Introduction
In Brazil, cactus cladodes are used as animal feed, especially during periods of drought, configuring a strategic forage resource to livestock in drylands (Pessoa et al., Reference Pessoa, Andrade, Magalhães, Teodoro, Santos, Araújo, Medeiros, Nascimento, Valença and Cardoso2020; Rocha Filho et al., Reference Rocha Filho, Santos, Véras, Siqueira, Novaes, Mora-Luna, Monteiro and Ferreira2021). Regardless of the genus, Opuntia or Nopalea, the cactus cladodes have a high metabolizable energy content (2.29–2.39 Mcal/kg dry matter) and represent an important source of water (Rocha Filho et al., Reference Rocha Filho, Santos, Véras, Siqueira, Novaes, Mora-Luna, Monteiro and Ferreira2021; Silva et al., Reference Silva, Batista, Guim, Souza, Carvalho, Silva Júnior, Arandas, Barros, Sousa and Silva2021a, Reference Silva, Araújo, Santos, Oliveira, Campos, Godoi, Gois, Perazzo, Ribeiro and Turco2021b).
Currently, the Miúda cactus cladodes (Nopalea cochenillifera Salm Dyck) and the Orelha de Elefante Mexicana (OEM) cactus cladodes (Opuntia stricta [Haw]. Haw.) are generally being used for livestock feeding in semiarid region of Brazil due to their resistance to carmine cochineal (Dactylopius opuntiae) (Vasconcelos et al., Reference Vasconcelos, Lira, Cavalcanti, Santos and Willadino2009), an important pest lowering the productivity of susceptible cactus cladodes varieties (Silva et al., Reference Silva, Ferreira, Oliveira, Santos, Gama, Chagas, Inácio, Silva and Pereira2018).
Several studies have revealed use of different varieties of cactus cladodes with satisfactory nutritional and productive responses in ruminant animals (Cardoso et al., Reference Cardoso, Carvalho, Medeiros, Guim, Cabral, Véras, Santos, Dantas and Nascimento2019; Moraes et al., Reference Moraes, Guim, Tabosa, Chagas, Almeida and Ferreira2019; Paula et al., Reference Paula, Véras, Guido, Chagas, Conceição, Gomes, Nascimento and Ferreira2019; Lopes et al., Reference Lopes, Ferreira, Batista, Maciel, Barbosa, Munhame, Silva, Cardoso, Véras and Carvalho2020). However, information is limited antinutritional factors that can compromise its quality and affect kidney function. The evaluation of urinary biochemicals in livestock, such as urea, creatinine and electrolyte levels, and renal excretion rates, reveal clinical signs indicating metabolic and nutritional disorders (Henriques et al., Reference Henriques, Gregory, Rizzo, Hasegawa and Meira Júnior2016; Pordeus Neto et al., Reference Pordeus Neto, Soares, Batista, Andrade, Andrade, Lucena and Guim2016).
According to Henriques et al. (Reference Henriques, Gregory, Rizzo, Hasegawa and Meira Júnior2016), emphasis on the kidney function of sheep is extremely necessary, in view of the high occurrence of diseases in the urinary tract of these animals. Silva et al. (Reference Silva, Araújo, Santos, Oliveira, Campos, Godoi, Gois, Perazzo, Ribeiro and Turco2021b) observed high concentration of crystals in the urine in sheep fed diets based on cactus cladodes silages. Low water intake is one the main causes of urolithiasis in sheep. Additionally, oxalates make the capture of minerals at the kidneys, which can cause renal failure and reduction in the ability to filter waste, tissue changes and even death of the cells (Rahman et al., Reference Rahman, Abdullah and Wan Khadijah2013; Barboza et al., Reference Barboza, Oliveira, Souza, Lima Júnior, Lima and Guerra2019).
Investigations with sheep and goats fed diets containing cactus cladodes have been showing increased diuresis (Vieira et al., Reference Vieira, Batista, Mustafa, Araújo, Soares, Ortolane and Mori2008; Gouveia et al., Reference Gouveia, Maciel, Soares, Silva Neto, Gonçalves, Batista and Carvalho2015; Pordeus Neto et al., Reference Pordeus Neto, Soares, Batista, Andrade, Andrade, Lucena and Guim2016; Cardoso et al., Reference Cardoso, Carvalho, Medeiros, Guim, Cabral, Véras, Santos, Dantas and Nascimento2019; Maciel et al., Reference Maciel, Carvalho, Batista, Guim, Maciel, Lima Júnior and Cardoso2019; Silva et al., Reference Silva, Munhame, Lopes, Souza, Guim, Carvalho, Soares, Barros, Arandas and Batista2021c) and there is little research who investigated the effects of the OEM cactus cladodes on urine parameters of lambs.
