Theriogenology 207 (2023) 110e122

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Theriogenology
journal homepage: www.theriojournal.com

Impact of antioxidant supplementation during in vitro culture of

ovarian preantral follicles: A review

ssio de Sousa Barberino,
Regina Lucia dos Santos Silva, Rica
*
Maria Helena Tavares de Matos
~o Francisco Valley, 56300-900, Petrolina, PE, Brazil
Nucleus of Biotechnology Applied to Ovarian Follicle Development, Federal University of Sa

a r t i c l e i n f o a b s t r a c t

Article history: The in vitro culture systems of ovarian preantral follicles have been developed for studying follicular and
Received 15 February 2023 oocyte growth, for future use of immature oocytes as sources of fertilizable oocytes and for screening
Received in revised form ovarian toxic substances. One of the key limitations of the in vitro culture of preantral follicles is the
10 May 2023
oxidative stress by accumulation of reactive oxygen species (ROS), which can impair follicular devel-
Accepted 27 May 2023
Available online 27 May 2023
opment and oocyte quality. Several factors are associated with oxidative stress in vitro, which implies the
need for a rigorous control of the conditions as well as addition of antioxidant agents to the culture
medium. Antioxidant supplementation can minimize or eliminate the damage caused by ROS, supporting
Keywords:
Antioxidants
follicular survival and development and producing mature oocytes competent for fertilization. This re-
Follicular growth view focuses on the use of antioxidants and their role in preventing follicular damage caused by oxidative
Oocyte stress in the in vitro culture of preantral follicles.
Oxidative stress © 2023 Elsevier Inc. All rights reserved.
Reactive oxygen species

1. Introduction
The in vitro follicle culture technology represents a valuable tool
to preserve the fertility in individuals subjected to cancer and
infertility treatment, to create gamete banks from endangered
species and breeds, to complement other reproductive technolo-
gies (e.g., in vitro embryo production), and to be used as models for
studies in reproductive toxicology [1,2]. The types of in vitro culture
systems available for preantral follicles include the culture of whole
ovary [3,4], ovarian cortical slices [5,6], isolated follicles [7,8] and a
“two-step” culture method, which associates the culture of frag-
ments of ovarian tissue or the ovary itself, followed by the isolation
of secondary follicles and the culture of these separately [9,10].
Studies on in vitro culture of preantral follicles in human, mice
and domestic animals are frequently performed to optimize the use
of immature gametes as sources of fertilizable oocytes [11e13]. In
addition, the follicle growth pattern in vitro can be used as a simple
and rapid method for screening toxic compounds [12,14,15].
* Corresponding author. Universidade Federal do Vale do Sa ~o Francisco (UNI-
VASF), Colegiado de Medicina Veterina
ria - Laborato
rio de Biologia Celular, Cit-
~o Nilo
ologia e Histologia. Rodovia BR 407, Km 12, Lote 543 - Projeto de Irrigaça
Coelho - S/N, C1. CEP, 56300-900, Petrolina, PE, Brazil.
E-mail address: helena.matos@univasf.edu.br (M.H. Tavares de Matos).
Despite the positive results obtained from preantral follicle culture,
such as production of oocytes in metaphase II (human [12]), em-
bryos (porcine: [16]; buffalo: [17]; sheep: [18]; goat [19,20]) and
live offspring (mouse [21]), one of the factors associated with low
quality oocyte is the generation of oxidative stress by the over-
production of reactive oxygen species (ROS) under in vitro condi-
tions [22]. ROS can be generated endogenously by enzymes of the
mitochondrial respiratory chain, nicotinamide adenine dinucleo-
tide phosphate oxidase (NADPH oxidase) or by xanthine oxidase, or
exogenously, including exposure to ultraviolet radiation, X and
gamma-rays, chemotherapy or in vitro environmental conditions
[22,23]. ROS is a collective term that defines a wide variety of
oxidant molecules with different biological properties and func-
tions, ranging from signaling to cell damage [24].
In the ovary, oxidative damage due to the excessive production
of ROS can affect steroidogenesis, oocyte quality and maturation,
ovulation, and luteolysis, and eventually leads to ovarian aging and
infertility [25]. In this sense, in addition to rigorous control of
culture conditions, there is still a need to use exogenous antioxi-
dants to facilitate the elimination of ROS, preventing the develop-
ment of oxidative stress and maintaining homeostatic balance in
each step of follicular development during in vitro culture of pre-
antral follicles in human, mice and domestic animal species
[8,25,26]. Overall, studies have found antioxidant supplementation
to be beneficial to prevent oxidative damage, mitochondrial

