Experiment I: A total of 528 WL layers with uniform body weight (1320-1328 g) were randomly distributed and housed in colony cages (4 birds/cage). Two adjacent cages with common feeder were considered as one replicate. Birds were allotted to 11 treatment groups of 6 replicates each with 8 hens. Two basal diets were prepared with 0.50% digestible lysine at 13.36% (LCP) and 15.78% (MCP) protein levels. Ten experimental diets were prepared by adding five different concentrations of crystalline lysine (0.50,0.55,0.60,0.65,0.70%) to the two basal diets. A diet with 17% CP, 0.70% lysine was prepared and fed to a group as control (Table 1). All the experimental diets were isocaloric with 2700 kcal/kg ME. The ratio between digestible M+C, Thr, Trp, Arg, Ile and Val to digestible Lys were 86, 66, 19, 114, 72 and 80 %, respectively and were maintained constant in all the diets.

Experiment II: A total of 390 WL layers which were having similar body weight (1315-1330 g) were randomly distributed and housed in colony cages (3 birds/cage). Two adjacent cages with common feeder were considered as one replicate. Birds were allotted to 13 treatment groups of 5 replicates each with 6 hens. Basing on the performance of birds in first trial lysine @0.65, 0.60 at 13.36 and 15.78% CP respectively were taken for preparation of basal diets.

Basal diet I is with 0.65 per cent supplemental lysine at 13.36 per cent CP(HL/LCP) and basal diet II is with 0.60 per cent lysine at 15.78 per cent CP (LL/MCP) and control diet with 0.70 per cent digestible lysine at 17.0% CP (Table 2). To these basal diets crystalline threonine was supplemented at six graded concentrations (60, 63, 66, 69, 72 and 75% of lysine as threonine). Energy levels in all 13 diets were maintained at 2700 kcal / kg ME. The ratio between essential amino acids like digestible M+C, Trp, Arg, Ile and Val to digestible Lys were 86, 19, 114, 72 and 80 per cent, respectively and were maintained constant in all the diets.

The dried and ground samples of the feed ingredients (Maize, Maize glutein meal, Soy meal, De oiled rice bran, Rape seed meal, Ground nut cake, Guar meal, Sun flower cake and Bajra, Cotton seed meal) were analyzed for proximate principles (AOAC, 2005) before diet formulation, while the amino acid content of individual ingredients was analyzed via NIR using Amino NIR^® by following the traditional wet chemistry (Amino lab^®) at Amino Degussa Evonik industries, Singapore. The experimental diets were prepared based on the results obtained and the complete feed samples were analyzed for proximate principles. The amino acid composition of composite feed is calculated based on the data arrived and quantity used for preparation of the ration.

The ratio of other digestible essential amino acids (EAA) to digestible lysine reported by Lemme (2009) was taken as the base for fixing the EAA profiles in the diets. The synthetic amino acids (AA) including methionine (Met), threonine (Thr), tryptophan (Trp) and lysine (Lys) were supplemented to adjust their dietary levels.

All the birds were managed in open sided houses in 3 tier cages under uniform environment and standard hygienic conditions throughout the experiment with a photoperiod of 16 L:8 D. The birds were offered feed and water ad libitum. The minimum and maximum ambient temperatures in the house were recorded every day at 06.00 and 14.30 h (IST), respectively.

The experiments were conducted as per the guidelines of Institutional Animal Ethics Committee.

Experiment 1: The study indicated that either concentration of d. lysine or protein in layer diet had no effect (p>0.05) on serum biochemical parameters viz. total protein, albumin, globulin, A:G, cholesterol, ALP, calcium and phosphorus (Table 3). No effects on total protein observed in the present study suggest that the protein quality was optimum at all concentration of d. lysine inclusion. Dibner and Ivery (1990) stated that during stress periods especially diets with deficit in amino acids at low proteins showed reduction in albumin content and suggested the lysine requirement @ 717 ± 22 (R² = 0.95) mg/b/d for maximum Egg mass.

Whereas Shahir et al. (2006) reported that, Serum albumin increased with increasing lysine intake up to 720 mg/hen/d and then remained relatively constant. Serum albumin is the major reserve protein of the laying hen that will be degraded in response to the needs of protein synthesis in the oviduct (Smith, 1978).

Significantly no variations in serum protein properties at various protein/lysine levels in this study indicate that the levels supplemented might be optimum enough.

Whereas, Garlich 2001 reported that increase in serum calcium concentration (20.84 to 24.63 mg/dl) with increase in lysine in diet (0.75 to 0.86%).

The HI titres were not influenced by increased concentration of lysine irrespective of protein (Table 3). In contrast to the current findings Panda et al. (2005 and 2007) and Rama Rao et al. (2003), reported increased HI titres with increased concentration of Methionine in the diet.

Experiment 2: Concentration of d. threonine in the diet had no effect (p>0.05) on serum bio chemical parameters viz. total protein (g/dl), albumin (g/dl), globulin (g/ dl), ALP (IU/L), cholesterol (mg/dl), calcium (g/dl) and phosphorus (g/dl) content (Table 4), in both the groups irrespective of protein / lysine level. The serum biochemical values for different parameters obtained in the present study were comparable with those reported earlier (Azzam et al., 2011; Waiebel et al., 2000). Further, present study findings are in line with Abdel-Wareth and Esmail (2014) and Azzam et al. (2011) who reported that supplementation with L-threonine had no effect (p>0.05) on serum calcium, phosphorus and alkaline phosphatase in laying hens. Wang et al. (2017) reported that various protein sources (Soya, cotton seed meal, rape seed meal) in diet of layers did not affect the plasma Glucose, total proteins, albumin and globulin levels in 36 to 40 weeks of age. This study inferred that various sources of proteins mean variation in availability of amino acids to the birds. Even though availability is varied the birds are not showing any alterations in biochemical parameters indicated that birds can maintain the health status by changing the feed pattern.

Xiao et al. (2017) reported that the level of serum Ca was higher (p<0.05) in MHA-Ca supplementation group than that of the other forms of Methionine and control when fed various forms of Methioine in Broiler breeders ration. No difference (p>0.05) was observed among the groups on P level. Richards et al. (2005) reported that diets with added DLM increased the contents of Ca and P in broiler chickens.

The concentration of d. threonine in diet had no effect on immunoglobulin concentration in both the groups (Table 4). In contrast to this Azzam et al. (2011) reported increased immunoglobulins with increase in threonine concentration (0.03%). Furthermore, Kadam et al. (2008) observed that injection of 10, 20, or 30 mg of threonine into the yolk sac significantly increased the humoral response to SRBC in broilers at 21d post hatch. Tyler et al. (1996) reported increased IgG concentration with increase in total protein concentration. Azzam et al. (2014) observed a higher level of IgG at 0.3% l-threonine and concluded that immune system of the laying hen has a specific requirement for threonine that is much higher than those required for egg production (0.3% vs 0.2%).

Serum biochemical parameters like serum total protein (g/dl), albumin (g/dl), globulin, A:G ratio, ALP (IU/L), cholesterol (mg/dl), Ca (g/dl) and phosphorus (g/dl) and serum HI titres against NDV were not influenced by increase in concentration of lysine or d. threonine irrespective of protein in diet.

Based on the present study it can be concluded that WL layers at 25-44 weeks of age require supplementation of 0.65 and 0.60 percent digestible lysine at 13.36 and 15.78 percent protein respectively in diet i.e. 598.8 and 570 mg/h/d and d. threonine at 60 per cent of d.lysine i.e., 458.3 and 517.1 mg/h/d respectively for better production performance.