Concentration of Digestible Threonine in Diet on Production Performance and Intestinal Morphometry of WL Layers

Gut health plays major role in poultry while exhibiting sustainable production (Salois et al., 2016).  Gut health of birds is subjective to systemic health of birds, animal welfare, the production efficiency of flocks, food safety and environmental impact (Oviedo-Rondon, 2019). Among the essential amino acids, threonine is the third limiting amino acid in corn-soy diets fed to layers (Kidd et al., 2007). It occurs at high concentration in  -globulin thus affecting immune function (Tenenhouse and Dentsch, 1996).  Threonine represents 30% of the total amino acid content in mucin. The ability of the small intestine to synthesize mucin is based on threonine availability. The mucous layer of intestine is important because it acts as a non-immune barrier. Decreased mucin protein synthesis was observed in rats when fed threonine deficit diets and the requirement of threonine in rats were found to be higher in gut infection situations than healthy condition (Faure et al., 2006). The enhanced requirement from 0.65 to 0.70 percent was observed on standard ileal digestibility basis of broilers in subclinical clostridium/eimeria challenged situations (Chung et al., 1996).
       
Whereas, Lelis et al., (2010) observed better egg production in dekalb brown layers at 25-37 weeks by supplementing threonine at 78% of Lysine. Nutrient requirement suggested by NRC, (1994) were the minimum values without any margin of safety. Parent firms recommended much higher lysine and other amino acids than NRC, (1994).
       
Currently, information regarding requirement of appropriate concentration of threonine in white leghorn (WL) layer diets for optimum performance, mucin production and gut integrity is scanty. Hence, the present study was undertaken to elucidate the effect of various concentrations of threonine at different protein levels on production performance, mucin synthesis and intestinal histomorphology.

One hundred and eighty commercial layers (Babcock) aged 25 weeks with uniform body weights were randomly distributed into six treatment groups of five replicates with six hens each. All the birds were managed under uniform environment and standard hygienic conditions for a period of 20 weeks (25 to 44 weeks of age). The birds were offered feed and water ad libitum. A constant photoperiod (16L:8D) in a day was maintained. The trial was conducted from December to April where the temperature varied from 22.49- 32.91°C with the Relative Humidity of 44.53-88.85%.
 
Experimental diet
 
Basing on the level of amino acid present in the ingredients (Table 1) analyzed at Amino Degusa Singapore NIRS, the basal diets were prepared (Table 2). Three basal diets containing low (13.46% CP with 0.65% lysine), medium (15.56% CP with 0.60% lysine) and high (17.05% CP with 0.70% lysine) protein with appropriate lysine combinations were prepared basing on the levels suggested by Kumari et al., (2017). Crystalline threonine with 98.5% availability was procured form local market (L-Threonine/Ajinomoto) supplemented at 63 and 66% of lysine as threonine to these three basal diets respectively. The diet with 17.05% CP, 0.70% lysine and 66% lysine as threonine was considered as control. With the help of the digestible amino acid levels in the ingredients the digestible amino acids levels in the compound diets were calculated and ratios were estimated. The ratio between digestible Methionine + Cystine, Tryptophan, Arginine, Iso leucine and Valine to digestible Lysine were 86, 19, 114, 72 and 80% respectively and were maintained constant in all the diets as per the management guide on Babcock BV-300. All the procedures performed in this study were in accordance with the ethical standards of the Institution (Guidelines of Institutional Animal Ethics Committee).
 

 

 
Production parameters
 
The body weight of the 50 percent of the birds per replicate (3 birds) was recorded at the beginning and at the end of each period (28 days). Feed intake and Feed conversion ratio [FCR (g/ g)] were measured weekly and pooled for the period. Egg weight was calculated as mean weight of eggs collected in 3 consecutive days of each period. Egg production and mortality were recorded daily. Basing on daily production hen day egg production percent was calculated. Egg mass was calculated period wise (egg production x average egg weight).
 
Gut health
 
Intestinal histomorphology, Goblet cell count and mucin (MUC2) gene expression were studied as detailed below.
Sample collection
 
A total of 30 birds (one bird per replicate) were sacrificed by cervical dislocation. Small segments of duodenum, jejunum and ileum were collected for histological studies and gene expression respectively.
 
