#### Meta-analysis identifies the effect of dietary multi-enzyme supplementation on gut health of pigs

Meta-analysis identifies the effect of dietary multi-enzyme supplementation on gut health of pigs

Gut health though is not well defned the role of gastrointestinal tract is vital if an animal must perform well. Apart from digestion, secretion, and absorption gut is harbored with consortium of microbiota which plays a key role in one's health. Enzymes, one of the alternatives for antibiotics with benefcial efects on digestion and consistency of food and its efect on gut health. The efect of enzyme supplementation on gut health is not well established and the objective of this meta-analysis is to investigate if the enzyme supplement has infuence on gut. This meta-analysis includes 1221 experiments which has single enzyme studies and or studies with multiple enzyme complexes but not challenged. The ratio of Lactobacillus and E. coli is related to ADFI which showed comparatively lower negative correlation coefcient, with −0.052 and − 0.035, respectively, whose I^{2} values are below 25%, showing that these studies show a signifcantly lower level of heterogeneity. Correlation between villus height, crypt depth, their ratio and fatty acid is also assessed, and it showed that when the animal is supplemented with two enzyme complexes resulted in positive gut health rather than the single or more than two enzymes.

The gastrointestinal tract (GIT) is the interface at which digestion, secretion, and absorption occurs. In, pigs, the digestive system is monogastric^{1}. The pig's primitive digestive system is formed when it is at twelve days of gestation period from the entoderm, however, the maturity of the GIT develops during the pig's perinatal life. Most of the digestive capabilities are observed during the perinatal stage^{2–6}. Hitherto, there is no known defnition for the health of the gut, or gut health. According to the World Health Organization, gut health is broadly defned as follows: (1) efective digestibility and absorption of food, (2) absence of gut illness, (3) normal and stable gut microbiota, (4) good immune status, and (5) a status of well-being^{7}.

The focus of global livestock industries is the use of economical, efcient, and sustainable production methods without compromising the quality or quantity of the products, as this determines the potential of obtaining good economic returns. Globally, swine is the most highly consumed meat and also serves as good animal model. Despite many milestones having been achieved in research, there is still room to improve the efciency of the production of swine^{8,9}. An efcient production using swine is obtained by maintaining the swine from the piglet stage to the slaughter stage with good health and good feed concomitantly without disturbing the animal's welfare or environment^{10}. During this process, pigs consume several types of feed and are subjected to various feeding strategies. Moreover, for economic reasons, many alternative feeds have been used that are difcult for the digestion (example, non-digestible fber rich feed) in a monogastric animal. Furthermore, the use of antibiotics in animal feed as a growth promotor should be restricted due to the possibility of arising antibiotic resistance and environmental issues.

The use of enzymes in feed supplement for promoting gut health in pigs is expected to increase as an alternative to antibiotics and a solution to economic concerns. Enzymes can also be used very efectively as an alternative to probiotics or essential oils^{11,12}. Due to this increasing interest, this study is conducted to meet the needs of long-term. Yet, no-good studies are made to investigate the efcacy of enzymes in feed supplement in terms of the gut health of pigs or any livestock animal.

**Table 1.**Enzyme supplement category based on feeding method.

**Figure 1.**Pie-chart showing the total percentage of the studies by each enzyme supplement category

Meta-analysis uses a statistical technique in a systematic review to derive and integrate the results from all the included studies^{13,14}. The efect size indices help to perform the quantifcation of all the collected study results using the same metrics. The assessment of heterogeneity is quantifed by Cochran Q statistic, I^{2}, H^{2}, and15 τ^{2}. Density plotting is a good approach for visualizing data in order to confrm whether the data is normally distributed or whether there is any skew or kurtosis.

**Results and discussion**

The titles and abstracts of 112 articles are screened before selecting 17 articles (Supplementary 1) for consideration in this study. Overall, 1221 experimental treatments were considered for this study afer an elaborate search and duplicates were excluded. The feed system categories are ad libitum, controlled, and restricted feed which are used in 1038, 157, and 26 treatments, respectively. Most of the animal studies involved ad libitum feeding systems. The treatments selected here has at least one type of enzyme supplements, ranging from single enzyme supplements to studies with multiple enzyme complexes. There are 349 treatments in which one type of enzyme is used, 213 treatments in which two types of enzyme were supplemented, 279 treatments in which three types of enzymes were supplemented, and 380 treatments in which more than three types of enzyme were supplemented, categorized as multi-enzyme complex. Table 1 shows the characteristics of the feeding methods with the type of enzyme supplement. The total percentage of the studies for each enzyme category is shown in Fig. 1, as follows: multi-enzyme complex studies made up 31% of the database, single enzyme supplementation 29%, two-enzyme supplementation 17%, and three-enzyme supplementation 23%.

