The base medium affects ultrastructure and survival of bovine preantral follicles cultured in vitro

Accepted Manuscript The base medium affects ultrastructure and survival of bovine preantral follicles cultured in vitro Carolina Rodriguez Jimenez, Valdevane Rocha Araujo, Jurandy Mauro PenitenteFilho, Jovana Luiza de Azevedo, Renata Gomes Silveira, Ciro Alexandre Alves Torres PII:

S0093-691X(15)00627-5

DOI:

10.1016/j.theriogenology.2015.11.007

Reference:

THE 13413

To appear in:

Theriogenology

Received Date: 11 May 2015 Revised Date:

4 November 2015

Accepted Date: 7 November 2015

Please cite this article as: Jimenez CR, Araujo VR, Penitente-Filho JM, de Azevedo JL, Silveira RG, Torres CAA, The base medium affects ultrastructure and survival of bovine preantral follicles cultured in vitro, Theriogenology (2015), doi: 10.1016/j.theriogenology.2015.11.007. 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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The base medium affects ultrastructure and survival of bovine

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preantral follicles cultured in vitro

3 Carolina Rodriguez Jimeneza*, Valdevane Rocha Araujob, Jurandy Mauro Penitente-

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Filhoa, Jovana Luiza de Azevedoa, Renata Gomes Silveiraa, Ciro Alexandre Alves

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Torresa

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Department of Animal Science, Laboratory of Physiology and Animal Reproduction,

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Federal University of Viçosa, Viçosa, 36.570-000, MG, Brazil. b

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Follicles (LAMOFOPA), State University of Ceará, Fortaleza, 60.714-903, CE, Brazil.

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Faculty of Veterinary Medicine, Laboratory of Manipulation of Oocytes and Preantral

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Corresponding author: Carolina Rodriguez Jimenez, Animal Nutrition Laboratory, Center of Nuclear Energy in Agriculture, University of São Paulo, Avenida

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Centenário, nº 303, Caixa Postal 96, Piracicaba, SP 13.400-970, Brazil; e-mail:

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[email protected]

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Running head: different base medium in cow preantral follicles

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The aim of this study was to determine the effectiveness of minimum essential medium

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alpha modification (α-MEM), tissue culture medium 199 (TCM-199), and McCoy's

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medium (McCoy's) on in vitro culture of preantral follicles included in the bovine

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ovarian cortex (in situ). Bovine ovarian fragments were cultured in α-MEM, TCM-199

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Abstract

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or McCoy supplemented (+) with glutamine, insulin, transferrin, selenium, ascorbic

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acid, bovine serum albumin (BSA), penicillin, streptomycin, and acid 4- (2-hidroxietil) -

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1-piperazinoetanossulfónico (HEPES) buffer in 24-well plates, at 37°C and 5% CO2 for

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1 or 7 days. The morphology of follicles, normal, primordial and development (primary

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and secondary), as well as viability and morphometric variables of follicles and oocytes

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were assessed. The morphology and morphometry of preantral follicles were analyzed

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by ANOVA followed by the Tukey and Dunnett tests and viability variables were

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determined by the Chi-square (χ2) test. The results showed that TCM-199+ reduced

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significantly (p<0.05) the percentage of morphologically normal and viable follicles

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after 7 days of culture compared to the (control). Similar results were observed in

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McCoy+, in which the percentage of viable follicles after 7 days of culture was

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significantly lower (p<0.05) compared to control. However, it was similar (P>0.05)

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between α-MEM+ and TCM-199+. Moreover, follicular diameters in McCoy+ and TCM-

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199+ were significantly smaller (p<0.05) compared to control and α-MEM+ after 7 days

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of culture. In addition, the ultrastructure of preantral follicles was similar between

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control and α-MEM+ after 7 days of culture. In conclusion, α-MEM+ showed to be the

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most effective medium to preserve morphology, morphometry and ultrastructure of

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bovine preantral follicles, ensuring their viability and growth after in situ culture.

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Keywords: oocyte; folliculogenesis; in situ; α-MEM; TCM-199; McCoy.

1. Introduction

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In view of the wide diversity of cellular metabolism, several types of culture

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media are used to meet the nutritional requirements of multiple cells, such as α-MEM,

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α-MEM glutamax, TCM-199, McCoy, Waymouth, Leibowitz and Menezo B2 [1]. In

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general, these medium have different concentrations of inorganic salts, amino acids,

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vitamins and nucleosides. Other substances are also added, such as antioxidants,

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proteins, micronutrients, glycoproteins, antibiotics, buffer agents, hormones and growth

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factors, which enrich growth medium to maintain cell viability in culture.

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The α-MEM is widely used for in vitro culture of preantral follicles in goat

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[2,3,4], bovine [5,6,7,8,11]; canine [9]; and murine [10]. Previous studies have shown

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that α-MEM is an important growth medium for isolated bovine preantral follicles

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[5,11] and enclosed in ovarian tissue [12]. However, Rossetto et al. [7] found that

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isolated bovine secondary follicles have difficulties to grow and maturate in vitro,

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differently from other species such as goat.