Histomorphology
 
The intestinal segments fixed in 10% neutral-buffered formalin were embedded in paraffin. The Hematoxylin and Eosin (H & E) stained sections (Kiernan, 2008) were visualized using Computer -Aided microscopy (DXM1200C, Nikon). Villus height and crypt depth were measured and analyzed using NIS-Elements BR software, version 2.20(Nikon). The number of positively H & E stained goblet cells were counted within 10 randomly selected villi using image-proplus software version 6.0 (Media Cybernetics).
 
Gene expression analysis of mucin: (Quantitative Real Time PCR analysis (MUC2 mRNA expression)
 
Process of isolation of total RNA from jejunum and ileum tissue was performed as per the manufacture’s protocol using TRIzol method (Qiagen, Invitrogen). Purity in terms of quantity and quality was assessed respectively by means of Genova nanodrop (OD 260/280 value) and gel electrophoresis. The OD values ranging from 1.9 to 2.2 were considered as good and were used further for the synthesis of cDNA. The synthesis was carried out using high capacity cDNA Reverse transcription kit (ABI, USA) in thermocycler (Mastercycler, Eppendorf) with the condition: 25°C (10 min), 37°C (120 min), 85°C (5 min) and 4°C hold. The resulted products were then stored at -20°C. The gene expression study was made using Step One Real Time PCR (Applied Biosystems®, Life Technologies v2.2.2 machine). cDNA prepared from respective jejunum and ileum tissues were used as template and SYBR® Green as a dye (Thermo scientific, #K0221). The primers (Horn et al., 2009) used in the study were (Table 3) amplified under the condition of 95°C (30s); {95°C (3s), 60°C (10s) and 72°C (32s)} 42 cycles; 72°C (10 min) and hold (4°C). Each sample was examined in triplicates and standard curve was made. The specificity of amplification was tested by means of melting curve analysis presented at the end of the real time PCR run. 2^-ΔΔCt (Livak and Schmittgen, 2001) method of analysis was performed to study the relative expression of target gene where GAPDH was used as reference gene. Further, fold change was calculated using the formula 2^Δ Δ Ct by taking n-fold difference relative to the calibrator. All the protocols have been done as per MIQE guidelines. 
 

 
Statistical analysis
 
The data was analyzed by two-way ANOVA and Duncan’s multiple range test (Duncan, 1955) was used to study significant differences (p<0.05) between means.

Production performance
 
Hen day egg production (HDEP), feed intake/bird/day (FI/b/d) were recorded regularly from 25 to 44 weeks (Table 4). Significant increase in egg production was observed with increase in threonine concentration at each protein level. The HDEP values were significantly (p<0.05) lower at 15.56% CP at both the threonine concentrations. Higher (p<0.05) HDEP was recorded at HCP group followed by LCP and MCP. High concentration of threonine had positive impact (p<0.05) than lower concentrations. This variation in production might be due to altered threonine availability, which was minimum in MCP group followed by LCP and HCP. Similar results were reported by Azzam et al., (2014) with threonine supplementation at 0.1 to 0.2% at 16% CP diet of Babcock brown layers. There was an increase in egg production with increase in threonine level. However, Figueiredo et al., (2012) reported no effect of incorporation of various levels of lysine (0.675, 0.743, 0.811 and 0.879%) in combination with different concentrations of threonine (0.542, 0.596 and 0.650%) in diets of Hy-Line W36 laying hens aged 42-58 weeks on egg production. Significant drop in egg production in low-protein fed birds was recorded by Roberts et al., (2007).  Whereas some researchers (Latshaw and Zhao, 2011; Rama Rao et al., 2011) reported that protein level in diet had no significant effect (p>0.05) on egg production. The results of the present study revealed that protein had an influence on egg production but ideal amino acid profile in low protein diets might ameliorated the effect.
 

       
Feed intake/bird/day ranged from 111.1-116.4g and was not influenced by threonine concentration. Egg weights were not influenced significantly either by protein/lysine level or by threonine concentration. Significantly better FCR was observed at LCP followed by HCP and MCP groups. This might be due to the fact that birds usually consume feed to satisfy energy needs. The diets fed to birds in the current study were iso-caloric. The ratio of all essential amino acids even in low protein groups were balanced with amino acids from natural and synthetic sources and it might have resulted in no effect on feed consumption. Similar findings were observed by (Roberts et al., 2007; Rama rao et al., 2011) by incorporating various levels of protein in layer diets. However, Latshaw and Zhao (2011) noticed higher feed consumption in birds fed 13.8% CP supplemented with amino acids compared to 15.5% and 17.0% CP. Results suggested that 13.46% CP with balanced amino acid supplementation in diet is sufficient for optimum production.

