Introduction
The developing in vitro follicle culture (IVFC) biotechnique aims to mimic ovarian folliculogenesis in vivo. This biotechnique could be used to maximize the number of potentially fertilizable oocytes for assisted reproductive technologies in human, and for in vitro embryo production in livestock (Figueiredo et al., Reference Figueiredo, Celestino, Faustino and Rodrigues2011). Also, IVFC may serve as an in vitro model for toxicological research (Stefansdottir et al., Reference Stefansdottir, Fowler, Powles-Glover, Anderson and Spears2014), limiting the number of experimental animals needed.
It has been shown that IVFC efficacy may be influenced by many variables, such as base medium composition and supplementation (Ferreira et al., Reference Ferreira, Maside, Sá, Guerreiro, Correia, Leiva-Revilla, Lobo, Araújo, Apgar, Brandão, Figueiredo and Campello2016), the animal model, and the follicular category (Cadenas et al., Reference Cadenas, Leiva-Revilla, Vieira, Apolloni, Aguiar, Alves, Lobo, Rodrigues, Apgar, Smitz, Figueiredo and Maside2017). Nonetheless, the vast majority of the studies have been performed using ovaries from a slaughterhouse. As a result, the ovaries used in IVFC are from animals of different ages, breeds, and physiological status (Silva et al., Reference Silva, Rossetto, Salles, Sá, Rosa e Silva, Rodrigues, Campello and Figueiredo2014). Under these conditions, it is impossible to determine, for instance, the effect of nutrition, another essential variable on in vitro follicle development.
In vivo, the association between nutrition and reproduction has been widely described, and it is accepted that a diet with a deficiency, excess, or imbalance in energy, such as proteins, vitamins, and minerals may compromise reproductive efficiency (Mostafa et al., Reference Mostafa, Bakr and Ayyat2020). In this sense, dietary fatty acids may positively affect reproduction in ruminants (Mattos et al., Reference Mattos, Staples and Thatcher2000). However, the relationship between the lipid content in the diet, specifically polyunsaturated fatty acids (PUFAs), and oocyte quality is still controversial (Fouladi-Nashta et al., Reference Fouladi-Nashta, Gutierrez, Gong, Garnsworthy and Webb2007; Fernandes et al., Reference Fernandes, Feltrin, Martins, Gaudêncio Neto, Aguiar, Silva, Oliveira, Silva, Silva, Bertolini and Rondina2014), although the presence of PUFAs in the follicular fluid has been positively correlated with oocyte competence (Matoba et al., Reference Matoba, Bender, Fahey, Mamo, Brennan, Lonergan and Fair2014). Nevertheless, despite the importance of PUFAs for reproduction, to the best of our knowledge, there has been no information about the effect of a diet rich in PUFAs on in vitro follicle development.
Among the different dietary supplementation protocols, several lipid sources have been tested to promote an increase in the energy density of the diet and positively influence reproduction. Flaxseed has been highlighted as an important source of lipids and its use in animal feed (c. 10% of dry matter) is mainly due to the high concentration of PUFA in its composition and the potential effects of this lipidemic profile on animal reproduction (Bernacchia et al., Reference Bernacchia, Preti and Vinci2014). Recently, it was demonstrated that supplementation with flaxseed in goats was able to increase the rate of in vivo follicular growth in goats (Alves et al., Reference Alves, Fernandes, Rossetto, Silva, Galvão, Bertolini and Rondina2019), as well as change the follicular fluid environment, the expression of oocyte mitochondrial genes and in vitro embryo development (Alves et al., Reference Alves, Fernandes, Calderón, Rossetto, Bertolini and Rondina2021).
Therefore, this study aimed to use the in vitro follicle culture (IVFC) biotechnique as a tool to evaluate the influence of whole flaxseed as a feed supplementation in the diet on the in vitro development of caprine early antral follicles (EAFs) and further embryo production. The endpoints evaluated were follicular daily growth and diameter, estradiol production, oocyte recovery rate and diameter, sperm penetration, pronuclei formation, and embryo development.
Materials and methods
Reagents
Unless mentioned otherwise, all chemicals and reagents used in the current study were purchased from Sigma-Aldrich Chemical Co. (St. Louis, Mo., USA).
Animals and experimental design
All procedures in this study were approved by the Ethics Committee in Animal Experimentation of the Ceará State University, Brazil (no. 3047564/2017, CEUA-UECE).
