Introduction
In modern commercial poultry production, a variety of environmental, technological, nutritional and biological stressors can intensify the biological formation of free radicals which in turn cause oxidative stress (Oke et al., Reference Oke, Akosile, Oni, Opowoye, Ishola, Adebiyi, Odeyemi, Adjei-Mensah, Uyanga and Abioja2024). This compromises animal health, as well as the productive, reproductive performance and quality of poultry (Surai, Reference Surai2016).
In this sense, from the peak of production, egg losses increase due to a series of physiological and hormonal changes (Amevor et al., Reference Amevor, Cui, Ning, Du, Jin, Shu, Deng, Zhu, Tian, Li, Wang, Zhang and Zhao2021). With increasing age of the bird, the cells of the intestinal mucosa weaken and there is a decrease in the height of the duodenum villi, which impairs the absorption of nutrients necessary for the formation of the eggshell (Sengor et al., Reference Sengor, Yardimci, Cetingul, Bayram, Sahin and Dogan2007). In addition, the oxidative stress to which birds are subjected also contributes, altering the balance of the gastrointestinal tract, impacting nutrient absorption (Surai and Fisinin, Reference Surai and Fisinin2015) and egg production (Wang et al., Reference Wang, Jia, Gong, Celi, Zhuo, Ding, Bai, Zeng, Yin, Xu, Liu, Mao and Zhang2021).
Among the strategies used to mitigate the effects of age in birds, phenolic compounds with antioxidant action and organic acids stand out (Mahfuz et al., Reference Mahfuz, Shang and Piao2021; Abd El-Hack et al., Reference Abd El-Hack, Salem, Khafaga, Soliman and El-Saadony2022, D’Alessandro et al., Reference D’Alessandro, Di Luca, Desantis and Martemucci2025). Anacardic acid is a phenolic compound present in different parts of the cashew tree (Anacardium occidentale L.) and is the main constituent naturally found in the liquid of the cashew nut shell, from the cold extraction, being commonly used under the salt form (calcium anacardate - CAn) in feed (Santos et al., Reference Santos, Freitas, Nepomuceno, Lima, Monteiro, Silva, Nascimento and Watanabe2022). Among the activities observed of anacardic acid, its anti-inflammatory, antibacterial (Olajide et al., Reference Olajide, Aderogba, Adedapo and Makinde2004; Behravan et al., Reference Behravan, Heidari, Heidari, Fatemi, Etemad, Taghipour and Abbasifard2012) and mainly antioxidant (Braz et al., Reference Braz, Freitas, Trevisan, Salles, Cruz, Farias and Watanabe2019) action is highlighted, promoted by the alkenyl chain, being associated with the hydrophobic bond of xanthine oxidase (Masuoka and Kubo, Reference Masuoka and Kubo2004). The antioxidant effects of CAn have been reported in studies with broilers, observing an effect on the lipid stability of meat from 0.75% inclusion in the diet (Freitas et al., Reference Freitas, Cruz, Nepomuceno, Gomes, Watanabe, Farias and Trevisan2022). However, no influence of CAn on the performance and egg quality of quails was observed (Santos et al., Reference Santos, Freitas, Nepomuceno, Lima, Monteiro, Silva, Nascimento and Watanabe2022), possibly associated with the higher level of calcium in the diets. In this sense, considering the greater buffering capacity of diets for laying hens, the hypothesis of the present study would be to combine the effect of an organic acid aiming at reducing gastric pH to provide greater dissociation of CAn into anacardic acid.
For this purpose, it was considered Citric acid (CAc), a weak organic tricarboxylic acid (2-hydroxy-1, 2,3-propanetricarboxylic acid) present in most fruits, especially citrus. It can be classified as a growth promoter, acidulant, bacterial inhibitor, but it is considered an antioxidant because it chelates metal ions (Salgado-Tránsito et al., Reference Salgado-Tránsito, Del Río-García, Arjona-Román, Moreno-Martínez and Méndez-Albores2011; Islam, Reference Islam2012). Although CAc presents a low antimicrobial effect, it could have a synergistic effect with antioxidants (Dibner and Buttin, Reference Dibner and Buttin2002; Özek et al., Reference Özek, Wellmann, Ertekin and Tarim2011) and its potential use in combination is observed with other additives, such as calcium anacardate (Freitas et al., Reference Freitas, Cruz, Nepomuceno, Gomes, Watanabe, Farias and Trevisan2023).