It was hypothesized that the use of Miúda cactus cladodes and OEM cactus cladodes in lamb diets may increase water retention and cause kidney dysfunction. Therefore, this study aimed to evaluate the water balance and urinary parameters of lambs fed diet without cactus (Tifton-85 hay as exclusive roughage) and diets containing cactus cladodes varieties.
Materials and methods
All procedures were performed with the authorization of the Committee of Ethics in the Use of Animals (License 142/2018).
Animals and experimental design
The study was conducted at Department of Animal Science at Federal Rural University of Pernambuco, Recife, Brazil (8°04′03″S and 34°55′00″W), at an altitude of 4 m. According to Köppen classification system, the climate is of the ‘Am’ type, which is hot and humid. The temperature range of 22–30°C and humidity of 80% (Alvares et al., Reference Alvares, Stape, Sentelhas, de Moraes Gonçalves and Sparovek2013; Chagas et al., Reference Chagas, Ferreira, Azevedo, Siqueira, Elins and Barros2015).
Thirty-six uncastrated male Santa Inês lambs, with an average body weight of 22.0 ± 2.9 kg and approximately six months old were distributed in a completely randomized design, with twelve lambs per treatment. The animals were housed in individual stalls (1.0 m × 1.8 m) containing feeders and drinkers, located in an open shed, with a cemented floor and covered with fibre cement tiles. The experimental period lasted for 75 days, with the first 30 days for the adaptation of the animals to the diets and the management and the remaining 45 days for evaluation and data collection. In the adaptation period, all animals were identified, vaccinated against clostridia and dewormed.
Treatments, management and chemical analysis
The diets were formulated based on NRC (2007) recommendations for gains of 200 g per day. The ingredients used were: Tifton-85 hay (Cynodon spp.), Miúda cactus cladodes (N. cochenillifera Salm Dyck), OEM cactus cladodes (O. stricta [Haw]. Haw.), ground corn, soybean meal, mineral mix and urea (Table 1). The experimental treatments were: (1) diet with Tifton-85 hay as exclusive roughage (control); (2) diet with partial replacement of Tifton hay by Miúda cactus cladodes; and (3) diet with partial replacement of Tifton hay by OEM cactus cladodes. The roughage:concentrate ratio was 60:40 (Table 2).
Chemical composition of ingredients of experimental diets (g/kg dry matter)
a Orelha de Elefante Mexicana cactus cladodes.
b g/kg natural matter.
c neutral detergent fibre assayed with a heat stable amylase and corrected for ash and nitrogenous compounds.
d non-fibrous carbohydrates; (-) not determined.
Ingredients proportion and chemical composition of the experimental diets
a Orelha de Elefante Mexicana cactus cladodes.
b g/kg natural matter.
c neutral detergent fibre assayed with a heat stable amylase and corrected for ash and nitrogenous compounds.
† Nutrients/kg of product: Ca = 110 g; Ca (max.) = 135 g; P = 87 g; S = 18 g; Na = 147 g; Mg = 20 g; Co = 15 mg; Cu = 590 mg; Cr = 20 mg; I = 50 mg; Mn = 2000 mg; Mo = 300 mg; Se = 20 mg; Zn = 3800 mg; F = 870 mg (max.); Fe = 1800 mg.
Diets were offered in the form of two daily meals at 08.00 h (0.60) and 15.00 h (0.40), as a total mixed ration, with ad libitum access to water. The cactus plants (subterminal cladodes) were manually harvested every two weeks when they were 2 years old. The cladodes were then stored in a shaded area and the processing was carried out immediately before each feeding in a forage machine (MC1n Laboremus®, Campina Grande, Brazil), with a 25-mm sieve, and the mixture of ingredients was conducted manually in the feeders. The hay was acquired in local commerce and ground in a forage machine with an 8-mm sieve.
