Impacts of dietary metabolizable energy content and limiting amino acids concentrations in low crude protein corn-soybean meal-based diets for broilers
Abstract
This study was conducted to investigate the effect of feeding different level of metablolizable energy (ME) in low crude protein (CP) and low essential amino acids (EAA) diets, supplemented by four limiting amino acids (LAA) including methionine+cystine, lysine and threonine on growth performance for different rearing phases (starter, grower and finisher), production index, nutritional profit, carcass and bone characteristics of broilers from 1 to 38 d of age. Blood metabolites, mortality, incidence of ascites and sudden death syndrome (SDS), relative weight of heart and right ventricle were measured in two steps (1-24d and 25-38d). Four experimental corn-soybean meal diets used in this experiment for starter, grower and finisher periods with following formula: T1 = 〖”ME” 〗_R + 〖”CP” 〗_R + 〖”EAA” 〗_R + 〖”LAA” 〗_R; T2 = 〖”ME” 〗_D + 〖”CP” 〗_D + 〖”EAA” 〗_D + 〖”LAA” 〗_(D ); T3 = 〖”ME” 〗_D + 〖”CP” 〗_D + 〖”EAA” 〗_D + 〖”LAA” 〗_R and T4 = 〖”ME” 〗_R + 〖”CP” 〗_D + 〖”EAA” 〗_D + 〖”LAA” 〗_R (D subscript stands for diluted with minimum 93% and R subscript stands for recommended with 100% of Ross 308 requirements for starter, grower and finisher periods). The results of this study indicate that there were no significant differences between T1 and T4 in feed intake, body weight gain and feed conversion ratio from 1 to 38 d of age. Highest live body weight and lower price due to lower CP level in compare to control diet resulted in better economical index in T4. Total mortality and SDS were significantly (P<0.05) higher in birds received T1 than those fed T2 and T3. Cholesterol and triglyceride levels were highest in T2 (P < 0.05) when low CP and ME levels were not supplemented by LAA. In conclusion, the results of this study showed that feeding low CP diet supplemented by ROS 308 recommend level of ME and LAA result in the same growth parameters, improved economical index and lower mortality in compare to ROSS 308 recommended level for ME and CP.
Key Words: Ascites, carcass trait, crude protein, economical index, limiting amino acids
Introduction
Protein is considered as one of the main costly components in poultry feed. During last years, there have been many reasons including progress in synthetic amino acid production, environmental pollution of nitrogen and feed cost to reduction of dietary crude protein (CP) level (Ferket, et al., 2002; Powers and Angel, 2008) . Numerous dietary strategies have been used to reduce the dietary CP concentration in poultry nutrition. In most of recent researches, nutritional strategies employed to address CP reduction in poultry diets have focused on amino acid (AA) composition, which is suggested that use of synthetic amino acid reduces the environmental pollution of nitrogen and, decreases the cost of diet (Al-Mayah, 2006; Corzo, et al., 2009b; Moran Jr, et al., 1992). However, there are some economical boundaries on how a nutritional strategy can cause an increase in level of one or some nutrients and this additional level might not be economically reasonable (Basurco, et al., 2015).
