes incubated under MO were not exposed to elevated P4 concentrations

413

during maturation. However, this condition did not affect the pattern of meiosis

414

progression. Our results clearly showed that the same proportion of oocytes reached the

415

MI and MII stages at similar times, regardless of the presence of oil overlay during

416

incubation. There is controversy about the effects of P4 on in vitro oocyte maturation.

417

Several findings have indicated that the P4 produced by cumulus cells is needed for the

418

meiotic resumption of bovine, ovine and porcine oocytes [54–57]. Moreover, the high

419

level of P4 produced by cumulus cells has been suggested to be responsible for the

420

acceleration of GVBD in porcine oocytes [58] by reducing gap junctional

421

communication in the cumulus cells [34]. However, other reports have shown no

422

significant stimulation of meiotic resumption by P4 in porcine [59] or mouse [60]

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17

ACCEPTED MANUSCRIPT oocytes. Moreover, P4 suppressed spontaneous meiotic maturation in mouse oocytes

424

[61] and decreased the rate of bovine oocyte maturation [62]. Whereas the significance

425

of P4 for maturation of oocytes in vitro continues to be a matter of debate, our results

426

demonstrated that the exposure of oocytes during IVM to high (cultures without oil

427

overlay) or low (cultures with oil overlay) concentrations of P4 did not directly affect

428

the maturation success in porcine oocytes. Moreover, the fertilization and embryo

429

development results were equal in HM and MO groups, suggesting no evident effects of

430

P4 or MO on oocyte developmental competence. These results are again in contrast to

431

those of Shimada et al [34]. Those authors found no influence of oil overlay on

432

fertilization parameters, but the rate of early embryonic development to the blastocyst

433

stage was greatly decreased in the presence of MO, suggesting that culture of pig COCs

434

in the MO-covered medium limits the developmental competence of oocytes. The

435

reasons for this discrepancy are unclear. However, several lines of evidence support our

436

results. The majority of laboratories working in IVP of porcine embryos use cultures

437

with an oil overlay during incubations, and the reported blastocyst formation rates after

438

maturation and fertilization are similar to those reached in the present study (30%–35%)

439

(reviewed in [16,19]). These percentages are much higher than those obtained by

440

Shimada et al [34] in their oil-covered system, where only a small proportion of oocytes

441

(3.3%) developed to the blastocyst stage after maturation and fertilization. The quality

442

of the oil might have contributed to the poor developmental outcomes obtained by

443

Shimada et al [34], as suggested by the same authors. In this line, several studies have

444

demonstrated that batches of oil can contain impurities and toxic contaminants. The

445

release of toxic components from the oil into the culture medium, such as Zinc [29],

446

Triton X-100 [63] and peroxides [32,33], has been associated with reduced embryo

447

development (reviewed in [20]).

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18

ACCEPTED MANUSCRIPT 4.1. Conclusions

449

In conclusion, despite the pronounced decrease in P4 concentration in the maturation

450

medium, the use of MO overlay during maturation, fertilization and embryo culture did

451

not affect the time-course of maturation or the developmental competence of pig

452

oocytes in vitro.

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448

453 Acknowledgments:

455

The authors are grateful to AIM Iberica (Murcia, Spain) and El Pozo (Murcia, Spain)

456

for supplying the boar ejaculates and the ovaries, respectively, used in this study. The

457

authors are grateful to Carolina Maside and Miguel Angel Angel for their assistance

458

throughout this work. This study was supported by: MINECO-FEDER (AGL2012-

459

38621 and AGL2012-39903), Madrid, Spain; MINECO (BES-2013-064087 and BES-

460

2013-064069), Madrid, Spain; Fundación Séneca (GERM 04543/07), Murcia, Spain.

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462

Conflicts of interest

463

None of the authors have any conflicts of interest to declare.

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464 465

Author contributions

All authors were involved in all phases of the research and writing of the paper.

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Figure legends

664 Fig. 1. Osmolality of in vitro maturation (IVM), fertilization (IVF) and embryo culture

666

(IVC) media at the beginning and end of each of incubation period. Incubations were

667

performed under mineral oil (MO) or without oil overlay under the typical humidity of

668

laboratory incubators (95–97%) (OF). Measurements of each sample were made in

669

duplicate, and values are the mean ± SD from two replicates. Different letters within

670

each incubation period indicate statistical differences (P < 0.001).

SC

RI PT

665

671

Fig. 2. Osmolality of in vitro maturation (IVM), fertilization (IVF) and embryo culture

673

(IVC) media at the beginning and end of each of incubation period. Incubations were

674

performed under mineral oil (MO) or without oil covering but surrounded by a humid

675

microenvironment (HM). Measurements of each sample were made in duplicate, and

676

values are the mean ± SD from four replicates. Different letters within each incubation

677

period indicate statistical differences (P < 0.03).

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678

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Fig. 3. Time course of progesterone concentration in the media during in vitro

680

maturation (IVM) of porcine oocytes. The oocytes were matured in medium (first 22 h

681

with hormones followed by 22 h without hormones) covered with mineral oil

682

(MO/oocytes) or without oil covering but surrounded by a humid microenvironment

683

(HM/oocytes). Maturation media with (MO) or without (HM) oil overlay not containing

684

oocytes were used as controls. Asterisk indicates statistical differences between

685

HM/oocytes and MO/oocytes groups within each time (P < 0.0001). Data are the mean

686

± SD from four replicates.