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Similarly, TCM-199 has been widely used for in vitro culture of bovine preantral

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follicles. Saha et al. [13] found that supplementing TCM-199 with follicle-stimulating

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hormone (FSH) and/or epidermal growth factor (EGF) to isolated follicles, they

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promoted an increase in follicular diameter. In addition, TCM-199+ has also been

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successfully used for in vitro growth and development of isolated preantral follicles (>

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150 µm) [14], leading to formation of antral follicles after in vitro culture [6]. Costa et

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al. [15] observed that the addition of FSH to TCM-199 did not influence the viability

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and growth of preantral follicles regardless of the presence of FSH.

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The McCoy medium is also highly used in studies on bovine preantral follicles.

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It is usually supplemented with ascorbic acid [16] or insulin-like growth factor-1 (IGF-

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1) [17]. Recently, McLaughlin et al. [18,19] found that the in vitro culture of bovine

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preantral follicles in two steps, firstly, preantral follicles included into ovarian tissue (in

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situ culture system), which leading to activation of primordial to secondary follicles,

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and secondly the secondary follicles were isolated from the ovarian tissue and

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individually cultured until antral follicles. In our study, the McCoy medium was

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supplemented with FSH and/or activin. However, based on literature reports, there has

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been no standard, reliable culture medium for bovine preantral follicles [1]. Moreover,

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in recent years, researchers have intensified their efforts in the culture of isolated

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advanced preantral follicles (> 150 µm).

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Since about 90% of preantral follicles are primordial, studies on base medium that

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allow activation, as well as in vitro development, contribute to obtaining a greater number

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of secondary follicles. These secondary follicles can be isolated and cultured in vitro

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again until complete their maturation. Thus, the aim of this study was to identify the

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effectiveness of α-MEM, McCoy and TCM-199 media on the development of bovine

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preantral follicles enclosed in ovarian cortical tissue (in situ).

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2. Material and methods

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The culture medium and chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

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2.2. Source of ovaries

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Ovarian cortical tissues (n = 24 ovaries) from mixed-breed cows at different

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ages and phases of the estrous cycle were collected from a local slaughterhouse.

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Immediately after slaughter, the ovaries were washed once in 70% alcohol for 10 sec

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followed by two washes with TCM-199 [20] supplemented with 20 mM HEPES, 2000

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µg/mL sodium bicarbonate [13, 21], 25 µg/mL pyruvic acid, 75 µg/mL penicillin and

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50 µg/mL streptomycin [18] been denominated TCM-199 to ovarian manipulation. The

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pairs of ovaries were transported in TCM-199 at 4°C [22] to the laboratory within a

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period of 2 h. The ovaries were divided into two groups, six pairs for the study of the

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morphology, morphometry and ultrastructure of preantral follicles in situ, and six pairs

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for the viability analysis of isolated preantral follicles.

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In the laboratory, ovarian cortical slices (3x3x1 cm) were cut from the ovarian

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surfaces using a sterilized surgical blade. Ovarian fragments were washed in a

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fragmentation medium, consisted of TCM-199 with 20 mM HEPES; 2000 µg/mL

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sodium bicarbonate [13]; 2 mM sodium pyruvate; 75 µg/mL penicillin, 50 µg/mL

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streptomycin and 10 % BSA [18]. The tissue pieces, control or placed into culture

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medium for 1 or 7 days were directly fixed for histological and ultrastructural analysis.

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Bovine tissues were transferred to 24-well plates containing 1 mL of culture medium. In

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vitro culture was performed at 37°C in a humidified incubator with 5% CO2.

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All culture media (α-MEM, TCM-199 or McCoy) were supplemented with 20

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mM HEPES; 3 mM glutamine; 10 ng/mL insulin; 2.5 µg/mL transferrin; 4 ng/mL

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selenium; 50 µg/mL ascorbic acid (α-MEM except); 0.1% BSA; 100 µg/mL penicillin

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and 100 µg/mL streptomycin as described by McLaughlin et al. [18]. After

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supplementation, the medium were called α-MEM+, TCM-199+ and McCoy's +.

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2.4. Morphological analysis and assessment of in vitro follicular growth

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Before culture (control) and after 1 and 7 days in culture, all pieces were fixed in

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Carnoy solution for 4 h and then dehydrated in increasing concentrations of alcohol,

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diaphonized in xylene and embedding in paraffin. The bovine tissue fragments were cut

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into 5 µm sections, and each section was mounted on a glass slide and stained using

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periodic acid Schiff – hematoxylin [23]. Follicular stages were assessed microscopically

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on serial sections. Slides were analyzed and examined under optical microscopy (400X

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magnification, Olympus BX50, Japan). Thirty preantral follicles were evaluated in each

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repetition, totaling 360 observations per treatment (180 at day 1 and 180 at day 7).

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Preantral follicles were classified as primordial and developing (primary and

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secondary). Primordial follicles were described as spherical or ovoid oocytes

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surrounded by a single layer of flattened and some cuboid-shaped granulosa cells

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(GCs). The primary follicles were described as spherical oocytes with a single layer of

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cuboid-shaped GCs and the secondary follicles as spherical oocytes with two or more

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layers of cuboid-shaped GCs [22]. Qualitative evaluation of the follicles was based on

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the integrity of the oocyte and GCs. The follicles were classified morphologically as:

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normal (an intact oocyte was present, surrounded by granulosa cells that were well

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organized in one or more layers and that had no pycnotic nuclei) or degenerated (Atretic

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follicles were defined as a retracted oocyte, pycnotic nucleus, and/or disorganized

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granulosa cells detached from the basement membrane [24].

