idade Digestible methionine + cystine levels for white-egg layers aged one to six weeks

The aim of this study was was to determine the nutritional requirements of digestible methionine+cystine (M+C) for white-egg layers aged one to six weeks. A completely randomized design with five methionine+cystine levels, six replicates, and 30 birds per experimental unit was adopted. Dietary treatments consisted of five diets supplemented with DL-Methionine with resulted in five levels of digestible methionine + cystine, 80% (0.516%), 90% (0.578%), 100% (0.640%), 110% (0.702%), and 120% (0.764%), based on Brazilian tables of nutritional requirements. Performance, serological blood, and histological data were evaluated. Feed intake, feed conversion, hepatic glycogen deposition, enzymatic activity of alanine aminotransferase and gamma-glutamyl transferase, and serum creatinine and albumin levels had showed a quadratic response to the levels of digestible M+C, with the respective requirements: 89.78% (0.575%), 114.33% (0.732%), 86.50% (0.554%), 100% (0.640%), 100.40% (0.643%), 104.30% (0.668%), and 111.88% (0.716%). Increasing levels of methionine+cystine elevated the relative liver weight and the deposition of hepatic glycogen, in addition to promote higher growth in pullets, with better body weight and body weight gain and feed conversion ratio. Our findings suggest that 0.732% digestible methionine+cystine is Research, Society and Development, v. 9, n. 8, e74985242, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5242 3 recommended, which corresponds to an intake of 151.20 mg/bird/d and a Met+Cys:Lys ratio 83%, for light pullets from one to six weeks.


Introduction
Among the essential nutrients directly influencing birds' performance is methionine, the noteworthy first limiting amino acid to these animals when corn-and soybean meal-based diets are supplied. Methionine also performs several functions in the organism of birds and affects the immune system, the deposition of protein, the lipid metabolism, and the energy metabolism (Zhu et al., 2019;Figueiredo Jr et al., 2020;Jankowski et al., 2020). Development, v. 9, n. 8, e74985242, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5242 4 Some other functions of methionine include the donation of methyl radicals, precursors of the biosynthesis of cysteine, which, bound to the pairs by a disulfide bond, forms cystine, which is why nutritional recommendations are expressed as methionine+cystine (Bin et al., 2017).
The proper methionine nutritional level not only boost productive performance rates, tissue formation, and the functioning of physiological systems, but also maximizes the preparation of pullets for the subsequent developer and egg-laying phases. However, there is little information about pullets, especially regarding amino acids. This may influence the maximization of production by birds in the phase subsequent to their growth, i.e., the egglaying phase, because it is clear that egg production in the laying phase will be closely linked to its period of development.
In this sense, Rostagno et al. (2017) have recently recommended 0.86% digestible methionine+cystine values for the starter phase (one to four weeks of age), and Dekalb White (2009) manual recommended levels of 0.75% digestible methionine+cystine for white-egg pullet layers from 1 to 5 weeks of age. D' Agostini et al. (2017), in turn, recommended 0.70% digestible methionine+cystine in diets for pullets aged one to six weeks. Therefore, the aim of the current study was to determine the requirement of digestible methionine+cystine for light pullets aged one to six weeks.

Material and Methods
This paper was an experimental research of a quantitative nature, which was used as a collection instrument the testing technique. According to Pereira et al. (2018) the quantitative method generates a set of data that can be analyzed using mathematical techniques, such as percentages, statistics and probabilities, numerical methods, analytical methods and generation of equations and / or mathematical formulas applicable to some process. A total of 900 Dekalb White pullets from the 1st day of age with an average initial live weight of 35.94 ± 0.10 g were housed in 1.0 × 1.5 m experimental pens. The floor was covered with sugarcane bagasse bedding, and each pen contained an incandescent 100 W light Development, v. 9, n. 8, e74985242, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5242 bulb for heating the birds in the first weeks of life, a tubular feeder, and a pendulum chick drinker. Water and feed were available ad libitum, and the vaccination and lighting programs adopted were those suggested for the sanitation challenge in the region and by guide of the lineage, respectively.
Concerning the growth curve, the pullets live weight was measured weekly in the period of one to six weeks of age, totaling five weightings, and the obtained data were submitted to the equation models proposed by Von Bertalanffy (1957), Brody (1945), Gompertz (1825), Logistic (Verhulst, 1845), and Richards (1959), using the GOSA Statistical Software, which gave the best adjustment of mathematical equations. Akaike's Information Criterion (AIC) (Akaike, 1987) was used to choose the mathematical model that best fit the pullets age-weight data on the growth curve.
The statistical analyses of the evaluated traits were performed using the SAS software (SAS, 2011). The digestible methionine+cystine requirement was estimated by regression analyses.