The overall performance of birds fed low protein diets in the present study was at par with high protein diet suggesting it as ideal CP level (13.46%) for economic production in commercial layer farming. This was in consonance with the result of Kumari et al., (2016) in WL Layers from 25-36 weeks of age.
 
Histomorphological changes in the intestine
 
H & E stained sections of duodenum, jejunum and ileum revealed non-significant increase in crypt depth (CD) and villus height (VH) with increases in threonine concentration from 63 to 66% irrespective of protein level (Table 5). Crypt depth (CD) and villus height (VH) were relatively higher for duodenum and jejunum in LCP group and for ileum in MCP group. Villus height was increased in MCP group but, not influenced by the concentration of either threonine or protein. This indicates the fact that threonine availability (410 and 429 mg) in LCP group up to 40 weeks  of age in WL layers is optimum for good intestinal health and mucus production and 66% threonine supplementation (429mg available threonine) was found to be the best. Similar results were obtained by Azzam et al., (2012) with threonine supplementation at 0.47, 0.66 and 0.74% levels in the diets of Babcock Brown layers aged 40 weeks. Abasi et al., (2014) found significant decrease in villus height and crypt depth in Jejunal epithelial cells of broilers with low protein diets but supplementation of threonine up to 110 and 120 times of lysine nullified these changes.
 

       
Goblet cell count decreased numerically from LCP to HCP fed birds (Table 5). Decrease in goblet cells with increase in protein/lysine level and also by increase in threonine concentration was observed. Goblet cells are mucin producing and secreting sites in digestive system. Reduced goblet cell numbers may be related to lower endogenous protein losses associated with lower CP levels. Interestingly, increasing dietary Thr level caused a marked increase in goblet cell numbers. Nicholas and Bentolo, (2008) indicated that the de novo synthesis of mucosal and mucin proteins appeared to be highly sensitive to luminal. The concentration, which demonstrates the importance of dietary Thr supply for gut metabolism. Our results suggest that after reduction of dietary CP level the jejunal VH, CD and goblet cell numbers were decreased, but Threonine supplementation to the diets at least 66% of lysine could compensate for these changes. Chen et al., (2017) reported feeding higher levels (1.08%) of threonine over strain recommendation (0.77%) increase in VH, reduced CD in broilers. Similarly, Zhang et al., (2016) also observed increased VH and decrease in CD in broilers fed with higher concentrations of threonine (0.90%) over the recommended (0.49%).
 
Mucin 2 mRNA expression
 
In current study expression of jejunal and ileal MUC2 mRNA was increased (p<0.05) with increasing level of threonine in LCP and MCP groups. The values were relatively higher in LCP group in both jejunum and ileum (Fig 1 and 2). Lower expression was recorded at control. The results linked positively with goblet cell number. Mucin 2 (MUC2) is secreted by goblet cells and is a major component of the protective mucin layer. Mucin layer protects infections by pathogenic bacteria and acts as a substrate and fixing medium for commensal bacteria. LCP with 66% threonine (429mg/b/d) can be suggested as ideal diet formulation for enhanced mucus production. At higher threonine (508 mg/b/d) availability with HCP diet, mucin 2 gene expression was lower and histological changes also revealed lower crypt depth, villus height and goblet cell number, whereas, productive performance did not differ significantly when compared to other groups. Wang et al., (2010) observed that 1.11% true ileal digestible (TID) threonine caused decreases in ileal acido mucins and duodenal sulpho mucin when compared to 0.74% TID threonine diet in piglets suggesting that excess dietary threonine affects intestinal mucosal barrier. However, no effect of dietary threonine restriction or supplementation was found on MUC2 mRNA expressions by (Horn et al., 2009; Chee et al., 2010) in broilers. This indicates that threonine availability at higher levels has deleterious effects on intestinal histomorphology and mucin2 gene expression in layers.
 

 

WL layers performed well at Low protein diets (i.e. 13.46% CP) supplemented with optimum concentrations of lysine (0.65%) and thronine (66% of lysine) without distressing the intestinal morphometry.