https://doi.org/10.1016/j.theriogenology.2023.05.027
0093-691X/© 2023 Elsevier Inc. All rights reserved.
R. Lucia dos Santos Silva, R. de Sousa Barberino and M.H. Tavares de Matos
dysfunction or support proper balance between oxidants and an-
tioxidants by influencing, for example, intracellular glutathione
(GSH) production, reducing ROS levels and increasing the cell's
total antioxidant capacity [11,27e30] This mechanism protects
follicles from toxic injury due to oxidative stress, maintains survival
and allows activation and growth of the ovarian follicles.
The present review summarizes the effects of oxidative stress on
ovarian follicle development and oocyte quality in vitro and the
importance of using antioxidant supplementation to improve the
in vitro culture of preantral follicles.
2. Oxidative stress during in vitro culture of preantral follicles
Oxidative stress is triggered by ROS accumulation in cells to
levels that exceed cellular antioxidant and repair capacity, directly
causing oxidative damage to all biomolecules in the cellular envi-
ronment, including proteins, lipids and DNA [25]. ROS include both
free radical and non-free radical oxygen-derived reactive molecules
[26], which appear as by-products of oxygen consumption and
cellular metabolism [31]. Physiologically, cells activate non-
enzymatic antioxidant mechanisms, such as GSH, selenium,
ascorbic acid and a-tocopherol, and/or enzymatic, such as super-
oxide dismutase (SOD), catalase (CAT), glutathione peroxidase
(GPx), and glutathione reductase (GR), to eliminate or suppress the
formation of oxidants and help cells repair damage caused by ROS
[32,33]. However, during in vitro follicular culture, handling and
lack of antioxidant defense mechanisms generate large amounts of
cellular oxygen and consequently increase ROS production [34].
In in vitro culture systems, follicles are maintained in a different
environment than in vivo and require adequate conditions to sur-
vive and develop [8]. Several factors are associated with oxidative
stress in vitro, such as exposure to ischemic conditions after col-
lecting ovaries, thus decreasing the ability of oocytes and somatic
cells to neutralize ROS, which are amongst the most prominent
physiological inducers of cell damage and apoptosis [35e37].
Furthermore, excessive exposure to light and manipulation before
and during in vitro culture are also factors that can stimulate DNA
damage and increase ROS production, impairing viability and the
development of oocytes and embryos [38e42]. Temperature vari-
ations, caused by rapid temperature drop in the culture plates
during oocyte preparation under the laminar flow hood [43] and
during pipetting [44], can make pH conditions unstable and harm
the oocytes, whereas pH variations can stimulate oxidative stress
[45]. In addition, considering that follicular fluid hinders O2 diffu-
sion, exposure to uncontrolled concentrations of O2 may limit
oxidative phosphorylation by reducing mitochondrial function and
ATP production, increasing production of toxic metabolites and ROS
in oocytes [46,47]. Variations in the culture medium such as high
glucose concentrations can increase intraoocyte ROS levels, reduce
GSH, and impair oocyte developmental competence, likely through
the promotion of glycolysis and oxidative phosphorylation [48].
The hyperosmolarity of the medium is also pointed out as a factor
inducing oxidative stress in the in vitro culture of oocytes and
embryos [36,49], possibly by stimulating the generation of ROS and
suppressing antioxidant enzymes such as SOD and GPX [50]. In
addition, considering that in vitro conditions, follicles are exposed
to higher levels of oxygen and lack of physiological defense
mechanisms against ROS [51], the absence of an efficient combi-
nation of antioxidants in the culture medium can result in oxidative
stress and compromise follicular development [52]. The increase in
ROS levels during in vitro culture is associated with a decrease in
follicular viability [53] and is recognized as a marker of follicle
health and efficiency of the culture [54,55] in various species (rats:
[56]; bovine: [57]; goats: [58]; equine: [59]; sheep [34]).
In this way, strategies have been developed to optimize and
111
Theriogenology 207 (2023) 110e122
improve the in vitro culture of preantral follicles, such as the sup-
plementation of culture media with antioxidant agents to prevent
or minimize cellular damages.
3. Supplementation of the culture medium with antioxidants
Antioxidants are molecules that help maintain the cell oxidant/
antioxidant balance by preventing or reducing excessive produc-
tion of ROS. Studies suggest that antioxidant supplementation of
the culture media can improve oocyte quality and the development
and survival of preantral follicles in vitro. Table 1 summarizes the
main effects of antioxidant agents during in vitro culture of pre-
antral follicles.
3.1. Alpha-lipoic acid
Alpha-lipoic acid (ALA; 5-[(3 R)-dithiolan-3-yl]pentanoic acid)
is a caprylic acid-derived antioxidant found in vegetables (e.g.
spinach, tomato, broccoli), and synthesized in animal tissues with
high metabolic activity, such as kidney and liver [60]. ALA is a
cofactor that plays a critical role in mitochondrial energy meta-
bolism [61], and due to the presence of two thiol functional groups,
it is beneficial in several oxidative stress-associated conditions by
eliminating ROS [62].
During in vitro culture of bovine ovarian tissue for 12 days, the
addition of ALA (100 ng/ml) to the base medium improved survival
and activation of primordial follicles compared to the control me-
dium [57]. In contrast, although ALA (50 and 100 mM) maintained
the granulosa cell proliferation similar to the base medium after six
days of culture of equine ovarian tissue, the follicular survival was
similar to the non-cultured tissues only until the second day of
in vitro culture [11]. These different results suggest that ALA con-
centrations that are required for maintaining follicular integrity
during ovarian tissue culture need to be defined according to the
days of culture and the animal species.
Supplementation with ALA during culture of bovine isolated
secondary follicles significantly increased follicular survival and
diameter compared to the control medium. These results can be
related to the increase in the transcription levels of genes and re-
ceptors of hormones and growth-related factors, such as insulin-
like growth factor 1 (IGF-1), growth differentiation factor-9 (GDF-
9), transforming growth factor beta (TGFb1), activin, bone
morphogenetic protein receptor type 1A (BMPR1a), transforming
growth factor beta receptor 1 (TGFbR1), FSH and activin receptor
type-2B (ActRIIB), and decrease in the transcription and protein
levels of Bax and c-Myc, pro-apototic molecules, in the follicles
cultured in medium containing ALA [63]. During in vitro culture of
mouse isolated follicles from fresh or vitrified ovaries, ALA
increased the follicular survival, antrum formation, the rate of oo-
cytes in metaphase II (MII), oocyte fertilization rate and embryonic
development [51,64] by promoting an increase in total antioxidant
capacity and a decrease in ROS levels in comparison to control
group [51].
3.2. Alpha-tocopherol
Alpha-tocopherol [vitamin E; (2S)-2,5,7,8-tetramethyl-2-
[(4S,8S)-4,8,12-trimethyltridecyl]-3,4-dihydrochromen-6-ol] is a
fat-soluble antioxidant that protect cellular membranes and lipo-
proteins from peroxidation [65]. Although the antioxidant action of
alpha tocopherol has been observed in the in vitro maturation of
oocytes (sheep: [66,67]; rabbit [68]), to our knowledge, only one
study has evaluated the effects of this vitamin during culture of
preantral follicles. After in vitro culture of goat ovarian tissue for 5
days, the highest concentration of a-tocopherol (15 mM) promoted
 Tabel 1

R. Lucia dos Santos Silva, R. de Sousa Barberino and M.H. Tavares de Matos
Main effects of antioxidant supplementation on in vitro culture medium of preantral follicles.

