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The Relationship Between Guanidino Acetic Acid and Metabolisable Energy Level of Diets on Performance of Broiler Chickens

This work aimed to investigate the effects on performance and carcass characteristics of broiler chickens of 2 levels of guanidino acetic acid (GAA) in a commercial form (CreAMINO®) and 4 levels of metabolisable energy. The eight dietary regimens were tested for starter (0-10 d), grower (11-22 d) and finisher (23-35 d) peri ods on a total of 200 male ROSS 308 broiler chickens in a factorial arrangements of treat ments (2 4), with experimental diet fed to 5 replicate pens with 4 birds each. The results revealed a positive effect for CreAMINO® supplementation on feed conversion ratio (FCR) for the starter, grower, finisher and cumulative periods (P<0.05, 0.05, 0.01 and 0.001, respectively); feed conversion was significantly improved in the treatment with CreAMINO® supplementation vs unsupplemented group. During the grower period, a significant difference in FCR was observed for metabolisable energy (ME) level (P<0.01). When examining the cumulative period of the trial (0 to 35 days of age), two-way interaction was significant for feed intake and body weight gain. Birds receiving the control level of energy, 75 or 50 kcal/kg lower ME than the control, consumed less feed when supplemented with CreAMINO®. Birds receiving 25 kcal/kg lower ME or the control energy levels gained more weight when supplemented with CreAMINO®. In summary, GAA (as CreAMINO) improved FCR of broilers in each period as well as cumulatively, while ME level also affected FCR. CreAMINO® supplementation to reduced-energy diets showed FCR improvements, while the best performance was detected for the diet with 50 kcal/kg lower ME.


Introduction
Metabolisable energy (ME) is considered as the standard measure used to describe energy requirements of the birds and dietary content of energy (National Research Council, 1994). The National Research Council recommended a level of ME of 3200 kcal/kg for broilers up to 56 days of age. However, according to Dozier et al. (2008) modern broilers consume less feed per unit of body weight gain and reach their target weights in fewer days due to genetic improvements compared to broilers used in previous years and as a result the level of nutrients has to be adjusted. The high cost of supplemental energy necessitates the optimisation of ME in the diets especially during the finisher period where the feed consumption is the greatest (Pesti et al., 2002). Nowadays, in commercial practices broiler diets contain 3000 kcal/kg in the starter and up to 3100-3200 kcal/kg in the grower/finisher. The dietary ME level is one of the major factors, which play an important role in regulating feed intake and feed efficiency in broilers (Lopez and Leeson, 2008).
In Saudi Arabia, there is a ban of animal byproduct use in the poultry industry, consequently lacking creatine as this semi-essential nutrient is only present in raw materials of animal origin (Ringel et al., 2008). Creatine is naturally synthesised mainly in the liver and kidney of avian from guanidino acetic acid (GAA). Guanidino acetic acid is synthesised in the liver and kidney from arginine and glycine then acted upon by the enzyme transamidinase and subsequently methylated by S-adenosyl-methione to creatine (Borsook and Dubnoff, 1940;Wyss and Kaddurah-Daouk, 2000). The need for creatine is age-dependent, higher amounts are needed by growing animals for muscle growth vs adults (Brosnan et al., 2009). Furthermore, it has an important role as an energy carrier in the cells (Walker, 1979). Guanidino acetic acid is considered the only immediate precursor for creatine in the body of animals; moreover, it is more stable and less expensive than creatine and it could be a good additive for poultry (Wietlake et al., 1954). However, the amount that is synthesised could be insufficient to meet the demand of the fast growing broilers and therefore it has the potential for being supplemented to broilers since it plays an important role in the cellular energy metabolism (Lemme et al., 2007b). Recently, positive effects on growth performance were noticed when broilers were administered GAA. The transformation of GAA into creatine was evidenced by an increase in creatine levels (in serum and muscle) (Lemme et al., 2007a(Lemme et al., , 2007bRingel et al., 2008;Michiels et al., 2012;Carvalho et al., 2013). Moreover, GAA could be beneficial in broiler diets because it may be able to spare arginine, which is considered to be the fifth limiting amino acid in typical corn-soybean diets for broilers (Baker, 2009;Waguespack et al., 2009). However, there is a lack of information about a possible interaction between GAA and ME in broilers.
Therefore, in the present study, the objective was to evaluate the efficacy of supplementing GAA (supplemented as CreAMINO ® ) to a typical corn soybean meal diets with graded levels of ME on performance and carcass characteristics of broiler chickens form 0 to 35 days of age.

