Inﬂ uence of low-protein diet with different levels of amino acids on laying hen performance, quality and egg composition

: The present study aimed to investigate the consequences of dietary levels of crude protein (CP) and total sulfur amino acids (TSAA, Met+Cys) on productive performance, egg quality and egg components for Lohmann Brown laying hens through 18-34 weeks of age. A 2×3 factorial design experiment was conducted using two levels of CP (16 and 18 %) and three levels of Met+ Cys (0.67, 0.72 and 0.77 %). A total of 144 Lohmann Brown laying hens at 18 weeks of age were randomly divided into 6 treatment groups. The best values of fi nal body weight and body weight change were recorded by the hens fed 18 % CP diet. Increasing dietary CP up to 18 % accompanied with the best (P> 0.01) feed conversion, egg weight and egg mass. The combination between low-CP diet (16 %) and 0.72 % Met+ Cys gave the best percentage of egg production. The majority of egg quality traits were not signifi cantly altered by dietary treatments. The interaction between CP and Met+Cys levels had a statistical (P>0.01) impact on egg content of moisture and total solids only. It could be concluded that the best production criteria could be obtained by using 0.72% Met+Cys. Moreover, dietary level of 16 or 18 % CP with 0.72% Met+Cys is recommended for feeding Lohmann hens.


INTRODUCTION
The first an important step to optimize performance and production in poultry is proper nutrition. Increasing egg yield profi ts is mainly depending on fl ock performance, feed and egg prices as well as farm management (Alagawany et al. 2014, Abou-Kassem et al. 2018. Several factors affect productive performance of laying hens. Nutrient levels in the diet should be optimized not to exceed the optimum level in order to optimize profi ts. Improving or maintaining laying hen performance, or both, could be achieved by maximizing the utilization of nutrients from current feedstuffs (Abd El-Hack et al. 2015). Reduction in production costs is needed although laying effi ciency and high egg production are characteristics of the modern laying hen. Soybean meal and corn are the main ingredients providing crude protein and energy in commercial poultry diets (Alagawany et al. 2016). The amino acid methionine is now available at competitive prices. It is now possible to formulate poultry diets which provide a nonprotein nitrogen and minimum excess of amino acids (Alagawany et al. 2015). However, we are unable to formulate diets with very low levels of crude protein that contain optimum levels of essential amino acids (Zeweil et al. 2011). But it is possible to readily reduce crude protein supply by 15 -20% using synthetic amino acids supplements if its cost could be economic and applicable (Aarnink et al. 1993). New feeding strategies in poultry production gave a new perception with the advent of environmental problems which always come from the oversupply of nutrients, particularly protein and amino acids (Meluzzi et al. 2001, Torki et al. 2016. So the present investigation designed as an attempt to reduce nutrient oversupply through decreasing dietary crude protein level and trying to compensate amino acid deficiency by supplementing graded levels of total sulphur amino acids. The objective of this study was to investigate quantitatively describe the effect of dietary levels of crude protein and total sulfur amino acids on productive performance, egg quality and whole egg composition for Lohmann Brown laying hens.

MATERIALS AND METHODS
The current experiment was consummated at Poultry Research Farm, poultry Department, Faculty of Agriculture, Zagazig University, Egypt. All the experimental procedures were conducted in accordance with the Local Experimental Animals Care Committee, and affirmed by the ethics of the institutional committee at Faculty of Agriculture, Zagazig University. Hens were breeding for utilizing rearing guidelines got from Zagazig University standard working methodology.

Experimental design, hens and diets
One Hundred and fourth four of Lohmann Brown laying hens, 18 wk of age, were procured from a local commercial layer farm. They were randomly allocated to six treatments groups, each of which included six repeats of four hens. Each replicate was housed in one layer cage. The current study was conducted in a 2×3 factorial design experiment including two (low and normal) levels of CP (16 and 18 %) and three levels of Met+ Cys (0.67, 0.72 and 0.77 %) through the experimental period from 18-34 weeks of age (Table I). The initial average live body weight of hens in all experimental treatment groups were nearly the same and had no statistically differences. All hens were housed in a windowed poultry house under the same managerial and hygienic conditions. The hens were exposed to photoperiod 17 h light: 7h darkness per day and were fed on ad-libitum basis and fresh water was supplied freely during the experimental period. Vaccination and medical program were done according to the different stages of age under supervision of a veterinarian.

