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Full Research Article

Influence of Dietary L-Threonine on Growth Response, Carcass Characteristics, Immune Function Response and Intestinal Histomorphology of Commercial Broilers

B
Bikas Ch Debnath1,*
https://orcid.org/0000-0001-8335-4522
B
Bikas Ch Debnath2
B
Barun Roy2
P
Paresh Nath Chatterjee2
J
Joydip Mukherjee2
N
Naresh Kurechiya3
1College of Veterinary Sciences and Animal Husbandry, Agartala-799 008, Tripura, India.
2West Bengal University of Animal and Fishery Sciences, Kolkata-700 037, West Bengal, India.
3College of Veterinary Sciences and Animal Husbandry, Mhow-453 441, Madhya Pradesh, India.

ABSTRACT

Background: Threonine is recognized as the third limiting amino acid for poultryin a maize-soybean meal. Insufficient levels of threonine can adversely affect the overall performance of the birds. The dietary requirements for L-Threonine as recommended by the National Research Council (NRC) may not adequately meet the needs of present-day commercial poultry birds.

Methods: Three hundred and thirty (330) day-old commercial unsexed broiler chicks of Vencobb-400 strain (45.05±0.4 g) were distributed in a completely randomized design (CRD) into 5 groups. The groups were based on varying doses of dietary L-Threoninein different rations, specifically: NRC recommendation, 100% Threonine of Vencobb-400 recommendation, 110% Threonine of Vencobb-400 recommendation, 120% Threonine of Vencobb-400 recommendation and 130% Threonine of Vencobb-400 recommendation for a 42-day trial period.

Result: The cumulative feed consumption exhibited both linear and quadratic increments; however, no significant difference was observed for body weight gain. The cumulative feed conversion ratio (CFCR) did not differ significantly (p>0.05) among the trial groups. The carcass weight, dressing percentage and relative yields of thigh and drumstick increased linearly, while the relative breast yield showed linear and quadratic trends. Conversely, abdominal fat% decreased in both linear and quadratic trends. The total serum immunoglobulin, ND-ELISA titre and mean neutrophil phagocytic activity index improved in both linear and quadratic manners, whereas the lymphocyte proliferation response (LPR) index linearly increased (p<0.001). The villus height (VH), crypt depth (CD), villus surface area (VSA), number of goblet cells per villus, VH:VW and VH:CD increased in both linear and quadratic manners, while villus width (VW) and crypt depth (CD) increased linearly. The highest Economic Index score (EIS) and relative European production efficiency factor (EPEF) were recorded in the group receiving 120% threonine.

INTRODUCTION

Threonine is the third limiting amino acid for broilers that are maintained on maize and soybean meal (Ayasan and Okan, 2006). To meet the specific nutritional needs of poultry, commercial dietary L-Threonine (98.5%) is commonly incorporated into broiler feed. Insufficient levels of threonine can adversely affect the overall performance of the birds (Dozier et al., 2001). For optimal growth performance of birds, the threonine levels in the diet must be sufficient (Kerr et al., 1999). Threonine plays an important role in the synthesis of uric acid, re-synthesis of pancreatic enzymes, protein synthesis, the turnover of body proteins and the production of collagen, elastin and antibodies (Sá et al., 2007).
       
Threonine maintains gastrointestinal health and optimizes the gut environment to help in preventing bacterial infections (Abbasi et al., 2013). The mucus layer that protects the intestinal lumen consists of mucins (glycoproteins in nature) secreted by goblet cells. Around 60-80% of the ingested threonine may remain in the portal drained viscera, where it is utilized for mucin production. Threonine constitutes around 30% of the total amino acids required for mucin synthesis. While primarily serving to protect the gut from digestive chyme, digestive juices and microorganisms, mucin also facilitates nutrient filtration, digestion and absorption within the gastrointestinal tract (Horn et al., 2009; Jiang et al., 2013). A deficiency in threonine may adversely affect immune function, while elevated levels of threonine in γ-globulin influence immunological responses (Sá et al., 2007; Kim et al., 2007). Threonine comprises 7-11% of all amino acids, highlighting its importance in immunoglobulins (Sandberg et al., 2007). Increasing dietary threonine intake has been linked to improved production of antibodies and serum IgG levels in swine (Wang et al., 2006). A higher level of threonine supplementation than that specified by NRC is necessary for improved growth performance, immune competence and disease prevention in broilers (Debnath et al., 2019). Therefore, further investigations are warranted to determine the influence of dietary threonine on the growth response, carcass characteristics, guthealth, immune response and economic viability of broilers.

