The effects of lactic acid bacteria and yeasts as probiotics on the growth performance, relative organ weight, blood parameters, and immune responses of broiler: A meta-analysis

Database development

A database was constructed based on peer-reviewed and published research articles which reported the use of probiotics in the broiler diet. Articles were selected based on the Systematic Review Center for Laboratory Animal Experimentation (SYRCLE') method⁷ and Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA)⁸. Articles selected were taken from PubMed, Web of science, Scopus, Google Scholar, and Science direct databases as well as individual journals such as World Poultry Journal Science, British Poultry Science Journal, and International Journal of Poultry Science using the keywords ‘probiotic’, ‘broiler’, ‘performance’, ‘organ weight’, ‘carcass’, and ‘blood serum’. In each article evaluated, the reference list was also searched for relevant articles. The raw database information from articles, authors, year of study, broiler (strain and sex), diet used in trial, length of trial, level of treatment, form and dosage of probiotic contained in the study was recorded in a spreadsheet following the referenced method⁷. After careful evaluation, the parameters included were growth performance, relative organ weight, carcass quality, blood parameters, and immune responses. While the economical parameters are not included due to insufficient data in the paper. The strains recorded on the raw database were Ross308, which dominated at 63.26%; Arbor Acres at 32.65%; and others at 4.09%.

Criteria for an article to be included in database were as follows: (a) article was published in a peer-reviewed with range 2008–2020, this paper length was chosen as related to journals aged last 10–12 years are often good⁹ (b) the broiler were modern-controlled-trial environment and management, (c) performed directly on broiler in vivo as the experimental animals, (d) The log concentration of lactic acid bacteria and yeast both powder and liquid form on the trial was transformation into 10¹⁰ in the database development, (e) non-probiotic treatment excluded from the database, (e) the articles written consistently in English were considered in studies, (f) the average duration of the study was minimum 0–21 days and the maximum at 0–53 days, (g) dosages given at the range 0–10 g/kg from total feed formulation. Moreover, the dependent and independent variables were selected with the aim of lactic acid bacteria and yeast related as probiotic on the broiler. Likewise, data extraction was completed in accordance with the task analysis to obtain the exact values from graphical data, the relevant figure from the papers were subjected to an online tool, WebplotDigitizer 4.4 (https://automeris.io/WebPlotDigitizer/), following the method¹⁰.

The final database consisted of 49 in vivo articles, 93 studies, and 225 n-total (3,375 n-total of total in this experiment). The details for the study selection included in this meta-analysis are provided in Figure 1. The search strategy is presented in Table 1. The summary of the final database is presented in Table 2, and PICOS criteria presented in Table 6.

Figure 1. Diagram flow of article selection included in meta-analysis adapted from PRISMA Method.

Data analysis

Statistical dataset analysis using a mixed-model approach was applied^11–14 with statement analysis in the system using the MIXED procedure of SAS (version 9.1, SAS Institute Inc., 2008), the following model was applied: The findings of a study was then taken as a random effect, while the supplementation concentration was taken as the fixed effect as follows^15–17:

where Yij = the expected output for dependent variable Y at level j from the variable X as a continuous variable in the study i,, B₀ = overall intercept across all studies (fixed effect), B₁ = linear regression coefficient of Y on X (fixed effect), B₂ = quadratic regression coefficient of Y on X (fixed effect), X_ij = value of the continuous predictor variable (probiotic supplementation level), s_i = random effect of study i, b_i = random effect of study i on the regression coefficient of Y on X in study i and e_ij = unexplained residual error. In the statement CLASS, the “study” variable was declared. Data were weighted by the number of replicates in each study. Additionally, an unstructured variance – covariance matrix (type = un) was performed at the random effect part of the model to avoid a positive correlation between intercepts and slopes. Significance of an effect was stated at the probability level of p < .05, and p < .1 was considered as a tendency of significance. In case that the quadratic model above was not significant, the model was changed into its corresponding linear model. The variable of the study was declared in the class statement as it did not contain any quantitative information. The regression equations are also presented with p-value, and root mean square error (RMSE).