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For the analysis of follicular and oocyte diameters, 10 morphologically normal

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preantral follicles per animal were observed and their images captured with an optical

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microscope (400X magnification, Olympus BX50, Japan). We evaluated 120 follicles

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per treatment, control (n = 60), day 1 (n = 60), and day 7 (n = 60). The diameters were

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calculated by Image J / Fiji 1:46 program [25].

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2.5. Ultrastructure analysis

152 To examine follicular morphology, Transmission Electron Microscopy (TEM)

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was performed to analyze ultrastructure of preantral follicles. A portion with maximum

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dimension of 1mm3 was cut from each fragment of the ovarian tissue and fixed in

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Karnovsky solution (2 % paraformaldehyde and 2 % glutaraldehyde in 0.1 M sodium

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cacodylate buffer pH 7.2) for 4 h at room temperature (25°C). After three washes in

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sodium cacodylate buffer, specimens were post-fixed in 1% osmium tetroxide for 1 h at

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room temperature. The samples were then dehydrated through a gradient of acetone

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solutions and thereafter embedded in Spurr resin. Semi-thin sections (3 µm) were cut in

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an ultramicrotome (RMC Products, Power Tome-X, Arizona, USA) and stained with

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toluidine blue for light microscopy studies. The ultrathin sections (60-70 nm) were

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contrasted with uranyl acetate and lead citrate, and examined under a Zeiss EM 900

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(Jena, Germany) transmission electron microscope. Based on histological results, the

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follicles observed (n=5 per treatment) in TEM were from the control, α-MEM+ and

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TCM-199+. Parameters such as density and integrity of ooplasmic and granulosa cell

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organelles, vacuolization and basement membrane integrity were evaluated.

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2.6. Viability analysis

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Before culture (control) and after 1 and 7 days in culture, preantral follicles were

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isolated by the mechanical method described by Figueiredo et al. [5]. Briefly, ovarian

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fragments were finely fragmented and transferred to 15 mL tubes containing 5 mL of

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TCM-199 with 20 mM HEPES, 2000 µg/mL sodium bicarbonate [13, 21], 2 mM

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sodium pyruvate, 75 µg/mL penicillin, 50 µg/mL streptomycin and 10 % BSA [18].

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Ovarian fragments were mechanically dissociated by repeated pipetting (1,600 and 600

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µm of diameter), the material obtained was filtered through nylon mesh 100 µm of

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diameter and subsequently the viability was analyzed with Trypan blue. We added 5 µL

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of Trypan Blue to each 100 µL of suspension medium containing isolated preantral

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follicles. Follicles were classified as viable or non-viable, when non-stained or blue-

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stained, respectively [26] under an inverted microscope (100X; Leica DFC295,

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Germany).

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The percentages of preantral follicles morphologically normal, primordial and

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developing (primary and secondary), as well as those with follicular and oocyte

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diameter were submitted to analysis of variance (ANOVA). Subsequently, comparisons

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between the means were made using the Dunnett test (control versus treatments) and the

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Tukey test (treatments versus treatments). The viability analysis of isolated preantral

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follicles was compared in contingency table by the Chi-square test (χ2). We used the

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software version 9.1 of the statistical program SAS. The values were considered

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statistically significant different at p < 0.05.

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3. Results

3.1. Morphology and viability of bovine preantral follicles cultured in situ in α-MEM+, TCM-199+ or McCoy+

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We analyzed 1,260 preantral follicles enclosed in ovarian tissue. Percentage of

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control and cultured preantral follicles at days 1 or 7 in α-MEM+, TCM-199+ and

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McCoy+ medium are presented in Figure 1. Was not found difference (p < 0.0.5)

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between control and medium base on day 1 of in vitro culture (Table 1). However, at

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day 7 of culture, TCM-199+ was the only treatment that significantly reduced the

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percentage of morphologically normal follicles compared to the control (p < 0.05).

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Comparing treatments at days 1 and 7, no differences were found (p > 0.05) regarding

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the percentage of morphologically normal follicles.

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Preantral follicles (n = 50 per treatment) were isolated from the ovarian stroma

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at days 0 (control), 1, and 7 in order to confirm the morphological results of (Table 1).

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Viable and non-viable follicles can be observed in Figure 2. The results showed that

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only follicles cultured in α-MEM+ at days 1 and 7 did not differ from the control (p >

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0.05). On the other hand, the rate of follicles cultured in TCM-199+ and McCoy+ was

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significantly lower than the control at day 7. At day 7 of culture, the percentage of

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viable follicles was significantly higher in α-MEM+ compared to TCM-199+. In

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addition, with the progression of culture, from day 1 to day 7, only the treatments using

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α-MEM+ and TCM-199+ kept the number of viable preantral follicles, whereas the

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McCoy+ medium significantly reduced the percentage of viable follicles after day 7 (p <

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0.05).