Laying productive performance and egg quality criteria
The layer hens were weighed at the beginning and the end day of experimental period, the live body weight change was then computed by the difference between the initial and final live body weights. On a hen-day basis, egg weight, egg production, and hen mortality were registered daily and feed intake was recorded weekly on a replicated basis. Egg mass was computed by multiplying by egg weight by hen-day egg number produced. Feed conversion ratio (FCR) was calculated as germ of feed intake/hen/day divided by gram of egg mass produced/hen/day. At the end of experimental period, 3 eggs from each replicate were chosen randomly to evaluate exterior and interior egg quality parameters such as (percentages of yolk, albumen, shell, and egg shape index (ESI), yolk index, shell thickness, Y:A ratio, Unit surface shell weight (USSW) and Haugh unit) which were measured according to Romanoff & Romanoff (1949).

Egg analysis
At 34 weeks of age (end of experimental period), 5 eggs from each treatment group were selected randomly for determination whole solids and

Statistical analysis
Data were statistically analyzed on a 2×3 factorial design basis according to Snedecor & Cochran (1982) using the following model: Where: Yijk = an observation, µ = the overall mean, Ai = effect of protein level (i=1 and 2), Sj = effect of Met+ Cys level (j =1 to 3), ASij =the interaction between the two variables and eijk= Experimental random error. Tukey's test was used for comparison among significant means.

Hen productive performance
Results in Table II  achieved the best results of the aforementioned parameters in comparison to other levels. Only egg production percentage and egg weight were significantly (P> 0.01) influences as a response to the interaction among CP and Met+ Cys levels.
The combination between low-CP diet (16 %) and 0.72 % Met+ Cys gave the best percentage of egg production. However, the best egg weight was found in the group fed normal-CP diet (18 %) supplemented with 0.77 % Met+ Cys comparing with other treatment groups. Results in Table II also illustrate the impact of low-CP diet, Met+ Cys supplementation and their combinations on body weight change during the experimental period. It is obvious that body weight change was significantly (P> 0.05 or 0.01) altered due to low-CP diet. The best values of final body weight (1790 g) and body weight change (139 g) were recorded by the hens fed normal-CP diet (18 % CP) comparing with those fed the low-CP diet. The different levels of Met+ Cys supplementation did not exert any significant effect on the values of body weight change. The combination between low-CP diet and Met+ Cys levels had a statistical (P> 0.05) impact on body weight change. It is noticeable that the interaction between 18% CP diet and 0.77 % Met+ Cys gave the heaviest body weight compared with other combinations. Meanwhile, the worst values were found by the combination between 16 % CP with 0.72 % Met+ Cys as compared with other treatment groups.

Egg quality criteria
Effects of low-CP diet, Met+ Cys supplementation and their interactions on external and internal egg quality traits are shown in Table III index, all external and internal egg quality traits were not significantly altered by Met+ Cys levels or the interaction among CP and Met+ Cys levels.