Furthermore, to determine (1) interaction between lactic acid bacteria and yeast; (2) interaction of type probiotic (powder and liquid) according to the following model¹⁶:

Where Yij = the expected output for dependent variable Y, μ = overall mean, Si = random effect of I study, τj = fixed effect of the j level, Sτij = random interaction between i study and the j level, and eij =residual error. A significant effect was declared at p<0.05 or there is a tendency when the p-value was between 0.05 and 0.10.

The effect of probiotics on growth performance

Our meta-analysis shows that probiotics positively affect growth performance. In terms of growth performance, we suggest that this finding is related to the ability of probiotics to induce intestinal mechanisms, resulting in a reduction in pathogenic bacteria. In the digestive system, intestinal pH, intestinal bacteria composition, and digestive activity are improved when probiotics are present in diets¹⁸. Some probiotics are known to produce enzymes, amylase, protease, and lipase to optimize nutrients’ breakdown^19,20. They can also increase specific enzymes in the host digestive tract to enhance nutrient absorption in the diet. In the poultry industry, probiotics are supplemented into the diet to maintain health by enhancing gut health, modulating the immune system, lowering glycaemic response, and improving various performances parameters^21–23. Moreover, the administering of probiotics has several ways in practice. The administering of probiotics can be included in the basal diet or combined with raw materials that contain prebiotics to enhance its effect^{20,30,50–54,67}. The probiotic can be given alone or with another additive without any negative effect such as acidifiers and phytogenics^{25,26,32,42,44}. Furthermore, probiotics can contain one or multiple microorganism strains that can be added to animals' diets^24,28,37.

There are previous studies that report that cell-wall components of yeast in dietary supplementation to lactic acid bacteria improve the growth rate, feed consumption, and feed efficiency in broilers^51,55. These positive and consistent results were due to yeast activating spores to reduce and remove potential pathogens in the gut which possibly increases body weight⁴⁴. Linearly, the factors related to synergism between yeast and lactic acid bacteria reported in the research could be related to environmental conditions in various experiments^45,46.

One study from Sugiharto et al., 2017⁴⁶ showed that rearing the broiler with a heat-stress environment at 35°C was more low weight than heat stress exposed; in agreement with Attia et al., 2017⁶⁸ that the heat stressed are successfully reduced the weight of broiler. Thus, yeast failed to alleviate heat-stress on the performance of broiler⁴⁶. The lower temperature in the chicken house may help increase feed intake to eat more of the experimental diets⁵⁷. In addition, other factors related due to being reared under a stocking density stress of 43 kg live weight per m² floor space⁵⁷. Another result⁴⁵ explains in more detail that yeasts in powder form significantly increase the body weight of broilers starting at 21 days old, with an increase in line with increasing feed intake and reducing FCR. These consistent results^57–59 explain that the use of lactic acid bacteria as probiotics in powder form help to increase the body weight of broilers, as a result of digestibility and metabolic process improvement caused by the bacteria, affecting energy partition and putting more energy into growth than maintenance⁴⁶. Conversely, we found in Attia et al., 2012⁶⁹ that the combination of the probiotic used in broiler after challenged with Salmonella enteritidis are insignificant differences.

Moreover, the positive use of probiotics both as powders and liquids was in line with the increasing level of treatment in broiler⁵⁷. The probiotics enhance liquid lactic acid bacteria synergism with yeast in the feed but suggested at an optimized level⁵⁷ of 0.8%. However, the dose-response relationship of probiotics in animal trials is rarely studied¹. At low doses a probiotic may be specific, for example bifidobacteria, due to the high specificity of bifidobacteria for that particular probiotic¹. In other hand, if the dose increased, this would leave some substrate for other probiotic strains able to ferment it. The outcome of high dose would show less specificity that that of the low dose¹. Treatment with both powder and liquid forms increases body weight and feed intake and reduces feed conversion^31,57,61. Moreover, probiotics for farm animals have positive effects on growth, efficiency of feed utilization⁷⁰. Determination of novel probiotic until meet animal requirement balancing are key for development of animal industry in future trends^71,72. Thus, utilization of feed is also reflected on the nutrient digestibility⁷³. In addition, the consistent result in studies⁴⁵ vs 50–52 show the relationship between both yeast and lactic acid bacteria working together to reduce potential pathogens in the gut of broilers but dose is dependent.