Results and Discussion
The data of variables, FBW, BWG and MCI fitted the linear regression models better, but variables FI and FCR adjusted better to the quadratic regression. The points of minimum found for FCR was 114.33% (0.732%) and the maximum point for FI were 89.78% (0.575%), estimated by the derivative of the regression equations (Table 2). Table 2-Effects of treatments on final live weight (FBW, g/bird), weight gain (BWG, g/bird), feed intake (FI, g/bird), feed conversion (FCR, g/g) and Met+Cys intake (MCI, mg/bird/d) in hens aged one to six weeks.  (2009) guide for the phase from one to six weeks of age, but bottom than the 0.859% described by Rostagno et al. (2017).
Likewise, Silva et al. (2009) utilized methionine+cystine levels for pullets aged one to four weeks varying from 0.650 to 0.850%, and a significant effect was observed on FI, BWG, and FCR. The optimum amino acid levels found in this study were 0.800% and 0.790% for BWG and FCR, respectively.
The methionine+cystine levels utilized in this experiment were not sufficient to cause an effective reduction in feed intake to reward for the excess amino acids in the diet.
According to Gietzen et al. (1998), there is evidence that alterations in the levels of limiting amino acids in relation to the total amino acids in the diet cause an imbalance detected in the prepiriform cortex of the brain, followed by behavioral alterations such as reduction in feed intake.
According to Keshavarz and Nakajima (1995), body weight has been considered the major factor for the egg weight in the laying phase, stated that for every 45 g of body weight below the expected weight, sexual maturity may be delayed by 3 to 3.5 d.
Additionally, Leeson & Summers (1997) emphasized the importance of maximizing weight gain until maturity, because the BW affect not only the egg weight, but delay onset of lay too.
According to D'Agostini et al. (2017), during the growth phase, it is essential to supply nutrients that meet the requirements for maintenance and gain, thereby ensuring the proper development of the immune and reproductive systems and body structure, which in turn provides greater productivity in the laying phase and the formation of uniform birds, which ultimately makes the activity more profitable.
Sulfur amino acids play an essential role in the performance and maturity of hens, as these acids are able to regulate the expression of numerous genes and thus control the metabolism of nutrients, cell functions, and the protein synthesis (Tesseraud et al., 2008).
Thus, optimal levels of dietary methionine is not only required for normal growth, but also for proper cellular and molecular functions (Fagundes et al., 2020).
A linear effect of the levels of digestible methionine+cystine (P < 0.05) was also found on EVC, LIV, SPL, and liver yield ( Table 3). The highest supplementation of methionine+cystine in the diet was considered the most efficient in terms of absolute and relative weights of the analyzed variables. decreased the weight of the liver and the hepatic protein, but elevated the synthesis of fat in the liver of layers reared in tropical conditions. The opposite was observed in this study, wherein the digestible methionine+cystine levels increased the absolute and relative weights of the birds' liver. Nevertheless, the above previous studies found these results with adult birds, and in the current study the tested birds were under growth, for which no scientific reports exist yet. Table 3-Effects of treatments on the absolute (g) and relative (%) weights of eviscerated carcass (EVC), liver (LIV), and spleen (SPL), of hens aged one to six weeks. In addition to the higher LIV, the absolute values of EVC and SPL were also higher, demonstrating the faster development of birds that received increasing levels of digestible methionine+cystine. Development, v. 9, n. 8, e74985242, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5242 Another important result was the increasing linear response shown by SPL with the levels of digestible methionine+cystine, which may indicate improvement of the immune system, since the weight of this organ is related to immunity and this organ is responsible for the processing of antigens (Ag), production of immunoglobulins (IgM), and the deposition and maturation of T lymphocytes (Turletti et al., 2008), corroborating studies that demonstrate this beneficial activity of methionine in the immune system (Kalvandi et al., 2019;Zhu et al., 2019).