This research is a part of a larger project with a methodology based on a previous study by Lopes et al. (Reference Lopes, Ferreira, Batista, Maciel, Barbosa, Munhame, Silva, Cardoso, Véras and Carvalho2020). Thus, further information regarding collection, processing and chemical analysis (dry matter – DM, ash, crude protein, ether extract, neutral detergent fibre – NDF and non-fibrous carbohydrates – NFC) of feed, leftovers and faeces, in addition to DM and nutrients intakes, and determination of total digestible nutrients (TDN), were reported by these authors. The determination of total oxalates in feed and leftovers samples was carried out according to Moir (Reference Moir1953). This method includes extraction steps, filtration, precipitation, centrifugation, dilution and titration with potassium permanganate.
Measurement of water intake
The water intake of the animals was measured over 10 days, in the middle and in the end of the experimental period. The water was provided to the animals ad libitum, in the morning, in plastic buckets with a capacity of 10 l. The intake of voluntary water was determined by means of daily measurement, recording the difference between the volume of the remaining water and the volume of the water supplied the previous day. The water evaporation rate was measured with six buckets distributed in different points in the shed.
Water intake via feed was estimated through difference between the natural matter intake and DM intake of the experimental diets. Total water intake was calculated as the sum of voluntary water intake and water intake from diet. The total water intake was also estimated from the equation proposed by the NRC (2007): 3.86 × DMI – 0.99, where DMI = dry matter intake. The surplus water was calculated by the formula: total water intake measured – estimated water requirement through equation proposed by NRC (2007). The water retention was calculated by the following equation: total water intake – (urinary volume + water excreted via faeces).
Urine analysis
Urine samples were obtained 45 days after feeding with the tested diets, by spontaneous urination of the animals, using colostomy bag, being applied to the preputial region. Immediately after urination, the pH was measured using a digital potentiometer and 10 ml of urine was immediately diluted into 40 ml of sulphuric acid at 0.036 N, and stored in 2 ml polyethylene tubes, maintained at −20°C for further analysis of urea, creatinine, uric acid, total protein, calcium (Ca), phosphorus (P) and magnesium (Mg).
Urine volume was estimated by dividing the average daily excretion of creatinine by sheep (23.2 mg/kg body weight) (Kozloski et al., Reference Kozloski, Fiorentini, Harter and Sanchez2005) by the average creatinine concentration (mg/l) in the urine spot sample, multiplying the result by the respective weight body of the animal (Carvalho et al., Reference Carvalho, Garcia, Pires, Silva, Pereira, Viana, Santos and Pereira2010). Urine analyses were performed using commercial kits Labtest® (Labtest Diagnóstica® S.A., Brazil) in an automated biochemical analyser Labtest® (Labtest®, Model Prestige 24i, Japan), following the manufacturer's instructions: urea (ref. 27; enzymatic-colorimetric system, urease), creatinine (ref. 96; kinetic), uric acid (ref. 140; enzymatic – Trinder), total protein (ref. 36; Sensiprot, pyrogallol red), Ca (ref. 90; cresolphthalein liquiform), P (ref. 42; phosphorus molybdate) and Mg (ref. 50; sulfonated magon). The urinary excretion index (UEI) and the fractional excretion rate (FER) were obtained using equations proposed by Garry et al. (Reference Garry, Chew, Rings, Tarr and Hoffsis1990).
Statistical analysis
The data were analysed using the GLM procedure in the statistical program SAS (2009) (version 9.4, SAS Institute Inc., Cary, NC, USA), with the initial body weight (BW) of the animals used as a covariate, according to the model below:
where Yij = the observed dependent variable; μ = general mean; Ti = effect of treatment; β(Xij − X) = covariate effect (initial BW); and eij = experimental error. The data were submitted to ANOVA and the Tukey's test, at 5% probability, was used to compare the averages between treatments.
Results
Lambs fed Miúda cactus cladodes showed higher intake of DM (g/day, g/kg BW and BW0.75; P < 0.050) and TDN (916.0 g/day; P = 0.001). The diet containing OEM cactus cladodes provided lower crude protein intake (168.0 g/day; P = 0.010). Diets containing cactus cladodes, regardless of variety, provided an increase in water intake via feed (P = 0.001), total water intake (P = 0.001), water in relation to the BW (P = 0.001), as a function of metabolic BW (P = 0.001), surplus water (P = 0.001) and water retention (P = 0.001), compared to the control diet. On the other hand, there was a decrease in voluntary water intake by animals that consumed this Cactaceae (P = 0.001) (Table 3).