Given the above, the objective was to evaluate the action of calcium anacardate associated with citric acid in diets of late-stage laying hens on performance, egg quality, serum biochemical profile, serum lipid peroxidation (TBARS) and catalase (CAT) in reproductive tissue.
Materials and methods
The project was approved by the Ethics Committee on the Use of Animals (CEUA-UFC) of the Federal University of Ceará, under protocol n° 9616090320. Prior to the study, cashew nut shell liquid was obtained by cold extraction in 99.5º GL ethanol, according to the methodology of Trevisan et al. (Reference Trevisan, Pfundstein, Haubner, Würtele, Spiegelhalder, Bartsch and Owen2006). Calcium anacardate was obtained from the reaction of anacardic acid in the cashew nut shell liquid with calcium hydroxide, through precipitation in the form of calcium anacardate, according to the methodology adapted from Paramashivappa et al. (Reference Paramashivappa, Kumar, Vithayathil and Rao2001). The proportion of anacardic acid in calcium anacardate was 66.65%. Citric acid monohydrate (C6H9O7) presented purity of 99.91%.
Animals and experimental design
A total of 432 Hy-Line W-36 (Hy-Line® do Brazil) laying hens with 63 weeks of age were used, selected based on weight (1.44 ± 0.100 kg) and egg production (80% laying) and uniformly distributed in the cages. During the experimental period, room temperature (maximum and minimum) and relative humidity in the shed were 26.5 and 32.8°C and 73 and 80%, respectively.
The birds were randomly distributed in cages, consisting of nine diets and six replicates of eight birds each. The diets consisted of: Control diet; Diet with 0.25% CAn; Diet with 0.25% CAn associated with 0.25% CAc; Diet with 0.50% CAn; Diet with 0.50% CAn associated with 0.25% CAc; Diet with 0.50% CAn associated with 0.50% CAc; Diet with 0.75% of CAn; Diet with 0.75% CAn associated with 0.25% CAc; Diet with 0.75% CAn associated with 0.50% CAc. The levels tested in the present study were based on previous research with calcium anacardate, with an antioxidant effect observed in chicken meat when included up to 0.75% and pro-oxidant from 1% in the diets (Freitas et al., 2022). Considering the higher calcium level and consequent buffering effect in laying hen diets, the reduction of pH was considered by adding citric acid aiming at the dissociation of calcium anacardate into anacardic acid.
Control diet (Table 1) was formulated, according to the nutritional requirements recommended by the strain manual (Hy-Line Internacional, 2016), with the other treatments obtained by replacing the inert according to the inclusion of CAn and CAc in the proportion of each treatment.
Ingredient composition of control diet
a DL-Methionine: Dextrarotatory and Levorotatory Methionine.
b L Lysine: Levorotatory Lysine.
c Composition per kg of product: Vit. A - 9,000,000.00 IU; Vit. D3 - 2,500,000.00 IU; Vit. E - 20,000.00 mg; Vit. K3 - 2,500.00 mg; Vit. B1 - 2,000.00 mg; Vit. B2 - 6,000.00 mg; Vit. B12 - 15.00 mg; Niacin - 35,000.00 mg; Pantothenic acid – 12,000.00 mg; Vit. B6 - 8,000.00 mg; Folic acid – 1,500.00 mg; Biotin – 100.00 mg.
d Composition per kg of the product: Iron – 100,000.00 mg; Copper – 20.00 g; Manganese - 130,000.00 mg; Zinc - 130,000.10 mg; Iodine - 2,000.00 mg; Selenium – 250.00 mg.
e Inert: Washed sand.
Performance of laying hens
The birds were fed during the 12 weeks of the experimental period, and the feed provided was weighed and the leftovers quantified. Eggs were collected daily, determining feed intake (g/b per d), egg weight (g), egg-laying percentage (%/b per d), egg mass (g/b per d) and feed conversion ratio egg mass (g of feed/g egg mass).
Egg quality
During the experimental periods, one day a week all eggs from each replicate were collected, identified and stored at 22ºC until the following day, when measurements were taken to calculate the quality variables of eggs, regarding the average egg weight (g), specific density (g/cm3), Haugh unit, yolk percentage, albumen percentage, shell percentage, shell thickness (mm) and yolk colour (Digital YolkFanTM, dsm-firmenich, Maastricht, Netherlands). Egg quality parameters were calculated as averages from the data obtained each week.