MATERIALS AND METHODS

Experimental design, trialbirds andtrial duration
 
The experiment was conducted at the Poultry Unit, Department of Animal Nutrition, West Bengal University of Animal and Fishery Sciences, Kolkata, India during the year 2022. All procedures used were in accordance with the approval of the ethical committee of WBUAFS, Kolkata, India. Three hundred and thirty (330) day-old commercial unsexed broiler chicks of Vencobb-400 strain (45.05±0.4 g) were distributed in a completely randomized design (CRD) into 5 trial groups. Everytrial group was further divided into six replicates, with eleven birds in each replicate.
 
Housing, light and diets
 
The broiler chicks were kept in deep litter by maintaining standard hygienic and bio-security protocols during the 42 days trial period. The birds were kept in 30 pens, each measuring 12 square feet i.e.,1.25 square feet/bird. L-Threonine (98.5% threonine) was incorporated into the basal diet at various levels (Table 1). Before formulation, each individual feed ingredient was analyzed for amino acid composition using High-Performance Liquid Chromatography (HPLC). Additionally, all isonitrogenous and isocaloric feeds were subjected to analysis for amino acid content, as shown in Table 2. The mash feed was provided to the experimental birds withad libitum access to clean drinking water. Routine vaccination was performed against Newcastle disease (NDB1 and Lasota strains) and Infectious Bursal Disease (intermediate strain).

Table 1: Allotment of various trial groups and threonine availability (%) in basal and trial diets.



Table 2: Composition of basal diets and proximate analysis (fulfilling 100% threonine requirement as per NRC, 1994 without L-Threonine).


 
Feed consumption, live weight, weight gain and feed conversion ratio (FCR)
 
The feed consumption was recorded weekly for each replicate and ultimately, the total ration intake (CFI) was calculated by the end of the trial. Individually bird from every replicate was weighed initially and subsequently at weekly interval to determine weekly body weight gain (WG). The weekly FCR for every group was determined based on the weekly live weight gain and feed consumption. The cumulative feed conversion ratio (CFCR) was calculated using the formula:

 
Metabolizability trial
 
Two birds, selected based on average body weight from each replicate, were moved to individual metabolic cages for the nutrient retention study. The daily feed consumption and faecal output of each bird were measured for five days. The preserved samples were analyzed for proximate according to A.O.A.C. (1995).
 
Blood sample collection
 
Blood samples were collected during the fifth week from the wing vein (two broilers per replicate) using EDTA. The total immunoglobulins level in serum was quantified using the Zinc sulfate turbidity (ZST) test as per McEwan et al., (1970) and Gawade et al., (2013). Beta Procedure of HA-HI test was conducted for antibody titre of Ranikhet Disease virus. The in vitro phagocytic activity of neutrophils and the process for assessing the Phagocytosis of Blood Neutrophils (in vitro) were performed using the NBT Reductive Method as per Das et al., (2011).
 
Slaughtering of birds, carcass quality and jejunal histo-morphometry study
 
Representative birds (two from each replicate) were randomly selected and sacrificed after 12 h fasting, for the intestinal histomorphometry and other carcass characteristics. A 5 cm segment from the midpoint of the jejunum of two chickens per replicate was used for the study. VH (Villus height = length of villus from crypt junction to the tip), CD (Crypt Depth  = depth from crypt-villus junction to the base of the crypt), VH to CD ratio (ratio between villus height and villus width) and the VSA were measured/calculated (Law et al., 2007);
 
Villus Surface Area = (π×mh×h)+ (π×mh/2)2 
 
Where:
‘mh’ represents the width at the mid and ‘h’ denotes height of the villus.
       
The density of goblet cells was considered as the number of goblet cellsper unit VSA.
 
Economic analysis
 
The performance index score (PIS) or european production efficiency factor (EPEF) were determined on day 42 by analyzing the records of broilers, their average live weight (kg/head), viability percentage and feed conversion ratio (FCR) as per (Van et al., 2003). Economic Index Score (EIS) was computed on the same day based on the cost of the experimental feed. Both relative and absolute EPEF were calculated as follow.



