Study area

The study was carried out on the poultry farm of the College of Veterinary Medicine and Animal Science of the Autonomous University of Tamaulipas, in Ciudad Victoria, Tamaulipas (dry subtropical area in northeastern Mexico) in the summer season. The area is located at 23°44′06″N and 97°09′50″W, at an altitude of 323 m. The mean annual rainfall is 926 mm, and the average temperature is 24 °C; in the summer, a maximum temperature of 40 °C is recorded, with thermal sensations of 45 °C and a minimum temperature of 22 °C (^{INEGI, 2017}). These climatic characteristics are typical for the dry subtropics (ACw). During the present study the average, maximum and minimum temperatures were 28 °C, 40 °C and 22 °C, respectively.

Management and diets

All procedures involving animal care and management were in accordance with and approved by the Bioethics Committee of the College of Veterinary Medicine and Animal Science of the Autonomous University of Tamaulipas.

Two hundred, 1-day-old male Ross broiler chickens weighing 42 g on average were obtained from a commercial hatchery. Each treatment (diet) included 50 birds randomly assigned with five replications of ten animals each. During the entire experiment, birds were housed in 20 floor pens with wood shavings as litter. Twenty-four hours of light per day were provided during the entire trial. Each pen had an automatic drinker and a manually-filled feeder. Space allocation was at ten birds per square meter. Water and feed were offered ad libitum. Birds were vaccinated on day 7 of the trial against fowl pox (wing puncture) and against Newcastle (ocular) using the La Sota strain.

The chickens were raised following standard commercial practice. Two feeding phases were used: 1-21 and 22-42 days of age, for starter and finisher phases, respectively. There were four treatments (T) for starter and finisher diets crude protein (CP; %) levels: 21.0 and 18.1 (T1); 21.4 and 18.5 (T2); 21.8 and 18.9 (T3); 22.2 and 19.3 (T4), respectively. The diets of T2 corresponds to standard diets formulated according to the NRC tables; the T1 correspond a low-CP level, while T3 and T4 are had higher CP levels over the standard diet (T2). The CP increments are equally spaced from T1 to T4. Dietary CP was increased by augmenting soybean meal in the diet, with small adjustments to the amounts of sorghum grain and vegetable oil to maintain the same levels of apparent metabolizable energy across diets. All diets were prepared according to National Research Council (^{NRC, 1994}) poultry recommendations and are shown in Tables 1 and 2.

Body weight and feed intake were measured weekly on an electronic scale with a capacity of 40 kg (±2 g; Rhino Model Bar-8, México) and feed conversion ratio (FCR; feed intake, g/weight gain, g) was calculated. At the end of the trial, two birds per cage, selected at random, were sacrificed by cervical dislocation (Mexican official law NOM 033-ZOO-1995) for carcass determinations and accessing chemical composition of breast and thigh meat. Carcass weight without viscera was used to estimate hot carcass yield. Then the carcass was dissected for main cuts: breast, thighs plus drumsticks, wings, back, trimmed back fat, liver, heart, and gizzard. These weights were recorded on an electronic scale with a capacity of 10 kg (±1 g; Rhino Model BAPO-10, México). A sample of approximately 150 g from the left breast and thigh from each carcass was obtained and frozen (-20 ºC) in nylon bags for 7 days, and then, laboratory analyses were performed. the samples were thawed at 4ºC for 24 h and subsequently at room temperature until thawed. Muscle samples were analyzed for dry matter (oven at 105 °C for 24 h), ash (furnace at 550 °C for 3 h), ether extraction and crude protein (^{AOAC, 1990}).

Statistical analyses

A completely randomized design with four treatments and five replicates was used in the statistical analysis. Dietary crude protein levels were the treatments compared. For growth performance (weight gain, feed intake, and feed conversion ratio) the replicate was the average of all broiler chickens in each pen. For carcass evaluation and meat chemical composition, the replicate was the average of two birds (selected at random) per pen. The percentage of dressed carcass yield was determined as carcass weight (g)/live weight (g)). An analysis of variance with Tukey’s test for mean comparisons was applied. Statistical significance was declared at P < 0.05. For statistical analyses, the GLM procedures of SAS (2007) were used.

Growth performance

Results of productive performance of broiler chickens are shown in Table 3. For the starter phase (1-21 days), there was no effect (P > 0.05) of dietary CP concentration on weight gain. Broilers in T2 had lower (P < 0.05) feed intake than those in T1 and T3. Feed conversion in T2 and T4 was similar (P > 0.05) and was better (P < 0.05) than that in T3. For the finisher phase (21-42 days), there was no effect (P > 0.05) of CP level on weight gain. Broilers in T3 had lower feed intake and better feed conversion than those in T1 (P < 0.05). Broilers in T2 and T4 showed intermediate values for these variables. Over the 42-day feeding period, there was no treatment effect (P > 0.05) on weight gain. Broilers in T1 had greater (P < 0.05) feed intake than those in T2 and T3, whereas broilers in T4 showed intermediate values. Broilers in T2 and T4 had better feed conversion ratios (P < 0.05) than those in T1; broilers in T3 showed intermediate values for this variable.

Carcass characteristics

Carcass characteristics of broiler chickens are shown in Table 4. There was no influence of treatment on weights of carcass, breast, drumsticks plus thighs, wings, or back fat (P > 0.05). Carcass yield was higher (P < 0.05) in T1 than T3. Chickens in T2 and T4 showed intermediate values. Likewise, there were no effects (P > 0.05) of dietary CP on the weights of liver, heart, or gizzard.

