The immunity-growth Link: How bacteria-derived beta-glucans shape broiler development

Introduction

Modern poultry farmers are heavily shifting towards a sustainable, clean and antibiotic free production. Due to rising demands of poultry producers and suggestions of policy makers, various immunomodulator natural feeds additives are in spotlight to be used as an alternative to antibiotics for their growth promoting and immunity enhancing properties. One such feed additive is β-glucan, a natural polysaccharide consisting of glucose molecules bound with β-1,3-linkages. Various forms of β-glucan have been identified and extracted from different sources, such as bacterial β-glucan consisting of linear β-1,3-linkages, yeast and fungal derived consisting β-1,3 backbone with 1,6 side chains and cereal derived β-glucan have β-1,3 backbone with 1,4 side chains (Murphy et al., 2010) [1] . These differences in branching, shape and molecular weight determine their biological function in the body. The cereal based β-glucans are primarily recognised for their ability to lower serum cholesterol, modulate gut microbiota and scavenge reactive oxygen species (Nakashima et al., 2018) [2] . These properties are largely attributed to their high viscosity and gel formation capability, which delays the gastric emptying, form a protective layer on the gut mucosa and slows down the cholesterol and carbohydrate absorption through enterocytes. This leads to low sugar spikes after a meal and controlled insulin levels thus helping in diabetes, blood sugar management, and cardiovascular functions. Additionally, they increase the bile secretion which helps in lipid metabolism (Henrion et al., 2019) [3] . On the other side, fungi, yeast and bacteria based β-glucans mainly work on immune function by enhancing the gut health, promoting immune cells (e.g. macrophage) activity, increasing the antibody titres following vaccination, and through anti-inflammatory and immune-modulatory effects (Singh et [4,5,6,7,8] .

Upon uptake via payer's patches in the GALT they subsequently move around the body and attach to specific receptors such as CR3 and Dectin 1 present on the surface of immune cells (e.g.macrophages). This interaction initiates intracellular signalling at DNA level which shifts the cells metabolic activity towards enhanced glycolysis, resulting in increased phagocytic activity, faster deployment rate and releasing more cytokines, antibodies and natural killer cells. Thus, by priming of cells, β-1,3-glucan stimulate both innate and adaptive immunity without any cell proliferation or auto immune complications, helping in combating any negative effects of pathogens, enteric infections or immune suppression by various stress factors in poultry rearing. (Jacobs et al., 2017; Schwartz et al., 2021) [9,10] . Yeast or fungal based β-1,3-glucan have long been used in the poultry sector but the bacterial derived glucans are new in the industry and has an edge above yeast/fungal or plant derived glucans in terms of yield, purity, bioavailability, cost, and efficiency (Kim, MK et al., 2003) [ 11] . By keeping the above benefits of linear β-1,3-glucan of bacterial origin in background knowledge, this study was conducted to evaluate the following objectives by supplementing betaspike (β-1,3-glucan) in broilers: 1. Growth performance parameters: Feed intake, body weight, body weight gain and feed conversion ratio 2. Hematological and serological parameters: Hemoglobin count and antibody titres against new castle disease (ND), low pathogenicity avian influenza (LPAI) and infectious bursal disease (IBD) vaccination protocols. [12] . The feed ingredients used and their nutrient composition as per AOAC 2013 [13] are presented in Table 1. Total Methionine (%) 0.50 0.52 0.45 * Trace Mineral Mixture: Each 1kg contains: Copper-15 g, Iron-80 g, Manganese-100 g, Zinc-80 g, Iodine-2 g, Cobalt-0.5 g, Selenium-0.5 g, Chromium 10 mg and Molybdenum 50 mg. ** Vitamin Premix: Each 500 g contains: Vit. A-12.50 mIU, Vit. D3-2.50 MIU, Vit. E-12 g, Vit. K-1.50 g, Thiamine (B1)-1.50 g, Riboflavin (B2)-5 g, Pyridoxine (B6)-2 g, Cyanocobalamin (B12)-0.015 g, Niacin-15 g, Cal D Pantothenate-10 g and Folic acid-0.50 g.

Materials and Methods

The birds were housed in deep litter system and were fed pre-starter, starter and finisher diets for 1-7, 8-21 and 22-42 days of age, respectively. Feed and water were provided adlib. The feed intake and body weight were recorded weekly for the calculation of feed conversion efficiency of birds. At the end of trial, 2 ml blood samples were collected from brachial wing vein per bird from 3 birds per replicate in EDTA vacutainer tubes for studying hematological and serological parameters which included hemoglobin count and antibody titres development against the different vaccination protocols, respectively. The antibody titres were analysed at Poultry Diagnostic Centre, Venkys India Ltd, Sector 16, Panchkula, Haryana. The results of the trial were statistically analysed by one way ANOVA using IBM SPSS software. Variability in the means were considered statistically significant at p < 0.05.