Antioxidant Concentration Species Culture system Control culture medium Culture Findings* Reference
period

Alpha-lipoic acid 100 ng/ml bovine ovarian tissue MEM supplemented with ITS, pyruvate, glutamine, 12 days [ survival and [ primordial follicle activation [57]
hypoxanthine, BSA, penicillin and streptomycin
50 and 100 mM equine ovarian tissue MEM supplemented with penicillin, streptomycin, BSA, 6 days Y follicular survival and maintenance of cell [11]
ITS, pyruvate, glutamine and hypoxantine proliferation
250 mM bovine isolated secondary TCM-199 supplemented with FSH and EGF, newborn 15 days [ follicular survival and growth; [ mRNA for IGF-1, [63]
sfollicles calf serum, sodium pyruvate, ITS, penicillin, GDF-9, TGFb1, BMPR1a, TGFbR1, FSH, ActRIIB; Y mRNA
streptomycin and sodium bicarbonate for Bax and C-Myc; [ protein levels of FSHR and IGF-1; ↓
protein levels of Bax
100 mM mice isolated secondary a-MEM supplemented with sodium pyruvate, rFSH, ITS, 12 days [ survival; [ antrum formation; [oocyte maturation; [ [64]
follicles r-EGF, FBS, sodium bicarbonate, penicillin and fertilization; [embryo development
streptomycin
100 mM mice isolated secondary a-MEM supplemented with sodium pyruvate, r-FSH, 12 days [ survival; [ antrum formation; [oocyte maturation; [ [51]
follicles ITS, penicillin, streptomycin, FBS and sodium fertilization; [embryo development; Y ROS; [ total
bicarbonate antioxidant capacity
Alpha-tocopherol 5, 10 or 15 mM caprine ovarian tissue MEM supplemented with penicillin, streptomycin, 5 days [ primordial follicle activation; Y follicular viability [69]
anphotericin B, BSA, ITS, pyruvate, glutamine and
hypoxanthine
Anethole 2000 mg/ml caprine isolated secondary MEM supplemented with BSA, ITS, glutamine, 18 days Maintenance of follicular survival and growth and [76]
follicles hypoxanthine and pyruvate mRNA for ICAM-1, CAV-1, PAI-1; Y TIMP-2; Y ROS;
[meiosis resumption
30 mg/ml caprine ovarian tissue MEM supplemented with BSA, ITS, glutamine, 7 days Maintenance of primordial follicle activation and [77]
hypoxanthine and pyruvate survival; [ follicular and oocyte growth; YROS; [ cell
proliferation
300 mg/ml caprine ovarian tissue a-MEM supplemented with BSA, ITS, glutamine and 7 days Maintenance of primordial follicle activation, levels of [30]
hypoxanthine H2O2 and thiol; mRNA for CAT, ERP29, KDM1AX1 and
112

KDM3A; [ follicular survival and growth

Ascorbic acid 50 mg/ml
1 bovine isolated secondary McCoy's 5a medium supplemented with bicarbonate, 12 days [ follicle survival and growth; [ MMP-9 expression [81]
follicles Hepes, BSA, l-glutamine, penicillin, streptomycin, ITS
and androstenedione
50 mg/ml caprine isolated secondary a-MEM supplemented with BSA, ITS, glutamine and 18 days [ follicular survival and growth; [antrum formation; [ [82]
follicles hypoxantine mRNA for MMP-9, TIMP-2
50 mg/ml bovine ovarian tissue MEM supplemented with ITS, pyruvate, glutamine, 8 days [ follicular survival; [ cell proliferation [83]
hypoxanthine, BSA, penicillin and streptomycin
50 mg/ml equine ovarian tissue MEM supplemented with penicillin, streptomycin, BSA, 6 days [ primordial follicle activation [84]
hypoxanthine, glutamine, pyruvate and ITS
50 mg/ml þ FSH caprine ovarian tissue MEM supplemented with ITS, pyruvate, glutamine, 14 days Maintenance of follicular survival and growth; [ [85]
hypoxanthine and BSA ultrastructural integrity of cells
b-Mercaptoethanol 10 mm buffalo isolated secondary MEM supplemented with steer se
rum, glutamine, 60 days [ follicular survival and growth; [antrum formation; [92]
follicles sodium pyruvate, hypoxanthine, FSH-P and gentamicin [oocyte growth
Coenzyme Q10 50 mM mice isolated secondary a-MEM supplemented with FBS, rhFSH, ITS, EGF, 4 days [ follicular survival and growth; [ antrum formation; [ [95]
follicles penicillin, streptomycin, sodium bicarbonate ovulation rate and oocytes and MII oocytes
Curcumin 10 and 25 mM sheep isolated secondary MEM supplemented with BSA, glutamine, sodium 4 days [ follicular survival and growth [101]
follicles pyruvate, hypoxanthine, ITS, gentamicin and FSH-P
Epigallocatechin-3- 1 mg/ml sheep ovarian tissue a-MEM supplemented with ITS, glutamine, 4 days [ follicular survival; [ follicle and oocyte growth; Y [5]

