Evaluation of Dietary Supplementation of Delta-aminolevulinic Acid and Chito-oligosaccharide on Production Performance, Egg Quality and Hematological Characteristics in Laying Hens

The effects of delta-aminolevulinic acid (DALA) or chito-oligosaccharide (COS) in layer diets on production performance, egg quality and hematological characteristics were investigated in this 8-week trial. Two hundred and seventy 27-week-old (Hy-Line brown) layers were randomly assigned to 5 treatments with 9 replications per treatment. Dietary treatments were i) Control (basal diet); ii) DALA1 (basal diet+DALA 2 mg/kg); iii) DALA2 (basal diet+DALA 4 mg/kg); iv) COS1 (basal diet+COS 100 mg/kg) and v) COS2 (basal diet+COS 200 mg/kg). In this study, no significant difference was observed in feed intake and egg shell quality (eggshell breaking strength and egg shell thickness) among treatments. The inclusion of DALA increased egg production during the first 4 weeks. Egg weight was increased in both DALA (quadratic, p<0.05) and COS (linear, p<0.05) treatments compared with the control treatment. Laying hens fed the DALA treatment had an increased yolk color unit (quadratic, p<0.05) and haugh unit (linear, p<0.05) compared with the control group. Similarly, these characteristics were also affected by COS treatments, with both values being linearly increased (p<0.05) in COS treatments compared with the control treatment. Additionally, birds fed DALA treatments significantly increased (quadratic, p<0.05) the number of RBC, WBC and lymphocytes compared with the control treatment. Dietary DALA supplementation linearly increased (p<0.05) the serum iron concentration at the end of the 8th week. The inclusion of COS increased (linear, p<0.05) the concentration of RBC, WBC and lymphocytes compared with the control treatment. In conclusion, dietary DALA at the lower dosage (2 mg/kg) could exert better effects in laying hens than higher dosage (4 mg/kg). Birds fed DALA supplemented diet had an increased iron availability, egg weight, eggshell quality and immunity. Moreover, the inclusion of COS (200 mg/kg) can increase egg weight, eggshell quality and immunity in laying hens. Therefore, both the utilization of COS and DALA could be considered as a new strategy for optimizing egg quality and health condition of laying hens. (


INTRODUCTION
Recently, the use of delta-Aminolevulinic acid (DALA) as a feed additive for livestock has received much interest for its possible utilization in the livestock feed industry.DALA is a precursor in porphyrin synthesis (Döring et al., 1998) and is a non-protein amino acid with a widespread distribution in living organisms.Mateo et al. (2006) suggested that DALA supplementation can increase RBC counts of weanling pigs.Our previous study also suggested dietary DALA supplementation at the level of 5 mg/kg can increase serum iron concentration and egg quality (Chen et al., 2008b).Therefore, investigation of dietary DALA may provide a new strategy for optimizing egg production and egg quality.
Chito-oligosaccharide (COS), which is easily obtained by chemical and enzymatic hydrolysis of poly-chitosan, was shown to reduce the establishment of pathogens in the intestine (Vishu Kumar et al., 2005) and to improve immune function (Okamoto et al., 2003).A previous study conducted by Huang et al. (2005) also suggested that dietary COS can increase nutrient digestibility and weight gain in broilers due to its antifungal and antimicrobial (Jeon et al., 2000) activities.Therefore, it can be postulated that, with its antimicrobial and antifungal function, COS supplementation can improve performance of laying hens.To the best of our knowledge, there has been no other research concerning the effect of COS in laying hens.
Therefore, the objective of the present study was to evaluate the effects of DALA or COS supplementation in layer diets on egg production, egg quality and hematological characteristics.

Animal care and use
The protocols used for the current experiment were approved by the Animal Care and Use Committee of Dankook University.

Preparation of COS and DALA
Delta-amino acid and chito-oligosaccharide were supplied by EASY BIO System Inc (South Korea).Briefly, DALA was produced by E. coli containing the R. capsulatus heme A gene under the control of the constitutive promoter at a maximum concentration of 21 mM in the absence of levulinic acid, which is an inhibitor of DALA dehydratase.The chito-oligosaccharide used in this study was the natural product of chitin, chitosan and chitosan oligosaccharides that were produced by fermentation using live probiotics, including Aspergillus oryzac, Bacillus subtillus, Saccharomyces cerevisiae and Lactobacillus acidophilus.The fermentation product was comprised of: 18.6% crude protein, 14.5% crude fat, 10.7% crude fiber, 22.6% crude ash, 9.0% moisture, 4.3% calcium, 1.7% phosphorus, 4.0% chitin chitosan and 3.0% chitosan oligosaccharide.

