Titration of Dietary Histidine during a 22-to 42-Day Feeding Phase following a 0-to 21-Day Feeding Phase with Variable Dietary Histidine Concentrations in Female Cobb 500 Broilers

: Further reductions of crude protein in broiler diets may cause essential amino acids traditionally considered to be supplied at adequate levels to become limiting. Requirement data for histidine are currently scarce and this amino acid is uniquely able to be stored within the body. Thus, the objectives of this study were to evaluate female Cobb 500 broiler growth performance and carcass characteristic responsiveness to increasing digestible histidine to digestible lysine ratios (dHis:dLys) during a 22-to 42-day feeding period and determine if dHis:dLys in preceding feeds influence the former responses. Starter diets were formulated to contain dHis:dLys of 33% or 38% and grower diets were formulated to contain dHis:dLys of 28%, 33%, 38%, or 43%, which gave rise to a 2 × 4 factorial arrangement of treatments. No interactions ( p > 0.05) between the starter or grower diet dHis:dLys occurred. Increasing the dHis:dLys from 22 to 42 days resulted in body weight gain, feed conversion ratio, and total breast meat yield quadratic responses ( p ≤ 0.05). Additionally, it appears that dHis:dLys between 33% and 38% fed during the starter phase does not influence broiler responsiveness to different dHis:dLys during the grower phase.


Introduction
A reduction of intact protein coupled with the fortification of feed-grade amino acids in broiler diets has shown to be efficacious in lowering feed costs [1], reducing nitrogen excretion [2], and improving welfare via litter conditions [3].Currently, feed-grade methionine, lysine (Lys), threonine, and valine are routinely supplemented in commercial corn and soybean meal diets fed in the United States [1].As production advancements continue to improve the cost and availability of feed-grade amino acids, other amino acids (e.g., isoleucine, arginine, glycine, and tryptophan) could be allowed to enter matrices during least-cost formulation [4].However, to further reduce intact protein, several key factors will need to be addressed including having knowledge of less limiting essential amino acid requirements [5].
Histidine (His) is an essential amino acid that is typically in excess in traditional corn and soybean meal diets, but supplementation of feed-grade arginine could result in diets with His in a limiting position [6,7].Early investigation of the amino acid needs of broilers revealed that adequate dietary His was required to maintain growth [8], and recent studies have noted the importance of His for breast muscle development [9,10].Histidine also fulfills functional roles in non-structural proteins, such as in hemoproteins (e.g., hemoglobin and myoglobin) and serine proteases (e.g., trypsin, chymotrypsin, and elastase) [11,12].Aside from protein synthesis, His may be decarboxylated to form histamine, a signal molecule that aids in gastric acid secretion, neurotransmission, and immune responses [13].Moreover, His or its methylated analogs can be condensed with β-alanine to form histidinecontaining dipeptides, which are thought to serve as hydrogen ion buffers, antioxidants, and neurotransmitters [14].
Histidine's essentiality was once debated on the grounds that it appeared to be similar to that of conditionally essential amino acids, such as glycine.Amino acid deletion studies utilizing a variety of growing animals species concluded His to be essential [8,15,16], whereas short-term nitrogen balance studies conducted on adults of the same species concluded the opposite [17][18][19].Further research revealed that carnosine (β-alanyl-histidine) acts as a form of endogenous His reserves and in times of dietary His deficiency, may be catabolized for the provision of free His for higher priority functions that prolong survival [20][21][22][23].However, the magnitude of dietary His deficiency needed to induce this response has not been characterized in broilers so it remains unclear whether carnosine poses nutritional implications under practical conditions.
In an attempt to precisely meet essential amino acid requirements, broiler diets are commonly formulated using the ideal protein concept.When implementing the ideal protein concept, dietary concentrations of essential amino acids are formulated as ratios relative to the dietary Lys concentration [24].Previous studies have produced varying optimal digestible His to digestible Lys ratios (dHis:dLys) within three weeks post-hatch and include 32% [25], 36% [26], and 38% [10].Even less understood are the broiler's His needs beyond three weeks post-hatch and whether endogenous His reserves influence His needs later in life.Therefore, the objectives of this study were to evaluate broiler responsiveness to increasing dHis:dLys based on growth performance and carcass characteristics during a 22-to 42-day feeding phase and determine if different dHis:dLys fed from 0 to 21 days impact later responses.

