Effect of Vernonia amygdalina leaf meal on growth performance, intestinal mucosa activity, digestive enzymes, absorption capacity, and immunity in broiler chickens

Gut health is multifaceted and is largely influenced by the rearing environment and the diet. The use of phytochemicals rich in phenolics and flavonoids can improve the digestive health of chickens and lead to better growth performance. The aim of this study was to examine the effects of dietary Vernonia amygdalina leaf meal (VALM) on growth performance, digestive enzyme activities, absorption function, organ weights and immunity of broilers. Two hundred and forty, one-day-old male Cobb 500 broiler chicks were randomly divided into four groups: an unsupplemented control and VA-1, VA-3 and VA-5 receiving VALM incorporation at concentrations of 1, 3 and 5 g/kg, respectively. Each treatment had six replicates of 10 chickens. On d 42, six chickens per replicate were isolated and euthanised. Digesta from the jejunal segments (10 cm) was collected for analysis of the digestive enzymes. The remaining digesta was then washed out with ice-cold phosphate-buffered saline before the jejunal segments (10 cm) were opened longitudinally to collect the mucosa by scraping. For the preparation of the homogenate, intestinal mucosa samples were homogenised with 154 mmol/l of ice-cold sodium chloride solution and centrifuged at 4 °C for 900 s. To determine immunoglobulins, glutathione and D-xylose, the supernatant was extracted and stored at -20 °C. Supplementation with VALM did not significantly influence the relative weights of organs in the different treatments. However, VALM at 3 g/kg caused a significant increase in amylase and trypsin concentration ( P <0.05). Immunoglobulin A and intestinal secretory immunoglobulin G concentrations were significantly improved ( P <0.05) in the birds fed 3 g/kg VALM. This supported the premise that 3 g/kg VALM in feed can improve gastric immunity status and digestive enzyme secretion.


Introduction
In the last 20 years, much work has been done on developing natural alternatives to antibiotic growth promoters (El-Hussein et al., 2008).For example, the use of Nigella sativa (Durrani et al., 2007), Lentinus edodes in combination with primalac (Willis et al., 2007), Curcuma longa and Aloe vera (Mehala and Moorthy, 2008), Cymbopogon citratus (Mmereole, 2010) have all been studied.These have been shown to significantly improve the productivity and health of poultry, especially in relation to their immune system.Similarly, Vernonia amygdalina, a medicinal plant, is widely used in the tropics for its hypolipidemic (Adaramoye et al., 2008), antibacterial (Agbankpe et al., 2016;Alo et al., Effect of Vernonia amygdalina leaf meal on growth performance, intestinal mucosa activity, digestive enzymes, absorption capacity, and immunity in broiler chickens  Abeokuta, 00234 Abeokuta, Nigeria;elomtokofai@gmail.com 2012) and hepatoprotective (Tokofai et al., 2021) properties.With such beneficial properties, V. amygdalina could be a relevant alternative to growth-promoting antibiotics.The findings of Owen et al. (2011); Osho et al. (2014); Oleforuh-Okoleh et al. (2015) and Tokofai et al. (2020) demonstrated the effects of V. amygdalina leaves on chicken productivity.However, studies on plant-based feed additives usually revolve around growth performance, without clearly elucidating the effects on digestive physiology, particularly the roles of enzymes involved in digestion, and immunity.In view of this, the following trial investigated the effects of Vernonia amygdalina leaf meal (VALM) on growth performance, digestive organs, digestive enzymes, nutrient absorption and the immune status of broilers.

Ethics statement
This study was carried out in strict compliance with the recommendations of the Guide for the Care and Use of Experimental Animals of the University of Lome, Togo.The protocol was approved by the Ethics of Animal Experimentation Committee of the same University.All efforts were made to minimise discomfort to the birds during the feeding trial.

Vernonia amygdalina leaf meal preparation
Fresh V. amygdalina leaves without the petioles were collected from Adéticopé village, located north of Lomé in Togo.The leaf blades were spread on tables at a temperature of 18 °C and were washed carefully.The leaves were turned over every 24 h.After 5 d, the leaves were crispy and pulverised using an electric mill equipped with a 2 mm mesh filter.The final powder was incorporated into the feed according to the treatments.

Bird management and feeding
Two hundred and forty, one-day-old male Cobb 500 broiler chicks were obtained from the hatchery of the Regional Centre of Excellence in Poultry Science (CERSA; Lomé, Togo) and were randomly assigned to four treatments.The treatments included a supplemented control and VA-1, VA-3 and VA-5, containing VALM at concentrations of 1, 3 and 5 g/kg respectively.Each treatment had six replicates of ten birds.The birds in each replicate were placed in 2 m2 wire mesh cages.Feed was offered from a conical sheet metal feeders and water was in plastic troughs, both supplied ad libitum.The birds were vaccinated against Newcastle and Gumboro diseases.Diets were formulated to meet NRC (1994) nutrient requirements, as shown in Table 1.During the experiment, the relative humidity ranged from 82 to 88% and the ambient temperature was 28.65±2.15°C.The lighting regimen was 23:1 light-dark cycle.The litter materials used were wood shavings and were changed weekly.