In their first weeks of age, supplementation of methionine+cystine increased hepatic glycogen deposition (HGD), which was higher in the treatment that met 120% (0.764%) of the requirements of digestible methionine+cystine. The point of minimum found for HGD was 86.50% (0.554%), estimated by the derivative of the regression equation (Table 4). The hepatic glycogen deposition was verified by the higher positivity to Periodic Acid Schiff staining (Figure 1), and no hepatic steatosis was detected in the studied treatments.
This deposition of hepatic glycogen can be seen in the higher frequency of pink coloration in the image. The hepatic deposition glycogen indicates an energy reserve, which in practical terms of poultry farm situation, allows a beneficial effect for the pullets as they could start laying phase in a better physiological state, through better formation, development and maturation of the reproductive system, and consequently an early age at first egg, and persistence of production. Liu et al. (2019) found that dietary levels of methionine can alter the levels of mRNA of genes related to the metabolism of glucose and lipids in the liver of laying hens, which in high amounts may result in a condition of hypercholesterolemia in birds . The increase in the levels of methionine+cystine had a quadratic effect on variables ALT, GGM, CRE, and ALB (Table 5), whose maximum obtained values were 100% (0.640%), 100.40% (0.643%), 104.30% (0.668%) and 111.88% (0.716%), respectively.
Regarding the serum protein levels (PTN), they showed a linear effect (P < 0.01) caused by the levels of digestible methionine+cystine.
Despite the linear response of the absolute and relative weights of the liver and the glycogen stock to the levels of digestible methionine+cystine, there were no biochemical alterations related to the activity of hepatic enzymes and creatinine, considering the standards for that species and age (Kaneko et al., 2008). Therefore, the hepatic changes observed in this study, increased LIV and HGDdid not cause unfavorable biochemical alterations, which might compromise the health and productive performance of birds in the laying phase. Research, Society and Development, v. 9, n. 8, e74985242, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5242 Table 5-Effects of treatments on the activity of enzymes alanine aminotransferase (ALT, U/L), aspartate aminotransferase (AST, U/L), gamma-glutamyl transferase (GGM, U/L) and serum creatinine (CRE, mg/dL), albumin (ALB, g/dL) and protein (PTN, g/dL) in hens aged one to six weeks. Albumin is a soluble protein that comprises approximately half of the protein from the blood serum. And, similarly to the findings of this study, Yalçin et al. (2011) stated that the ALB and PTN levels increased as the supplementation of methionine+cystine in the birds' diet was increased. Because ALB and PTN are elements produced in the liver, these results also corroborate the previous ones, in which better liver functioning was observed with greater supplementation of methionine+cystine.
Likewise, Jankowski et al. (2020) found an increase in plasma protein and albumin levels, as there was an increase in methionine levels in the diets. This is explained by the fact that albumin is responsible for 55-60% of the total plasma protein, being composed of 585 amino acids arranged in helices that are retained by 17 sulfur-containing particles disulfide bridges: cystine, cysteine and Methionine.
To build the growth curve, the data of the growth variable of birds aged one to six weeks fitted the mathematical models proposed by Gompertz and Richards better; however, the parameters estimated by the Gompertz function showed the lowest AIC (1362.937) ( Table   6), and consequently best described of the average growth curves of hens aged one to six weeks ( Figure 2). Source: own research. Cuello et al. (2018) worked with performance, growth, and productive traits of pullets, and determined the estimates of sexual maturity rate and body growth of birds through equation adjustments to the mathematical model proposed by Gompertz. Likewise, Neme et al. (2006) studied growth curves of different pullets strains and found maturity and growth rates based on the parameters estimated by the Gompertz function.
The application of the growth curve is aimed at modeling the response pattern of data such as age-weight throughout the life of an animal (Freitas, 2005), and the Gompertz model is the most widely employed to obtain the growth curve of birds and to study nutritional "trends" and requirements during growth (Hruby, 1994).

Conclusion
Increasing dietary methionine+cystine levels elevated the relative liver weight and the deposition of hepatic glycogen. In addition, they promoting higher growth in pullets, with better body weight and body weight gain and feed conversion ratio.