In total, 18 Anglo-Nubian crossbred, non-lactating adults, between second and third birth, were selected for cyclicity.
Goats were allocated to two diet groups: control and flaxseed, according to homogeneity in body weight, (33.4 ± 3.6 kg; overall mean ± standard deviation (SD)), body condition scores (2.7 ± 0.3, from 1 to 5), and ages (45.5 ± 7.25 months). In the control treatment (n = 9), the diet consisted of elephant grass hay (Pennisetum purpureum) and commercial concentrate with ground corn grain, soybean meal, wheat bran, and mineral mixture. In the flaxseed treatment (n = 9), it was added at 30% to the concentrate dry matter basis of whole full-fat linseed. The total lipids of the diets were 2.8 % and 8.5% on a dry matter basis, respectively, for the control and flaxseed groups, and were isonitrogenous (13% crude protein to dry matter basis). The composition of the experimental diets and their chemical composition are presented in Table 1.
Table 1. Ingredient composition of the concentrate-based diets
Animals from each group, homogeneously segregated for use in three replicates were kept in collective stalls (Fernandes et al., Reference Fernandes, Feltrin, Martins, Gaudêncio Neto, Aguiar, Silva, Oliveira, Silva, Silva, Bertolini and Rondina2014), receiving mineral salt and water ad libitum. In total, seven, six, and five animals were used in the first, second, and third replicates, respectively. In all the experimental groups, diets were provided twice a day (07:00 h and 15:00 h) for 30 days, up to follicle recovery. All animals received the diets to satisfy their energy requirements for maintenance and breeding according to the National Research Council (NRC, 2007) for adult non-dairy does.
Collection of ovaries, isolation, selection and culture of early antral follicles
After 30 days of feeding, the animals were randomly selected for slaughter. The goats’ reproductive tracts were collected immediately post mortem. Ovaries from both groups were collected and transported to the laboratory as previously described (Chaves et al., Reference Chaves, Martins, Saraiva, Celestino, Lopes, Correia, Verde, Matos, Báo, Name, Campello, Silva and Figueiredo2008). For the IVFC and oocyte in vitro maturation (IVM) we used the methodology previously described by our group (Cadenas et al., Reference Cadenas, Leiva-Revilla, Vieira, Apolloni, Aguiar, Alves, Lobo, Rodrigues, Apgar, Smitz, Figueiredo and Maside2017). Briefly, in the laboratory, EAFs (300–400 µm) from each dietary treatment, i.e. control and flaxseed were isolated and individually cultured in vitro in 100-µl drops of α-MEM (pH 7.2–7.4), supplemented with 3 mg/ml bovine serum albumin (BSA), 10 ng/ml insulin, 5.5 μg/ml transferrin, 5 ng/ml selenium, 2 mM glutamine, 2 mM hypoxanthine, 50 μg/ml ascorbic acid, and 50 ng/ml growth hormone (GH) for 18 days at 38.5°C and 5% CO2 in air under mineral oil. The fresh medium was prepared and pre-equilibrated overnight before use. The medium was replaced partially (60 μl) every other day.
Morphological evaluation of follicle development: follicle survival, daily growth, and levels of estradiol
The classification of normal or degenerated follicles was performed every 6 days based on their morphological aspects, and those showing morphological signs of degeneration, such as darkness of the oocytes and surrounding cumulus cells, or misshapen oocytes, were classified as degenerated. The average of two measurements (height and length) of the follicle was used as a measure of follicle diameter. The daily growth rate was calculated based on the diameter changes over the culture period (18 days).
At the end of the culture period, levels of estradiol were measured in spent medium using a competitive immunoassay commercial kit (enzyme linked fluorescence assay VIDAS, Biomerieux, Marcy l’Etoile, France). The analytical sensitivity of the E2 was 9 pg/ml (range, 9–3000 pg/ml) and the intra-assay coefficient of variation was 5%.
In vitro maturation (IVM) and fertilization (IVF)
All follicles were mechanically opened and only those oocytes ≥ 110 µm in diameter (zona not included) were submitted to IVM in groups of 10 cumulus–oocyte complexes (COCs)/100-µl drop) for 30 h at 38.5°C and 5% CO2 in air under mineral oil. The IVM medium consisted of tissue culture medium 199 supplemented with 1 µg/ml 17β-estradiol, 5 µg/ml luteinizing hormone, 0.5 µg/ml rFSH (bovine), 10 ng/ml epidermal growth factor, 1 mg/ml BSA, 1 mM pyruvate, 50 ng/ml, insulin-like growth factor 1, and 100 mM cysteamine.