The selected articles are reported with the diferent enzyme supplementation which are listed in the Table 2. The supplemented enzymes are categorized into carbohydrase, protease, phytase and lipase. Though many studies included single type of enzyme which are carbohydrase, protease and lipase the individual enzymes are little varied based on the choice of feed, herein carbohydrase are α-Galactosidase and xylanase supplemented as single enzymes, and xylanase was always added in combination with other carbohydrase is β-Glucanase in case of single type of enzyme supplement. Apart from this, some treatments included protease and lipase as individual enzyme supplement. In combination supplemented articles with carbohydrase and protease, xylanase is present in most of the treatments. In case of two types of enzyme supplements, it is 3 diferent enzymes are feed where 2 enzymes are carbohydrase which are xylanase and amylase. The other type added is protease which is extracted from bacterial source. Phytase is feed in two or three enzyme type combination only.

**Table 2.**Types of enzymes as feed supplement utilized in the meta-analysis. Reference in the table are in the supplementary document.

Most of the experiments are carried using commercial enzymes, accounting for 66.83% of the treatments, followed by enzymes of bacterial and fungal origin, with 23.58%. These two types of enzymes account for 90.41% of the database created. On the other hand, treatments where the enzymes of bacterial and fungal origin are individually supplemented accounted for with 5.24% and 5.48%, respectively, along with other types of feed administered to the pigs in the diferent studies, as presented in Table 3.

**Table 3.**Consolidated table with feed type and method, enzyme type and its origin. Flow of information through the stages of the meta-analysis.

**Efects of enzymes in gut health. **An in-depth meta-analysis of digestion, a gut health response variable processed with subclasses average daily feed intake (ADFI), average daily gain (ADG), crude protein (CP), gross energy (GE), gain to feed ratio (G:F) and the Lactobacillus : E. coli (La:Ec) ratio, is conducted. In the case of ADFI, the estimates showed a signifcantly lower heterogeneity with a total heterogeneity and a standard error (SE) of 0.0055 and 0.0043, respectively, whose total heterogeneity by total variability, I^{2}, is 18.81%. The heterogeneity of Q-statistic is 69.0841, with a p-value of 0.4068 (Table 4). The model estimate is −0.0525 with a standard error (SE) of 0.0242, whose z-score is −2.1726 and a 95% class interval (CI) upper bound and lower bound of −0.0051 and −0.0998, respectively. Te analysis shows that the studies are not diferent and hence the results are valid. Overall, the enzyme supplement in the feed, irrelevant on the type and number of enzymes, showed a negative correlation (Table 5). On the other hand, the ADFI and La:Ec subclasses had a lower negative correlation coefcient, with −0.052 and −0.035, respectively, and a 95% CI upper bound of −0.005 and 0.011, respectively, with a lower bound of −0.099 and −0.08, whose I^{2} values are below 25%, indicating that these studies show a very signifcant lower level of heterogeneity. Moreover, it is evident that the ratio of Lactobacillus and E. coli is related to the ADFI. This heterogeneity is dependent on animal age, gender, feed, and other conditions. These results are consistent with other available experimental studies displaying that pigs when fed with enzyme supplemented diet has presented with better gut health even when the pigs are challenged with other pathogens^{16}. Hence, the outcomes coherent and indicate that feed intake with specifc enzymes plays a critical reduction in pathogens (ex. E.coli) or leaky gut, in other words, this can maintain the gut fora with benefcial microbial abundance (ex., Lactobacillus sp.) with the static efects towards pathogenic bacteria which is a protective mechanism for maintenance or enhancement of gut health.