The low CP AA-supplemented diets are one of the main strategies for reducing CP concentration. Adjustment in essential AA to meet or exceed the NRC (1994) or commercial strains recommendations (Alagawany, et al., 2014; Bregendahl, et al., 2002), and addition of non-essential AA (Corzo, et al., 2005) are most considered challenges to improve broiler growth performance when feeding low CP diets to broilers. Among ten essential amino acids (EAA), emphasis of many researches was on main limiting AA [methionine + cysteine (Met+Cys), lysine (Lys) and threonine (Thr)] due to their crucial impacts on growth performance. In most commercial diets, the total sulfur amino acids (TSAA) met+cys are first limiting AA (Fatufe and Rodehutscord, 2005), which makes it essential to supplement the diet with synthetic methionine for better performance and economical diets. Al-Mayah (2006) showed that addition of methionine may reduce the concentration of crude protein without a noticeable change in diet cost. Similar improvement in feed efficiency was also observed in reduced CP diets with addition of varying levels of TSAA (Powell, et al., 2009). Cys can be considered as a non-essential amino acid, because it can be supplied via transsulphuration of methionine (Pillai, 2005). Lys is known as second limiting amino acid in broiler diets and considering its function as a reference amino acid in ideal protein concept has a crucial impact to support optimum growth of fast-growing commercial broilers. Waguespack, et al. (2009) concluded that, as long as crystalline dl-Met, Thr , and Glycine (Gly) are supplemented and are not limiting, supplementation of 0.25% L-Lys-HCl to a corn-soybean meal-based (CSBM) diet will support broiler growth performance equivalent to broilers fed a control or commercial CSBM diet with 22%. In addition to its utilization for protein synthesis, Thr has active influence in many biological functions such as gut integrity and immunity. Sigolo, et al. (2017) showed that Thr inclusion at 10% more than Ross recommendations in the low protein diet improved average daily gain (ADG), average daily feed intake (ADFI), eviscerated carcass weight, and consequently carcass yield. Moreover, Thr inclusion improved serum lipid profile. Furthermore, many researches have been conducted to evaluate the effect of other essential amino acids (EAA) in the low CP diets. Corzo, et al. (2005) showed that broilers fed a low CP diet (18%) supplemented by Gly or Isoleucine (l-Leu) had equal Feed conversion ratio (FCR) to that of broilers fed a control diet with higher CP concentration (22%). Dean et al. (2006) reported that broilers fed the low CP AA-supplemented diets had growth performance equal to broilers fed conventional corn-soybean meal-based diets as long as crystalline Gly was supplemented to provide 2.32% total Gly + Serine (Ser). However, experimental diet (contained 160g CP/kg) which was used by Dean, et al. (2006) cannot be considered as a practical commercial diet because they used additional crystalline AA other than the commercially available AA. Whereas Aletor, et al. (2000) demonstrated that there is no significant effect in performance parameters between broiler chicken in grower phase fed decreasing dietary CP from 225 to 153g kg-1 and when such diets are supplemented with EAAs to meet the minimum NRC requirements.
Dietary energy content might affect AA utilization pathway and consequently might alter total digestion and absorption of AA reference book. It has been proven that energy to protein ratio is one of main factors in CP metabolism, and also has remarkable effect on optimal FI, ADG, FCR and nutrients digestibility (Hidalgo, et al., 2004; Kamran, et al., 2008; Moosavi, et al., 2011). The gastrointestinal tract consumes approximately 20% of all dietary energy to support digestive and absorptive processes (Vaugelade, et al., 1994). Therefore, energy availability for small intestine also affects the supply of other nutrients (Cant, et al., 1996). Moreover, role of amino acids concentrations in controlling feed intake (FI) can be another considerable part of dietary energy and energy concentration relationship with AA (Classen, 2013). However, there are a limited number of reports looking at the influence of different level of metabolizable energy (ME) on amino acid absorption while CP concentration is diluted. Zhai, et al. (2014) reported that low apparent metabolizable energy (AME) and high AA density diets significantly decreased FI of broiler and their body weight (BW) on d 35, 42, and 54.
On the other hand, one of common problem at high altitudes (more than 1000m) is elevated ascites incidence which is a cardiovascular metabolic disorder (Cisar, et al., 2005; Khajali, et al., 2007). Right ventricular hypertrophy (RVH) and ascites are a response to increased workload by the right ventricle as the result of atmospheric oxygen and PH (Izadinia, et al., 2010). Farms which are located in high altitude, approximately 1000m above sea level, may increase mortality due to the incidences of ascites (Beker, et al., 2003). There are few researches to evaluate the impact of low CP diets on productivity and ascites incidence in broiler chickens in high altitude. Most researches have focused on effects of some essential amino acids on incidence of ascites. Izadinia, et al. (2010) indicated that dietary protein source was associated with pulmonary hypertension and ascites in broiler chickens reared under cold stress at high altitude. They showed that broiler fed diets including canola meal protein had higher rate of ascites incidence compared to broilers fed soybean meal protein. In addition, there is some evidence about effect of AA supplementation on ascites incidence. Some researchers showed that broilers fed diets supplemented with high concentrations of tryptophan, an essential amino acid and precursor for serotonin, developed higher pulmonary arterial pressures than broilers fed diets containing adequate concentrations of tryptophan (Kluess, et al., 2012).