After the IVM, all COCs were washed and transferred in groups of 10 into 100-µl drops of fertilization medium under mineral oil. The fertilization medium consisted of IVF-TALP (Parrish et al., Reference Parrish, Susko-Parrish, Leibfried-Rutledge, Critser, Eyestone and First1986) supplemented with 30 μg/ml heparin (Calbiochem 375095), 15 μM hypotaurine, and 5 μg/ml gentamicin. The IVF-TALP medium was pre-equilibrated for at least 2 h before use. Refrigerated semen diluted in extender from two fertile bucks were pooled and motile sperm were selected by the swim-up procedure (Fukui et al., Reference Fukui, Kikuchi, Kondo and Mizushima2000) in Sperm-TALP medium (Papa et al., Reference Papa, Papa, Oliveira, Guasti, Castilho and Giometti2015). Viable sperm were diluted in the appropriate volume of fertilization medium to achieve a final concentration of 2 × 106 sperm/ml. Spermatozoa and COCs were co-incubated for 18 h at 38.5°C in a humidified atmosphere of 5% CO2.
In vitro embryo culture, transfer and pregnancy diagnosis
After the IVF, all presumptive zygotes were washed and transferred to microdrops of embryo culture medium (G1TM, Vitrolife, Gothenburg, Sweden) (10 zygotes: 10 µl medium) under mineral oil, and incubated for 2 days at 38.5°C in a humidified atmosphere of 5% CO2 and 5% O2. On day 3 post-IVF, all cleaved embryos were surgically transferred into the oviduct of three synchronized recipient goats, as previously reported by Sá et al., (Reference Sá, Ferreira, Sousa, Duarte, Paes, Cadenas, Anjos, Fernandes, Rosseto, Cibin, Alves, Rodrigues, Rondina, Gastal and Figueiredo2020). Pregnancy diagnosis was performed on day 36 after transfer by transrectal ultrasonography.
Statistical analysis
Statistical analysis was carried out using Sigma Plot 11 (Systat Software Inc., USA). Comparison of means (follicle and oocyte diameters, estradiol and daily growth rate) between treatments were analyzed using t-test. One-way repeated measures analysis of variance (Holm–Sidak post-hoc test) was performed to compare the effect of treatment among days of culture. When appropriate, chi-squared or Fisher’s exact tests were used to evaluate the percentage variables (in vitro fertilization parameters, intact, degenerated, and extruded follicles) between treatments. Data are presented as mean [± standard error of the mean (SEM)] and percentage, and the statistical significance was defined as P < 0.05 (two-sided).
Results
Follicle morphology and growth after in vitro culture
The results of follicle morphology, growth rate, and estradiol production are summarized in Table 2.
Table 2. Percentages of morphologically normal, extruded and degenerated follicles, daily growth, and estradiol production at the end of in vitro culture (day 18)*
*There was no difference between control and flaxseed treatments
Abbreviations: Deg, degenerated follicles; E2, estradiol; Ext, extruded follicles; MIF, morphologically intact follicles.
There was no influence of the administration of the whole flaxseed in the diet on follicle morphology, growth rate, and estradiol production. Both treatments, control, and flaxseed, increased (P < 0.05) follicle diameter from day 0 (368.12 ±7.8 and 376.28 ± 8.1 µm, respectively) to day 18 (779.70 ± 25.6 and 783.71 ± 25.7 µm, respectively). Also, follicle daily growth rate was lower (P < 0.05) in the first 6 days of culture than from day 6 onwards (10.36 ± 1.1 and 10.90 ± 1.1 vs. 34.76 ± 2.2 and 34.35 ± 2.0 µm/day, respectively).
Oocyte parameters, evaluation of in vitro embryo development, transfer, and pregnancy diagnosis
The results of oocyte diameter, percentage of oocytes ≥ 110 µm, sperm penetration, male pronucleus formation, two pronuclei formation, and cleaved embryos after the IVFC of caprine EAFs are summarized in Table 3.