**Assessment of publication bias.** The publication bias is assessed using funnel plots and further regression tests for funnel plot asymmetry is done in every case (Fig. 2a–g). Using Egger's mixed efects meta regression model, the publication bias is predicted based on the standard error and rank correlation test for asymmetry, using Kendell's Tau statistics for ADFI, ADG, CP, GE, G:F, and La:Ec (Table 6). Egger's test for fatty acids (FA) and La:Ec showed no signifcance, which indicates that there is no evidence for publication bias in this case. However, although the ADFI and G:F results are non-signifcant, they are comparatively lower than the former ones. Although, there is no consensus for the benefciary efects of ADFI, ADG, GE, CP, or G:F in feed supplemented with enzymes, in the case of La:Ec and FA, the efects of enzyme supplementation are observed in some studies^{17–19}.

**Growth relation with ADFI.**The ameliorating efect of enzyme supplementation on feed appears to be based on the substrate which is added. In enzyme supplementation strategy, as the addition of enzymes is assumed to increase animal performance, in this study we carried out variance–covariance analysis to decipher the enzyme supplement efcacy in animal gut health. Polynomial regression analysis is performed using the ADFI with ADG values. Initially, the data distribution is visualized using density plots (Fig. 3a–c). To fit the model, the x variable is cubed when the y represented ADFI with an intercept (α) of 0.001 (p=0.01) and where β1 is 1.118, β2 is 1.346, and β3 is 0.117 (Fig. 4a). Tis ftted model had a residual standard error (RSE) of 0.2383 and 56 degrees of freedom (DOF), a multiple R-squared (RSD) of 0.1965, an adjusted R-squared (Rad) of 0.1534 with a F-statistic of 4.564 (p-value=0.006256). Reduction in ADG is ascribed to a reduced ADFI, however in the gut health challenges, growth may be impaired due to an increase in the requirements for the metabolic and digestive processes. Apart from gut health conditions, the G:F ratio also is impaired by other factors like diet composition, age of pigs, other environmental factors also infuence. However further studies are needed considering all the factors.

**Table 4.**Random-efects model with the τ

^{2}estimator as restricted maximum-likelihood estimator. Signifcant codes: 0 ‘***' 0.001 ‘**' 0.01 ‘*' 0.05 ‘.' 0.1 ‘ ' 1.

**Table 5.** Summary of correlation for Predicted Fisher's r-to-z scores transformed to correlation coefcients.

**Infuence of enzymes on Lactobacillus versus E.coli. **Many subjects have demonstrated that under dysbiosis conditions, the abundance of E. coli is higher and the amount of benefciary bacteria belonging to the genera Firmicutes (e.g. Lactobacillus) is reduced. Lactobacillus is highly associated with gut health factors and animal performance^{20}. Thus, the ratio of Lactobacillus and E. coli has played an important role in the animal's gut health and overall performance. Here, when Lactobacillus is increased, the abundance of E. coli is found to decrease. The α is −0.057 when y is Lactobacillus, with the x squared to fit the regression model (Fig. 4b). The estimated coefcients are β1 is −0.575 (p-value=0.008) and β2 is −0.39024. RSE is 0.309 with 41 DOF. The RSD and Rad are 0.4088 and 0.38, respectively, with an F-statistic of 14.18 (p-value=0.00002). Thus, it is very clear that the population of Lactobacillus negatively regulates the pathogenic E. coli population, and the use of enzymes can sometimes enhance the Lactobacillus count in the intestine to improve gut health.

Gut health is associated with microbial abundance and diversity as the presence of certain types of microbes are benefcial, since the metabolites produced by these bacteria assist other benefcial bacteria in the microbial network^{20–23}. However, the infuence of the types or numbers of enzymes on the structure of microbes and fatty acids production in gut has not yet been well established. The changes in substrate availability for microbes is a determining factor in the role of microbes in gut health. In the case of E. coli, model ftting did not show any trend or signifcance regarding fatty acids in the GIT (Fig. 4c). On the other hand, Lactobacillus showed a trend and model ftting (Fig. 4d). The intercepts for E. coli and Lactobacillus are 0.2437 (p-value=0.000547) and −0.3829 (p-value = 0.000004), respectively, indicating that when E. coli is at zero, the fatty acid intercept is positive, meaning that the production of FA is not dependent on E. coli. On the other hand, the Lactobacillus intercept is predicted to be a negative value, indicating that if Lactobacillus is absent, the production of fatty acid is also absent, which could lead to signifcant problem. The results are consistent with other published articles where the supplementing the animals with enzyme resulting in reduced pathogens even during challenged conditions and in non-challenged conditions^{16,24}.