Despite many studies had been conducted for evaluating the effect of low CP diet on broiler performance, very few studies have been done comparing the different ratio of ME:AA for broiler chickens growth and body composition in different LAA concentration. Also, previous studies did not report economic considerations based on diet cost or chicken meat prices. Our hypothesis is that the effect of AME concentration on performance is more pronounced with low CP LAA-supplemented diets. The objective of this study was to determine the influence of low dietary protein and LAA concentrations at the different level of ME in CSBM diets fed to birds from 1 to 38 d of age and also effect of low CP LAA-supplemented diets on growth performance, and incidence of ascites in high altitude. Growth performance, total mortality and the incidence of ascites and SDS, production index and nutritional profit, carcass characteristics, weight of heart and right ventricle, relative weight and relative length of intestine, bone characteristics such as breaking strength and bone ash, and serum parameters were all determined, and these measurements were examined to determine the differences between experimental diets.
Material and Methods
Birds and husbandry
Three hundred and sixty day-old male Ross 308 broiler chicks were randomly distributed in groups of 15 birds in 24 litter-floored pens (1.5×1.5 m). Mean body weight of the chicks in all pens was similar (39±0.5g). Birds were housed in an environmentally controlled room and given ad libitum access to water and mash diets and exposed to a 23L: 1D cycle as lighting program. Room temperature was kept at 32◦C during the first 3 d of life and then, it was reduced gradually according to age until reaching 22◦C at 24 d of age, and maintained until d 38.
Experimental diets
Broiler response to dietary CP and ME with different EAA supplementation and two levels of LAA were evaluated from 1 to 10 d (starter period), 11 to 24 d (grower period) and from 25 to 38 d of age (finisher period). The treatments consisted of diets containing 2 different dietary CP levels, 〖”CP” 〗_D and 〖” CP” 〗_R (D subscript stands for diluted with 93% and R subscript stands for recommended with 100% of Ross 308 requirements for starter, grower and finisher periods; Ross, 2014) and 2 dietary ME concentrations, 〖”ME” 〗_D and 〖”ME” 〗_R with combination of 2 dietary essential amino acid concentrations, 〖”EAA” 〗_D and 〖”EAA” 〗_R. Also, diluted EAA diets were divided into two types, first type contains of recommended concentrations of LAA 〖”(LAA” 〗_R) and second type with 7% diluted concentrations of LAA 〖” (LAA” 〗_D).
Dietary CP concentrations to meet 93 and 100% of Ross 308 recommendations were respectively 22.15 and 23.80% (to meet the minimum requirement for valine, it was needed to raise CP content in starter phase from 23 to 23.8%) of the diet for starter period, 20.25 and 21.5% for grower period, 18.5 and 19.5% for finisher period. Experimental diets were formulated to meet or exceed the nutritional requirements of broilers as suggested by Aviagen (Ross, 2014) for starter, grower and finisher periods. Ingredients, chemical composition and energy concentration of the experimental diets for starter, grower and finisher periods are set out in the Table 1, 2 and 3, respectively.