Chemical composition of whole egg
Data presented in Table IV

Hen productive performance
Insignificant improvements in daily feed intake and egg production percentage were noticed with increasing dietary CP level (Table II). In partially agreement, Novak et al. (2006) who indicated that feed consumption was affected by protein intake, where feed consumption linearly decreased as protein intake decreased Results of the present study confirmed that adding 0.72 % Met+Cys to the diet exerted the best feed conversion, egg production and egg mass (Table III). The enhancement of feed efficiency may be due to the increase of egg mass and may be attributed to more balanced amino acids. The obtained results coincided with those found by Koreleski &Świątkiewicz (2011) andZeweil et al. (2011) who reported that methionine supplementation significantly improved feed conversion per kg eggs. Feed conversion ratio, egg production, egg weight and egg mass were significantly affected by an increase in digestible sulphur amino acids intake (Kakhki et al. 2016). Contradicting results obtained by Hassan et al. (2000) who pointed out that feed efficiency was not significantly improved with increased TSAA intake during the first phase of feeding (20 to 40 wk of age). For egg production, our results are in line with those reported by Liu et al. (2005) who reported that increasing dietary levels of total sulphur amino acids (TSAA) from improved (p<0.05) egg number for laying hens. Also, Abd El-Maksoud et al. (2011) stated that egg production of laying hens fed methionine supplemented diet was significantly increased versus hens fed diet without methionine supplementation. Our findings concerning the positive effect of Met+ Cys supplement on egg mass are consistent with Harms &Russell (1996) andZeweil et al. (2011) who found that egg mass increased as methionine level increased from 0.23 to 0.31% in the first productive cycle for laying hens. On the other hand, Solarte et al. (2005) reported that increasing total sulphur amino acids level from 0.684 to 0.734% had no further improvement on egg mass.
In the current study, positive responses were detected on the percentage of produced egg and values of egg weight due to the interaction between dietary CP levels and Met+Cys levels. With the same trend, Pavan et al. (2005) found significant differences for egg weight with the combination of 15.5 and 0.71;17 and 0.71;15.5 and 0.64;14 and 0.71;17 and 0.64 of CP and TSSA,respectively, showing the highest values. Also, the interaction between protein and methionine revealed that the best average egg weight was produced by hens fed 16% CP supplemented by 2.74 methionine % of CP (Zeweil et al. 2011). On the contrary to our results, Zeweil et al. (2011) indicated that the interaction effect between protein and methionine showed insignificant differences in egg production at the end of experimental period.
An improvement in body weight was noticed due to normal-CP diet in the present study (Table II). This improvement may be due to the bioavailability and balanced amino acids which provided by the tested diet. However, lower body weight due to feeding the lowest dietary treatment (16% CP) was cleared by Cole (1996) who confirmed that amino acids may be absorbed in unavailable form because of fructose-lysine complex production. This would inhibit the release of trypsin and lysine utilization remains only 10% which leads to slower absorption of amino acids and consequently poor laying performance. Moreover, the reservation of body protein gradually depletes in birds fed on reduced CP diet. Our results disagree with those data reported by Abd El-Maksoud et al. (2011) andZeweil et al. (2011) who reported insignificant difference in the body weight for laying hens fed different levels of protein. According to our findings, the combination among the highest levels of both dietary CP and supplemental Met+Cys produced the heaviest hen weight (Table  II). This result agree with those found by Novak et al. (2006) who postulated that body weight gain elevated side by side with increasing dietary CP levels and with methionine supplementation. In contrast, Abd El-Maksoud et al. (2011) andZeweil et al. (2011) showed that the interaction impact between protein and methionine revealed insignificant differences in live body weight at the end of experiment.

Egg quality criteria
As explained in Table III, no significant impacts were detected on the majority of egg quality criteria due to dietary treatments. Likewise, Novak et al. (2006) reported no significant (p<0.05 or 0.01) difference in albumen, yolk, shell percent and Haugh unit when increasing or decreasing CP in layer diet during 20-43 wk of age. Moreover, Zeweil et al. (2011) revealed that the most of egg quality traits had not been affected by protein levels. Koreleski & Świątkiewicz (2011) indicated that egg shell characteristics were unchanged when methionine content in the diet was increased. Similar to our results, Pavan et al. (2005) found no significant differences in internal egg quality with the interaction between protein and TSAA in layer diets. Also, Novak et al. (2006) reported no significant (p<0.05 or 0.01) difference in albumen, yolk, shell percent and Haugh unit due to interaction effect between CP and TSAA in layer diet during 20-43 wk of age. On the other hand, Haugh unit score was significantly lower in layers fed a 13% CP diet in comparison with control, but yolk colour index was higher (P < 0.05) for the layers fed the 14% and 13% CP diets than those fed the normal diet (Torki et al. 2016).

Chemical composition of whole egg
No significant impacts were recorded due to CP or Met + Cys levels on the chemical composition of the egg as shown in Table IIII. Similar results were obtained by Gabriel & Babatunde (1976) who reported that both the total moisture and crude protein percentages were significantly increased as the dietary protein levels increased (20%) but the differences in total ash, shell and total lipid percentages were not significantly differed and were remarkably constant in white leghorn egg. Garcia et al. (2005) suggested that Met + Cys levels had no effect on egg contents.

CONCLUSIONS
In view of the aforementioned results and discussion, conclusion could be drawn that the best production criteria could be obtained by using 0.72% Met+Cys. Moreover, dietary level of 16 or 18 % CP with 0.72% Met+Cys is recommended for feeding Lohmann hens.