  
Statistical analysis
 
One-way analysis of variance (ANOVA) as described by Snedecor and Cochran (1994) was used for data analysis utilizing the SPSS (2002) (SPSS 21.0, Chicago, IL, USA). Tukey’s test was applied to compare the treatment means when significant differences were observed (P<0.05). Both linear and quadratic responses via polynomial contrasts were also assessed. No statistical analysis was conducted for the cost assessment of various experimental diets.

RESULTS AND DISCUSSION

Influence of dietary L-Threonine on feed consumption, weight gain and FCR
 
The cumulative feed consumption of broilers was higher (p=0.001) in the 110%, 120% and 130% threonine supplementation group compared with the NRC recommended group (Table 3). The NRC recommendation group recorded numerically lower body weight gain; however, differences among treatment groups were not statistically significant (P >0.05).No statistical differences in CFCR were observed among the experimental groups.

Table 3: The Influence of L-Threonine on feed consumption, weight gain, FCR and carcass parameters of Vencobb-400 broilers.


       
The current findings are in agreement with Ayasan and Okan (2014) and Debnath et al., (2019), who indicated that the supplementation with L-Threonine significantly increased feed consumption. This increase may be attributed to the improved amino acid balance in the experimental diets and the associated enhancement in feed consumption. Furthermore, the numerical improvement of growth rate observed in the threonine-supplemented groups could be associated with the essential roleof threonine in protein synthesis and the continuance of tissue protein turnover (Ton et al., 2013).
 
Carcass characteristics
 
The average carcass weight was significantly (p<0.05) higher in the 120% threonine group (Table 3). With supplemental L-Threonine, the 120% threonine group showed a higher (p<0.001) relative dressed yield (%)dressing percentage. The breast yield (%) was significantly (p=0.001) higher. The 120% threonine group exhibited a higher (p<0.05) relative thigh yield and a higher (p=0.02) relative drumstick yield. A lower (p=0.05) abdominal fat percentage was observed in the 120% threonine group.
       
Estalkhzir et al., (2013); Mazraeh et al., (2013) and Khan et al., (2006) reported that the supplemental threonineled to an increase (p<0.05) in relative carcass weight (%). Adequate threonine might have played a role in both nitrogen retention and energy recovery, resulting in higher carcass yield. Increasing dietary threonine improved breast meat yield, although body weight gain remained similar across the threonine supplemented groups (Abbasi et al., 2013). Additionally, Estalkhzir et al., (2013) demonstrated that supplemental threonine led to an increase in breast meat. The drumstick weight in broilers also increased significantly (Abbasi et al., 2013). Kidd and Kerr (1997) found that increasing amounts of L-Threonine supplementation led to an optimal relative thigh yield (%) alongside a reduction in abdominal fat percentage.
 
Immunity
 
Total immunoglobulin level was significantly (p<0.001) higher in the 130% threonine group (Table 4). Similarly, the ND titre (ELISA) was significantly (p=0.001) higher in the 130% threonine group. The lymphocyte proliferation response (LPR) index was also found to be elevated (p<0.001) in the 130% threonine group. This index further showed a significant linear increase. Moreover, the neutrophil phagocytic activity index was significantly (p<0.001) higher in the 130% Threonine group.

Table 4: The Influence of L-Threonine on immune status and jejunal histo-morphometry of Vencobb-400 Broilers (n=60).


       
Threonine constitutes 7-11% of immunoglobulin proteins, thus, supplemental threonine may positively influence the effective synthesis of immunoglobulins (Sandberg et al., 2007). Broiler diets enriched with threonine demonstrated a higher antibody titre against Newcastle disease (Rezeipour et al., 2012). By promoting lymphocyte proliferation and reducing apoptosis, threonine plays a role in modulating the immune system (Wang et al., 2006; Li et al., 2007 and Debnath et al., 2019). The addition of L-Threonine to culture media may have enhanced cell proliferation, increased antibody production in lymphocytes and reduced apoptosis through protein synthesis and cellular signaling (Duval et al., 1991). As a result of threonine supplementation, chicks exhibited a remarkable enhancement in proliferation response of leukocyte to the concanavalin A (MacDougall and Klasing, 1998).
 
Intestinal histomorphometry
 
The VH, VW, CD, VH: VW and VH: CD were significantly increased in the treatment groups (Table 4). The villus surface area (VSA) and the goblet cell number per villus were significantly (p<0.001) increased in the threonine-supplemented groups. However, the density of goblet cells permm2 was not significantly (p>0.05) affected.
       