Feeding regimen
Two hundred and seventy 27-week-old (Hy-line brown) layer chickens were used in this 8-week trial.All layers were randomly assigned to 5 treatments with 9 replicates per treatment.Each replicate consisted of 3 adjacent cages, with 2 hens per cage.The replicates were equally distributed into the upper and lower cages to minimize the effect of cage level.The hens were housed in a windowless laying house under a 17 h light: 7 h dark photo period at approximately 21°C.There was a 7-day adjustment period prior to the start of the experiment, during which the hens were provided with the control (CON) diet.All cages were equipped with nipple drinkers and a common trough feed.Feed and water were provided ad libitum throughout the experimental period.

Experimental design and diets
Dietary treatments were i) Control (basal diet); ii) DALA1 (basal diet+DALA 2 mg/kg); iii) DALA2 (basal diet+DALA 4 mg/kg); iv) COS1 (basal diet+COS 100 mg/kg) and v) COS2 (basal diet+COS 200 mg/kg).All diets used in the present study were formulated to meet or exceed the nutrient recommendations of the NRC (1994) based on corn, soybean and wheat.Treatment additives were included in the diet by replacing the same amount of corn and each treatment was made isolysinic and isocaloric.

Samples and measurements
Daily records of egg production and weekly records of feed consumption were kept throughout the experimental period.Egg production was expressed as an average henday production, which was calculated from the total number of eggs divided by the number of days and summarized on an average basis.A total of 36 saleable eggs (no shell defects, cracks or double-yolks) were randomly collected from each treatment at 17:00 h (4 eggs per replicate) on a weekly basis.The egg quality of the collected eggs was then determined at 20:00 h on the day of collection.Egg weight was measured using an egg multi tester (Touhoku Rhythm Co. Ltd., Tokyo, Japan).Eggshell breaking strength was evaluated using a model II egg shell force gauge (Robotmation Co., Ltd., Tokyo, Japan).A dial pipe gauge (Ozaki MFG.Co., Ltd., Tokyo, Japan) was used to measure egg shell thickness, which was determined based on the average thickness of the rounded end, pointed end, and the middle of the egg, excluding the inner membrane.Finally, egg weight, egg yolk color and haugh unit were evaluated using an egg multi tester (Touhoku Rhythm Co. Lt., Tokyo, Japan).At the beginning of the experiment, two birds per replicate were randomly selected and 5 ml of blood were collected from the left jugular vein using a sterilized needle.The samples were then transferred into a K 3 EDTA vacuum tube (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA).At the end of the experiment, blood was collected from the same laying hens.The samples for serum analysis were then centrifuged at 3,000×g for 15 min, and aliquots were stored at -4°C until analyzed for serum total protein, albumin, hemoglobin (Hb), iron and total iron binding capacity (TIBC) using an automatic blood biochemistry analyzer (HITACHI 747, Hitachi, Tokyo, Japan).Red blood cell (RBC), white blood cell (WBC) and lymphocyte counts of the whole blood samples were determined using an automatic blood analyzer (ADVIA 120, Bayer, Tarrytown, NY, USA).

Statistical analysis
All data were evaluated by analysis of variance following the GLM procedure for a completely randomized design.Analyses were conducted using the SAS software program (SAS Institute, 1996), with pen serving as the experimental unit.For the blood profile data, the initial data was used as a covariate.Significant differences among the means of the treatment groups were determined by Duncan's multiple-range test.Variability in the data was expressed as standard error (S.E.) and a probability level of p<0.05 was considered as statistically significant.Orthogonal comparisons were made using polynomial regression to measure the linear and quadratic effects of increasing dietary concentrations of supplemental COS and DALA.

Egg production and egg weight
Through the entire experiment, no significant differences were observed in feed intake and egg shell quality (eggshell breaking strength and egg shell thickness) among treatments (Tables 2 and 3).The inclusion of DALA increased (p<0.05)egg production during the first 4 weeks.Egg weight was increased in both DALA (quadratic effect, p<0.05) and COS (linear effect, p<0.05) treatments compared with the control treatment.Laying hens fed DALA had an increased yolk color unit (quadratic effect, p<0.05) and haugh unit (linear effect, p<0.05) compared with the control treatment.Similarly, yolk color unit and haugh unit were also affected by COS treatments, with both values being linearly increased compared with the control treatment (p<0.05).