Four experimental diets used for this experiment for starter, grower and finisher periods with following formula:
T1= 〖”ME” 〗_R + 〖”CP” 〗_R + 〖”EAA” 〗_R + 〖”LAA” 〗_R
T2= 〖”ME” 〗_D + 〖”CP” 〗_D + 〖”EAA” 〗_D + 〖”LAA” 〗_D
T3= 〖”ME” 〗_D + 〖”CP” 〗_D + 〖”EAA” 〗_D + 〖”LAA” 〗_R
T4= 〖”ME” 〗_R + 〖”CP” 〗_D + 〖”EAA” 〗_D + 〖”LAA” 〗_R
Growth performance and Production index
Body weight and feed intake were measured on d 10, 24 and 38, respectively at the end of the starter, grower and finisher period. On the same days, ADFI, ADG, and FCR were calculated and corrected for mortality. Production index (PI) and Nutritional Profit (NP) were calculated using the following formula:
Production index=(livability in percentage × (weight gain + body weight at 1 d in kg))/(period length in days × FCR)×100
Nutritional profit=[(live body weight at 38 d in kg×body weight price in dollar/kg) – (total feed consumed in kg×feed price in dollar/kg)] per bird
Mortality
All floor pens were checked daily for mortality, and dead birds weighed and examined to identify the cause of death. If they had normal appearance (bird was not sick) and well-grown body for age, crop fully filled with feed, empty gall bladder, and no clinical sign of other disease, their death was documented as the result of SDS (Hulan, et al., 1980; Ononiwu, et al., 1979). A diagnosis of clinical ascites was based on the following criteria: lethargy and reluctant to move, dark red combs signifying cyanosis, and the presence of excess fluid in the abdomen as felt by palpation (Beker, et al., 2003). On d 39, 3 birds per pen (18 birds per treatment) was randomly slaughtered, then hearts were removed, atria, major vessels and gross fat were cut off and the right ventricle (RV) was cut away from the left ventricle and septum. The RV was weighed and RV/BW and RV/HW ratios were computed.
Carcass traits
On the last day of the experiment (d 39), 3 bird per pen was chosen randomly, weighed, and slaughtered to evaluate carcass traits. The weights of the carcass (defeathered) with and without head and drumsticks were recorded. Carcass parts (breast, thigh and drumsticks), abdominal fat and internal organs (liver, pancreas and spleen), gut tracts (duodenum, jejunum, ileum, cecum and colon) were weighed and expressed as relative weight (as a percentage of live weight and eviscerated carcass).
Bone characteristics
Tibia and tarsometatarsus samples were obtained for more laboratory analyzes (18 birds/ treatment). The tissue was stripped off the bone, and the tibias were dried overnight at 100° C, defatted using 6 h ether extraction and ashed in a muffle furnace for 15 h at 540°C. Tibia ash (TA) percent was calculated as [TA weight/dry defatted tibia weight] × 100. Tarso-metatarsus bones have been used for bone-breaking strength. Bone-breaking strength was measured using an Instron universal testing machine (STM-250- SANTAM) with automated materials test system software version 4.2. The deformation rate was 5 mm/min. Tracing of force was recorded at a constant rate. The graphs showed plateau curves of maximal force (N) reached to measure of the energy stored in the bone.
Blood samples
On 24d and 38d, blood samples were collected from the brachial vein of 3 birds per replicate. The collected samples were kept on ice and centrifuged at 3000g for 10 min at 4°C. Plasma samples were stored at −80°C until analysis. The frozen plasma samples were thawed and plasma concentrations of albumin, uric acid, triglyceride, cholesterol and total protein were measured using commercially available kits (Pars Azmoon, Tehran, Iran) according to Tietz, et al. (1995).
Statistical analysis
The data were analyzed as a completely randomized design with 4 dietary treatments using the GLM procedure of SAS (2003). Experimental unit was replicate pen (n = 6) for all parameters. Differences between treatments were analyzed using Fisher’s least significant difference (LSD) and differences among treatments were considered significant at P < 0.05.
Results and discussion
To our knowledge, there are no reports in the literature of similar experiments and all similar experiments used constant level of ME in different level of EAA or LAA. Therefore, the purpose of our research was to determine the optimum level of ME into low CP CSBM diet using Met+Cys, Lys and Thr as limiting amino acid that would result in no negative effect on growth performance. Also, focus of earlier researches was on ME: CP ratio, but results of this study shows that there might be relationship between ME: AA ratio.
Growth performance
Effect of dietary treatments on growth performance in each phase is shown in Table 4. During d 1 to d 10 period, performance traits was significantly influenced by experimental diets. Diluted CP and EAA diet with lower ME level (T2), resulted in reduced ADG, and increased FCR. Adding synthetic LAA to low CP and ME diets resulted in significantly improved growth performance compared to diet 2 in all rearing periods. There was no significant difference for FCR between control and T4 groups in the grower and finisher phases. At overall (1–38 d), reducing the ME content of the diet negatively affected ADG of broiler chickens (P < 0.05). On the other hand, reducing the CP content of the diet by 7% of strain recommended level did not affect ADG and FCR of chickens when low CP diets were combined by 〖”ME” 〗_R + 〖”EAA” 〗_D + 〖”LAA” 〗_R at overall.