Bird Husbandry
A total of 672 vent-sexed female Cobb MV × 500 broiler chicks were obtained from a commercial hatchery (Cobb-Vantress Inc., Siloam Springs, AR, USA) and transported to the University of Arkansas Poultry Research Farm to be reared in a solid-walled, tunnelventilated research house.Upon arrival, birds were group weighed and randomly allocated into 56 floor pens measuring 0.9 × 1.2 m with 12 chicks per pen (0.09 m 2 per bird).Each pen was equipped with a hanging feeder, a section of nipple drinker line, and built-up, topdressed litter composed of pine shavings.Feed and water access were provided ad libitum for the duration of the experiment.Water flow rates were set at 21 mL per minute and were increased by 7 mL weekly ending at 56 mL per minute according to Miles et al. [27].The initial house temperature was set to 32.8 • C and gradually decreased to 18.3 • C throughout the experiment.A lighting schedule of 24L:0D from days 0 to 1, 23L:1D from days 2 to 7, and 18L:6D from days 8 to 42 was used.Lighting intensity was gradually reduced from 32 to 5 lux throughout the experiment and was verified at bird level (Extech LT300 lightmeter, Extech Instruments, Waltham, MA, USA).

Experimental Design and Diets
This study entailed two subsequent feeding phases: a starter phase, which lasted from 0 to 21 days, and a grower phase, which lasted from 22 to 42 days.Starter diets were formulated to contain dHis:dLys of 33% or 38% and grower diets were formulated to contain dHis:dLys of 28%, 33%, 38%, or 43%.The 2 dietary treatments in the starter phase and the 4 dietary treatments in the grower phase gave rise to a 2 × 4 factorial arrangement.

Measurements 2.3.1. Live Performance
Mortality was collected twice daily and recorded.Cumulative body weights (BW) and feed consumption were collected at 0, 21, and 42 days and used to calculate individual BW gain, feed intake (FI), and feed conversion ratio (FCR) for each phase.Body weight gain was calculated by subtracting the average initial BW from the average final BW for each pen.Feed intake was calculated based on bird days as described by Greenwood et al. [30].Feed conversion ratio, represented as a gram of feed per gram of BW gain, was calculated and corrected for mortality by dividing pen feed consumption by the summation of pen BW gain and recorded pen mortality weight.

Blood Plasma and Muscle Tissue Analysis
At 21 and 42 days, 7 and 4 birds per treatment, respectively, were randomly selected for sampling of blood and Pectoralis major (breast filet).After recording each bird's live weight, 3 mL of blood were drawn from the brachial vein via syringe and transferred to sodium heparinized vacutainers (Becton, Dickinson and Company, 1 Becton Drive, Franklin Lakes, NJ, USA).Birds were then euthanized via cervical dislocation where, after verification of mortality, approximately 10 g of tissue were collected from the left cranial region of each breast filet.All samples were placed on ice and immediately transferred from the research house to the laboratory (University of Arkansas, Fayetteville, AR, USA).Blood was then centrifuged at 2000 RPM for 10 min and plasma was removed.Blood plasma and breast filet tissue samples were then stored at −80 • C until analysis.Blood plasma was analyzed for concentrations of L-histidine and β-alanine and breast filet tissue was analyzed for concentrations of carnosine (β-alanyl-histidine) and anserine (β-alanyl-Nπmethyl-histidine) (ATC Scientific, North Little Rock, AR, USA) (methods 982.300) [29].

Carcass Characteristics
After 42-day BW were recorded, 6 birds per pen were randomly selected and tagged for processing.On day 43, tagged birds were transported to the University of Arkansas Pilot Processing Plant following an overnight (10 h) feed withdrawal.Upon arrival, birds were individually weighed at the plant, electrically stunned (11 V, 11 mA for 11 s), and exsanguinated via jugular vein cut.Birds were then soft scalded (55 • C for 2 min) and de-feathered, and the head, neck, and feet were removed.Carcasses were then mechanically eviscerated via an evisceration machine (Baader Linco 1396, BAADER LINCO, Inc., Kansas City, KS, USA) and followed by the collection of peritoneal fat pads according to Waldroup et al. [31].Hot carcass and fat pad weights were recorded, and carcasses were chilled in ice water for 4 h.Chilled carcasses were weighed and deboned on a debone line to collect weights of breast filet, Pectoralis minor (tenders), wings, and legs (bone-in skin-on drumstick and thigh).Part weights for each bird were divided by the corresponding individual live BW to determine relative percentage yields for each part.Deboned breast filets were subjectively evaluated for woody breast myopathies.Breast filets were palpated by a trained individual to derive scores which were ranked on a whole number increment scale with 0 being no signs of woody breast, 1 being mild to moderate woody breast, and 2 being severe woody breast as described by Maynard et al. [32].