**Changes in VH:CD ratio.** Digested food is absorbed through brush borders, also known as projected villi, and invaginated crypts, which are microanatomical structures that play a crucial role in gut health. These structures are lined with a mucus layer that provides a mucosal barrier. The height of the projected villus and the depth of the crypts are also indicators of GIT health. Many factors infuence the villus height and crypt depth ratio (VH:CD), including the type of feed, viscosity, pH, and pathological conditions, among others. Thus, a balance between the physiological and pathophysiological is essential for an adequate VH:CD ratio. The supplementation feed with enzymes may modulate the VH:CD levels, with certain studies reporting that enzyme supplementation increases viscosity, leading to changes in this ratio which result in a leaky bowl18. In the case of VH:CD, the intercept coefcient is −0.33308 with an RSE of 0.1921 and DOF of 37 (Fig. 4e). The RSD is 0.1706, Rad is 0.1257, and the F-statistic is 3.805 (p-value=0.03). The correlation between VH and fatty acid is predicted to have an intercept of −0.20688 (p-value=0.0000007) with an RSE of 0.174 and a DOF of 37 (Fig. 4f). The RSD is 0.01201, Rad is −0.04139, and the F-statistic is 0.225 (p-value=0.79).

This study will serve as an important contribution to the body of literature on the impact of enzyme used in animal feed. Here upon compiling the gut microbial structure (Fig. 5), VH, CD, E. coli, Lactobacillus, and fatty acids against the diferent types of enzyme complex, and using Hedge's g for comparison, it is very clear that the usage of two types of enzymes, based on the substrate provided, is more benefcial than the use of a single enzyme or three enzymes and above (Fig. 6). It is also clear that the choice of enzymes based on the substrate is critical in case of animal health. For instance, phytase with carbohydrase supplement improves animal performance by increasing mineral and nutrient absorption. the use of enzymes by animal weight category also plays a role

^{25}. In case of single enzyme supplement for example xylanase (carbohydrase), animals below 10 kg has showed reduced gut health conditions, where the animals showed reduced V:C ratio mediated by a proinfammatory cytokine tumor necrosing factor alpha (TNFα)

^{26}. TNFα is well known to trigger the infammatory responses

^{27}. Whereas, addition of two enzymes like protease and carbohydrase has beneftted the animals by modifcations in digestibility and microbial consortium in contrast

^{24}. Infammation in gut is mediated through the upregulation of infammatory cytokines which tends to leaky gut, where the changes in mucosal barrier function, gut immunity, and microorganisms are observed.

**Figure 2.** Funnel plots to assess the publication bias. (a) ADFI. (b) ADG. (c) CP. (d) GE. (e) GF. (f) La:Ec. (g) Fatty acids. ADFI: Average daily feed intake; ADG: Average daily gain; LB: Lactobacillus; Ec: Escherichia coli; V/VH: Villus height, C: Crypt depth, CP: Crude protein; GE: Gross energy; GF: Gain to Feed ratio, and La:Ec: Lactobacillus : E. coli La:Ec ratio.

**Table 6.**Tests for publication bias.

**Figure 3.**Density plot. (a) Density plot of ADFI and ADG data. (b) Density plot of fatty acids. (c) Density plot of microbes.

**Figure 4.** Polynomial regression analysis. (a) Polynomial regression analysis of ADFI versus ADG. (b) Polynomial regression analysis of Lactobacillus versus E.coli (c) Polynomial regression analysis of E.coli versus fatty acids. (d) Polynomial regression analysis of Lactobacillus versu Fatty acids. (e) Polynomial regression analysis of V:C versus fatty acids. (f) Polynomial regression analysis of Villus height versus fatty acids. ADFI: Average daily feed intake; ADG: Average daily gain; LB: Lactobacillus; Ec: Escherichia coli; V/VH: Villus height, C: Crypt depth, FA/SCFA: fatty acids.

**Figure 5.**Change in the composition of the gut bacterial population upon enzyme supplement.

**Figure 6.** Gut health parameters assessment based on number of enzyme supplement: EC1: single enzyme; EC2: two enzyme complexes; EC3: three or more enzymes.