In general, reducing the level of protein in the diet without AA supplementation reduced the productive performance of the birds. Supplementation of low CP diets by LAA could improve growth performance of broilers. Results of this experiment showed that LAA supplementation in presence of higher level of ME has more impact on growth performance of broilers fed the low CP diets. Also, according to results of this study we can suggests that ADFI can be significantly altered by the LAA balance of a diet in Starter phase. Finding of this study support the hypothesis of Lipstein, et al. (1975) that birds will “overeat” in an attempt to consume amounts of limiting EAA required for maximum growth. Results of our finding is in line with other researches that reported that broilers fed an lower CP diet with supplemental LAA had feed conversion equal to that of broilers fed a control CP diet (Corzo, et al., 2005; Dean, et al., 2006). Whereas other researchers indicated that Supplementation of low CP diets with synthetic EAA to the amounts recommended by the NRC resulted in a significantly lower body weight, BWG and a poorer FCR compared with birds fed on the control diet (Aftab, et al., 2006; Deschepper and DeGroote, 1995).
Some researchers have shown that ME levels can have influence on Lys digestibility (Rutherfurd, et al., 2007) and also suggested that in low CP diets formulated to reduce production costs, higher concentrations of dietary Thr can compensate for marginal concentrations of some EAA (Corzo, et al., 2009a). Batal and Parsons (2002) Showed that an increase in digestibility of the cornstarch and soybean oil caused increased MEn of the crystalline amino acid diet. One possible explanation for better performance of birds fed low CP diet, supplemented by higher level of ME can be improvement of digestibility of LAA and also non-supplemented EAA by higher level of ME in this experiment.
Production index
Effect of dietary treatments on body weight, production index and nutritional profit of are presented in Table 6. Interestingly, the highest live body weight belongs to birds in T4 which fed low CP and EAA diet with recommended concentration of LAA and ME. Because of highest live body weight and lower price due to lower CP concentration in compare to control diet, highest production index and nutritional profit were obtained from T4 (P < 0.05). Lower price for low CP and EAA diets in T2 and T3, could not result in better production index and nutritional profit because of major effect of these diets on growth performance. The profitability can be enhanced when the profits and cost are equally considered in broiler diets. Calculated economical index in this study suggest that, although supplementing of low CP diet with LAA and higher ME level can increase the price of diet/kg-1 in compare to non-supplemented low CP diets, compensation in growth performance results in higher income.
Jiang, et al. (1998) showed that at the commercial broiler, the base of evaluation of economic value is per marketable broiler bird. Body weight, feed consumption, and mortality are three important production traits in their evaluation model. Higher body weight at harvesting day gives higher economic values of carcass yield and breast meat, but leads to lower economic values of feed consumption and mortality. Results of this study is in line with report of other researchers which had shown that feeding low CP AA-supplemented diets confers potential economic advantage (Aletor, et al., 2000).
Mortality
There was no significant effect for total mortality and mortality caused by ascites and SDS in starter and grower periods (Table 5). Increases in total mortality and mortality from ascites and SDS were observed by different concentration of CP, ME and AA in the diets for finisher periods (P < 0.05). Total mortality and mortality from SDS were higher in birds fed recommended concentration of CP, ME and AA (T1) than those fed low CP diets (P<0.05). Birds fed low CP, AME and EAA with recommended level of LAA (T4) had the lowest level of total mortality (P<0.05). Buys, et al. (1998) concluded a higher protein content of the feed reduces the incidence of triiodothyronine (T3)-induced ascites mortality and this could be due to different causal mechanisms. They also showed that the initial growth-depressive effect of intermittent lighting schedule was more pronounced in birds fed a normal protein ration (23.5% CP) than in those receiving feed with a subnormal protein content (20.5% CP). Furthermore, It has been proven that high energy diets can raise the incidence of SDS and ascites (Druyan, 2012; Leeson, 1995). It was expected that incidence of SDS and ascites would be more in birds with higher BWG and higher growth performance. Our results are in line with finding of other researchers who reported that low CP diets caused reduction in SDS and ascites incidence (Buys, et al., 1998; Izadinia, et al., 2010). Noticeably, results of this experiment shows that low CP and EAA diets supplemented with LAA (T3 and T4), caused a significant reduction in SDS and total mortality in compare to control (P<0.05). Effect of dietary treatments on the relative weight of heart (HW) and right ventricle (RV) are in line with our finding in mortality causes. Relative weight of RV/BW or RV/HW in T1 and T4 were significantly higher that other groups (P<0.05). Feed a high protein or high energy diet leads to an increase oxygen demand in the growing broiler and consequently leads to a pressure overload in the right ventricle and, hence, right ventricular hypertrophy (Khajali, et al., 2007).