The significant increase in the jejunal VH, VW, CD, VH:VW and VH:CD could be attributed to the increased availability of this amino acid. A high threonine diet might have contributed to an increase in jejunal VH, VW, CD, VH: VW and VH:CD as the gut utilizes threonine more frequently. Threonine not only provides energy for normal intestinal functions but also helps to maintain small intestinal bulk and vitality (Zaefarian et al., 2008). Among all amino acids, threonine is particularly important as an essential nutrient due to its high metabolism in the portal-draining viscera (Schaart et al., 2005). The small intestine directly utilizes between 30 and 50% of threonine in its tissues (Wu, 1998). Considering the rapid turnover of mucosal tissues, it is plausible that an increment in supplementary threonine levels made optimum amounts of this critical limiting amino acid. Mitchell and Carlisle (1992) noted that the increase in surface area resulting from villus growth can explain the enhanced absorptive capacity. This may elucidate the larger surface area of jejunal villus observed in broilers that were fed a high dietary threonine. Regardless of the experimental groups, the number of goblet cells in the present study might have increased in proportion to the enhanced villus surface area associated with higher levels of threonine supplementation.
 
Nutrient metabolizability and economicefficiency
 
The nutrient metabolizability parameters were non-significant (p>0.05) (Table 5). The group with the highest relative European production efficiency factor (EPEF%) and the most favorable Economic Index score (EIS) was in the 120% threonine group (numerically).

Table 5: The Influence of dietary L-Threonine on metabolizability (%) (n=60) and economic efficiency (n=330) of Vencobb-400 Broilers.

CONCLUSION

Supplementation of dietary threonine at 120% of the Vencobb-400 recommendation improved carcass characteristics, immune parameters, intestinal histo-morphology and economic efficiency in broilers. However, higher threonine supplementation (130% of the Vencobb-400 recommendation) showed beneficial effects on certain immune response parameters.

ACKNOWLEDGEMENT

The present study was supported by West Bengal University of Animal and Fishery Sciences, Kolkata.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Full Research Article

    Influence of Dietary L-Threonine on Growth Response, Carcass Characteristics, Immune Function Response and Intestinal Histomorphology of Commercial Broilers

    B
    Bikas Ch Debnath1,*
    https://orcid.org/0000-0001-8335-4522
    B
    Bikas Ch Debnath2
    B
    Barun Roy2
    P
    Paresh Nath Chatterjee2
    J
    Joydip Mukherjee2
    N
    Naresh Kurechiya3
    1College of Veterinary Sciences and Animal Husbandry, Agartala-799 008, Tripura, India.
    2West Bengal University of Animal and Fishery Sciences, Kolkata-700 037, West Bengal, India.
    3College of Veterinary Sciences and Animal Husbandry, Mhow-453 441, Madhya Pradesh, India.

    ABSTRACT

    Background: Threonine is recognized as the third limiting amino acid for poultryin a maize-soybean meal. Insufficient levels of threonine can adversely affect the overall performance of the birds. The dietary requirements for L-Threonine as recommended by the National Research Council (NRC) may not adequately meet the needs of present-day commercial poultry birds.

    Methods: Three hundred and thirty (330) day-old commercial unsexed broiler chicks of Vencobb-400 strain (45.05±0.4 g) were distributed in a completely randomized design (CRD) into 5 groups. The groups were based on varying doses of dietary L-Threoninein different rations, specifically: NRC recommendation, 100% Threonine of Vencobb-400 recommendation, 110% Threonine of Vencobb-400 recommendation, 120% Threonine of Vencobb-400 recommendation and 130% Threonine of Vencobb-400 recommendation for a 42-day trial period.

    Result: The cumulative feed consumption exhibited both linear and quadratic increments; however, no significant difference was observed for body weight gain. The cumulative feed conversion ratio (CFCR) did not differ significantly (p>0.05) among the trial groups. The carcass weight, dressing percentage and relative yields of thigh and drumstick increased linearly, while the relative breast yield showed linear and quadratic trends. Conversely, abdominal fat% decreased in both linear and quadratic trends. The total serum immunoglobulin, ND-ELISA titre and mean neutrophil phagocytic activity index improved in both linear and quadratic manners, whereas the lymphocyte proliferation response (LPR) index linearly increased (p<0.001). The villus height (VH), crypt depth (CD), villus surface area (VSA), number of goblet cells per villus, VH:VW and VH:CD increased in both linear and quadratic manners, while villus width (VW) and crypt depth (CD) increased linearly. The highest Economic Index score (EIS) and relative European production efficiency factor (EPEF) were recorded in the group receiving 120% threonine.