Results

Growth performance

The weekly and cumulative feed intake data as presented in Table 2 suggests no significant differences among different groups (p = 0.18) in overall feed intake during the 42day period. However, during first week period feed intake values showed a tendency towards variance (p = 0.052) with numeric values of feed intake ranging from 167.92±0.67 g in T 2 to 175.46±0.20 g in group T 3 . At week 6 the feed consumption ranged from highest (909.61±38.00 g) in continuous supplemented group (T 4 ) to lowest (787.38±57.32 g) in low dose supplemented group (T 2 ). Similarly, the overall feed intake of 42 days period was highest in T 4 and lowest in T 2 group. There was no significant difference among all the treatment groups (p = 0.18). However, the results showed that numeric values of continuous supplemented group (T 4 ) were highest (3999.04±62.56 g) followed by control (3928.87±31.20 g), competitor (3911.20±7.70 g), high dose (3905.33±22.23 g) and low dose (3839.97±55.65 g) groups. Initial body weights across all the treatments were comparable, with no significant differences (p = 0.725). The overall body weight of broiler birds ranged from lowest in T 2 (2658.05±67.86) to highest in T 4 (2880.67±61.35). The statistically analysed data for overall period revealed that the numeric values for continuous supplemented group (T 4 ) resulted in highest body weight followed by high dose group (T 3 ), control (T 1 ), competitor (T 5 ) and low dose group (T 2 ). However, throughout the experimental period, either weekly or cumulative body weights showed no significant differences (p > 0.05) among all the treatment groups (Table 2). During the first week body weight gain was highest in group T 1 (176.77±2.01) and T 3 (176.39±5.09). As the trial progressed further, the results showed numerically highest body weight gain in continuous supplemented group (T 4 ) in comparison to control, competitor and rest of the beta-spike supplemented groups. The overall body gain was also highest in group T 4 with the mean value of 2836.36±61.50 g. However, the statistical analysis was not significant (p > 0.05) across all the groups during all the individual weeks or overall period (Table 2). The mean values of weekly and cumulative feed conversion ratios are summarised in Table 2. The results showed that no significant difference in FCR was observed throughout the individual weeks or whole 42 days period among different treatment groups. The cumulative FCR values recorded were as 1.48±0.02 (T 1 ), 1.47±0.02 (T 2 ), 1.47±0.05 (T 3 ), 1.41±0.03 (T 4 ) and 1.48±0.01 (T 5 ) with T 1 , T 2 , T 3 and T 5 showing very close and consistent values. Although, continuous supplemented group (T 4 ) demonstrated a superior (lowest) overall FCR of 1.41±0.03 compared to the control and other treatment groups, these variations were all non-significant with each other (p > 0.05). The statistical analysis for mean hemoglobin count (Table 3) among different treatment groups revealed no significant differences (p = 0.664) among birds of control group (T 1 ), low dose supplementation (T 2 ), high dose supplementation (T 3 ), continuous supplementation (T 4 ) or the competitor product group (T 5 ). Although, a slight numerically decreasing trend in the values was observed in the supplemented groups relative to control (T 1 ).

Humoral immune response

The mean serological antibody titres determined across different vaccination protocols are presented in Table 4. At day 28 post vaccination the new castle disease titres (log 2 ) ranged from 4.68 (T 2 ) to 6.25 (T 4 ). The continuous supplemented group (T 4: 6.25) and competitor group (T 5: 6.0) showed numerically higher values of new castle disease antibody titres than control (T 1: 4.8). The low pathogen avian influenza (LPAI) titres (log 2 ) at day 28 showed consistency among all groups ranging from 6.82(T 5 ) to 7.41 (T 3 ). However, at day 42 LPAI titres numeric values were increased in all the supplemented groups (T