Proximate composition of meat

Results of the chemical composition of broiler chicken breast and thigh meat are shown in Table 5. Breast meat had similar (P > 0.05) percentages of water, ash, and ether extract; however, crude protein was greater (P < 0.05) in breast meat of birds in T2 than in T3. Chickens in T1 and T4 showed intermediate values. Thigh meat between treatments had similar (P > 0.05) water and ether extract percentages. Crude protein was highest (P < 0.05) for T2 and lowest (P < 0.05) for T3. With respect to ash, treatments T2 and T3 had higher values (P < 0.05) than in T1.

Growth performance

Both the starter and finisher diets had increasing crude protein levels from T1 to T4. Broiler weight gain was not influenced by the treatment; however, birds of T1 exhibited increased feed intake that negatively influenced feed conversion. Animals in T2, T3, and T4 showed roughly similar feed conversion ratios. Reports on productive performance of broiler chickens have been variable when comparing different levels of dietary crude protein. ^{Houshmand et al. (2012)} observed better growth performance of broiler fed diets containing 23.4% and 20.2% CP for starter and finisher diets, respectively, than birds fed the low protein diets. ^{Abdel-Maksoud et al. (2010)} reported maximum body weight and feed efficiency in starter broilers at 21% CP, particularly when supplemented with essential amino acids; broilers fed 19% and 23% CP diets showed lower productive parameters. ^{Boonsinchai et al. (2016)} observed no effect on growth performance in broilers (1-42 days) when dietary crude protein had a small reduction (5%) from the recommended requirement; however, reductions of 10% and 15% negatively affected feed efficiency.

Contrary to the observations of the present study, ^{Ghazanfari et al. (2010)} observed that in broilers that incremental changes in dietary protein levels did increase feed intake and improved weight gain and feed conversion, compared with a low level of protein in the diet. ^{Jafarnejad et al. (2010)} also reported improved weight gain and feed conversion in broilers (1-21 days) with high dietary protein levels (23% vs. 21% CP). Using mathematical models, ^{Faridi et al. (2012)} found that 24.1% dietary CP was the optimal level for feed efficiency in starter broilers. With a meta-analysis, ^{Faridi et al. (2015)} established 24.5% CP in the diet as the optimal level for feed efficiency for starter broilers 1-21 days of age. However, ^{Laudadio et al. (2012a)} reported that CP protein levels at 18.5%, 20.5%, and 22.5% did not affect growth performance of broilers during hot temperature conditions. In a three-week study with broiler chickens raised under a tropical hot and humid environment, ^{Awad et al. (2014)} observed reduced growth performance of broilers fed low-CP diets.

Improved productive performance of broiler chickens has been observed when increased over NRC recommendation the levels of lysine (^{Oliveira et al., 2013}; ^{Bernal et al., 2014}; ^{Quadros et al., 2019}; ^{Zarghi et al., 2020}) and methionine plus cystine (^{Goulart et al., 2011}). In the current study, the increase in crude protein increased the lysine concentration in the diets over the NRC recommendations; however, the crude protein increase in diets did not have evident increase on the levels methionine plus cystine in the used diets over the NRC recommendations. It is possible that methionine plus cystine were not in the level required to optimize the productive performance of broiler chickens in the present study. The crude protein increase in diets was made by the increase in soybean meal that increased lysine; nevertheless, the increase in methionine plus cystine was less evident. Further research considering these adjust in amino acids should be made in this subtropical area of México, particularly in the summer when the average, maximum and minimum temperatures are 28 °C, 40 °C and 22 °C, respectively. Extra methionine supplementation has caused positive effects on health and antioxidant status of broiler chickens under heat stress (^{Liu et al., 2019}).

Carcass characteristics

In agreement with the results from the present study, ^{Mandal et al. (2010)} and ^{Oliveira et al. (2013)} reported that carcass characteristics and breast yields were not influenced by dietary CP level. Also, ^{Miranda et al. (2015)} did not report effects on carcass yield of broilers fed diets with different CP levels supplemented with L-valine and L-isoleucine; however, the proportion of abdominal fat was reduced without dietary supplementation of these amino acids.

Contrary to the information observed in the present study, ^{Gheorghe et al. (2013a)} observed greater carcass yields in broilers fed a diet with high vs. low protein levels. ^{Gheorghe et al. (2013b)} found that the increase in dietary protein did not affect carcass yield, but did improve breast and thigh yields and reduced abdominal fat. In agreement, ^{Laudadio et al. (2012b)} obtained higher muscle yields (breast and thigh) in broilers with a high protein diet than those with a medium protein or low protein diet. Likewise, ^{Hada et al. (2013)} and ^{Dehghani and Jahanian (2016)} reported reduced carcass and breast yields using low protein diets; however, abdominal fat was increased with low protein diets.

^{Dozier III et al. (2008)} recognized the importance of lysine in breast muscle formation; higher lysine levels than NRC recommendations promoted breast meat yield (^{Bernal et al., 2014}; ^{Zarghi et al., 2020}). In the current study all diets were supplemented with the same lysine level and covered the NRC recommendation and the increase in CP level in diets had small increases in lysine or methionine plus cystine. Further research with higher levels of these amino acids in diets for broiler chickens to establish optimum productive efficiency and carcass yield is necessary in the dry subtropics of Mexico.

Proximate composition of meat

Contrary to the observations from the present study, increased dietary protein levels in other studies boosted protein and reduced lipid deposition in broiler chicken muscle (^{Marcu et al., 2012}; ^{Marcu et al., 2013}). ^{Nagata et al. (2011)} reported that high dietary energy also requires adequate CP to ensure protein deposition in the muscles of birds. In contrast, ^{Gómez et al. (2011)} found no effect of dietary CP on carcass protein deposition, although low crude protein diets were supplemented with amino acids that were used for muscles growth in the birds.