Serum evaluation
After completing 74 weeks of age, two birds per plot were selected based on the average weight of the experimental unit for blood sampling. Blood samples from two birds per replicate were collected by cardiac puncture, placed in appropriate tubes and left at room temperature for coagulation and subsequent centrifugation at 2,000 g for 15 min. After centrifugation, the supernatant (serum) was removed, and each sample was divided so that each aliquot was packaged and subsequently used in the respective determinations.
Creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol, triacylglycerides were carried out by the automation method (Metrolab 2300 plus) with kinetic kits from Weiner, according to the manufacturer’s instructions. Malondialdehyde (MDA) was determined according to the methodology described by Draper and Hadley (Reference Draper and Hadley1990), in which 250 μl of serum were added in glass tubes followed by 400 μl of 35% perchloric acid and heated in a hot water bath for one hour (37ºC). The mixture was centrifuged (1,400 g for 10 min) and then 600 μl of the supernatant was removed and 200 μl of thiobarbituric acid (1.2%) was added. This mixture was heated in a hot water bath for 30 min (95ºC). Then, the solution was cooled and the reading was performed in a spectrophotometer (Bioscience Ultrospec 6300 PRO) at 535 nm. The standard curve was obtained using 1,1,3,3-tetramethoxypropane and the results obtained were expressed in nmol MDA/ml of serum.
Catalase (CAT) in reproductive tissue
After blood sampling, the birds were euthanised by electrical stunning and slaughtered by jugular vein bloodletting, to remove the reproductive organs (ovary, magnum and uterus), which were identified and placed in resistant plastic bags and immediately frozen in liquid nitrogen. Then, the bags were stored at –80°C until analysis of enzymatic activity related to lipid oxidation processes in the body. Extracts for determination of antioxidant enzyme activity catalase (CAT) and soluble protein content were prepared from 50 mg of sample from each organ and macerated in liquid nitrogen with 4.0 ml of 0.1 M potassium phosphate buffer (pH 7.0), containing 0.1 mM EDTA. The homogenate was centrifuged at 12,000 g for 15 min at 4°C and, until analysis, the supernatant was stored at –25°C. CAT activity was determined according to the method described by Havir and McHale (Reference Havir and McHale1987). The reaction medium consisted of 0.1 M potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.5 M H2O2 and 150 μl of conveniently diluted crude extract, in a final volume of 1.5 ml. Activity was determined by the decrease in absorbance at 240 nm, due to H2O2 consumption. The results were expressed in nmol/μg of protein and represent the average of four repetitions, with each extract dosed in duplicate.
Soluble proteins were determined according to the method described by Bradford (Reference Bradford1976), using Coomassie blue reagent. For 1.0 L of this reagent, 100 mg of Coomassie Brilliant Blue G-250 (Sigma Chemical Company) was dissolved in 50 ml of 95% ethanol plus 100 ml of 85% phosphoric acid. The final volume of the solution was completed with deionised water. To an aliquot of 0.1 ml of the suitably diluted extract, 1.0 ml of Coomassie reagent was added. The mixture was left to rest for 15 min, and then subjected to absorbance reading in a spectrophotometer (Bioscience Ultrospec 6300 PRO) at 595 nm. As a standard, bovine serum albumin was used. The results of soluble protein contents were expressed in mg g–1 MF. The results represented the average of four replications, with each extract dosed in duplicate.
Statistical analysis
Data were analysed using the General Linear Models (GLM) procedure from the Statistical Analysis System 9.4 (SAS Inst. Inc., Cary, NC, USA). The model was:
$${\rm{Yij}} = {\rm \mu }{\rm{Y}} + {\rm{Ti }} + {\rm \varepsilon ijk}$$
where Ƴijk = value observed at the treatment i (i = Control diet, 0.25% CAn, 0.25% CAn + 0.25% CAc, 0.50% CAn, 0.50% CAn + 0.25% CAc, 0.50% CAn + 0.50% CAc, 0.75% CAn, 0.75% CAn + 0.25% CAc, 0.75% CAn + 0.50% CAc) on replicate j (j = 1 to 6); µƳ = population mean; Ƭi = effect of treatment i; Ɛijk = experimental error associated with the observed Ƴijk value. The means were compared using the Student Newmann-Keuls test at 5% of significance.