Theriogenology 207 (2023) 110e122

gallate hypoxanthine, ascorbic acidand BSA apoptosis; [ p-Akt immunoexpression
Eugenol 5 mM bovine isolated secondary TCM-199 supplemented with BSA, penicillin, 18 days Maintenance of follicular viability and growth, antrum [116]
follicles streptomycin, ITS, ascorbic acid, FSH, glutamine, formation, mRNA for SOD, CAT and PRDX6; [ mRNA for
hypoxanthine GPX1
40 mM/ml caprine ovarian tissue a-MEM supplemented with BSA, ITS, glutamine and 7 days [ follicle survival and activation; [ follicle and oocyte [30]
hypoxanthine growth; [ total antioxidant activity; [ mRNA for ERP29
and calreticulin
Gallic acid 100 mM replacing sheep isolated secondary a-MEM supplemented with BSA, glutamine, 12 days Maintenance of morphology and fully grown oocytes; [ [13]
transferrin, selenium follicles hypoxanthine, ITS and ascorbic acid follicular diameter and antrum formation; [ GSH levels;
and ascorbic acid [ mitochondrial activity; [ meiosis resumption
Hesperidin 50 mmol/L mice 12 days [29]
 R. Lucia dos Santos Silva, R. de Sousa Barberino and M.H. Tavares de Matos
isolated secondary a -MEM medium supplemented with ITS, r- FSH, [ follicular survival and growth; [ MII oocytes and
follicles (3D) penicillin, streptomycin and FBS fertilization; [embryo development; [ mRNA for Bcl-2,
[ cell proliferation, [ FSH-R; Y mRNA for Bax
Kaempferol 1 mM kaempferol sheep isolated secondary a -MEM supplemented with BSA, glutamine, 12 days Maintenance of follicular growth, fully grown oocytes [28]
replacing transferrin, follicles hypoxanthine, ITS and ascorbic acid and GSH; [ follicular survival; [antrum formation; [
selenium and ascorbic follicular growth, [ mitochondrial activity
acid
10 mM sheep ovarian tissue a-MEM supplemented with ITS, glutamine, 7 days Maintenance of follicular survival and growth; [ [131]
hypoxanthine and BSA primordial follicle activation; Y DNA fragmentation; [
cell proliferation
Melatonin 1000 pM caprine ovarian tissue MEM supplemented with ITS, pyruvate, glutamine, 7 days Maintenance of survival, growth and primordial follicle [138]
hypoxanthine and BSA activation
100 pg/ml sheep ovarian tissue a-MEM supplemented with ITS, glutamine, 7 days [ primordial follicle activation and survival; Y [139]
hypoxanthine, ascorbic acid and BSA apoptosis; [ p-Akt and p-FOXO3a immunoexpression;
1000 and 2000 pM bovine ovarian tissue a-MEM supplemented with ITS,glutamine, 6 days Maintenance of follicular survival; [ primordial follicle [140]
hypoxanthine, penicillin, streptomycin) and BSA activation;
100 pg/ml from day 0 to caprine isolated secondary a-MEM supplemented with BSA, ITS, glutamine, 12 days Maintenance of antrum formation and follicular [141]
day 6 and, 1000 pg/ml follicles hypoxanthine and ascorbic acid growth; [ follicular survival; [ fully grown oocyte
from day 6 to day 12
1000 pg/ml sheep isolated secondary a-MEM supplemented with BSA, ITS, glutamine, 18 days [ follicular survival; [ antrum formation and growth; [ [142]
follicles hypoxanthine and ascorbic acid fully grown oocytes; [ mitochondrial activity and Y ROS
10 pg/ml mice isolated secondary a-MEM supplemented with FBS, ITS, rFSH, penicillin 7 days [ follicular survival and growth [143]
follicles and streptomycin
10 pg/ml mice Isolated secondary a-MEM supplemented with FBS, rFSH, ITS, mrEGF, 10 days [ follicular survival; Y p53 expression [27]
follicles penicillin, streptomycin
10
7 M bovine isolated secondary TCM-199 supplemented with ITS, BSA, glutamine, 18 days [ antrum formation and follicular growth; maintenance [144]
follicles hypoxanthine, ascorbic acid and FSH of mRNA for CAT, SOD, PRDX-6
N-acetyl-cysteine 1 mM bovine isolated secondary TCM-199 supplemented with FSH, ITS, BSA, glutamine, 18 days Maintenance of follicular ultrastructure, antrum [147]
follicles hypoxanthine, penicillin, streptomycin and HEPES formation; [ follicular growth; [ follicular viability
25 mmol/L human ovarian tissue a -MEM supplemented with ITS, r-FSH and human 32 weeks Maintenance of ultrastructural integrity, [ GDF-9, Y [148]
113

serum sBcl-2
Protocatechuic acid 56.25 mg/ml replacing sheep isolated secondary a-MEM supplemented with BSA, ITS, ascorbic acid, 12 days [ follicular survival; maintenance of antrum formation, [151]
transferrin, selenium follicles glutamine and hypoxanthine fully grown oocyte, GSH levels and mitochondrial
and ascorbic acid activity
Quercetin 25 mg/mL bovine ovarian tissue MEM supplemented with ITS, pyruvate, glutamine, 18 days [ growing follicles; [ total antioxidant capacity [160]
hypoxanthine, BSA, penicillin, streptomycin
Resveratrol 2 mM sheep isolated secondary a-MEM supplemented with BSA, ITS, glutamine, 18 days Maintenance of antrum formation, DNA fragmentation, [165]
follicles hypoxanthine, ascorbic acid GSH levels and mitochondrial activity
2 mM sheep ovarian tissue a-MEM supplemented with ITS, glutamine, 7 days [ primordial follicle activation; Y DNA fragmentation; [ [166]
hypoxantine, ascorbic acid and BSA cell proliferation
Rutin 1 mg/ml sheep ovarian tissue a-MEM supplemented with BSA, ITS, glutamine, 7 days [ follicular survival; [ follicle and oocyte growth; [ [169]
hypoxanthine and ascorbic acid primordial follicle activation; Y apoptosis; [ p-Akt
immunoexpression
0,1 mm/ml rutin sheep isolated secondary a-MEM supplemented with BSA, glutamine, ITS and 12 days [ follicular survival and growth; [ fully-grown oocytes; [34]
replacing transferrin, follicles ascorbic acid [ GSH levels
selenium and ascorbic
acid
Sodium selenite 10 ng/ml mice isolated secondary TCM-199 supplemented with sodium pyruvate, r- FSH, 12 days [ follicular survival and growth; [ MII oocytes [171]
follicles ITS, penicillin and streptomycin

Theriogenology 207 (2023) 110e122

10 ng/ml mice isolated secondary TCM-199 supplemented with sodium pyruvate, r- FSH, 14 days [ follicular survival and growth; [ antrum formation; [ [172]
follicles ITS, penicillin and streptomycin MII oocytes; Y ROS; [ activity of Se-GPx; [ total
antioxidant capacity