Statistics
Pen was considered the experimental unit for all analyses and dietary treatments were assigned to pens in a randomized complete block design with pen location serving as the blocking factor.Each of the 8 dietary treatment combinations were represented by 7 replicate pens.All data were analyzed using JMP Pro 17 (SAS Institute, Cary, NC, USA) statistical software package.Data for live performance from 0 to 21 days and 21-day blood plasma and breast tissue were subjected to a two-tailed t-test to assess the effect of the starter diet dHis:dLys ratio.Data for live performance from 22 to 42 days, 42-day blood plasma and breast tissue, and 43-day carcass characteristics were subjected to a two-way ANOVA to assess the main effect of the starter diet dHis:dLys ratio, the main effect of the grower diet dHis:dLys ratio, and their interactions.Means were separated using a Tukey's honest significant difference (HSD) test when appropriate.Additionally, all 22to 42-day live performance, 42-day blood plasma and breast tissue, and 43-day carcass characteristic data were subjected to regression analyses.The initial model fit included the linear term for the starter diet dHis:dLys ratio, the linear and quadratic terms for the grower diet dHis:dLys ratio, as well as any possible interactions.Data were considered significant at p ≤ 0.05 in all cases.

Results
Increasing the starter diet dHis:dLys from 33% to 38% resulted in minor numerical reductions (i.e., 20 g) for 0-to 21-day BW gain (p = 0.067) and 21-day BW (p = 0.064) for both parameters, albeit they were not statistically significant (Table 3).Feed intake and FCR were also uninfluenced (p > 0.05) by the starter diet dHis:dLys.Increasing the starter diet dHis:dLys from 33% to 38% increased 21-day plasma His concentrations (p = 0.003) by 67% and tissue carnosine concentrations (p = 0.017) by 79% (Table 4).Twenty-one-day concentrations of plasma β-alanine and tissue anserine were uninfluenced (p > 0.05) by the starter diet dHis:dLys.No interactive effect (p > 0.05) was found between the starter and grower diet dHis:dLys for any 22-to 42-day live performance (Table 5) or 43-day carcass characteristic (Tables 6 and 7) parameters assessed.Likewise, the main effect of the starter diet dHis:dLys did not influence (p > 0.05) any of those same parameters.The main effect of the grower diet dHis:dLys influenced 42-day BW (p = 0.017), 22-to 42-day BW gain (p = 0.014), and 22-to 42-day FCR (p = 0.003).Birds fed a grower diet dHis:dLys of 38% had an improved BW gain by approximately 178 g and FCR by 16 points compared to those fed a dHis:dLys of 28%.The main effect of the grower diet dHis:dLys also influenced total breast meat yield (p = 0.024) and peritoneal fat pad yield (p = 0.015).Birds fed a grower diet dHis:dLys of 38% had the highest total breast meat yield and was 0.86 percentage units higher than birds fed a dHis:dLys of 28%.Moreover, birds fed a grower diet dHis:dLys of 38% had a 0.19 percentage unit decrease in fat pad yield compared to those fed a dHis:dLys of 28%.