Results
The addition of CAn associated or not with CAc in the diets did not affect the laying hen’s performance (Table 2). Regarding egg quality, there was no effect of CAn and CAc in the diets on the percentage of yolk, albumen and shell, specific density, Haugh units and eggshell thickness (Table 3). On the other hand, it was observed that the addition of 0.75% of CAn and its association with CAc (0.25 and 0.50) in diets resulted in more pigmented yolks compared to birds fed control diet and diet with 0.25% of CAn. For TBARS, it was observed that the isolated addition of CAn at the level of 0.75% resulted in lower values in the yolk compared to the control diet and diet with 0.25% CAn and 0.75% CAn associated with 0.25 and 0.50 CAc, although not differing from the other treatments.
Performance of late-stage laying hens (63–74 weeks) fed diets with calcium anacardate associated or not with citric acid
FCR – feed conversion ratio per egg mass; CAn – calcium anacardate; CAc – citric acid; SEM – standard error of means.
Egg quality of late-stage laying hens (63–74 weeks) fed diets with calcium anacardate associated or not with citric acid
Alb – albumen; SD – specific density; HU – Haugh unit; EST – Eggshell thickness; TBARS – thiobarbituric acid reactive substances; MDA – malondialdehyde; CAn – calcium anacardate; CAc – citric acid; SEM – standard error of means. Means followed by different letters in column differ by Student Newmann-Keuls test at 5% of significance.
The values of creatine, alanine aminotransferase, aspartate aminotransferase, total cholesterol and triglycerides in blood were not significantly influenced (P >0.05) by the treatments (Table 4). However, the serum lipid oxidation determined for birds fed with 0.50% CAn, associated with 0.50% CAc and 0.75% CAn, did not differ from each other and was significantly lower in relation to those determined for the other treatments. In turn, birds fed the control diet showed the highest values of TBARS, with a reduction in values with the addition of isolated CAn up to the level of 0.75% or in the association of 0.50 CAn with 0.50% of CAc. However, TBARS values showed an increase with the use of 0.75% CAN associated with 0.25% or 0.50%, with no difference in the results obtained in relation to the birds fed with the control diet.
Biochemical profile and lipid peroxidation in blood of late-phase laying hens (63–74 weeks) fed diets with calcium anacardate associated or not with citric acid
Cr – Creatinine; ALT – Alanine aminotransferase; AST – Aspartate aminotransferase; TAG – triacylglycerides; TBARS – thiobarbituric acid reactive substances; MDA – malondialdehyde; CAn – calcium anacardate; CAc – citric acid; SEM – standard error of means. Means followed by different letters in a column differ by Student Newmann-Keuls test at 5% of significance.
For catalase activity in magnum (Table 5), greater activity was observed in laying hens fed with a diet containing isolated levels of 0.50 or 0.75 % CAn and its associations with 0.25 or 0.50% of CAc and lower activity for birds fed with a control diet and 0.25% CAn alone or associated with 0.25% of CAc.
Catalase in the reproductive tissue of late-phase laying hens (63–74 weeks) fed diets with calcium anacardate associated or not with citric acid
CAn – calcium anacardate; CAc – citric acid; SEM – standard error of means. Means followed by different letters in column differ by Student Newmann-Keuls test at 5% of significance.
Discussion
Considering that the experimental diets were formulated to be isoenergetic and isonutritive, it can be inferred that CAn associated or not with CAc did not influence the use of energy and nutrients in the diet, meeting the requirements of metabolisable energy, protein and amino acids.
The absence of significant effects for organic acids (Kaya et al., Reference Kaya, Kaya, Gül and Çelebi2013; Cruz et al., Reference Cruz, Freitas, Aguiar, Braz and Trevisan2019) or plant extracts used in broiler feed (Freitas et al., Reference Freitas, Borges, Trevisan, Watanabe, Cunha, Pereira, Abreu and Nascimento2012; De Melo et al., Reference De Melo, Gomes, Faria, Freitas, Watanabe, Watanabe, Souza and Fernandes2020) and laying hens diets (Özek et al., Reference Özek, Wellmann, Ertekin and Tarim2011; Bozkurt et al., Reference Bozkurt, Kamil, Mehmet, Metin, Ahmet and Abdullah2012) on performance is common and can be seen as an indicator that they did not interfere the nutrients absorption, allowing the maintenance of the flora beneficial to the digestive tract, thus excluding the possibility of toxic effects of such additives (Vasconcelos et al., Reference Vasconcelos, Bastos-Leite, Gomes, Goulart, Sousa and Fontenele2016).