*Compared with Control culture medium; Activin, bone morphogenetic protein receptor type 1A (BMPR1a); activin receptor type-2B (ActRIIB);bovine serum albumin (BSA); catalase (CAT); endoplasmic reticulum protein 29
(ERP29); Epidermal growth fator (EGF); fetal bovine serum (FBS); human follicle stimulating hormone (FSH); follicle stimulating hormone Receptor (R-FSH); follicle stimulating hormone pituitary (FSH-P); glutathione (GSH);
glutathione peroxidase 1 (GPX1); growth differentiation factor-9 (GDF-9); insulin-like growth factor 1 (IGF-1); insulin, transferrin, selenium (ITS); intercellular adhesion molecule 1 (ICAM-1); in vitro maturation (IVM)metaphase
II (MII); metalloproteinase
9 (MMP-9); Minimum essential medium eagle (MEM); Minimum essential medium eagle - Alpha Modification (a-MEM); peroxiredoxin-6 (PRDX-6); phosphorylated Akt (p-Akt); phosphorylated
forkhead box class O 3a (p-FOXO3a); plasminogen activator inhibitor-1 (PAI-1); reactive oxygen species (ROS); recombinant human follicle stimulating hormone (rhFSH); superoxide dismutase (SOD); tissue inhibitor of
metalloproteinases-2 (TIMP-2); Tissue culture medium-199 (TCM-199); transforming growth factor beta receptor 1 (TGFbR1).
R. Lucia dos Santos Silva, R. de Sousa Barberino and M.H. Tavares de Matos
primordial follicle activation, however, reduced follicular viability
compared to the control medium [69]. One possible explanation is
that a-tocopherol may act as a pro-oxidant at high concentrations
[70].
3.3. Anethole
Anethole (1-methoxy-4-[(E)-prop-1-enyl]benzene) is a com-
pound abundantly found in essential oils of various plants [71].
In vivo and in vitro studies confirmed that the antioxidant activity of
anethole is due to its ability to decrease ROS concentrations
[72e74]. In the female reproduction, anethole increased the rates of
bovine blastocyst formation when used as a supplement in the
in vitro maturation and in vitro embryo production media [74,75].
During in vitro culture of goat secondary follicles, anethole
(2000 mg/ml) maintained survival, promoted follicular growth, and
expression of genes associated with follicular development [inter-
cellular adhesion molecule 1 (ICAM-1)], and remodeling of base-
ment membrane (caveolin-1 (CAV-1) and plasminogen activator
inhibitor-1 (PAI-1), reduced tissue inhibitor of
metalloproteinases-2 (TIMP-2)] expression gene and ROS levels
compared to the anethole-free medium and increased the rate of
oocytes that resumed meiosis [76]. Furthermore, supplementation
of the culture medium of goat ovarian tissue with anethole (30 mg/
ml) maintained follicle survival, induced activation of primordial
follicle, increased follicular and oocyte growth and cell prolifera-
tion, and reduced ROS levels when compared to the anethole-free
medium [77]. In addition, although supplementation of the cul-
ture medium of goat ovarian tissue with 300 mg/ml anethole
improved follicle survival and follicular and oocyte growth, it
maintained markers of oxidative activity (levels of hydrogen
peroxide, thiol, and CAT) similar to the observed in the control
medium [30]. Despite the promising results of anethole during
in vitro culture of goat follicles, there is a lack of research focused on
its use as a supplementation of culture medium of preantral folli-
cles in other species.
3.4. Ascorbic acid
Ascorbic acid [(2R)-2-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxy-
2H-furan-5-one] is also known as vitamin C and is widely found in
citrus fruits, strawberries, tomatoes, broccoli, green peppers, red
peppers and leafy vegetables; small amounts have also been found
in fish and milk [78,79]. The ascorbic acid is a powerful antioxidant
and free radical scavenger that protects tissues, cell membranes,
and DNA from oxidative damage [80].
The addition of ascorbic acid in the culture medium of goat and
bovine isolated preantral follicles increased follicular survival,
antrum formation, growth and the expression of matrix
metalloproteinases-9 (MMP-9) [81,82] and its inhibitor TIMP-2, en-
zymes associated with the turnover of collagen on the basement
membrane during follicle growth, reducing oocyte extrusion [82]
when compared to the control medium without this antioxidant.
During in vitro culture of preantral follicles enclosed in bovine
ovarian tissue, ascorbic acid supplementation maintained normal
oocyte ultrastructure with uniform distribution of organelles and
increased cell proliferation [83]. Supplementation of the culture
medium of equine ovarian tissue with ascorbic acid increased pri-
mordial follicle activation [84]. In goat ovarian tissue culture,
ascorbic acid supplementation alone or combined with FSH
maintained follicle survival and growth similar to ascorbic acid-free
medium after in vitro culture for 7 days. However, after long-term
in vitro culture (14 days), maintenance of the ultrastructural
integrity of cells was observed only in follicles cultured with
ascorbic acid associated with FSH [85]. Although ascorbic acid is a
114
Theriogenology 207 (2023) 110e122
recognized antioxidant, one of the limitations of these studies is
that they did not analyze specific markers of oxidative stress after
culture, which would be important to evaluate oocyte quality.
Supplementation of in vitro culture of preantral follicles with
ascorbic acid is often used in association with other antioxidants
such as transferrin, a metal-binding protein, and selenium (sheep:
[86]; dog: [87]; caprine: [20]; mice [88]). In this association,
transferrin and ascorbic acid prevent the formation of the hydroxyl
radical and selenium acts as an essential component of seleno-
proteins such as glutathione peroxidase (GSH-Px), responsible for
the decomposition of peroxides in non-toxic and non-radical
products (Se-GSH-Px) [34,89].
3.5. b-Mercaptoethanol
Beta mercaptoethanol (2-sulfanylethanol) is a low molecular
weight thiol that contributes to intracellular GSH synthesis and acts
as an antioxidant and antiapoptotic factor in ovarian folliculo-
genesis [90,91]. Supplementation of in vitro culture medium of
buffalo isolated secondary follicles with b-mercaptoethanol
significantly increased follicular survival, antrum formation rate
and oocyte growth when compared to the control group [92].
However, no marker of oxidative stress was analyzed in this study.
3.6. Coenzyme Q10
Coenzyme Q10 (CoQ10) is an oil-soluble component of almost
all cell membranes and functions as a diffusible electron carrier in
the mitochondrial respiratory chain. CoQ10 supplementation has a
significant protective effect on many tissues, including reproduc-
tive organs [93] and in the in vitro maturation of oocytes, reducing
the number of oocytes with aberrant spindles (human [94]), ROS
levels and increased the percentage of fertilized oocytes (mice
[95]). Although the literature still lacks studies evaluating its
effectiveness in preantral follicle culture, specially its antioxidant
action, CoQ10 supplementation in the culture medium of mouse
isolated secondary follicles increased the survival rates, follicular
growth, antrum formation, ovulation rate and MII oocytes when
compared to follicles cultured in medium without CoQ10 [96].
These results may be associated with the influence of higher levels
of CoQ10 on improving mitochondrial function and ROS neutrali-
zation, thus improving oocyte quality [97].
3.7. Curcumin
Curcumin [(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-
1,6-diene-3,5-dione] is a polyphenolic compound with recognized
anti-inflammatory, anti-apoptotic and antioxidant properties [98].
Curcumin can minimize the effects of oxidative stress by chelating
heavy metals or by regulating the activity of antioxidant enzymes
[98,99]. In vitro supplementation with curcumin has already been
tested in the culture of granulosa cells (human [100]), preantral
follicles (sheep [101]), as well as in oocyte maturation (mouse:
[102]; swine [103]) and embryo culture (buffalo [104]).
In the in vitro culture of isolated ovine secondary follicles, sup-