    INTRODUCTION

    Threonine is the third limiting amino acid for broilers that are maintained on maize and soybean meal (Ayasan and Okan, 2006). To meet the specific nutritional needs of poultry, commercial dietary L-Threonine (98.5%) is commonly incorporated into broiler feed. Insufficient levels of threonine can adversely affect the overall performance of the birds (Dozier et al., 2001). For optimal growth performance of birds, the threonine levels in the diet must be sufficient (Kerr et al., 1999). Threonine plays an important role in the synthesis of uric acid, re-synthesis of pancreatic enzymes, protein synthesis, the turnover of body proteins and the production of collagen, elastin and antibodies (Sá et al., 2007).
           
    Threonine maintains gastrointestinal health and optimizes the gut environment to help in preventing bacterial infections (Abbasi et al., 2013). The mucus layer that protects the intestinal lumen consists of mucins (glycoproteins in nature) secreted by goblet cells. Around 60-80% of the ingested threonine may remain in the portal drained viscera, where it is utilized for mucin production. Threonine constitutes around 30% of the total amino acids required for mucin synthesis. While primarily serving to protect the gut from digestive chyme, digestive juices and microorganisms, mucin also facilitates nutrient filtration, digestion and absorption within the gastrointestinal tract (Horn et al., 2009; Jiang et al., 2013). A deficiency in threonine may adversely affect immune function, while elevated levels of threonine in γ-globulin influence immunological responses (Sá et al., 2007; Kim et al., 2007). Threonine comprises 7-11% of all amino acids, highlighting its importance in immunoglobulins (Sandberg et al., 2007). Increasing dietary threonine intake has been linked to improved production of antibodies and serum IgG levels in swine (Wang et al., 2006). A higher level of threonine supplementation than that specified by NRC is necessary for improved growth performance, immune competence and disease prevention in broilers (Debnath et al., 2019). Therefore, further investigations are warranted to determine the influence of dietary threonine on the growth response, carcass characteristics, guthealth, immune response and economic viability of broilers.

    MATERIALS AND METHODS

    Experimental design, trialbirds andtrial duration
     
    The experiment was conducted at the Poultry Unit, Department of Animal Nutrition, West Bengal University of Animal and Fishery Sciences, Kolkata, India during the year 2022. All procedures used were in accordance with the approval of the ethical committee of WBUAFS, Kolkata, India. Three hundred and thirty (330) day-old commercial unsexed broiler chicks of Vencobb-400 strain (45.05±0.4 g) were distributed in a completely randomized design (CRD) into 5 trial groups. Everytrial group was further divided into six replicates, with eleven birds in each replicate.
     
    Housing, light and diets
     
    The broiler chicks were kept in deep litter by maintaining standard hygienic and bio-security protocols during the 42 days trial period. The birds were kept in 30 pens, each measuring 12 square feet i.e.,1.25 square feet/bird. L-Threonine (98.5% threonine) was incorporated into the basal diet at various levels (Table 1). Before formulation, each individual feed ingredient was analyzed for amino acid composition using High-Performance Liquid Chromatography (HPLC). Additionally, all isonitrogenous and isocaloric feeds were subjected to analysis for amino acid content, as shown in Table 2. The mash feed was provided to the experimental birds withad libitum access to clean drinking water. Routine vaccination was performed against Newcastle disease (NDB1 and Lasota strains) and Infectious Bursal Disease (intermediate strain).

    Table 1: Allotment of various trial groups and threonine availability (%) in basal and trial diets.



    Table 2: Composition of basal diets and proximate analysis (fulfilling 100% threonine requirement as per NRC, 1994 without L-Threonine).


     
    Feed consumption, live weight, weight gain and feed conversion ratio (FCR)
     
    The feed consumption was recorded weekly for each replicate and ultimately, the total ration intake (CFI) was calculated by the end of the trial. Individually bird from every replicate was weighed initially and subsequently at weekly interval to determine weekly body weight gain (WG). The weekly FCR for every group was determined based on the weekly live weight gain and feed consumption. The cumulative feed conversion ratio (CFCR) was calculated using the formula:

     
    Metabolizability trial
     
    Two birds, selected based on average body weight from each replicate, were moved to individual metabolic cages for the nutrient retention study. The daily feed consumption and faecal output of each bird were measured for five days. The preserved samples were analyzed for proximate according to A.O.A.C. (1995).
     