The effect of probiotic on the relative organ weight and carcass quality

The meta-analysis results show limited effects on the carcass and organ weight of broilers. In agreement from 52,62 the carcass quality shows no significant difference after administering probiotics of both lactic acid bacteria and yeast. The one factor can be caused reduced of percentage carcass are heat-stress environmental. The carcass heat-stress was associated with the reduced of carcass quality⁵². In line, from Attia et al., 2017⁶⁸ that the high temperature was insignificant effect with organ weigh and carcass quality. Conversely, that broiler exposed with 32°C was reported higher abdominal fat, relative organ weight, and breast meat compared with control group 22°C. Apart from physiological adjustment derived from depressed feed intake, the increased Corticosterone level may be responsible lower percentage of carcass⁵². Carcass percentage was reported to increase by one study⁴⁵, with the saleable product in terms of edible portions. Reported⁴⁵ the carcass quality can be affected from physiological and genetic potential, feed formulated, strain of the broiler rearing⁷³. The excess fat deposition in carcass of broiler is undesirable to producers because of reduced carcass yield and to consumers that prefer a leaner product¹.

Likewise, one study⁶² the use of yeast as probiotic reduced abdominal fat of broiler. Reported⁶² yeast help to reduce fat deposition because, modern-broiler-farming were intensive feeding (ad-libitum), fatter caused limb defects, and sudden death syndrome. The probiotics reported reduced fat of broiler compared without probiotic⁶⁵. The mode of action was that probiotics decreased the activity of acetyl-CoA carboxylase⁶⁶. Acetyl-CoA carboxylase has been widely suggested as the rate-limiting enzyme in fatty acid synthesis⁶⁶. The decline in the synthesis of fatty acids, in turn, would decrease their availability for esterification to triglycerides for deposed in the adipose tissue⁶⁶. Furthermore, the minimum dose of probiotic to stimulate fatty acids are currently unknown^63,64,66. Differences in the broiler line/breed and conditions, as well as microorganism strains (highly species-and strain specific), origin species, concentrations, and methods of administration of the probiotic bacteria, may explain these results⁶⁵. However, our study can’t exactly suggest the dose optimum for using this probiotic. But, Adli & Sjofjan, 2020⁷⁴ suggested that using probiotic as replacement of antibiotic growth promoters (AGPs) are not more than 1% from total feed formulation.

In one study⁴², the effects of probiotics on relative weights of liver and spleen were not significant (P > 0.05), while bursa of Fabricius relative weight increased (p < 0.05). Supplementing diets with probiotics could help to prevent necrotic enteritis which associated with degeneration of hepatocytes and immune system of the broiler⁷⁵. The smaller liver in broiler may indicate a higher resistance to pathogen microorganism such as Clostridium perfrigens⁷⁶. The IGF-1 can produce short-fatty acids (SCFAs), which act either directly or indirectly on the liver and adipose tissue to promote growth of organ and skeletal development⁶⁵. The report from 77 at the end of the feeding trial showed that the development of gizzard weight was decreased, dateable irregularities in the gizzard are a sensitive index to reduce anti-nutritional factors in the basal diet after exposure to toxic substance not to amount of lactic bacteria⁷⁷. In light of a previous study⁶⁹, the probioic was effective treatment in decreasing the shedding of Salmonella in faeces and in decreasing the re-isolation of Salmonella from the liver, spleen and cecum. Moreover, increased weight of this lymphoid organ may indicate a higher immunity achieved in treated broiler, which could be explained by probiotic anti-microbial activity^18,42. The factor affected by significant differences in the relative organ weight is the ability to absorb substances from probiotics^18,43,44. 18 stated that variances between the broilers result from impacts on absorption and other capacities of the relative organ weight. The growth factors correlate with age, while the broiler's uses in the relative age cause the same internal organ’s growth. In instances, an increased relative organ weight may be in line with an increase in lymphocyte concentration¹⁸. Reported from Al-Sagan et al., 2020⁷⁸ the addition of probiotic are significant differences influences until 29.3% lymphoid organs which are correlated with immune response compared with supplemental group.