Forty-two day blood plasma amino acid and breast tissue dipeptide data are found in Table 8.The grower diet dHis:dLys main effect influenced plasma His (p = 0.014) and β-alanine (p = 0.045) concentrations.Birds fed a grower diet dHis:dLys of 43% had a 70% increase in plasma His concentrations and a 44% decrease in plasma β-alanine concentrations compared to those fed a dHis:dLys of 28%.Moreover, increasing the grower diet dHis:dLys from 28% to 43% resulted in a positive linear response (p < 0.001) for plasma His concentrations and a negative linear response (p = 0.007) for β-alanine concentrations.An interactive effect between the starter and grower diet dHis:dLys occurred for breast tissue concentrations of carnosine (p = 0.001) and anserine (p = 0.007).Tissue carnosine concentrations of birds fed a starter diet dHis:dLys of 33% were statistically similar regardless of the grower diet dHis:dLys.However, when birds were fed a starter diet dHis:dLys of 38%, tissue carnosine concentrations were higher in those fed a grower diet dHis:dLys of 43% compared to those fed dHis:dLys of 28% and 33%.Breast tissue anserine concentrations were less interoperable, but birds fed a starter and grower dHis:dLys combination of 38% and 33% had lower tissue anserine concentrations than those fed combinations of 33% and 33% as well as 38% and 43%.Additionally, a starter dHis:dLys linear × grower dHis:dLys quadratic response was observed for both breast tissue anserine (p = 0.003) and carnosine (p = 0.003).   1 Values represent least-square means of replicate pens with 6 birds selected per pen for processing at 43 days post-hatch. 2 Treatments identified by the digestible histidine to digestible lysine ratio (100%). 3Total breast meat (Pectoralis major + minor).a,b Means within a column that do not share a common superscript differ (p ≤ 0.05). 1 Values represent least-square means of replicate pens with 6 birds selected per pen for processing at 43 days post-hatch. 2 Treatments identified by the digestible histidine-to-digestible lysine ratio (100%). 3Breast filets with a score of 0 exhibited no hardness in the caudle region of the filet, breast filets with a score of 1 exhibited hardness in the cranial and caudle regions of the filet but remained flexible in the mid-region, breast filets with a score of 2 exhibited hardness throughout the filet.Increasing the grower diet dHis:dLys from 28% to 43% resulted in linear and quadratic responses for 42-day BW (p = 0.018 and 0.023, respectively), 22-to 42-day BW gain (p = 0.015 and 0.019, respectively), 22-to 42-day FCR (0.004 and 0.006, respectively), and day 43 total breast meat yield (p = 0.003 and 0.004, respectively).Additionally, increasing the grower diet dHis:dLys linearly decreased (p = 0.013) fat pad yield.Asymptotes for 42-day BW, 22to 42-day BW gain, and 22-to 42-day FCR were all found at a dHis:dLys of 37%, while day 43 total breast meat yield at a dHis:dLys of 36%.The requirement equations and estimates can be found in Table 9. Optimum dHis:dLys were calculated at 95% of the quadratic asymptote when a particular parameter elicited a quadratic response.Thus, BW gain and FCR were optimized at a dHis:dLys of 35% and total breast meat yield at 34%. 1 Prediction equation based on digestible histidine to digestible lysine ratio (100%). 2 Requirement estimated to be 95% of the maximum digestible histidine to digestible lysine ratio (100%) response. 3Pectoralis major + minor.