To our knowledge there is no meta-analysis reporting the gut health of pigs based on the enzyme supplement. Also, there is a need for more experiments to be proven regarding the activity of enzymes relation with all the gut health attributes. Based on these results, we propose that use of two enzyme supplement in pig feed programs rather than multiple enzyme supplement which may beneft the pig gut health. However further experiments should be conducted to decipher the correlation between enzyme supplement and all the gut health.

**Methods**

Search strategy. An intensive computerized literature search is performed to retrieve studies between 2000 and 2018. The search is conducted on PubMed, Web of Science, Google Scholar, and Scopus. The key terms used are "enzymes," "feed additives," "feed supplement," "Swine," "pig," "health," "microorganisms," and "gut health." Upon completing the search, all duplicates are removed, and the articles' titles and abstracts are screened for selection. The full text is then accessed for the knowledge retrieval by two researchers individually. Afer completing the literature search as per PRISMA guidelines, a fow chart for identifcation, screening, eligibility, and inclusion is prepared

^{28}(Fig. 7).

**Figure 7.** Flow of information through the stages of the meta-analysis.

Literature inclusion and exclusion criteria. Only articles where swine feed is supplemented with enzymes or enzyme complexes and fed to animals that had never been challenged are considered. The article selection criterion is as follows: (1) a weaning period between 17 and 28 days; (2) a commonly used feed, i.e. soybean meal, corn-soybean meal, barley, or wheat, supplemented ad libitum or via a controlled system; (3) feed is supplemented with at least a single type of enzyme or with a multi-enzyme complex; (4) articles reporting animal performance, gut health parameters (such as SCFA), villus and crypt sizes or ratios, and microorganism studies; (5) articles with mean and SEM are only considered to avoid bias. The exclusion criteria are: (1) studies where animals are challenged with pathogens or toxins; (2) feed supplemented with probiotics or prebiotics.

**Database characteristics.** The database is created using Microsof Access. A form is created to input the data extracted from each article. Each form represented an experimental study from the selected article which ft as a single row in the database. Initially, the database had a total of 1221 rows and 39 columns. Upon including the data from the articles, the efect sizes are computed and added as separate columns. The calculated efect sizes are Cohen's d, Hedges' g, Fisher's z, Cohen's f, odds ratio, Cox-odds ratio, log odds, Pearson's r, Cox-log odds, efect size eta square, the standard error of the efect size, the variance of efect size, the lower and upper confdence limits, and the weight factor. As a result, the fnal database had a total of 55 columns afer the addition of the alculated efect sizes for meta-analysis. Every experimental study is encoded with a unique number which is generated automatically when a form is generated freshly, which is used as a general code. Each article is given a sequence number. For inter- and intra-studies, another set of sequence number are allocated, such that a unique code is available for each study retrieved from the selected publications. Thus, the rows in the database represented the treatment and the columns represented the exploratory variables or parameters.

**Data extraction.** All the eligible study data are extracted by data entry using the Microsof Access form, where the publication year of the article, the title of the article, the author names, the sample size, and the mean and SEM are recorded. Moderating variables such as age, weaning period, initial weight, fnal weight, enzyme type, number of enzymes, enzyme source, enzyme concentration, sample type and time, experiment period, number of animals per pen, genetic background of the animals, feed type, and time are also recorded for analysis. The researchers extracting the data independently cross-verifed the data for typographic errors and accuracy.

**Data analysis. **The unique codifed data is classifed based on the following five health response variables: absorption and digestion, gut illness, microbiota, immune status, and status of wellbeing^{7}. To determine their efects across multiple studies, as well as within individual studies, the models suggested by^{29,30} are utilized. Dependent and independent variables are defned as per the^{29} approach. R programming version 3.5.1 is used for all data analysis, where the "esc" package is used to calculate the efect size^{31}. The calculated efect size is used for the meta-analysis. The packages "metaphor," "robumeta, "meta," "metagear," and/or "mvmeta" are used to conduct the meta-analysis^{13,32}. The data is initially explored via graphical analysis to observe the data distribution to check for any heteroscedasticity. Density plots are generated using the Cohen's d efect size values of the experimental variables.