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Table 2, presented the average of percentage of primordial and developing

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follicles and their variation represented as standard error of the mean (SEM). After 7

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days of culture, all treatments reduced the percentage of primordial follicles compared

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to the control (p < 0.05), although the percentage of developing follicles was not

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concomitant in cultured treatments. The percentage of developing follicles was

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significantly higher in α-MEM+ than in TCM-199+ at day 1 of culture. Comparing the

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treatments at different culture periods, there was a significant reduction in the

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percentage of primordial follicles in all treatments from day 1 to day 7. However, the

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developing follicles kept the same percentages during the in vitro culture (p > 0.05).

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3.2. Morphometry of bovine preantral follicles cultivated in situ in α-MEM+, TCM-199+

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or McCoy+

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For the morphometric analyses, we measured the dimensions of follicles and

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oocytes (n = 210) enclosed in ovarian tissue, totaling 30 measurements per treatment.

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The averages of follicular diameter and oocyte at days 1 and 7 of in vitro culture can be

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observed in Table 3. At day 1 of in vitro culture, no differences were found (p > 0.05)

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between follicular dimensions of the control and the culture media tested (α-MEM+,

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TCM-199+ and McCoy+). However, at day 7, TCM-199+ and McCoy+ significantly

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reduced the follicular diameter when compared to the control. In addition, all treatments

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showed oocyte diameters significantly lower than those of the control (p < 0.05),

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although they did not differ from each other. Comparing the culture media, no

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differences were found in the follicular and oocyte dimensions after 1 day of culture (p

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> 0.05). However, at day 7 of in vitro culture, the dimensions of follicles cultured in

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McCoy+ and TCM-199+ were significantly lower than those in α-MEM+. Moreover,

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TCM-199+ and McCoy+ reduced the follicular diameter as culture progressed from day

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1 to day 7 (p < 0.05).

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3.3. Ultrastructural analysis

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Transmission electron microscopy was used as a qualitative technique and

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complementary for histological analysis and follicular viability to evaluate the integrity

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of control (Figure 3A,B) preantral follicles and follicles cultivated for 7 days in α-

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MEM+ (Figure 3C,D) and TCM-199+ (Figure 3E,F). Confirming the results obtained in

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histological studies, preantral follicles cultured in α-MEM+ showed similar

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ultrastructure to the control. In both of treatments, follicles showed nucleus and

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organelles uniformly distributed in the cytoplasm such as mitochondria, endoplasmic

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reticulum and some vacuoles. Moreover, granulosa cells and basal and nuclear

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membranes were preserved and intact.

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Follicles cultured in TCM-199+ were characterized by a high number of

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vacuoles scattered throughout the cytoplasm, showing a very low density of organelles,

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as well as irregular or fragmented nuclear and cytoplasmic membranes. In addition, it

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was observed fragmentation in granulosa cells, pycnotic oocytes with a dark color in

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most cells, as well as increased volume and disruption of the nuclear envelope along

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some segments of the nucleus contour (Figure 3E,F).

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3.4. Differences in α-MEM, TCM-199, and McCoy

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For a better understanding of the present study, we carried out a comparative

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analysis of the components of the base media. The α-MEM and TCM-199 have greater

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variety of minerals salts than the McCoy medium (Figure 4). In addition, α-MEM

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incorporates higher concentrations of amino acids L-arginine, L-asparagine, L-glutamic

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acid and L-Histidine when compared to TCM-199 and McCoy (Figure 5). The α-MEM

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has a greater number of nucleosides, nucleoside triphosphate type (deoxyadenosine 0.01

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g/L; deoxycytidine 0.011 g/L and deoxyguanosine 0.01 g/L) and ribonucleosides

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(adenosine 0.01 g/L, guanosine 0.01 g/L and uridine 0.01 g/L).

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The McCoy medium shows a concentration of glucose (3 g/L) threefold greater

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than α-MEM or TCM-199 (1.0 g/L). Although α-MEM does not show high glucose

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concentrations, it has pyruvic acid (0.11 g/L), an intermediate organic compound

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extremely important for glucose metabolism, Krebs cycle, synthesis of fatty acids,

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steroids and amino acids [27].

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TCM-199 contains cholecalciferol or vitamin D (100 ng/mL). It also has

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menadione sodium disulfide (0.000016 g/L), which is a synthetic form of vitamin K3, a

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product of low stability in light, heat, humidity and pH. Most vitamins of α-MEM and

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TCM-199 show concentrations equal and/or similar. However, the McCoy medium has

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higher vitamin concentration compared to α-MEM and TCM-199 (Figure 6).

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4. Discussion

In the present study, we evaluated the efficiency of commercial media α-MEM,

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TCM-199 and McCoy`s for in-vitro culture of preantral follicles in bovine ovarian

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tissue. For parameters of analysis, we considered the percentage of morphologically

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normal and viable follicles, as well as the percentage of follicular activation (primordial

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and developing follicles) and follicular and oocyte diameters. We also investigated the

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composition differences in the medium used, such as concentration of mineral salts,

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amino acids, vitamins and nucleosides. The results showed that α-MEM+ had a

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percentage of morphologically normal and viable follicles similar to that of the control,

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and these percentages were higher than those in TCM-199+. In addition, α-MEM+ was

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able to increase the follicular diameter compared to TCM-199+ and McCoy+.