Table 3. Oocyte diameter, percentage of oocytes ≥ 110 µm, sperm penetration, male pronucleus formation, two pronuclei formation, and cleaved embryos after the IVFC of caprine EAFs
*Only oocytes ≥ 110 µm were submitted to IVM, IVF and IVC.
†Values calculated out of the overall fertilized oocytes.
A,BWithin a column (P < 0.05).
Abbreviations: 2PN, two pronuclei; EAFs, early antral follicles; IVFC, in vitro follicle culture; MPN, male pronucleus.
After the IVFC, the recovery rate of oocytes ≥ 110 µm was higher (P < 0.05) in the flaxseed treatment when compared with control group. However, oocytes from goats fed with flaxseed based diet showed lower (P < 0.05) overall sperm penetration with no further effect on the cleavage rate.
Regardless of the diet, most of the fertilized oocytes showed only one pronucleus (MPN) and only five embryos were produced on day 3 post-IVF, three at the 4-cell to 6-cell stage, and two at the 2-cell to 3-cell stage. All five embryos were surgically transferred into the oviduct of three recipient goats. At 36 days after the embryo transfer, none of the recipient showed signs of estrous behaviour, however no pregnancy was detected after ultrasonography examination.
Discussion
It is well established that lipid supplementation in the diet of ruminants affects ovarian activity (Robinson et al., Reference Robinson, Pushpakumara, Cheng, Peters, Abayasekara and Wathes2002). In this context, different lipid sources have been tested in animal feed, as the composition of fatty acids seems to have a crucial role in specific reproductive processes, including the follicular fluid environment (Alves et al., Reference Alves, Fernandes, Rossetto, Silva, Galvão, Bertolini and Rondina2019), follicular development (Childs et al., Reference Childs, Lynch, Hennessy, Stanton, Wathes, Sreenan, Diskin and Kenny2008), hormonal production (Robinson et al., Reference Robinson, Pushpakumara, Cheng, Peters, Abayasekara and Wathes2002), oocyte quality (Fernandes et al., Reference Fernandes, Feltrin, Martins, Gaudêncio Neto, Aguiar, Silva, Oliveira, Silva, Silva, Bertolini and Rondina2014) and embryo development (Leroy et al., Reference Leroy, Vanholder, Mateusen, Christophe, Opsomer, de Kruif, Genicot and Van Soom2005). Our study aimed to evaluate for the first time the efficiency of the use of whole flaxseed, an oilseed rich in polyunsaturated fatty acids, in the diet of goats, on the in vitro development of isolated EAFs and subsequent embryo production. The current results showed that there was no influence of the administration of the whole flaxseed in the diet on follicle morphology, growth rate, and estradiol production. However, a higher recovery rate of oocytes ≥ 110 µm was observed in the flaxseed treatment, with lower overall sperm penetration in the oocytes. Despite this, no effect of the diet was observed on the embryo cleavage rate.
Recently, several studies have highlighted the composition of flaxseed and its potential for animal feed as a lipid source, especially in the possible effects on reproduction (Alves et al., Reference Alves, Fernandes, Rossetto, Silva, Galvão, Bertolini and Rondina2019, Reference Alves, Fernandes, Calderón, Rossetto, Bertolini and Rondina2021). To date, studies have been limited to the effects on follicular dynamics, oocyte quality, embryo development, metabolic parameters in plasma and follicular fluid, and oxidative stress, with no study evaluating the possible effects on in vitro follicular development. In the present study, the use of whole flaxseed in the diet of goats did not significantly affect follicle morphology and growth after in vitro culture and estradiol production. Similar results were shown in vivo where dietary PUFAs did not affect either follicular dynamics or steroid production in bovine (Childs et al., Reference Childs, Lynch, Hennessy, Stanton, Wathes, Sreenan, Diskin and Kenny2008). Also, PUFAs had no effect on granulosa cell proliferation and steroidogenesis during in vitro culture in ovine (Wonnacott et al., Reference Wonnacott, Kwong, Hughes, Salter, Lea, Garnsworthy and Sinclair2010) and bovine (Lammoglia et al., Reference Lammoglia, Willard, Hallford and Randel1997). Conversely, several authors have shown that increasing dietary PUFAs increased the size of preovulatory follicles and the total number of follicles (Beam and Butler, Reference Beam and Butler1997).