    Blood sample collection
     
    Blood samples were collected during the fifth week from the wing vein (two broilers per replicate) using EDTA. The total immunoglobulins level in serum was quantified using the Zinc sulfate turbidity (ZST) test as per McEwan et al., (1970) and Gawade et al., (2013). Beta Procedure of HA-HI test was conducted for antibody titre of Ranikhet Disease virus. The in vitro phagocytic activity of neutrophils and the process for assessing the Phagocytosis of Blood Neutrophils (in vitro) were performed using the NBT Reductive Method as per Das et al., (2011).
     
    Slaughtering of birds, carcass quality and jejunal histo-morphometry study
     
    Representative birds (two from each replicate) were randomly selected and sacrificed after 12 h fasting, for the intestinal histomorphometry and other carcass characteristics. A 5 cm segment from the midpoint of the jejunum of two chickens per replicate was used for the study. VH (Villus height = length of villus from crypt junction to the tip), CD (Crypt Depth  = depth from crypt-villus junction to the base of the crypt), VH to CD ratio (ratio between villus height and villus width) and the VSA were measured/calculated (Law et al., 2007);
     
    Villus Surface Area = (π×mh×h)+ (π×mh/2)2 
     
    Where:
    ‘mh’ represents the width at the mid and ‘h’ denotes height of the villus.
           
    The density of goblet cells was considered as the number of goblet cellsper unit VSA.
     
    Economic analysis
     
    The performance index score (PIS) or european production efficiency factor (EPEF) were determined on day 42 by analyzing the records of broilers, their average live weight (kg/head), viability percentage and feed conversion ratio (FCR) as per (Van et al., 2003). Economic Index Score (EIS) was computed on the same day based on the cost of the experimental feed. Both relative and absolute EPEF were calculated as follow.



      
    Statistical analysis
     
    One-way analysis of variance (ANOVA) as described by Snedecor and Cochran (1994) was used for data analysis utilizing the SPSS (2002) (SPSS 21.0, Chicago, IL, USA). Tukey’s test was applied to compare the treatment means when significant differences were observed (P<0.05). Both linear and quadratic responses via polynomial contrasts were also assessed. No statistical analysis was conducted for the cost assessment of various experimental diets.

    RESULTS AND DISCUSSION

    Influence of dietary L-Threonine on feed consumption, weight gain and FCR
     
    The cumulative feed consumption of broilers was higher (p=0.001) in the 110%, 120% and 130% threonine supplementation group compared with the NRC recommended group (Table 3). The NRC recommendation group recorded numerically lower body weight gain; however, differences among treatment groups were not statistically significant (P >0.05).No statistical differences in CFCR were observed among the experimental groups.

    Table 3: The Influence of L-Threonine on feed consumption, weight gain, FCR and carcass parameters of Vencobb-400 broilers.


           
    The current findings are in agreement with Ayasan and Okan (2014) and Debnath et al., (2019), who indicated that the supplementation with L-Threonine significantly increased feed consumption. This increase may be attributed to the improved amino acid balance in the experimental diets and the associated enhancement in feed consumption. Furthermore, the numerical improvement of growth rate observed in the threonine-supplemented groups could be associated with the essential roleof threonine in protein synthesis and the continuance of tissue protein turnover (Ton et al., 2013).
     
    Carcass characteristics
     
    The average carcass weight was significantly (p<0.05) higher in the 120% threonine group (Table 3). With supplemental L-Threonine, the 120% threonine group showed a higher (p<0.001) relative dressed yield (%)dressing percentage. The breast yield (%) was significantly (p=0.001) higher. The 120% threonine group exhibited a higher (p<0.05) relative thigh yield and a higher (p=0.02) relative drumstick yield. A lower (p=0.05) abdominal fat percentage was observed in the 120% threonine group.
           