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After 7 days of culture, only the follicles cultured in α-MEM+ and McCoy+

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showed a percentage of morphologically normal follicles similar to that in the control.

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However, only in α-MEM+, the percentage of viable follicles remained similar to that in

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the control after 7 days of culture. Similar results were observed when α-MEM+ was

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used for in vitro culture of isolated bovine preantral follicles, which can replace TCM-

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199+ [1,11]. In contrast, in a similar experiment, Rossetto et al. [6] verified that TCM-

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199+ was better to keep the morphology and viability of isolated bovine preantral

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follicles compared to α-MEM+ and McCoy+. In the current study, however, TCM-199+

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was less efficient than α-MEM+. These results may be attributed to the differences in

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their compositions. The α-MEM has a greater number of nucleotides and amino acids.

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In addition, α-MEM and TCM-199 have equal and, or similar concentrations of

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vitamins. TCM-199 was not efficient in the follicular viability or morphometry,

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possibly because of the presence of cholecalciferol and menadione sodium disulfide

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absent in α-MEM. Cholecalciferol, or vitamin D3, is a fat-soluble vitamin, and one of

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the most toxic and slowly metabolized vitamins [28]. Premaor [29] showed that vitamin

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D levels below 3.2×10-5 g/L (32 ng/mL) can be characterized as deficient. However,

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Castro et al. [3] showed that high doses (0.0002 g or 100 ng) can cause calcium

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destabilization. Menadione sodium disulfide, also found in TCM-199, is not used

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directly as a nutritional supplement due to its high toxicity and low stability [31]. Lima

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[32] stated that the storage of high concentrations of vitamins can lead to toxic

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symptoms and large doses may interfere in the metabolism of other vitamins. Therefore,

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bovine preantral follicles respond differently to conditions and, or culture systems used,

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enclosed (in situ) or isolated from the ovarian tissue.

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The morphometric responses showed that at day 7 of culture, the dimensions of

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the follicles cultured in McCoy+ and TCM-199+ were lower than those in the control

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and α-MEM+. Similar to our findings, Previous researches showed that the diameter of

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bovine preantral follicles increases after in vitro culture in α-MEM+ with [12] or without

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the addition of hormones (follicle-stimulating, luteinizing and estradiol) [8]; vascular

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endothelial growth factor (VEGF) [1, 11]. On the other hand, Rossetto et al. [6] showed

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that the addition of FSH and activin promoted follicular growth and antral formation in

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secondary follicles cultured in TCM-199+. Using McCoy+ in the presence of FSH, EGF

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and IGF-1, for the first time in the literature, Gutierrez et al. [33] obtained the first

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bovine antral follicles cultured in vitro from preantral secondary follicles. Years later,

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these results were confirmed by McLaughlin et al. [18], who added FSH and, or activin

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to McCoy+ to a culture of bovine preantral follicles in two phases (in situ and isolated).

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In this study was observed that McCoy medium has higher concentrations of vitamins.

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than α-MEM and TCM-199. The TCM-199 has contents of vitamins similar to those of

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α-MEM, however, with some additional vitamins that may interfere negatively on the

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follicles. Nevertheless, it can be inferred that the smaller follicular diameter observed

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from day 1 to day 7, and the low viability observed, both in TCM-199 and McCoy, may

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be a result of a high concentration of vitamins in preantral follicles. Thus, the efficiency

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of α-MEM can be associate with to additional amino acids and nucleosides, as well as

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mineral salts and vitamins, which collaborated to the positive response in in vitro

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culture of preantral follicles.

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It has been a challenge to produce an ideal medium to activate bovine primordial

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follicles from its natural quiescence until the later stages in situ and,or isolated culture

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systems. It is known that several factors may interfere in this process, such as the

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culture system performed (in situ or isolated culture system), the concentrations of

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substances added to culture media (growth factors, hormones etc.), as well as the

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metabolites produced by the follicle cells through the culture. Considering that about

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90% of the follicular population is composed by the primordial follicles, the

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optimization of the in vitro culture systems to promote the development of this follicle

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is very important. However, it is still difficult to develop those follicles in most species,

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including bovine. Based on difficulty of promoting their in vitro activation and

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development, this study tested three base media. The base medium is the first source of

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the nutrients that come into contact with the follicle. Thus, this study have proved that

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α-MEM may be the medium of choice to maintenance of the follicular integrity and

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development as previously shown by Araújo et al. (1, 11). On the other hand, both

354

TCM-199 and McCoy media besides not promote follicular activation, did not show any

355

influence about development and viability. Thus, these results are a starting point to

356

begin an organized series of research with substances that could improve the in situ

357

culture system and promote follicle development using α-MEM as a base medium.

SC

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349

Therefore, the knowledge the action of the medium in the culture system is of

359

vital importance because of the great variety and the medium act differently in terms of

360

species, cells and culture types. Although in recent years, researchers have worked more

361

intently in the culture of isolated preantral follicles (> 150 µm). In addition, several

362

studies have succeeded in using α-MEM to culture preantral follicles in caprine [34, 35,

363

36, 37, 4] canine [9]; murine [38, 10]; primate [39]; and even bovine [5, 6, 7, 8, 1, 11].