Both treatments, control and flaxseed, increased (P < 0.05) follicle diameter from day 0 to the end of the culture period, and also follicle daily growth rate was lower (P < 0.05) in the first 6 days of culture than from day 6 onwards. These growth patterns (first third of culture vs. second and last third) are in agreement with previous published papers under the same culture conditions (Cadenas et al., Reference Cadenas, Maside, Ferreira, Vieira, Leiva-Revilla, Paes, Alves, Brandão, Rodrigues, Wheeler and Figueiredo2018). However, compared with a previous study performed by our group, the end points: percentage of morphologically normal follicles (∼80 vs. 65%), follicle diameter (∼780 vs. ∼550 µm) and growth rates (∼23 vs. ∼11 µm/day) showed higher figures when using ovaries from controlled well nourished animals (present study) compared with ovaries from slaughterhouse (Cadenas et al., Reference Cadenas, Leiva-Revilla, Vieira, Apolloni, Aguiar, Alves, Lobo, Rodrigues, Apgar, Smitz, Figueiredo and Maside2017). It is well known that animal nutritional status (Abecia et al., Reference Abecia, Sosa, Forcada and Meikle2006) and age (Ottolenghi et al., Reference Ottolenghi, Uda, Hamatani, Crisponi, Garcia, Ko, Pilia, Sforza, Schlessinger and Forabosco2004) can profoundly affect in vivo follicle development. Therefore, the use of a homogenous group of animals (same body weight, body condition score, and age) could contribute to provide a high quality starting material for the IVFC.
There are vast reports in the literature about how the composition of follicular fluid affects follicular development, as well as the quality of the oocyte and embryo (Sinclair et al., Reference Sinclair, Lunn, Kwong, Wonn, Linforth and Craigon2008). Moreover, there is strong evidence that maternal metabolic condition and diet are one of the factors that most influence the follicular microenvironment (Valckx and Leroy, Reference Valckx, Leroy and Watson2015). Fat supplementation with unsaturated fatty acids, for example, is considered one of the main strategies for modulating the lipidic composition of follicular fluid (Zachut et al., Reference Zachut, Arieli, Lehrer, Argov and Moallem2008). According to Wathes et al. (Reference Wathes, Abayasekara and Aitken2007), the amount and proportion of different unsaturated fatty acids in the reproductive tissues reflect the animal’s dietary consumption, and these unsaturated fatty acids influence reproductive processes through a variety of mechanisms, including the provision of precursors for the synthesis of prostaglandins, as well as the modulation of the expression patterns of many key enzymes involved in the metabolism of steroids. Using a diet contained an amount of lipids similar to that of present study, Fernandes et al. (Reference Fernandes, Feltrin, Martins, Gaudêncio Neto, Aguiar, Silva, Oliveira, Silva, Silva, Bertolini and Rondina2014) observed a higher concentration of total cholesterol in the follicular fluid of goats compared with the control group. Also, previous studies have shown that a diet rich in lipids, with flaxseed as a lipid source, was able to change the lipid profile of the plasma, as well as affect the expression of genes related to the accumulation of lipids in the granulosa cells (Alves et al., Reference Alves, Fernandes, Rossetto, Silva, Galvão, Bertolini and Rondina2019) and the concentration of glutathione peroxidase in the follicular fluid (Alves et al., Reference Alves, Fernandes, Calderón, Rossetto, Bertolini and Rondina2021).