Animals, husbandry and treatments
A total of 200, one-day-old Ross 308 male broiler chicks were obtained from a commercial hatchery (Al-Wadi Poultry Farm Co., Riyadh, Saudi Arabia) and grouped by weight in such a way as to reduce variation in mean body weight. Then chicks were allotted into 40 experimental cages with four chicks per cage (50 cm length, 60 cm width and 36 cm depth) in a four-deck cage system and received the experimental diets in electrically heated battery brooders with raised wire floors. Environmental temperature in the first week of life was kept at 35°C and then decreased to 22°C until the end of the experiment. The chicks were vaccinated against Marek's disease, Newcastle disease and infectious bronchitis. The growth experiment was carried out from 0 to 35 days of age and samples were obtained at the end of the trial. Feed and water were provided ad libitum and birds were maintained a 24-h light schedule. The study was conducted under a protocol approved by King Saud University and complied with the current laws of Saudi Arabia. Typical starter (0-10 d), grower (11-22 d) and finisher (23-35 d) diets based on corn-soybean meal were formulated which met or exceeded the recommendations in commercial practice in Saudi Arabia except for metabolisable energy for each period and provided in mash form. The control diets contained 3000, 3075 and 3150 kcal/kg for starter, grower and finisher, respectively (Tables 1-3). The increments of ME were achieved by lowering the amount of oil added to the diets. Chicks received one of 8 treatments for each growing period (starter, grower and finisher) as follows: i) 75 kcal/kg less ME than the control; ii) 50 kcal/kg less ME than the control; iii) 25 kcal/kg less ME than the control; iv) commercial ME level (control); v) 75 kcal/kg less ME than the control+0.06% CreAMINO ® (CreAM); vi) 50 kcal/kg less ME than the control+0.06% CreAM; vii) 25 kcal/kg less ME than the con-trol+0.06% CreAM and viii) commercial ME level+0.06% CreAM. All the chemical analyses for the experimental diets were performed by Evonik Industries AG Feed Additives, Hanau, Germany. The metabolisable energy content of the diets was based on calculation.

Measurements
Measurement of body weight (BWG), feed intake (FI) and feed conversion rate (FCR) were taken weekly. Mortality was checked daily and weights of dead broilers were used to adjust FCR (mortality-corrected FCR). At day 35, seven birds per treatment were selected for calculation of dressing percentage and parts yield. After euthanasia, feather, heads and shanks were removed, and the remaining carcasses were dissected to breast and leg (thigh and drum stick) and were weighed. The percentage of yield of each part was calculated on the basis of dressed weight.

Statistical analysis
Data were analyzed by using the general linear model procedure of SAS (2002) for randomised complete block design with 4 2 factorial arrangements of treatments, in which each experimental diet was fed to 5 replicate pens. The data were tested for the main effects of energy level (4 levels), CreAM (with or without) and for interaction effect for energy CreAM. The experimental unit was the pen mean. Orthogonal contrasts (linear, quadratic and cubic) were used for further investigating the significant energy level in cases where the term was significant. The overall level for statistical significance was set at P<0.05. All values were expressed as statistical means±standard error of the mean (SEM).