However, other studies have shown beneficial effects (Soltan, Reference Soltan2008; Youssef et al., Reference Youssef, Hassan, Ali and Mohamed2013) in the use of additives in poultry feed. The divergence in the results is due to the different characteristics of the existing organic acids and the need to adjust the level of each acid or their mixture considering the diversity of conditions in poultry farming.
The absence of differences among treatments for egg quality may be related to meeting the requirements of the birds since the diets were formulated to be isonutritious and there was no significant variation in feed intake. In turn, even if the intensity of yolk pigmentation is not an indicator of the nutritional value of egg, the greater yolk pigmentation from supplementation with 0.75% of CAn and its association with CAc (0.25 and 0.50) could be an attractive factor in terms of consumer preferences, since it affects perception by associating yolk colour with age and state of animal health and egg quality, thus affecting the purchase decision (Loetscher et al., Reference Loetscher, Kreuzer and Messikommer2013).
In this sense, phenolic compounds represent one of the most important and diverse groups among products of plant origin and include most of the substances responsible for the characteristic of taste, odour and colour of fruits, leaves and seeds (Ignat et al., Reference Ignat, Volf and Popa2011), and may be responsible for the change in yolk colour. This result corroborates with Braz et al. (Reference Braz, Freitas, Trevisan, Salles, Cruz, Farias and Watanabe2019), who also observed improvement in yolk colour with the inclusion of cashew nut shell liquid in laying hen diet. Kaya et al. (Reference Kaya, Kaya, Gül, Çelebi, Timurkaan and Apaydin2014) also observed an improvement in egg yolk colour when using 0.2% CAc in laying hen diet, evidencing better pigmentation due to increased absorption of carotenoids from corn associated with reduced pH of intestinal content.
The lower lipid oxidation observed from the lower values of TBARS in the eggs of late-stage laying hens fed with 0.75% of CAn in the diet results from the ingestion of compounds with antioxidant action, corroborating the results observed by Zhao et al. (Reference Zhao, Yang, Yang, Wang, Jiang and Zhang2011) and Zhang et al. (Reference Zhang, Li, Miao, Zhang and Zou2018). Thus, the results observed on the lipid oxidation of the yolk in the present study are in agreement with the literature, since the benefits of the antioxidant action of anacardic acid in the yolk of eggs have been demonstrated by other researchers with the dietary addition of cashew nut shell liquid (Abreu et al., Reference Abreu, Pereira, Freitas, Trevisan, Costa and Braz2017; Braz et al., Reference Braz, Freitas, Trevisan, Salles, Cruz, Farias and Watanabe2019), as well as its pro-oxidant action, when CAn was added at level higher than 0.75% in the diet of broilers (Abreu et al., Reference Abreu, Pereira, Freitas, Trevisan, Da Costa and Cruz2019).
The serum biochemical profile is an important indicator of the physiological, pathological and nutritional status to which the animals were subjected (Minafra et al., Reference Minafra, Marques, Stringhini, Ulhoa, Rezende, Santos and Moraes2010; Jiwuba et al., Reference Jiwuba, Ikwunze, Dauda and Ugwu2016), and can therefore be used as an indicator of the productive performance of birds and metabolic diseases. Creatinine is a byproduct of phosphocreatine breakdown in skeletal muscle and is considered an important index of protein metabolism and kidney function (Huang et al., Reference Huang, Fu, Lan, Rehman and Tong2017). As the blood levels of creatinine in the birds were not influenced by the isolated inclusion of calcium anacardate and its association with citric acid in the diet, it can be inferred that there was no impairment of renal functions (Konan et al., Reference Konan, Bacchi, Lincopan, Varela and Varanda2007).
The serum concentration of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) indirectly indicate the health status of the liver (Gu et al., Reference Gu, Chen, Jin, Wang, Wen and Zhou2021). These enzymes are present in hepatocytes and, when they are elevated in the serum, it means that there has been some damage to the tissue (Huang et al., Reference Huang, Choi, IM, Yarimaga, Yoon and Kim2006). Therefore, as no changes were observed in the activities of AST and ALT enzymes, it can be concluded that liver functions were not compromised either, since these enzymes are important physiological markers of cell damage in liver tissue (Georgakouli et al., Reference Georgakouli, Manthou, Fatouros, Deli, Spandidos, Tsatsakis, Kouretas, Koutedakis, Theodorakis and Jamurtas2015).