The effect of lipid supplementation, including a PUFA-rich diet, on oocyte quality has been widely studied in ruminants. There is a consensus that the effects are mediated by changes in follicular fluid fatty acids composition (Childs et al., Reference Childs, Lynch, Hennessy, Stanton, Wathes, Sreenan, Diskin and Kenny2008; Fouladi-Nashta et al., Reference Fouladi-Nashta, Gutierrez, Gong, Garnsworthy and Webb2007). However, according to Santos et al. (Reference Santos, Bilby, Thatcher, Staples and Silvestre2008), changes in the composition of oocyte fatty acids are relatively small, suggesting a possible mechanism of selective uptake of PUFAs. After the IVFC, the recovery rate of oocytes ≥ 110 µm was higher (P < 0.05) in the flaxseed treatment. The beneficial effect of the whole flaxseed in the diet on oocyte growth may be due to the fact that, among other important functions, fatty acids are stored within the oocyte and cumulus cells, providing a potent source of energy via β-oxidation (Dunning et al., Reference Dunning, Russell and Robker2014). However, oocytes from goats fed with flaxseed based diet showed lower (P < 0.05) overall sperm penetration. This result is consistent with other studies that associated elevated fatty acid concentration in diet with low oocyte competence (O’Callaghan et al., Reference O’Callaghan, Yaakub, Hyttel, Spicer and Boland2000; Wakefield et al., Reference Wakefield, Lane, Schulz, Hebart, Thompson and Mitchell2008). Conversely, Cardoso et al. (Reference Cardoso, Lima, Silva, Souza-Cáceres, Pereira, Oliveira, Nogueira and Melo-Sterza2019) feeding cows with flaxseed for a long period found no differences in oocyte viability. In general, the relationship between fatty acids and oocyte quality is still controversial, as some studies showed positive effects (Fouladi-Nashta et al., Reference Fouladi-Nashta, Gutierrez, Gong, Garnsworthy and Webb2007; Matoba et al., Reference Matoba, Bender, Fahey, Mamo, Brennan, Lonergan and Fair2014), while in others no effect was observed (Fernandes et al., Reference Fernandes, Feltrin, Martins, Gaudêncio Neto, Aguiar, Silva, Oliveira, Silva, Silva, Bertolini and Rondina2014). Percentages of normal fertilization (2PN) and cleaved embryos on day 3 post-IVF were not affected by the presence of the whole flaxseed in the diet. Alves et al. (Reference Alves, Fernandes, Calderón, Rossetto, Bertolini and Rondina2021), feeding goats with a similar amount of flaxseed for the same period, also found no difference in the cleavage rate and proportion of blastocysts compared with the control group. Similar results were described in caprine when another important source of PUFAs, cashew walnuts, was added to the diet (Fernandes et al., Reference Fernandes, Feltrin, Martins, Gaudêncio Neto, Aguiar, Silva, Oliveira, Silva, Silva, Bertolini and Rondina2014). The low embryo production when using in vitro grown caprine oocytes has been described before (Saraiva et al., Reference Saraiva, Rossetto, Brito, Celestino, Silva, Faustino, Almeida, Bruno, Magalhães, Matos, Campello and Figueiredo2010; Silva et al., Reference Silva, Rossetto, Salles, Sá, Rosa e Silva, Rodrigues, Campello and Figueiredo2014). However, those previous studies showed low oocyte nuclear maturation as well, unlike our system, which was able to produce a considerable amount of metaphase II oocytes (Cadenas et al., Reference Cadenas, Leiva-Revilla, Vieira, Apolloni, Aguiar, Alves, Lobo, Rodrigues, Apgar, Smitz, Figueiredo and Maside2017). In this current study, 40% of the in vitro grown oocytes were fertilized, even though most of the fertilized oocytes formed only MPN. To our knowledge, this is the highest fertilization rate described in this species so far for in vitro grown oocytes. These findings indicated that the oocytes were penetrated by the spermatozoa and were able to contribute toward MPN formation. However, the second meiotic resumption did not occur appropriately, preventing normal female pronuclei formation and subsequent cleavage. Taken together, these facts suggest that oocyte developmental competence (oocyte cytoplasmic maturation) should be improved.
In conclusion, except for the increase in the percentage of fully grown oocytes, in general, dietary flaxseed did not affect goat in vitro folliculogenesis. However, it did negatively affect the sperm penetration rate, even though with no further effect on the cleavage rate. Also, our current IVFC system was able to produce a considerable amount of meiotically matured oocytes that were able to be fertilized. Nevertheless, the low percentage of oocytes showing normal fertilization and embryo production highlights the need for further research to improve oocyte cytoplasmic maturation.
Financial support
This research was supported by grants from the National Council for Scientific and Technological Development (CNPq-79/2013 linha 3 – Rede Nordeste de Biotecnologia (Rede de pesquisa do ovário artificial) – Processo No. 407594/2013-2). J. Cadenas is the recipient of a grant from FUNCAP/CE (Brazil). The authors thank Saul Gaudêncio Neto for his valuable help during embryo transferences.
Ethical standards
All procedures in this study were approved by the Ethics Committee in Animal Experimentation of the Ceará State University, Brazil (no. 3047564/2017, CEUA-UECE).
Conflicts of interest
The authors declare no conflicts of interest.