Nutrient and oxalates intake, and water balance in lambs fed cactus cladodes-based diets
a Orelha de Elefante Mexicana cactus cladodes.
b standard error of the mean.
c body weight.
d metabolic body weight.
e total digestible nutrients.
f natural matter.
* values obtained by Lopes et al. (Reference Lopes, Ferreira, Batista, Maciel, Barbosa, Munhame, Silva, Cardoso, Véras and Carvalho2020).
The diets based on both cactus cladodes varieties provided a total intake of 6.98 kg/day of water, higher than that observed in the control treatment, which was 3.71 kg/day. Lambs fed with OEM cactus cladodes showed higher intake of total oxalates (6.21 g/day; P = 0.001) and produced greater urinary volume (1.86 kg/day) than those which received the control diet (1.14 kg/day), equivalent to 0.31 of the total water intake. In addition, they lost more water through the faeces (1.78 kg/day) compared to those consumed Miúda cactus cladodes (1.17 kg/day), so that this faecal water excretion represented 0.30 of the total water intake (Table 3).
The urinary pH was influenced by the diets, being alkaline in the animals fed with the Miúda cactus cladodes (pH = 8.14), in comparison to control and OEM diets. The urinary concentrations of urea, creatinine, total proteins, Ca and P, as well as UEI of urea and P and the FER of P, decreased significantly with the use of cactus cladodes varieties. The urinary content of Mg and the UEI of uric acid and Mg increased with the inclusion of cactus in the diets, being the urinary concentration of Mg was higher in lambs fed with OEM diet (1.85 mmol/l; P = 0.001). The Miúda cactus cladodes caused a reduction in UEI and FER of Ca (Table 4).
Urinary parameters of lambs fed with cactus cladodes-based diets
a Orelha de Elefante Mexicana cactus cladodes.
b Standard error of the mean.
Discussion
The increase in the DM and nutrients intakes is usually reported in studies involving lambs fed cactus-based diets, due to its high acceptability, low NDF content, high NFC level and high passage and degradation rate (Costa et al., Reference Costa, Ferreira, Campos, Guim, Silva, Siqueira, Barros and Siqueira2016; Cardoso et al., Reference Cardoso, Carvalho, Medeiros, Guim, Cabral, Véras, Santos, Dantas and Nascimento2019; Lopes et al., Reference Lopes, Ferreira, Batista, Maciel, Barbosa, Munhame, Silva, Cardoso, Véras and Carvalho2020; Mora-Luna et al., Reference Mora-Luna, Herrera-Angulo, Siqueira, Conceição, Chagas, Monteiro, Véras, Carvalho and Ferreira2022). On the other hand, the low level of DM in diets containing cactus cladodes provides high water intake via diet. The reduction in voluntary water intake was 68.35 and 77.03% between animals fed with Miúda cactus cladodes and OEM cactus cladodes, respectively, when compared to animals submitted to the control diet (Table 3). The greater water supply provided by diets based on cactus cladodes demonstrates the importance of using this feed in dry regions of the world, where there are quantitative and often qualitative limitations on water to meet the requirements of different ruminant animals' species.
These results corroborate with those reported by Pordeus Neto et al. (Reference Pordeus Neto, Soares, Batista, Andrade, Andrade, Lucena and Guim2016) and Cardoso et al. (Reference Cardoso, Carvalho, Medeiros, Guim, Cabral, Véras, Santos, Dantas and Nascimento2019), when analysed the inclusion of Miúda cactus cladodes in the sheep diets. According to Silva et al. (Reference Silva, Araújo, Santos, Oliveira, Campos, Godoi, Gois, Perazzo, Ribeiro and Turco2021b), succulent forages can provide part of the lamb's water requirement, contributing to the supply of quality water for the animals. In addition, the higher water intake caused by diets with cactus cladodes represents one of the reasons for the low DM content in the faeces of animals that receive this feed. Silva et al. (Reference Silva, Batista, Guim, Souza, Carvalho, Silva Júnior, Arandas, Barros, Sousa and Silva2021a), observed strong and significant correlation between faecal dry matter and water intake (r = − 0.91; P = 0.001), so that these two variables were negatively related.