    Estalkhzir et al., (2013); Mazraeh et al., (2013) and Khan et al., (2006) reported that the supplemental threonineled to an increase (p<0.05) in relative carcass weight (%). Adequate threonine might have played a role in both nitrogen retention and energy recovery, resulting in higher carcass yield. Increasing dietary threonine improved breast meat yield, although body weight gain remained similar across the threonine supplemented groups (Abbasi et al., 2013). Additionally, Estalkhzir et al., (2013) demonstrated that supplemental threonine led to an increase in breast meat. The drumstick weight in broilers also increased significantly (Abbasi et al., 2013). Kidd and Kerr (1997) found that increasing amounts of L-Threonine supplementation led to an optimal relative thigh yield (%) alongside a reduction in abdominal fat percentage.
     
    Immunity
     
    Total immunoglobulin level was significantly (p<0.001) higher in the 130% threonine group (Table 4). Similarly, the ND titre (ELISA) was significantly (p=0.001) higher in the 130% threonine group. The lymphocyte proliferation response (LPR) index was also found to be elevated (p<0.001) in the 130% threonine group. This index further showed a significant linear increase. Moreover, the neutrophil phagocytic activity index was significantly (p<0.001) higher in the 130% Threonine group.

    Table 4: The Influence of L-Threonine on immune status and jejunal histo-morphometry of Vencobb-400 Broilers (n=60).


           
    Threonine constitutes 7-11% of immunoglobulin proteins, thus, supplemental threonine may positively influence the effective synthesis of immunoglobulins (Sandberg et al., 2007). Broiler diets enriched with threonine demonstrated a higher antibody titre against Newcastle disease (Rezeipour et al., 2012). By promoting lymphocyte proliferation and reducing apoptosis, threonine plays a role in modulating the immune system (Wang et al., 2006; Li et al., 2007 and Debnath et al., 2019). The addition of L-Threonine to culture media may have enhanced cell proliferation, increased antibody production in lymphocytes and reduced apoptosis through protein synthesis and cellular signaling (Duval et al., 1991). As a result of threonine supplementation, chicks exhibited a remarkable enhancement in proliferation response of leukocyte to the concanavalin A (MacDougall and Klasing, 1998).
     
    Intestinal histomorphometry
     
    The VH, VW, CD, VH: VW and VH: CD were significantly increased in the treatment groups (Table 4). The villus surface area (VSA) and the goblet cell number per villus were significantly (p<0.001) increased in the threonine-supplemented groups. However, the density of goblet cells permm2 was not significantly (p>0.05) affected.
           
    The significant increase in the jejunal VH, VW, CD, VH:VW and VH:CD could be attributed to the increased availability of this amino acid. A high threonine diet might have contributed to an increase in jejunal VH, VW, CD, VH: VW and VH:CD as the gut utilizes threonine more frequently. Threonine not only provides energy for normal intestinal functions but also helps to maintain small intestinal bulk and vitality (Zaefarian et al., 2008). Among all amino acids, threonine is particularly important as an essential nutrient due to its high metabolism in the portal-draining viscera (Schaart et al., 2005). The small intestine directly utilizes between 30 and 50% of threonine in its tissues (Wu, 1998). Considering the rapid turnover of mucosal tissues, it is plausible that an increment in supplementary threonine levels made optimum amounts of this critical limiting amino acid. Mitchell and Carlisle (1992) noted that the increase in surface area resulting from villus growth can explain the enhanced absorptive capacity. This may elucidate the larger surface area of jejunal villus observed in broilers that were fed a high dietary threonine. Regardless of the experimental groups, the number of goblet cells in the present study might have increased in proportion to the enhanced villus surface area associated with higher levels of threonine supplementation.
     
    Nutrient metabolizability and economicefficiency
     
    The nutrient metabolizability parameters were non-significant (p>0.05) (Table 5). The group with the highest relative European production efficiency factor (EPEF%) and the most favorable Economic Index score (EIS) was in the 120% threonine group (numerically).

    Table 5: The Influence of dietary L-Threonine on metabolizability (%) (n=60) and economic efficiency (n=330) of Vencobb-400 Broilers.

    CONCLUSION

    Supplementation of dietary threonine at 120% of the Vencobb-400 recommendation improved carcass characteristics, immune parameters, intestinal histo-morphology and economic efficiency in broilers. However, higher threonine supplementation (130% of the Vencobb-400 recommendation) showed beneficial effects on certain immune response parameters.

    ACKNOWLEDGEMENT

    The present study was supported by West Bengal University of Animal and Fishery Sciences, Kolkata.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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