Feed consumption and efficiency
Data related to chick's performance such as FI, BWG and FCR for starter, grower, finisher and cumulative periods are presented in Tables 4-7. Table 4 shows the performance results for the starter period (0 to 10 days of age). No significant differences in BWG, FI and FCR were found related to the ME level of the diet, which may be explained by the small quantity of feed consumed during the first ten days. However, a significant difference in FCR was observed due to CreAM supplementation (P<0.05), birds, which had received CreAM converted feed to body weight more efficiently as compared to the other group (1.45 vs 1.51). However, Stahl et al. (2003) demonstrated that creatine monohydrate did not significantly affect FCR of broilers during weeks one and two. When examining the chemical analyses of the diets used in this period, it was noted that diet which contained 50 kcal lower ME had lower methionine and lysine content due to unknown reason. Table 5 shows the performance results for the grower period (11 to 22 days of age). A significant two-way interaction (CreAM energy) was observed for FI (P<0.05), chicks consumed less feed at all levels of energy when diets were supplemented with CreAM except at the level of 3050 kcal/kg. Chicks in this group consumed more feed when the diet was sup- However, there was a trend due to CreAM on BWG (P=0.06), such that birds received CreAM gained 39 g more body weight as compared to the group, which did not receive CreAM. As a result, birds which had received the diets supplemented with CreAM had a better FCR (P<0.005) as compared to the other group (1.71 vs 1.80, respectively). This is in line with observations of Stahl et al. (2003) who found improvement of FCR from weeks three to four after creatine monohydrate supplementation, they linked that improvement to muscle cell hydration and increase in weight gain as GAA plays a significant role in muscle tissues in animal tissue. When examining the cumulative period of the trial (0 to 35 days of age) (Table 7), two-way interactions were significant for FI and BWG (P<0.05 and P<0.05, respectively). Birds, which had received the control level of energy, 75 and 50 kcal/kg lower ME than the control consumed less feed when these diets were supplemented with CreAM as compared to the unsupplemented diets. On the contrary, CreAM supplementation to the diet which had 25 kcal/kg less of ME increased feed intake by 144 g. On the other hand, birds which had received diets lower in 75 or 50 kcal/kg ME gained less weight when these diets were supplemented with CreAM as compared to the other groups. Birds, which received (25 kcal/kg) lower ME or the control energy levels had gained more weight when these diets were supplemented with CreAM. However, the magnitude of BWG  change was the greatest for birds which had received (25 kcal/kg) lower energy as compared to all other levels of energy (195 g difference between CreAM supplemented and unsupplemented group). Feed conversion ratio was influenced by the energy level and the CreAM (P<0.05 and P<0.001, respectively). A cubic trend for energy level on FCR was observed (P<0.05). Birds, which received the control energy level had the best FCR followed by those received 50, 25 and 75 kcal/kg less ME. This could explain the cubic trend. Supplementing the diet with CreAM improved FCR by more than 7 points as compared to the unsupplemented group (1.61 vs 1.68, respectively; P<0.0001). Ringel et al. (2008) showed similar weight gain in birds which had received vegetable diet that was supplemented with CreAM and those which received meat and bone meal diet for the period from 14 to 35 days. However, FCR was improved to a higher degree in birds which had received CreAM supplemented diet. Positive improvements in broiler's performance due to CreAM supplementation were reported by Lemme et al. (2007a); however, a hierarchy of GAA requirements was reported in their experiment. Males requirement for maximum gain was lower than that for FCR (0.08 vs 0.12%, respectively) for both starter and grower periods while, best performance for females was achieved at 0.12 % dietary GAA for the starter (0 to 21 days) and 0.08% for the grower period (22 to 42 days of age). Energy is an expensive component of broiler's diet due the relative high price of inedible fats. Dietary energy content influences the intake of all other nutrients; however, broilers display an exceptional ability to control their energy intake by regulating their feed intake as diet energy concentration changes (Lopez and Leeson, 2008). Saleh et al. (2004) reported that decreasing the amount of energy in the diet decreases growth rate and feed efficiency, however their experiment involved a wider range of ME (270 kcal/kg) as compared to this study (75 kcal/kg). However, in this trial ME content of the diet was changed in small increments (25 kcal/kg). The results are in line with those obtained by Noy and Sklan (2002). Feed intake was not influenced by the ME level of the diet, implicating that the energy requirement may not have been low enough to affect feed intake.

Carcass characteristics
The mean percentage of carcass parts is documented in Tables 8 and 9. Energy level had a significant effect on leg and thigh percent (P<0.05), both leg and thigh responded to the level of energy in a cubic trend (P<0.05). CreAM, on the other hand, had no influence on all parameters measured except for liver percent, CreAM supplementation decreased liver percent from 3.07 to 2.84% (P<0.05). Previous studies that involved creatine supplementation to swine (O'Quinn et al., 2000) and broilers (Stahl et al., 2003) found that dietary treatment had little to no effect on dressing percentage or fat deposition.

Conclusions
Guanidino acetic acid improved FCR for each growing period and for the cumulative period for broilers. On the other hand, dietary ME level could be adjusted in small increments to lower the cost of feed without losing performance. Based on that, it is recommended to lower the ME level by 50 kcal/kg for each growing period and supplement diets with CreAM.