plementation with 10 and 25 mM of curcumin significantly
increased the survival rate and follicular growth compared to the
control medium. These results were associated with the antioxi-
dant capacity of curcumin and its phytoestrogenic effect [101].
Furthermore, the authors observed that supplementation with low
(1 mM and 5 mM) and high (100 mM) concentrations of curcumin had
an inhibitory effect on follicular survival and growth. These results
are directly related to the concentration of curcumin which can,
instead of inhibiting oxidative effects, stimulate the production of
ROS [103,105].
R. Lucia dos Santos Silva, R. de Sousa Barberino and M.H. Tavares de Matos
3.8. Epigallocatechin-3-gallate
Epigallocatechin-3-gallate (EGCG; [(2R,3R)-5,7-dihydroxy-2-
(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-chromen-3-yl] 3,4,5-
trihydroxybenzoate) is an abundant polyphenolic component
from green tea extract [106]. The EGCG possesses various biological
functions, including antitumor and antioxidant functions [107]. In
female reproduction, supplementation of IVM and/or in vitro
fertilization (IVF) media with EGCG improves the percentage of
fertilized oocytes (porcine [108]) and reduced oxidative and
endoplasmic reticulum stress in oocytes, thereby mitigating cyto-
plasmic abnormalities (mice [109]). However, supplementation of
culture medium of preantral follicles with EGCG is little described
in the literature. After in vitro culture of sheep ovarian tissue, EGCG
reduced follicular cell apoptosis and increased follicle growth
compared to the follicles in ovarian tissue cultured in the control
medium, keeping these parameters similar to those observed in
non-cultured tissues [5]. Although no oxidative marker has been
evaluated in the referred study, the authors suggested that modu-
lation of phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt)
signaling pathway, which appears to be the main pathway that
regulates the growth and survival of primordial follicles [110], is
important for the maintenance of in vitro follicular survival by
EGCG.
3.9. Eugenol
Eugenol (2-methoxy-4-prop-2-enylphenol), commonly ob-
tained from natural essential oils in several plants [111,112], is well
described for its antioxidant ability to scavenge free radicals, to
inhibit the generation of ROS, to prevent the production of reactive
forms of nitrogen and increase the cellular antioxidant potential
[112]. The antioxidant capacity of eugenol has been studied with
the aim of providing protection to the reproductive organs of fe-
males in vivo [113,114] and in vitro [115].
The addition of eugenol in the culture medium of isolated
bovine secondary follicles maintained the follicular viability and
growth, and expression levels of SOD, CAT and peroxiredoxin 6
(PRDX6) enzymes compared to the control medium without
eugenol, and significantly increased the expression of GPX1 [116].
Supplementation of culture medium of goat ovarian tissue with
eugenol increased primordial follicle activation and antioxidant
capacity and maintained follicular survival similar to uncultured
tissue [30]. In addition, mRNA relative expression of ERP29, a
metabolic stress-inducible endoplasmatic reticulum protein [117],
and the expression of the protein calreticulin, responsible for
regulating the proteins in the endoplasmic reticulum [118],
increased in the tissue cultured in the presence of eugenol [116],
indicating that this antioxidant protected ovarian tissue by inhib-
iting oxidative stress in the endoplasmic reticulum [30].
3.10. Gallic acid
Gallic acid (3,4,5-trihydroxybenzoic acid) is a phenolic com-
pound naturally found in various plants, vegetables, nuts and fruits
[119]. The main therapeutic effects of gallic acid are derived from
antioxidant properties, which are exerted by removing ROS or
inactivating enzymes responsible for ROS production, and by
increasing the activity of antioxidant enzymes, such as GSH
[120,121]. In vitro preservation of ovarian tissue stored in extract of
Amburana cearensis has already been associated with the presence
of gallic acid in its composition [122]. However, only one study has
described the use of gallic acid in the in vitro culture of preantral
follicles.
During culture of sheep secondary follicles, supplementation of
115
Theriogenology 207 (2023) 110e122
the medium with gallic acid in replacement of three antioxidants
(ascorbic acid, transferrin and selenium) maintained the follicular
morphology similar to the control medium (supplemented with
ascorbic acid, transferrin and selenium) and increased follicular
growth. This study showed that gallic acid protects oocytes against
oxidative stress through its ability to increase GSH and mitochon-
drial activity and consequently inhibiting cell apoptosis and
improving follicular development in vitro. Furthermore, using a
PI3K pathway inhibitor (LY294002), the authors demonstrated the
possible involvement of the PI3K pathway in follicle and oocyte
development under the influence of gallic acid [13].
3.11. Hesperidin
The flavanone glycoside hesperidin [(2S)-5-hydroxy-2-(3-
hydroxy-4-methoxyphenyl)-7-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-
6-[[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy-
methyl]oxan-2-yl]oxy-2,3-dihydrochromen-4-one] is a bioflavo-
noid that is predominantly found in citrus fruits [123]. Due to its
strong cellular antioxidant protection, in vivo administration of
hesperidin has already been shown to improve reproductive per-
formance in rodent models [124e126]. Hesperidin also protected
porcine oocytes during IVM by reducing ROS levels and conse-
quently minimizing oocyte deterioration during in vitro aging [127].
During in vitro culture of mouse preantral follicles, hesperidin
significantly increased survival, follicular diameter and antrum
formation, due to the upregulation of Bcl-2, r-FSH and PCNA genes.
In addition, it inhibited the expression of the pro-apototic Bax gene,
which allowed complete follicular development followed by oocyte
maturation and embryonic development of these oocytes after
fertilization [29].
3.12. Kaempferol
Kaempferol (3,5,7-trihydroxy-2-(4-hydroxyphenyl)chromen-4-
one) is a yellow-colored dietary flavonoid, present in numerous
fruits and vegetables [128,129]. During in vitro embryo culture,
kaempferol reduced ROS production and increased the expression
of anti-apoptotic gene Bcl-2 [130].
Supplementation of in vitro culture medium of sheep secondary
follicles with kaempferol increased follicular survival, antrum for-
mation, and levels of active mitochondria and maintained follicular
growth, fully grown oocytes and GSH concentration similar
compared to follicles cultured in medium with the addition of se-
lenium, transferrin and ascorbic acid [28]. During sheep ovarian
tissue culture, kaempferol maintained follicular survival and
growth, increased primordial follicle activation and the cellular
proliferation, and reduced DNA fragmentation [131]. The authors of
these studies suggest that survival and follicular development
promoted by kaempferol supplementation are related to its phy-
toestrogenic activity and the control of cellular redox balance due
to increased mitochondrial activity.
3.13. Melatonin
Melatonin (N-[2-(5-methoxy-1H-indol-3-yl)ethyl]acetamide) is
a neurohormone secreted by the pineal gland of vertebrates and
partially by other peripheral organs such as intestine, retina,
immunocompetent cells and gonads [132]. In ovarian tissue,
melatonin acts by multiple mechanisms, such as maintenance of
telomeres, stimulation of SIRT expression and antioxidant activity
[133]. The antioxidant action of melatonin is due to its ability to
directly eliminate toxic oxygen derivatives and to reduce ROS,
protecting the DNA of granulosa cells and oocytes (bovine: [134];
mouse [135,136]). In addition, supplementation of sheep ovarian
R. Lucia dos Santos Silva, R. de Sousa Barberino and M.H. Tavares de Matos Theriogenology 207 (2023) 110e122