Carcass traits
Effect of dietary treatments on carcass characteristics is presented in Table 6. Dilution of ME and EAA in the diet caused reduced relative breast weight at 38 d (P < 0.05). Some researcher reported that high CP and AA density diets may increase meat yield, especially breast weight (Dozier, et al., 2008; Nahashon, et al., 2005) while others showed that there is no significant difference in carcass characters and breast yield in lowering of dietary CP or AA supplementation (Aletor, et al., 2000). There was no significant difference in relative weight of abdominal fat pad (AFP), liver, pancreas and spleen among dietary treatment. Because there was no major dilution of CP, ME or AA in this experiment, it is perhaps not too unexpected that neither carcass weight nor abdominal fat were affected by diet dilution. However, there are some evidences that show supplementation with synthetic AA for decreasing the total CP concentration of the diet results in greater fat deposition in commercial broilers because of less available surplus protein to be metabolized (Deschepper and DeGroote, 1995).
Intestinal tracts
Effect of dietary treatment on intestinal relative weight was not significant (Table 9). Intestine development can be affected by nutritional manipulation while chickens fed a higher nutrient density diet showed faster development in gastrointestinal tract throughout all growing phases (Nahashon, et al., 2005; Zhai, et al., 2013).
Bone parameters
Effect of dietary treatment on tibia ash content and minimum breaking strength of tarsometatarsus was not significant (Table 10). Skinner, et al. (1991) showed that the higher AA levels in their study supported normal linear growth of the tibia but influenced the rate of calcification. Also they concluded that in diets that are marginal in Calcium, higher AA levels may decrease bone calcification. Coto, et al. (2009) indicated that increasing levels of Lys improved toe ash and reduced total phosphorus in excreta but increased the water soluble phosphorus to total phosphorus ratio in excreta. Whereas Araújo, et al. (2006) reported that there were not interactions between the amino acids and calcium levels, and amino acid or calcium levels did not affect bone density and tibia variables in both strains.
Blood samples
The effect of CP and ME with different AA levels on plasma metabolites are shown in Table 11. In starter and grower periods, plasma albumin levels were affected by dietary CP and ME content, being lower (P < 0.05) in chickens fed the diluted AA and ME diet (T2 and T3). There was no significant difference between control diets and low CP supplemented by LAA diets for plasma albumin levels.
The level of total proteins and uric acid in the plasma of Chickens was not significant. The low CP diets with recommended ME and LAA levels (T4) resulted in higher blood serum concentrations of triglyceride in 24 and 38d (P < 0.05). The cholesterol level was highest in T2 (P < 0.05) when low CP and ME levels were not supplemented by LAA. The observed increase in the levels of cholesterol and total proteins in plasma could be related with higher lipogenesis rate in chickens fed increased ME to CP ratio diets (Rosebrough and Steele, 1985) .
Kamran, et al. (2010) indicated that birds fed on low CP diets preferentially use carbohydrates as an energy source rather than free fatty acids, resulting in higher plasma triglyceride levels. In our study, the reduction of the dietary CP content at higher ME level increased the blood serum concentrations of triglycerides. Cholesterol level was significantly higher in birds in group 2 those fed on diluted level of ME, CP and AA. Sigolo, et al. (2017) reported that the reduction of the dietary CP content at constant ME increased the concentrations of total cholesterol, HDL cholesterol and LDL cholesterol. These researchers have also showed that Thr supplementation reduced the concentrations of total cholesterol and triglycerides.