It is important to mention that although all diets provide enough water to attend the water requirements of the animals, estimated at 3.37; 3.99 and 3.53 kg/day depending on the DM intake (NRC, 2007) for the lambs that received the control diets, with Miúda cactus cladodes and OEM cactus cladodes, respectively, there was still water intake in the drinking fountain. The amplitude in excess values between quantified water intake and that obtained from the NRC (2007) was greater in diets with cactus cladodes, regardless of the variety (P = 0.001; Table 3), which can cause hypervolemia. It is possible that the diuretic effect and the occurrence of osmotic diarrhoea caused by intake of cactus cladodes increased the animals' water requirement. In addition, this fact may show a mechanism used by the body to assist in the excretion of oxalates, an anti-nutritional component contained in the diets (Table 2) and consumed in greater quantity by animals submitted to the diet containing OEM cactus cladodes (Table 3).
The increase in diuresis caused by the intake of diets with cactus cladodes has been justified by the greater water supplies provided to animals that receive this feed as a dietary ingredient, thus being a mechanism for regulating body water volume (Vieira et al., Reference Vieira, Batista, Mustafa, Araújo, Soares, Ortolane and Mori2008). However, this result can still be attributed to the high concentration of minerals in the cactus cladodes, such as potassium (K) (Batista et al., Reference Batista, Ribeiro Neto, Lucena, Santos, Dubeux and Mustafa2009; Cordova-Torres et al., Reference Cordova-Torres, Mendoza-Mendoza, Bernal-Santos, García-Gasca, Kawas, Costa, Mondragon Jacobo and Andrade-Montemayor2015), which has recognized diuretic effects (Galati et al., Reference Galati, Tripodo, Trovato, Miceli and Monforte2002). Additionally, the greater volume of urine generated by the OEM cactus cladodes may be related to the higher intake of total oxalates, being a way of satisfactorily excreting these substances via urine. Cardoso et al. (Reference Cardoso, Carvalho, Medeiros, Guim, Cabral, Véras, Santos, Dantas and Nascimento2019), when including 450 g/kg of Miúda cactus cladodes (based on DM) in sheep diets, observed urinary production of 1.70 l/day, a volume similar to that excreted by the animals that received the two varieties of cactus evaluated in the present study (Table 3).
The greater elimination of water via faeces by animals fed with OEM cactus cladodes, in comparison with Miúda cactus cladodes, may be related to higher faecal production and lower faecal DM content, and the higher content of oxalates in the diet based on this variety (Table 3). According to Lucena (Reference Lucena2011), it is possible that the presence of organic acids increases the osmolarity of the intestinal digest, which increases the loss of water through the faeces. In addition, Silva et al. (Reference Silva, Batista, Guim, Souza, Carvalho, Silva Júnior, Arandas, Barros, Sousa and Silva2021a) reported lesions in the epithelium of the large intestine of lambs fed cactus cladodes (Nopalea and Opuntia), such as inflammation, what can decrease the efficiency of water reabsorption.
The alkaline urinary pH caused by the intake of Miúda cactus cladodes is caused possibly due to the higher Ca, Mg and K concentration in this variety of cactus cladodes in relation to the OEM cactus cladodes (e.g.: 17.0 g/kg DM v. 8.2 g/kg DM de K in the Miúda and OEM varieties, respectively) (Germano et al., Reference Germano, Costa, Barbosa, Medeiros and Carvalho1999; Batista et al., Reference Batista, Mustafa, McAllister, Wang, Soita and McKinnon2003; Silva, Reference Silva2018), since diets with high levels of cations can cause metabolic alkalosis in ruminants (Goff et al., Reference Goff, Ruiz and Horst2004; Correa et al., Reference Correa, Zanetti, Del Claro and Paiva2009), thus causing an increase in plasma pH and bicarbonate concentration. Thus, urinary bicarbonate excretion can occur to maintain electroneutrality, which increase the pH of the urine. However, all values are within the range (6.0–8.5) reported by Hendrix (Reference Hendrix2005) for sheep.