For conducting a meta-analysis of the efect sizes, the corresponding sample variance are calculated using Cohen's d and Pearson's r (r). Here, the r is transformed into Fisher's z score^{33}. The correlation coefcient quantifes for both the direction and strength of the linear relationship between the two quantitative variables and is therefore used ofen as the resulting measure for meta-analyses. Here, the Fisher's r-to-z transformed correlation coefcient is used for the analysis, as this alternative measure is a bias-corrected version of the previous coefcient^{34}. The meta-analysis is carried using the random-efects model for measuring between two quantitative variables. Sequentially, the classifed data is explored using graphical analysis with Cohen's d, efect size, Fisher's transformed r-to-z, the estimated sample variance, and Hedge's g. Heterogeneity is estimated using the restricted maximum-likelihood estimator method with random-efect model ftting^{35}. The study weights are estimated as inverse-variance, which is a default setting in the "metafor" package.

A quantifed heterogeneity is reported based on the Q-statistic, I^{2}, H^{2}, and τ^{2} data. A Q-statistic is reported with a DOF with a p-value of the test, which is null hypothesis signifcance test, which tells us the overall heterogeneity between the studies, but cannot tell us the extent of true heterogeneity^{36}. To overcome the limitations of the Q-statistic, τ^{2} is reported to estimate the total amount of true heterogeneity. In the case of τ^{2}, it is only dependent on a specifc type of efect size estimates, as such, it cannot be used to compare diferent meta-analyses with diferent efect sizes. However, the I^{2} index can be obtained with a diferent number of studies and with several types of efect size metrics, which are comparable. I^{2} indices of 25%, 50%, and 75% are interpreted as low, moderate, and high, respectively. As shown in previous studies, the between-study variance, τ^{2}, and the I^{2} are directly related^{15,37}. The summary of efect size is derived from the estimated model coefcient using the standard error, z-score, p-value, and upper and lower bounds of the confdence interval (CI). For every subgroup metaanalysis is onducted, the results are obtained by further transformation of Fisher's r-to-z scores into correlation coefcients estimates along using the upper and lower bound 95% CI to interpret the results.

**Data visualization.** Although a possible heterogeneity is suggested, the data did not point out the specifc studies which are infuencing this heterogeneity. Thus, Bajaut graphical plotting is carried as a diagnostic plot in an attempt to sort the heterogeneity data. The data used for plotting is the squared Pearson's residuals of the studies and the infuencers, i.e. the standardized squared diference between the ftted value for each study. The data points in the plot that identify the infuencing data are the study ID numbers. The most infuential studies are observed in the top right quadrant of the plot^{38}. Another set of diagnostic plots is also generated and visualized for the potential infuencers and outliers using infuence plots. Upon computing, we produced a tabular data displaying the diference in the fts, covariance ratios, Cook's distances, and the diagonal elements of the hat matrices. Any infuencing data is marked with an asterisk and the infuencers are visualized by diferent colors in the plot^{39,40}.

The point estimates of the study, with the CI and the summary of efect sizes, is plotted on a forest plot to visualize the meta-analysis of the gut health subgroups, or response variables (forest plots are not shown rather the data is presented in table form). The edges of the polygon represent the 95% confdence limit. The publication bias is evaluated based the funnel plots, the Egger's regression test, and the rank correlation test^{41}. Moderator analysis is carried out to estimate the heterogeneity using meta-regression models.

Variance–covariance analysis are performed to determine the efect of enzyme supplementation on the gut health response variables within individual studies and between diferent studies in which residual analysis is initially conducted. The residual analysis is further graphically verifed and observed for normal distribution. The wellbeing of the animals is correlated with performance, hence, the correlation between the coefcients of average daily feed intake is initially regressed against the average daily weight gain to evaluate the biological relevance^{29}. Furthermore, to infer the correlation between other gut health factors, polynomial regression model analysis is conducted, as discussed earlier. Factors included comparing Lactobacillus to Escherichia coli, whose populations indicate the status of the health and dysbiosis condition, since gut health can also be characterized by the villus height (VH), crypt depth (CD), concentration of short chain fatty acids (SCFA), and VH:CD ratios. These parameters are mechanistically inter-related in the support of gut health, hence, we verifed their relationship and their associated regulatory mechanisms in the presence of enzyme supplements. The formula for the polynomial regression ft is provided along with the representative plots^{30}.

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**Article made possible through the contribution of Sungkwon Park et al.**