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687

28

ACCEPTED MANUSCRIPT Fig. 4. Kinetics of nuclear progression of immature porcine oocytes. The oocytes were

689

matured in medium (first 22 h with hormones followed by 22 h without hormones)

690

covered with mineral oil (MO) or without oil overlay but under a humid

691

microenvironment (HM). Data are the mean ± SD from 4 replicates. Under the present

692

culture conditions, the same proportion of oocytes reached the metaphase I (MI) and II

693

(MII) stages at similar times, regardless of the presence or absence of oil overlay during

694

incubation. The majority of the oocytes were in MI stage at 28 h and 34 h of maturation.

695

At 40 h and 44 h of culture, approximately 60% and 75% of oocytes, respectively, had

696

progressed to the MII stage. A total of 1,414 oocytes were analyzed with a minimum of

697

65 oocytes per group for each time point.

M AN U

698 699 700 701

706 707 708 709 710 711 712

EP

705

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704

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702 703

SC

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688

713 714 715 716 717

29

ACCEPTED MANUSCRIPT 718

Table 1

719

In vitro fertilization parameters of porcine oocytes cultured in media overlaid with

720

mineral oil (MO) or without an oil cover under the typical humidity of laboratory

721

incubators (95–97%) (OF).

722

(%) Group

RI PT

Oocytes Efficiency&

Oocytes Penetrated#

Monospermic*

MO

78

87.7 ± 6.0

53.2 ± 6.5

OF

80

87.2 ± 5.0

(%)

M AN U

SC

(N)

48.7 ± 4.5

28.0 ± 4.1

34.5 ± 6.9

723

#

724

containing only one male pronucleus/total of oocytes penetrated.

725

monospermic oocytes/total of oocytes inseminated. Data are presented as the mean ±

726

SD (two replicates).

730 731 732 733 734 735 736

&

Number of

TE D

729

Number of oocytes

EP

728

*

AC C

727

Number of oocytes penetrated/total inseminated oocytes.

737 738 739 740 741 742 30

ACCEPTED MANUSCRIPT 743

Table 2

744

In vitro development of porcine embryos cultured in media overlaid with mineral oil

745

(MO) or without an oil cover under the typical humidity of laboratory incubators (95–

746

97%) (OF).

747

(N)

2,4-cells

Blastocysts

(%)

(%)

#

155

57.7 ± 7.6a

47.7 ± 3.9a

OF

159

32.5 ± 7.3b

25.5 ± 4.8b

(%)

30.7 ± 5.9a

SC

MO

Efficiency*

RI PT

Group

Embryos developed to

Oocytes

7.7 ± 2.5b

748

#

749

oocytes cultivated. Data are presented as the mean ± SD (two replicates).

750

a,b

M AN U

Number of blastocysts/ total of 2,4-cells embryos. *Number of blastocyst/total of

Different letters in the same column indicate differences (P < 0.003).

751 752

756 757 758 759 760 761 762

EP

755

AC C

754

TE D

753

763 764 765 766 767 768 31

ACCEPTED MANUSCRIPT 769

Table 3

770

In vitro fertilization parameters of porcine oocytes cultured in medium overlaid with

771

mineral oil (MO) or without oil but surrounded by a humid microenvironment (HM).

772 Oocytes

Group

RI PT

(%) Efficiency&

Oocytes Penetrated#

Monospermic*

(%)

MO

146

77.7 ± 11.9

53.0 ± 13.0

36.7 ± 7.6

HM

159

68.4 ± 16.2

51.4 ± 14.1

M AN U

SC

(N)

773

#

774

containing only one male pronucleus/total of oocytes penetrated.

775

monospermic oocytes/total of oocytes inseminated. Data are presented as the mean ±

776

SD (four replicates).

Number of oocytes penetrated/total inseminated oocytes.

780 781 782 783 784 785 786 787

Number of

EP

779

&

AC C

778

Number of oocytes

TE D

777

*

30.6 ± 9.3

788 789 790 791 792 793

32

ACCEPTED MANUSCRIPT 794

Table 4

795

In vitro development of porcine embryos incubated in cultured in media overlaid with

796

mineral oil (MO) or without oil but surrounded by a humid microenvironment (HM).

797 Embryos developed to 2,4-cells (%)

Blastocysts#

per

blastocyst

41.4 ± 4.5

34.2 ± 10.7

34.6 ± 16.4

MO

309

61.0 ± 12.9

55.7 ± 8.2

HM

310

62.4 ± 17.5

53.2 ± 7.1

43.9 ± 3.3

#

799

oocytes cultivated. Data are presented as the mean ± SD (four replicates).

M AN U

Number of blastocysts/ total of 2,4-cells embryos. *Number of blastocyst/total of

800 801

807 808

EP

806

AC C

805

TE D

802

804

(%)

(%)

798

803

Efficiency*

RI PT

(N)

numbers

SC

Group

Oocytes

Total cell

33

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

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 Highlights

•

We evaluated the effect of mineral oil overlay on porcine IVP outcomes.

•

Progesterone level in the IVM medium was profoundly decreased in presence of

•

RI PT

oil.

However, oil did not affect IVM time-course or oocyte developmental

AC C

EP

TE D

M AN U

SC

competence.