364

Therefore, studies on culture medium to provide activation and development of these

365

follicles contribute to the recovery of a higher number of developing follicles that can

366

be used in two-phase culture.

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The result of the morphological analysis was confirmed by ultrastructural

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367

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358

368

analysis. This technique is considered an important tool because it provides more

369

information about the quality of preantral follicles and is able to detect damage to cell

370

membranes and organelles. Transmission electron microscopy studies showed that

371

granulosa cells and basal and nucleus membranes were preserved, as well as important

372

organelles, such as mitochondria and endoplasmic reticulum, even after 7 days of

373

culture in the presence of α-MEM+. Similar results were described by Araujo et al., [2]

16

ACCEPTED MANUSCRIPT 374

who observed that after 7 days of in vitro culture using only α-MEM+, the follicles

375

showed preserved ultrastructural integrity. Similarly, Chaves et al. [40] and Lopes et al.

376

[41] suggested that the use of α-MEM at 4°C preserves the quality of caprine and canine

377

preantral follicles, respectively, for further in vitro culture. Rossetto et al. [7] verified that the ultrastructure was preserved in bovine

379

preantral follicles cultured in both α-MEM+ and TCM-199+, after 16 days of culture,

380

although hormones were added to these medium. Silva et al. [24] observed by

381

transmission electron microscopy the presence of vacuoles and, consequently, the

382

decrease of organelles, such as mitochondria. These vacuoles are possibly comprised of

383

changed organelles that were phagocytized, but remained in the cells, characterizing one

384

of the first signals of follicular degeneration [24]. Such observations have already been

385

reported in preantral follicles in caprine [41]. Thus, we highlight the importance of the

386

ultrastructural analysis before and after in vitro culture of preantral follicles in different

387

species as a valuable tool for quality evaluation of follicles (bovine: [26]; murine: [42,

388

43]; and goats: [34, 2].

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378

390

Conclusion

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389

The results showed that α-MEM+ is an effective medium to preserve

392

morphology, morphometry and ultrastructure of bovine preantral follicles, ensuring

393

their viability and growth after short term in situ culture system.

394

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391

395 396

Acknowledgments

397

The authors wish to thank: Federal University of Viçosa (UFV), CAPES/CNPq

398

– IEL National – Brazil – for the scholarship granted, to FAPEMIG and CNPq, for

17

ACCEPTED MANUSCRIPT 399

financial support and NAP/MEPA (ESALQ/USP) and NMM-CCB (UFV) for technical

400

Support.

401 402

Conflict of interest

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403 404

There is no conflict of interest that could be compromise the impartiality of the research

405

reported.

407

SC

406 Figure legends

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Figure 1 – Bovine preantral follicles (control; a, b) and cultivated after 1 and 7 d in the

410

presence of α-MEM+ (c, d); TCM-199+ (e, f) and McCoy+ (g, h), respectively. O =

411

oocyte; n = nucleus; GC = granulosa cells; PF = primordial follicles; PaF = primary

412

follicle; and SF = secondary follicle. 400X magnification; PAS-hematoxilin.

413

Figure 2 – Bovine preantral follicles mechanically isolated before the control (a, b) and

414

after 1 day (c, d) or 7 day (e, f) of culture in α-MEM+, stained with Trypan blue (dead)

415

and not stained (alive).

416

Figure 3 - Ultrastructural analysis (n=5 follicles) of control and cultured bovine

417

preantral follicles after 7 days of in vitro culture. (A, B) normal preantral follicles

418

control; C, D) normal preantral follicles after 7 d of culture in vitro in α-MEM+ (E, F)

419

degenerate preantral follicles after 7 d of culture in vitro in TCM-199. O = oocyte; Nu =

420

oocyte nucleus; m = mitochondria; GC = granulosa cells; ER=endoplasmic reticulum;

421

Mb = basement membrane; oocyte membrane (white arrow); vacuoles (black arrow).

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18

ACCEPTED MANUSCRIPT

Figure 4 – Minerals in minimal essential medium (α-MEM-●), tissue culture medium

423

(TCM-199-◌) and McCoy medium (McCoy-▼) 199.

424

Figure 5 – Amino acids in minimal essential medium (α-MEM-●), tissue culture

425

medium (TCM-199-◌) and McCoy medium (McCoy-▼) 199.

426

Figure 6 – Vitamins in minimal essential medium (α-MEM-●), tissue culture medium

427

(TCM-199-◌) and McCoy medium (McCoy-▼) 199.

429

SC

428 References

432

M AN U

430 431

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Silva JR, Figueiredo JR, Rodrigues APR. Effect of bone morphogenetic protein 7

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Lucci CM, Báo SN, Figueiredo JR. Degeneration rate of preantral follicles in the

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2000; 62;1322-1328.