Fig. 1. Antioxidants for the development of preantral follicles. The use of antioxidants allows the redox balance in follicles cultured in vitro by increasing the expression of anti-
oxidant genes, mitocondrial activity, glutathione (GSH) levels and total antioxidant capacity. With antioxidant protection, follicular cells reduce the expression of pro-apoptotic
factors (Bax, c-Myc and p53) and increase the production of growth factors [insulin-like growth factor 1 (IGF-1), growth differentiation factor-9 (GDF-9), transforming growth
factor beta receptor 1 (TGFbR1)], hormones [follicle stimulating hormone (FSH)] and proteins (tissue inhibitor of metalloproteinases-2 (TIMP-2), activin, bone morphogenetic
protein receptor type 1A (BMPR1a); activin receptor type-2B (ActRIIB), phosphorylated Akt (p-Akt), metalloproteinase
9 (MMP-9), phosphorylated forkhead box class O 3a (p-
FOXO3a)] involved in follicular development.

tissue transport medium with melatonin improved oocyte
competence for development to the blastocyst stage [137].
During culture of goat ovarian tissue, melatonin supplementa-
tion maintained follicular activation, survival and follicular growth
similar to that observed in tissues cultured without melatonin
[138]. However, supplementation of the medium of sheep ovarian
tissue culture with melatonin increased activation of primordial
follicles and follicular survival compared to control medium. These
results were attributed to lower immunoexpression of cleaved
caspase-3 in follicular cells and regulation of the PI3K pathway
[139]. Furthermore, melatonin supplementation increased the
activation of bovine primordial follicles, but follicular survival
remained similar to the control group [140]. The difference be-
tween the findings of these studies may be related to intrinsic
differences in the species. In addition, in these studies, techniques
to measure the antioxidant action of melatonin in vitro culture of
ovarian tissue were not used or mentioned.
In the in vitro culture of goat secondary follicles, the use of
medium supplemented sequentially with melatonin, i. e, 100 pg/ml
melatonin from day 0 to day 6 and 1000 pg/ml melatonin from day
6 to day 12, increased follicular survival and the percentage of fully
grown oocytes compared to melatonin-free medium [141]. How-
ever, supplementation of the culture medium of sheep secondary
116
follicles with 1000 pg/ml melatonin throughout 18 days increased
survival, follicular and oocyte growth, antrum formation, and the
percentage of fully grown oocytes. Furthermore, there was a sig-
nificant increase in the levels of active mitochondria and a reduc-
tion in ROS concentrations in oocytes after IVM [142]. In the culture
medium of preantral follicles from mice vitrified ovaries, melatonin
supplementation increased follicular survival and follicular growth
[143] and decreased the expression of the apoptotic gene p53
compared to groups cultured without melatonin [27]. Bovine sec-
ondary follicles cultured with melatonin had a higher rate of
antrum formation and follicular growth, and maintained mRNA
levels of CAT, SOD, and PRDX-6 [144].
3.14. N-acetyl-cysteine
N-acetyl-cysteine (NAC; (2R)-2-acetamido-3-sulfanylpropanoic
acid) is a potent antioxidant that can be used to optimize in vitro
conditions during follicle growth, oocyte maturation and cryo-
preservation [145]. NAC acts directly in the donation of electrons to
reactive species, and indirectly as a glutathione precursor that in-
tegrates the intracellular antioxidant system [146].
During in vitro culture of isolated bovine secondary follicles,
supplementation with NAC increased follicular growth and the
R. Lucia dos Santos Silva, R. de Sousa Barberino and M.H. Tavares de Matos
percentage of viable follicles compared to the control medium
[147]. The authors suggested that a possible combination of NAC
with other antioxidants with different mechanism of action could
improve ultrastructure of follicular cells, reducing vacuoles in the
ooplasm and signs of degeneration in the mitochondria and
endoplasmic reticulum, compared to control medium.
Supplementation of long-term in vitro culture (32 weeks) of
human ovarian tissue with NAC maintained follicular cell integrity,
increased GDF-9 expression and reduced Bcl-2 expression. The
authors suggested that NAC supplementation prevent the apoptotic
system from being activated and consequently prevent the activa-
tion of pro-life mechanisms such as the expression of Bcl-2 [148].
3.15. Protocatechuic acid
Protocatechuic acid ([tert-butyl (dimethyl)silyl] 3,4-bis [[tert-
butyl (dimethyl)silyl]oxy]benzoate) is a widely distributed natu-
rally occurring phenolic acid and normally found at high concen-
trations in vegetables and fruits and are readily absorbed by
animals and humans [128,149]. The ovarian protection promoted by
protocatechuic acid has been little investigated either in vivo or
in vitro. Its antioxidant and cytoprotective effects have already been
demonstrated against cisplatin in the ovary of mice [150]. Proto-
catechuic acid supplementation was able to replace three antioxi-
dants (transferrin, selenium and ascorbic acid) in the culture
medium of sheep secondary follicles, increasing follicle survival
compared to medium supplemented with transferrin, selenium
and ascorbic acid and maintaining antrum formation and fully
grown oocytes, mitochondrial activity and GSH concentrations
similar to control [151].
3.16. Quercetin
Quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-
4-one) is a type of naturally occurring flavonoid compound that is
ubiquitous in multiple vegetables, fruits and leafy, displays anti-
oxidant properties against various diseases [152,153]. Quercetin is
recognized for its ability to directly donate hydrogen atoms and
quench ROS and for promoting the activation of the endogenous
antioxidant defense system, directly interacting with intracellular
signaling cascades related to antioxidant function [154,155]. Some
effects of quercetin supplementation have already been described
on granulosa cell culture, such as increased cell viability and