The results obtained in the present study agree with those reported by Toyomizu et al. (Reference Toyomizu, Okamoto, Ishibashi, Nakatsu and Akiba2003) and Cruz et al. (Reference Cruz, Freitas, Braz, Salles and Silva2018) when evaluating the supplementation of anacardic acid (0.1%) and calcium anacardate (0.25, 0.50, 0.75 and 1%) in rat and broiler diets, respectively, which did not observe a harmful effect on the biochemical profile of the animals’ blood, suggesting that renal or hepatic dysfunction would be unlikely to occur through dietary supplementation of anacardic acid. Based on these results, it can be suggested that the inclusion of up to 0.75% of CAn and its associations with CAc (0.25%; 0.50%) in the diets are not toxic for laying hens in the period from 63 to 74 weeks old.
Malondialdehyde (MDA) is one of the end products of lipid peroxidation and the level of MDA can indicate the degree of lipid peroxidation in the body, which is directly proportional to increased oxidative stress (Cheng et al., Reference Cheng, Song, Zheng, Zhang, Zhang, Zhang, Zhou and Wang2017). Likewise, the decrease in the concentration of MDA in plasma or tissues indicates a decrease in lipid peroxidation (Seven et al., Reference Seven, Aksu and Seven2010; Echeverry et al., Reference Echeverry, Yitbarek, Munyaka, Alizadeh, Cleaver, Camelo-Jaimes, Wang and Rodriguez-Lecompte2016).
In this sense, the antioxidant characteristics of anacardic acid can be attributed to phenolic and flavonoid compounds (Correia et al., Reference Correia, David and David2006; Trevisan et al., Reference Trevisan, Pfundstein, Haubner, Würtele, Spiegelhalder, Bartsch and Owen2006), which reduce lipid peroxidation through their free radical scavenging properties, through the antioxidant system and hydrogen supplying abilities of phenolic compounds. In this scenario, it was observed that, by improving lipid metabolism through the use of antioxidants, the antioxidant status of the body benefits, preventing lipid peroxidation, with consequent improvement in the demand for egg production in laying hens, through hepatic lipogenesis by oestrogens (Qi et al., Reference Qi, Wu, Zhang, Yue, Xu, Ji and Qi2011). Thus, it is suggested that the reduction in serum MDA concentration in birds that a consumed diet with isolated doses of CAN, combined or not with AC, had an antioxidant effect, being effective in improving the lipid stability of the serum of birds in this study.
The increase in CAT concentrations in the magnum and the decrease in SOD concentrations in the ovary, magnum and uterus of birds that received isolated doses of CAN and associated with AC suggest that the phenolic compounds present in the CAN may have different mechanisms of antioxidant action in the various tissues and need to be further studied. According to Papadopoulou et al. (Reference Papadopoulou, Petrotos, Stagos, Gerasopoulos, Maimaris, Makris, Kafantaris, Makri, Kerasioti, Halabalaki, Brieudes, Ntasi, Kokkas, Tzimas, Goulas, Zakharenko, Golokhvast, Tsatsakis and Kouretas2017), the main polyphenols present in olive residue, hydroxytyrosol and tyrosol, were able to increase catalase activity through oxidative phosphorylation and also reduce SOD activity, and this effect is associated with the action of polyphenols on uptake direct release of superoxide anion, with a reduction in enzyme activity, as a kind of compensation mechanism.
Conclusions
The addition of 0.75% CAn increases yolk pigmentation, reduces lipid oxidation in the yolk and blood plasma, and increases CAT activity in the magnum in late-stage laying hens. These benefits can also be obtained with the combination of 0.50% CAn and 0.50% CAc.
Data availability
The datasets generated during and/or analysed during the current study are not publicly available due to being part of a study with other research institutions but are available from the corresponding author on reasonable request.
Acknowledgements
The authors want to acknowledge Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação Cearense de Apoio ao Desenvolvimento Científico (FUNCAP).
Author contributions
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by GCAW, ERF, MCAM, EOS and CNC. The first draft of the manuscript was written by RCN, and the final version was performed by PHW. All authors read and approved the final manuscript.
Funding statement
The current work was supported by the Fundação Cearense de Apoio ao Desenvolvimento Científico - FUNCAP.
Competing interests
None.
Ethical standards
The study has been approved by the Ethics Committee on the Use of Animals (CEUA-UFC) of the Federal University of Ceará, under protocol n° 9616090320.