The effect of probiotic on the blood parameters and immune responses

The meta-analysis of different probiotic levels on some blood parameters showed red and white blood cell concentration increased (p <0.05) with increasing probiotic supplementation levels in the feed. The increased of the white blood cells had correlated with yeast reduce the uric acid (UA) content in the blood⁷⁹. The uric acid is a metabolite of protein that has an antioxidant function, but is converted to a pro-oxidant in the cell or cytoplasm⁷⁹. In contrast, lymphocyte, and hemoglobin were not significantly different (p >0.05)¹⁵. Linearly, blood serum rose in line with probiotic increase. Additionally, one¹⁸ shows that, the lactic acid bacteria that help reduce avian-pathogenic bacteria were Escherichia coli and Clostridium perfrigens. The beneficial action indicates that lactic acid bacteria produced extracellular enzymes to enhance the nutrient digestibility of feed and synthesize immune function using endogenous anti-microbial²⁰. In terms of negative linear response in studies²¹ there was no positive result on red and white blood cells.

Moreover, probiotics could be related to a lowered recycling of bile salts in the gut or inhibited hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity²¹. The mechanism operating in lactic acid bacteria, as probiotics to elicit their hypocholesterolemic effect is interference with intestinal bile acid transport and absorption, leading to an increase in bile acid excretion²¹. The potential pathogens reduce but are not eliminated, thus, probiotics balance the intestinal environment to enhance the broiler’s immune systems^45,50–52. Although, the lactic acid bacteria do not produce butyric acid themselves, they stimulate the proliferation of butyric acid and cell-wall of yeast in the blood circulation by the mechanism that is called cross-feeding⁶⁵. Continued research⁴² shows probiotics help to increase the white blood cell count as level of probiotic is increased. One study⁴² stated that the increase of white blood cells and immune response was due to the level increase of B and T lymphocyte production. In line with the 41 studies the amount of red blood cells, hemoglobin, and white blood cells consistently tends to increase compared to controls. The positive effect from yeast as a probiotic could derive from its outer cell wall components namely: chitin, mannan, and glucan which have an immunostimulant effect. Moreover, these outer wall components promote lactic acid bacteria activity, which is activated by producing enzymes that cause disintegration of bile salts, making them unconjugated⁷⁰. The yeast can enhance the immune response by promoting growth of lactic acid bacteria and thus simultaneously producing antibacterial substances and stimulating the production of immunoglobulin³³. Thus, yeast acts as a supporting agent of lactic acid bacteria, which adhere to the endogenous epithelial cells to initiate colonization³³.

Underlying data

All data underlying the results are available as part of the article and no additional source data are required.

Extended data

Figshare: Extended data for ‘The effects of lactic acid bacteria and yeasts as probiotics on the growth performance, relative organ weight, blood parameters, and immune responses of broiler: A meta-analysis’. https://doi.org/10.6084/m9.figshare.14060414⁸⁰.

This project contains extracted data of outcome measures (BW: body weight; BWG; body weight gain; FCR: feed conversion ratio; FI; feed intake; RBC: red blood cell; WBC: white blood cell).

Reporting guidelines

Figshare: PRISMA checklist for ‘‘The effects of lactic acid bacteria and yeasts as probiotics on the growth performance, relative organ weight, blood parameters, and immune responses of broiler: A meta-analysis’’. https://doi.org/10.6084/m9.figshare.14060501⁸¹.

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC BY 4.0)