regulation of antioxidant genes (SOD, CAT and GSSH) (rats: [156];
goats [157]), and in IVM, such as reduction of ROS and age-related
mitochondrial impacts of oocytes (swine: [158]; human and mice
[159]). During culture of bovine preantral follicles in the ovarian
tissue, supplementation with quercetin increased the percentage of
normal developing follicles by improving the total antioxidant ca-
pacity [160].
3.17. Resveratrol
Resveratrol (5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-
diol) is a natural polyphenol produced in several plants and
fruits, especially in grapes, berries and peanuts [161]. It has anti-
inflammatory, anti-neoplastic anti-microbial and anti-oxidative
properties. The beneficial antioxidant action of resveratrol on fe-
male reproduction have already been demonstrated in the pro-
tection of granulosa cells against chemotherapeutic agents in vitro
[162], in IVM (bovine: [163]; sheep [164]) and in preantral follicle
culture [165].
During culture of sheep isolated secondary follicles, low con-
centrations (2 mM) of resveratrol did not interfere with the rates of
antrum formation, DNA fragmentation, GSH concentrations and
117
Theriogenology 207 (2023) 110e122
mitochondrial activity. However, the highest concentration (30 mM)
proved to be toxic and harmful to follicle development [165].
Nevertheless, during sheep ovarian tissue culture, the lowest con-
centration of resveratrol (2 mM) increased primordial follicle acti-
vation, reduced DNA fragmentation and stimulated the
proliferation of granulosa cells through the activation of the PI3K
pathway [166]. Therefore, resveratrol can act as a prooxidant as
well as an antioxidant agent depending on the concentration
administered to the cells.
3.18. Rutin
Rutin (3,30 ,40 ,5,7-pentahydroxyflavone-3-rhamnoglucoside) is
an important flavonoid, also known as vitamin P or quercetin-3-O-
rutinoside, that is consumed in the daily diet, being found in fruits,
vegetables or in medicinal plants [167e169].
Rutin supplementation during in vitro culture of sheep ovarian
tissue significantly increased primordial follicle activation, follicle
and oocyte growth, and Akt protein expression [169]. Furthermore,
addition of rutin to the culture medium of sheep secondary folli-
cles, replacing three antioxidants of the medium (transferrin, se-
lenium and ascorbic acid), increased follicular survival and GSH
concentrations, and maintained the percentage of fully grown oo-
cytes and active mitochondria similar to the group cultured in
medium with transferrin, selenium and ascorbic acid together.
These results demonstrate that the use of rutin as the only anti-
oxidant can contribute to preantral follicle development in vitro
[34].
3.19. Sodium selenite
Sodium selenite is an inorganic form of selenium endowed with
antioxidant capacity [170]. In mouse secondary follicles isolated
from vitrified or non-vitrified ovarian tissue, sodium selenite sup-
plementation increased follicular survival, antrum formation, and
the percentage of MII oocytes compared to follicles cultured in
sodium selenite-free medium [171,172]. Furthermore, sodium
selenite reduced ROS concentrations and increased total antioxi-
dant capacity and selenium-dependent glutathione peroxidase (Se-
GPx) activity in preantral follicles at the end of a 96 h culture period
compared to control [172].
4. Final considerations
Supplementation of in vitro culture medium of preantral follicles
with antioxidants has diverse effects, including maintenance of
follicular survival, primordial follicle activation, secondary follicle
development, better oocyte quality and higher percentage of oo-
cytes capable of resuming meiosis. Overall, antioxidant supple-
mentation can reduce ROS levels, regulate the expression of
antioxidant genes, such as GSH, CAT and SOD, mitochondrial ac-
tivity, thus maintaining the cellular redox balance, decreasing DNA
fragmentation and apoptosis of cultured preantral follicles (Fig. 1).
These positive effects may vary according to the animal species, the
type of culture and cells and the concentration of antioxidant used.
Too much antioxidant supplementation can have pro-oxidant ef-
fects and impair follicular development and oocyte quality in vitro.
In this sense, knowing the mechanisms of action of antioxidant
substances is crucial for determining the best antioxidant and/or
combination of antioxidants to supplement the in vitro culture
medium of preantral follicles. This information may indicate
whether two or more antioxidants that act through different
mechanisms of action may or may not interact and potentiate the
control of oxidative stress in vitro.
Despite the beneficial effects of antioxidants on the in vitro
R. Lucia dos Santos Silva, R. de Sousa Barberino and M.H. Tavares de Matos
culture of preantral follicles, some substances, such as a-tocoph-
erol, ascorbic acid, hesperidin, b-mercaptoethanol and CoQ10, still
remain little explored. In addition, the mechanisms and antioxidant
effects are less explored in the in vitro culture of preantral follicles
enclosed in ovarian tissue, thus limiting accurate evaluation of the
antioxidant effect of each substance. Furthermore, most of the
antioxidants tested in the in vitro culture of follicles are natural,
bioactive compounds especially found in food and medicinal
plants. Therefore, the use of plant-based culture medium or plant-
derived antioxidants could serve as an inexpensive and effective
alternative supplement for the in vitro culture of preantral follicles.
Further studies are still needed to determine the antioxidant
mechanisms and their contributions, and the best antioxidant or
the best combination of antioxidants for the in vitro culture of
preantral follicles.
Acknowledgments
R.L.S. Silva holds a scholarship from Foundation for Science and
Technology Support from Pernambuco State (FACEPE, Brazil; nº
IBPG-0284-5.05/19). M.H.T. Matos is supported by a grant from the
National Council for Scientific and Technological Development
(CNPq, Brazil; nº 317582/2021-6).
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