Discussion
The His needs of female broilers have not been previously characterized for any growth period, but Franco et al. [10] reported an optimal dHis:dLys of 38% for Cobb 500 male broilers from 8 to 17 days post-hatch.The current findings suggest that the His needs of female Cobb 500 broilers to achieve maximum live performance could be lower than that of their male counterparts.However, amino acid dose-response experiments typically utilize 5 to 7 treatments to estimate requirements [33].Therefore, a more in-depth investigation within 3 weeks post-hatch is warranted to obtain an accurate depiction of early His requirements for female Cobb 500 broilers.
Carnosine and anserine are abundant His-containing dipeptides found in chicken skeletal muscle.Carnosine is synthesized from the precursors His and β-alanine via carnosine synthase [34,35].Anserine appears to be primarily synthesized through carnosine methylation via carnosine N-methyltransferase [36][37][38][39].Several researchers have demonstrated that dietary His supplementation increases breast tissue carnosine concentrations, while concentrations of anserine respond variably [9,[40][41][42][43].In this study, day 21 blood plasma concentrations of His were significantly increased, while β-alanine approached significance and was numerically decreased.This suggests that feeding a higher starter diet dHis:dLys promoted the accumulation of breast tissue carnosine at 21 days.
An objective of this experiment was to determine if dietary His concentrations in earlier feeding phases altered responses to dietary His in later feeding phases.Although day 21 carnosine concentrations were significantly different between the two starter dHis:dLys groups, interactions between the starter and grower diet dHis:dLys did not occur for any 22to 42-day live performance or 43-day carcass characteristic parameters.It is possible that the lowest grower diet dHis:dLys (i.e., 28%) was not so deficient to generate a detectable response within a 3-week feeding period.The interactive effect between the starter and grower diet dHis:dLys approached significance (p = 0.100) for 22-to 42-day FCR where the largest difference (i.e., 14 points) was observed between birds fed a starter and grower diet dHis:dLys combination of 38% and 28% and birds fed a combination of 33% and 28%.However, reducing the dHis:dLys ratio below 28% while maintaining dietary conditions applicable to the industry could prove challenging.Thus, it appears that standard doseresponse experiments utilizing practical ingredients are adequate to assess the His needs of broilers in later growth periods.
Robbins et al. [40] determined that breast muscle carnosine did not begin to accumulate until the concentration of dietary His that resulted in approximately 95% of maximal growth was fed.Interactions between the starter and grower diet dHis:dLys occurred for both 42-day breast tissue carnosine and anserine concentrations.When birds were fed a starter diet dHis:dLys of 33%, carnosine concentrations were not statistically different among grower diet dHis:dLys.However, when birds were fed a starter diet dHis:dLys of 38%, a grower diet dHis:dLys of 43% promoted higher carnosine concentrations as opposed to 28% and 33%.If the starter diet dHis:dLys of 33% was marginally deficient of the true His requirement for maximal growth, dietary His during the grower phase may have been partitioned for other physiologic functions that support growth rather than carnosine formation.This supports the findings of Robbins et al. [40], which are that dietary His is prioritized for weight gain over carnosine formation.Regression models generated for day 42 carnosine and anserine suggest that their concentrations within breast tissue are dynamic depending on the dietary His status at a given age.Thus, factorial designs on a larger scale seem necessary to fully understand His dipeptide accumulation within the skeletal tissue of broilers.
Previous research by Dorigam et al. [26] reported an optimal dHis:dLys of 35% for male Cobb 500 broilers for a 22-to 37-day feeding period by implementing a nitrogen balance technique.The optimal dHis:dLys of 38% proposed by Franco et al. [10] and the results of the current study suggest that the optimal dHis:dLys for Cobb 500 broilers lies between 35% and 38%, with the differences in requirement estimates possibly arising from the tested age range and sex.Thus, the preliminary data presented herein gives a starting point for an investigation into His needs during later growth periods and will allow future researchers to further hone the specific His needs of broilers utilizing complete dose-response experiments.

Table 1 .
Calculated and analyzed composition of experimental starter diets fed to female Cobb MV × 500 broilers from 0 to 21 days post-hatch.

Table 2 .
Calculated and analyzed composition of experimental grower diets fed to female Cobb MV × 500 broilers from 22 to 42 days post-hatch.

Table 3 .
Live performance of female Cobb MV × 500 broilers from days 0 to 21 fed different digestible histidine to digestible lysine ratios in the starter phase (0 to 21 days).1 1Values represent least-square means for 28 replicate pens with 12 birds per pen at 0 days post-hatch.2Treatmentsidentified by the digestible histidine to digestible lysine ratio (100%).

Table 4 .
Blood plasma and breast tissue amino acid and dipeptide concentrations of female Cobb MV × 500 broilers on day 22 fed different digestible histidine to digestible lysine ratios in the starter phase (0 to 21 days).1

Table 5 .
Live performance of female Cobb MV × 500 broilers from days 22 to 42 fed different digestible histidine to digestible lysine ratios in both the starter (0 to 21 days) and grower (22 to 42 days) phases.1

Table 6 .
Carcass characteristics of female Cobb MV × 500 broilers processed on day 43 fed different digestible histidine to digestible lysine ratios in both the starter (0 to 21 days) and grower (22 to 42 days) phases.1

Table 7 .
Woody breast incidence and severity of female Cobb MV × 500 broilers processed on day 43 fed different digestible histidine to digestible lysine ratios in both the starter (0 to 21 days) and grower (22 to 42 days) phases.1

Table 8 .
Blood plasma and breast tissue amino acid and dipeptide concentrations of female Cobb MV × 500 broilers on day 42 fed different digestible histidine to digestible lysine ratios in both the starter (0 to 21 days) and grower (22 to 42 days) phases.1