Discussion

Growth performance and better immunity status derive the poultry farms productivity and most direct indicators for its economic value. In this study, the evaluation of dietary supplementation of betaspike (β-1,3-glucan) across various treatment groups (T 2 -T 4 ) and with a competitor product (T 5 ) demonstrated no significant differences (p > 0.05) in overall feed intake, body weight, body weight gain and feed conversion ratio (FCR) compared to the control (T 1 ) over the 42 days trial period. These findings suggests that inclusion of the supplement across different dietary regimens did not have any profound effects on body growth, feed intake or palatability compared to the control or competitor product. However, the numerical data suggests that product was good enough to bring modulations in the body growth, feed intake and FCR trajectories from a commercial standpoint. The continuous supplementation of betaspike in group T 4 had numerically the highest body weight gain (2880.67±61.35 g) and better FCR values (1.41±0.03) than control (T 1 ) or competitor product (T 5 ). In modern broiler farming where feed only accounts for the approximately 70% of the cost of running, a numerically 177.14 g increase in final body weight and a 7 point reduction (1.48 to 1.41) in FCR than control group still provides a meaningful economic benefit for the commercial scale poultry farmers. The lack of statistical significance may be due to sample size limitations, inherent physiobiological variations among birds of different groups, baseline conditions or management conditions of the birds during the trial. Often, the significant effects of these feed additives are demonstrated under the stress, high pathogen load, or disease conditions. Under good sanitary practices and farm management, the baseline physiology or performance of the control group remains high, which can mask the statistical significance. Our body growth results are in line with the findings of Sun Hee Moon et al. (2016) [14] , who reported that β-1,3-glucan supplementation in broilers did not have any significant effect on fed intake, body weight gain and feed conversion ratio (p > 0.05). However, similar to our findings, he stated a numerical increase in mean values of these parameters in the β-1,3glucan supplemented groups when compared to control. Our data also corroborate with the findings of Zhang et al.

(2012) [15] who reported that bacterial derived β-1,3-glucan supplementation had no significant (p > 0.05) effect on growth of broilers. However, supplementation of β-1,3glucan have been shown to increase body weight, average daily gain and FCR at significant levels (p < 0.05) in comparison to control (Wei Guo et al., 2023 and Zhang et al., 2020) [16,17] suggesting its use as a potential growth promoting feed additive in broiler production. Hemoglobin count is a vital factor that indicate physiological status, biological stress or nutritional health status of birds. In the present study, mean hemoglobin count of birds under different treatment groups remained in normal physiological range and had no statistically significant differences (p > 0.05). Although, the product did not show any increase in hemoglobin count, but it also had no hemolytic or toxic effects ensuring the safety and biocompatibility of the additive across all the dietary groups. [18] reported that β-1,3-glucan supplementation enhanced both cellular and humoral immune responses, and reduced mortality in NDV challenged broilers compared to the control group. Consistent with our results, Cao L. et al. (2023) [19] and Horst G. et al. (2019) [20] also reported a remarkable increase in ND antibody titres in the β-1,3-glucan supplemented group compared with the control. The antibody titre response to LPAI demonstrated numerically elevated humoral response in betaspike supplemented groups (T 2 , T 3 and T 4 ) in comparison to control(T 1 ) and competitor supplemented group (T 5 ) both at day 28 and day 42 of the production cycle. While ND titres peaked early (day 28) and subsequently declined due to rapid replication and clearance kinetics of live attenuated vaccines, LPAI on the other hand showed persistent elevation till market age (day 28 to day 42). This is possibly due to the fact that inactivated LPAI vaccines are typically formulated with oil adjuvants which forms a depot in body tissues that releases viral antigens slowly over a long period in the bloodstream. This suggests that betaspike supplementation worked synergistically with the vaccine protocol and sustained the titres over a long period of time without decay. In agreements with our results, Abd Al-Tawab et al. (2019) [21] and Zhen et al. (2020) [22] reported that broilers supplemented with β-1,3-glucan improved the humoral immunity by increasing AIV and NDV antibody titres.

The IBD titre evaluation at day 42 revealed that low dose (T 2 ; 1281, CV 32%) and continuous supplemented (T 4 ; 1158, CV 39%) groups demonstrated numerically superior antibody titres compared to both control (T 1 ; 1000, CV 46%) and competitor (T 5 ; 483, 24%). The higher titre in T 2 and T 4 were also accompanied by lower coefficient of variation relative to control, indicting both strong humoral immune response and uniformity of immune protection in birds. Aligning with our findings, Van Hamme et al. (2019) [6] reported that β-1,3-glucan increased antibody titre against IBD vaccination and decreased the CV% compared to the control group. Similarly, Ezzat et al. (2025) [23] found that β-1,3-glucan supplemented birds showed significantly increased (p < 0.05) IBD and ND antibody titres compared to the control. Overall, the continuously supplemented T 4 group showed the balanced, broad-spectrum protection across all pathogens, indicating that continuous supplementation of betaspike (β-1,3-glucan) supports broader and more persistent immune function in broiler production.

Conclusion

This study suggests that bacterial derived β-1,3-glucan (Betaspike) offers an effective alternative to conventional yeast or fungal sources, offering superior purity, bioavailability, production efficiency and cost. Although, growth performance parameters were statistically nonsignificant (p > 0.05), continuous supplementation at commercial scale could offer economic benefits. Demonstration of positive immunomodulatory and numerically enhanced growth performance positions Betaspike (β-1,3-glucan) as a functional feed additive for better immune function and overall production in poultry.

Acknowledgement

AcknowledgementsAll the financial and logistic support to carry out this research trial was provided by Carus Laboratories Pvt. Ltd., Sector 3, Karnal, Haryana

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