Urinary excretion and the UEI of urea were lower in animals that received cactus cladodes, which can be attributed to the lower production of ammonia in the rumen (Bispo et al., Reference Bispo, Ferreira, Véras, Batista, Pessoa and Bluel2007). According to Ferreira et al. (Reference Ferreira, Silva, Ramos, Véras, Melo and Guimarães2009), urinary excretion of urea reflects the serum concentration of this nitrogenous compound. Similarly, there was a decrease in the total protein content in the urine due to the presence of cactus cladodes in the diets, which can be explained by the more efficient use of nitrogen in the rumen environment (Cardoso et al., Reference Cardoso, Carvalho, Medeiros, Guim, Cabral, Véras, Santos, Dantas and Nascimento2019).
The reduction in the concentration of urinary creatinine provided by diets containing cactus cladodes is due to the increase in water intake (overhydration) and, therefore, in the production of urine (González and Scheffer, Reference Gonzaléz and Scheffer2002; Pordeus Neto et al., Reference Pordeus Neto, Soares, Batista, Andrade, Andrade, Lucena and Guim2016). The higher UEI of uric acid observed in animals fed with varieties of cactus cladodes is due to the increase in microbial protein synthesis, a result already found in studies using cactus cladodes in the sheep diets (Cardoso et al., Reference Cardoso, Carvalho, Medeiros, Guim, Cabral, Véras, Santos, Dantas and Nascimento2019; Santos et al., Reference Santos, Carvalho, Magalhães, Silva, Cardoso, Sousa, Silva, Nascimento and Batista2020).
The lower urinary Ca concentrations caused by diets containing cactus cladodes, in addition to the lower UEI and FER caused by Miúda cactus cladodes, are probably related to the high absorption rates of this element by sheep that receive cactus-based diets (Santos et al., Reference Santos, Guim, Batista, Soares, Souza and Araújo2009; Lucena, Reference Lucena2011; Silva, Reference Silva2018), causing animals to excrete less Ca in the urine. This behaviour signals that this mineral is bioavailable, even with the possible complexation by oxalic acid.
As for the urinary excretion of P, Silva (Reference Silva2018), evaluating increasing levels of replacement of Buffel hay (Cenchrus Ciliaris L.) by OEM cactus cladodes (up to 500 g/kg DM) in sheep feed, found an increasing linear effect on the absorption of this mineral (g/day), which justifies the lower levels of P excreted via urine by animals submitted to diets containing cactus cladodes. Urine Mg and the UEI of Mg were higher in the lambs fed cactus cladodes, although Lucena (Reference Lucena2011) and Silva (Reference Silva2018) have shown superior absorption of Mg in diets with the participation of cactus cladodes, which should be associated with excess of Mg consumed, since this cactus has high levels of this mineral in its composition (Batista et al., Reference Batista, Mustafa, McAllister, Wang, Soita and McKinnon2003; Santos et al., Reference Santos, Guim, Batista, Soares, Souza and Araújo2009).
No significant increases in the renal excretion of urea, creatinine and electrolytes, for example, were observed, which are important markers of kidney function. Therefore, our results indicate that kidney functioning is not compromised by diets tested in this study. However, long-term studies are needed to better elucidate the renal function of lambs that receive diets based on different varieties of cactus cladodes.
Conclusion
The cactus cladodes, Miúda and Orelha de Elefante Mexicana varieties, increased water intake and retention, and diuresis in lambs, altered urine parameters, without compromising kidney function, and may be used as a forage resource in drylands.
Author's contributions
SILVA, T. G. P.: Conceptualization, Investigation, Formal analysis and Writing – Original Draft Preparation; LOPES, L. A.: Investigation, Methodology and Data curation; CARVALHO, F. F. R.: Conceptualization, Supervision and Funding acquisition; GUIM, A.: Methodology and Supervision; SOARES, P. C.: Methodology and Supervision; SILVA JÚNIOR, V. A.: Methodology, Supervision; BATISTA, A. M. V.: Conceptualization, Methodology, Supervision, Data curation and Funding acquisition; All authors read and approved the manuscript.
Financial support
This research was supported by Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) – Recife, PE, Brazil (Grant: APQ-0425-5.01/14), and by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001.
Conflicts of interest
The authors declare there are no conflicts of interest.
Ethical standards
This study was conducted in strict compliance with the Brazilian legislation for research and experimentation with animals and was approved by the Committee of Ethics in the Use of Animals of the Federal Rural University of Pernambuco, located in Recife, PE, Brazil (License No142/2018).