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Ruminant Res 2011a; 95:139 -143. [37] Araújo VR, Silva GM, Duarte ABG, Magalhaes DM, Almeida AP, Gonçalves

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ACCEPTED MANUSCRIPT Table 1 – Percentages of morphologically normal preantral follicles (mean ± standard error of the mean) and viable follicles (%) cultivated in α-MEM+, TCM-199+ and McCoy’s+ medium during 1 and 7 days. Morphologically normal Viability follicles


Day 1 Control

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preantral follicles


Treatments

Day 7

Day 1

70 ± 7

Day 7

44/50 (88%)

66 ± 2 aA

48 ± 9 aA

41/50 (82) abA

41/50 (82) aA

TCM-199+

52 ± 7 aA

39 ± 5 *aA

36/50 (72) bA

26/50 (52) *bA

McCoy’s+

52 ± 5 aA

44 ± 8 aA

46/50 (92) aA

35/50 (70) *abB

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α-MEM+

* Significant difference for the treatment-control (P < 0.05); letters in the same column are different (P < 0.05);

A, B

a, b

superscript lowercase

superscript uppercase in the

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rows indicate a statistically significant difference between the days of culture (P < 0.05); Cont = control; α-MEM+ = minimum essential medium supplemented; TCM-

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199+ = tissue culture medium supplemented 199; and McCoy’s+ = McCoy’s medium supplemented.




The percentage of morphologically normal follicles and viable

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follicles represents all the stages of preantral follicles (primordial, primary and secondary follicles).

ACCEPTED MANUSCRIPT Table 2 – Percentages of primordial and developing follicles (average ± standard error of the mean) cultured in α-MEM+, TCM-199+ and McCoy’s+ medium during 1 and 7 days. Primordial

Developing

Treatments

Control

Day 7

Day 1

Day 7

RI PT

Day 1 49 ± 8

22 ± 5

28 ± 6 aA

8 ± 1 *aB

38 ± 6 aA

39 ± 10 aA

TCM-199+

35 ± 6 aA

13 ± 5 *aB

17 ± 3 bA

26 ± 6 aA

McCoy’s+

31 ± 7 aA

18 ± 4 *aB

22 ± 5 abA

26 ± 7 aA

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α-MEM+

* Significant difference for the treatment-control (P < 0.05); letters in the same column are different (P < 0.05);

A, B

a, b

superscript lowercase

superscript uppercase in the

rows indicate a statistically significant difference between the days of culture (P <

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0.05); Control = non cultured tissue; α-MEM+ = minimum essential medium supplemented; TCM-199+ = tissue culture medium supplemented 199; and McCoy's+

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= McCoy’s medium supplemented.

ACCEPTED MANUSCRIPT Table 3 – Mean ± standard error of the mean (SEM) of the follicular diameter/oocyte in micrometers (µm) of bovine preantral follicles cultured in α-MEM+, TCM-199+ and McCoy’s+ medium during 1 and 7 days. Mean ± SEM Day 1

Primordial

Primary

Secondary

Control

36 ± 1.1 (6)

47 ± 1.5 (16)

75 ± 2.9 (8)

α-MEM+

35 ± 0.6 (12)

48 ± 1.4 (14)

66 ± 3.1 (4)

TCM-199+

37 ± 0.6 (9)

44 ± 0.9 (17)

73 ± 5.2 (4)

McCoy’s+

38 ± 0.6 (4)

47 ± 1.0 (21)

62 ± 0.8 (5)

45 ± 2.0 (30) aA 46 ± 2.2 (30) aA

48 ± 1.4 (30)

aA

Mean ± SEM Day 7

M AN U

Follicles ± SEM (n) - Day 7

52 ± 2.8 (30)

SC

Treatment

RI PT

Follicles ± SEM (n) - Day 1

36 ± 1.1 (6)

47 ± 1.5 (16)

75 ± 2.9 (8)

52 ± 2.8 (30)

α-MEM+

34 ± 0.9 (16)

45 ± 2.8 (4)

77 ± 5.4 (10)

50 ± 4.1 (30) aA

TCM-199+

34 ± 0.7 (19)

48 ± 1.8 (10)

69 ± 0.0 (1)

39 ± 1.7 (30) *bB

McCoy’s+

34 ± 0.5 (22)

49 ± 2.8 (7)

60 ± 0.0 (1)

39 ± 1.5 (30) *bB

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Control

Oocyte ± SEM (n) - Day 1

Mean ± SEM Day 1

24 ± 0.7 (6)

27 ± 1.0 (16)

35 ± 3.2 (8)

28 ± 1.2 (30)

α-MEM+

19 ± 0.8 (12)

24 ± 1.1 (14)

30 ± 5.0 (4)

23 ± 1.0 (30) *aA

TCM-199+

20 ± 1.5 (9)

22 ± 0.7 (17)

27 ± 4.3 (4)

22 ± 0.8 (30) *aA

McCoy’s+

22 ± 2.1 (4)

22 ± 0.6 (21)

24 ± 1.3 (5)

22 ± 0.5 (30) *aA

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Control

Oocyte ± SEM (n) - Day 7

Mean ± SEM Day 7

Control

24 ± 0.7 (6)

27 ± 1.0 (16)

35 ± 3.2 (8)

28 ± 1.2 (30)

α-MEM+

19 ± 0.5 (16)

21 ± 2.0 (4)

26 ± 2.2 (10)

22 ± 1.0 (30) *aA

TCM-199+

20 ± 1.0 (19)