Hematological characteristics
Total protein, albumin, hemoglobin (Hb) and total iron binding capacity were not influenced by dietary treatment (Tables 4 and 5).The numbers of RBC, WBC and lymphocytes were increased (quadratic effect, p<0.05) by DALA treatments compared with the control treatment (Table 4).Dietary DALA supplementation linearly increased (p<0.05) the iron concentration at the end of the 8th week (Table 5).Moreover, the inclusion of COS linearly increased (p<0.05) the concentrations of RBC, WBC and lymphocytes compared with the control treatment.

Effects of oligosaccharides on laying hens
Recently, various oligosaccharides are being added to livestock feed as prebiotics to improve animal performance, enhance immune ability, and influence the gut microflora (White et al., 2002;Lemieux et al., 2003).Several studies have suggested that COS has antifungal (Hirano and Nagao, 1989) and antimicrobial (Jeon et al., 2000) activities.Du et al. (2001) reported that COS with degrees of polymerization between 3 and 8 enhanced immunity and growth performance in livestock.Thus, it was hypothesized that laying hen performance may be improved by COS supplementation.In the current study, although COS supplementation did not exert any effect on feed intake and egg production, it increased egg weight at the end of the study.The reason may be the increased nutrient digestibility as a result of antimicrobial and antifungal activities of COS.This assumption is supported by Li et al. (2007), who suggested that chito-oligosaccharide supplementation can increase nutrient digestibility in broilers.Moreover, studies using mannan-oligosaccharides (MOS) suggest that MOS supplementation improves egg production in broiler breeder hens (Stanley and Sefton, 1999;Berry and Lui, 2000).The reason for the improvement has been attributed to the ability of MOS to maintain gut health via adsorption of pathogenic bacteria.Therefore, we suggest that COS may operate in a similar fashion.However, since there is a scarcity of work in the literature on the effect of dietary COS in laying hens, it is difficult to speculate as to the exact mechanisms whereby COS exerts its effects.
Furthermore, measurement of blood concentration is routinely conducted to evaluate the response of animals to various prebiotics.In our study, the RBC and WBC counts and the lymphocyte numbers were linearly increased by COS supplementation, indicating that COS may have exerted beneficial effects on the immune system.This is in partial agreement with Okamoto et al. (2003), who suggested the COS supplementation may increase the lymphocyte concentration.Similarly, our previous studies (Wang et al., 2009;Zhou et al., 2009) showed that lymphocyte number and RBC count were linearly increased by COS supplementation in pigs and broilers, respectively.The discrepancy among these studies may possibly be due to the different hygienic conditions employed.Moreover, Chen et al. (2009) suggested that COS supplementation did not influence immunity under non-challenge conditions, but significantly increased WBC and lymphocyte counts when pigs were challenged with a lipopolysaccharide.Therefore, it is reasonable to speculate that COS supplementation increased immunity in the current study.

CONCLUSION
In the current study, dietary DALA supplementation at lower dosage (2 mg/kg) exerted a better effect on laying hens than higher dosage (4 mg/kg).Birds fed the DALA supplemented diet had an increased iron availability, egg production, egg weight, eggshell quality and immunity.Moreover, the inclusion of COS (200 mg/kg) increased egg weight, eggshell quality and immunity in laying hens.Therefore, both the utilization of COS and ALA could be considered as a new strategy for optimizing egg quality and

Table 2 .
Effects of DALA and COS supplementation on egg production and egg weight in laying hens 1

Table 4 .
Effect of DALA and COS on RBC, WBC and lymphocyte concentrations in laying hens 1

Table 5 .
Effects of DALA and COS supplementation on Hb, total protein, albumin, iron and TIBC concentrations in laying hens 1

Table 3 .
Effect of DALA and COS on yolk color unit, egg yolk index and Haugh unit in laying hens 1 Each mean represents 27 pens with 54 chicks each per treatment.Control = Basal diet; DALA1 = Basal diet with DALA 2 mg/kg; DALA2 = Basal diet with DALA 4 mg/kg; COS1 = Basal diet with COS 100 mg/kg; COS2 = Basal diet with COS 200 mg/kg.