22 ± 1.0 (10)

29 ± 0.0 (1)

21 ± 0.79 (30) *aA

McCoy’s+

18 ± 0.7 (22)

24 ± 1.8 (7)

41 ± 0.0 (1)

20 ± 1.0 (30) *aA

*

Follicular difference or oocyte of the control treatment with cultivated treatments (P <0.05);

a, b

superscript lowercase letters indicate follicular or oocyte difference in

ACCEPTED MANUSCRIPT cultivation day between treatments (P < 0.05);

A, B

superscript uppercase letters

indicate follicular or oocyte difference between days 1 and 7 of cultivate and treatments (α-MEM+, TCM-199+ or McCoy's+ - P < 0.05); α-MEM+ = minimal essential medium supplemented; TCM-199+ = tissue culture medium 199 and

RI PT

McCoy's+ = medium McCoy's+ supplemented.

* Diferença folicular ou oocitaria do tratamento controle com os tratamentos cultivados a,b

Letras minúsculas sobrescritas indicam diferença folicular ou oocitaria

no dia de cultivo entre os tratamentos (P<0.05);

SC

(P < 0.05);

A,B

Letras maiúsculas sobrescritas

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indicam diferença folicular ou oocitaria entre os dias de cultivo 1 e 7 e tratamento (αMEM+, TCM-199+ ou McCoy’s+ - P < 0,05); α-MEM+ = meio essencial mínimo suplementado; TCM-199+ = meio de cultivo de tecido 199 suplementado e McCoy’s+

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= meio McCoy’s+ suplementado.

ACCEPTED MANUSCRIPT Figure 1

(a)

PaF

(b)

PaF

n

GC GC

RI PT

n

(c)

PF

(d)

O

n

M AN U

GC

SC

O

O

O

GC

(e)

O

n

(f)

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PF

PF

PF

n

o n

GC

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EP

GC

PaF

(g)

(h)

PF

GC

O

n

o GC

n

ACCEPTED MANUSCRIPT Figure 2

(b)

(c)

PF (alive)

(d)

(e)

SF (dead)

PaF (alive)

RI PT

SF (alive)

PF (dead)

AC C

EP

TE D

M AN U

SC

(a)

(f)

SF (alive)

ACCEPTED MANUSCRIPT Figure 3

A

B

m

O

O GC

C

GC

O

m

D

M AN U

ER

SC

Nu

m

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Nu

m

mb

GC

Nu

E

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Nu

F

m

EP

O

GC

O

Nu

AC C

ER

GC

Nu

ACCEPTED MANUSCRIPT Figure 4 – Minerals

10

4 1 2 3 4 5 6 7 8 9

2

1

2

3

4

5

6

7

9

EP

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Minerals

8

Minerals CaCl2*H2O Fe(NO3)3 *9H2O MgSO4 (anhydrous) KCl KH2PO4 Na * Acetate (anhydrous) NaHCO3 NaCl Na2HPO4 (anhydrous)

M AN U

0

AC C

[1x] g / L

6

α-MEM

0,2 0 0.09767 0.4 0 0 2.2 6.8 0.122

SC

8

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Alpha - MEM TCM-199 McCOY

TCM-199

0.1396 0.00072 0.09767 0.4 0.06 0.05 0.35 8.0 0.04788

McCOY

0.1324324 0 0.0976876 0.4 0 0 2.2 0 0.504

ACCEPTED MANUSCRIPT Figure 5 Amino acids

0.12 0.10

0.06 0.04 0.02 0.00

4

6

8

10 12 14 16 18 20 22 24 26

L-Alanine L-Arginine * HCL L-Asparagine * H2O L-Aspartic Acid L-Cysteine L-Cysteine *HCL*H2O L-Cystine *2HCL L-Glutamic Acid Glycine L-Histidine *HCL*H2O Hydroxy-L-proline L-isoleucine L-Leucine L-Lysine L-Lysine L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tryptophan L-Tyrosine * 2Na * 2H2O L-Valine

M AN U

2

EP

TE D

0

AC C

g/L

0.08

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

RI PT

Alpha-MEM TCM-199 McCoy's

SC

0.14

ACCEPTED MANUSCRIPT Figure 6

Vitamin B12 Thiamine *HCL

Alpha-MEM TCM-199 McCoy's

RI PT

p-Amino Benzoic Acid Nicotinic Acid Niacinamide

SC

myo-Inositol Menadione sodium bisulfite Folic Acid Choline Chloride Calciferol

0.000

M AN U

D-Biotin L-Ascorbic Acid * Na 0.002

0.004

0.006

0.008

EP

TE D

g/L

AC C

Vitamins

Riboflavin Pyridoxine *HCL Pyridoxal * HCL D-Panthothenic Acid

0.010

0,020

0,050

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Highlights • Bovine preantral follicles were cultured in vitro in α-MEM+, TCM-199+ and McCoys+. • We Analyzed the different components of α-MEM+, TCM-199+ and McCoys+. • We evaluated the morphological, morphometric, viability and ultrastructure parameters. • The α-MEM+ preserved the morphology, morphometric and ultrastructure of preantral follicles. • The α-MEM+ maintained the viability and promoted the growth after cultivation in situ.