Sample collection and procedures
On d 42, six birds (one per cage replicate) were selected at random and weighed after 12 h of feed withdrawal.After euthanasia via cervical dislocation, the chickens were immediately dissected.Then, the small intestine was carefully removed without the mesentery and stored in a refrigerated stainless-steel tray.The intestinal segments (10 cm) were cut from the middle of the jejunum.Digesta from these segments was rinsed out with ice-cold phosphate-buffered saline (Fisher Scientific, Waltham, MA, USA) collected in plastic tubes for digestive enzyme analysis.The 10 cm jejunal segments were then opened longitudinally and mucosa collected by scraping.Using a sterile glass microscope slide, jejunal mucosa was directly scraped off at 4 °C, immediately frozen in liquid nitrogen and kept at -80 °C until analysis.The weights of the large and small intestine, gizzard and proventriculus (without contents), liver and pancreas (without the gallbladder) were determined separately.

Preparation of jejunal mucosa and digesta homogenate
The homogenate was prepared using 0.3 g of intestinal mucosa sample.Using a homogeniser, 154 mmol/l of icecold sodium chloride solution was added to both samples which were then centrifuged at 4,450×g for 15 min at 4 °C.After that, the supernatant was collected and kept at -20 °C until analysis.

Determination of jejunal digestive enzyme
Trypsin, amylase and lipase activities were measured using commercial kits according to the manufacturer's instructions (MyBioSource, Inc., San Diego, CA, USA).For better inter-sample comparison, all results were normalised to the total protein concentration in each sample.The Bradford (1976) method was used to determine the total protein content, with bovine serum albumin serving as the reference protein.

Determination of intestinal absorptive function
UV spectroscopy was used to measure the amount of D-xylose that was absorbed across the intestinal mucosa (Furton et al., 1995).After the chickens in each group were deprived of feed (but not water) for 2 h, a 10% D-xylose solution was administered by oral gavage at a dose of 1 ml/ kg on days 2, 5, 7, 9, 12, and 15.After 1 h, 2 ml of blood was drawn into a centrifuge tube containing 30 μl of 2% heparin anticoagulant.After centrifugation at 1,360×g at 4 °C for 10 min, the serum was collected, and the D-xylose content was determined.

Glutathione absorption
The colorimetric method was used to measure absorption of glutathione (GSH) from the intestinal mucosa (Sun et al., 2003).On d 2, 5, 7, 9, 12, and 15 1 g/kg GSH was administered by oral gavage to all birds after they had been deprived of food (but not water) for 3 h.After this, 2 ml of blood was drawn after 1 h into a centrifuge tube containing 30 ml of 2% heparin anticoagulant.The serum was prepared after being incubated at 1,360×g for 10 min at 4 °C.The GSH content was assessed using a kit, according to the manufacturer's instructions (MyBioSource, Inc.).

Mucosal immune parameters
Immunoglobulin (Ig) G and secretory IgA (SIgA) concentrations in the intestinal mucosa were measured using ELISA quantification, according to the manufacturer's instructions (MyBioSource, Inc.).To enable inter-sample comparison, all data were normalised to the total protein content in each sample.

Statistical analysis
All data were analysed using R version 4.1.2(R Core Team, 2021).Each replicate was taken as the experimental unit for analysis of performance data.The linear and quadratic effects of VALM inclusion were assessed using regression analysis.Data were expressed as mean and pooled SEM.
The regression model was as follows: Where Yij was the response variable; α the intercept (indicators with the basal diet); β1 and β2 the regression coefficient; Xi was the studied factor effect as the inclusion of VALM (i = 0, 1, 3, 5 g/kg), and eij was the observational error for (ij) th observation.

Growth performance
The effects of dietary VALM supplementation on broiler performance are presented in

Organ weights
Table 3 shows the relative weights of organs in birds fed with VALM.No differences were found for the proventriculus, liver, pancreas, gizzard, small intestine, and large intestine (P>0.05).

Digestive enzymes
The effects of dietary VALM supplementation on broiler digestive enzymes are presented in Table 4. VALM supplementation in the diet decreased amylase and trypsin linearly and quadratically.Lipase levels were not linearly and quadratically affected by the inclusion of VALM in the diet (P>0.05).

Mucosal immune parameters
VALM supplementation in the diet linearly and quadratically decreased SIgA and IgG (Table 5).

Absorptive functionality in intestines
A significant effect of treatments (P<0.05) was observed in the intestinal mucosa (Figure 1). 1 VA-1, VA-3 and VA-5: groups of birds fed with Vernonia amygdalina leaf meal (VALM) incorporated at a rate of 1, 3 and 5 g/kg respectively.2SEM = standard error of the mean; Linear and quadratic effects of VALM inclusion were evaluated using regression analysis. 1VA-1, VA-3 and VA-5: groups of birds fed with Vernonia amygdalina leaf meal (VALM) incorporated at a rate of 1, 3 and 5g/kg respectively. 2SEM = standard error of the mean; Linear and quadratic effects of VALM inclusion were evaluated using regression analysis.

Effect of VALM on GSH absorption in the intestinal mucosa of broilers chicken at different ages
Multiple comparisons showed that the absorption of GSH in VA-3 group was significantly higher than that in the control on d 9 (13.15 vs 11.50; P<0.05).VA-1 and VA-5 showed no significant (P>0.05)difference compared to the control group (Figure 2).

Discussion
The positive effects of VALM on growth performance were mainly observed at a supplementation up to 3 g/kg.Tokofai et al. (2020) reaffirmed that dietary supplementation with VALM at 3% in the diet significantly increased average daily gain (ADG) and improved FCR in broilers.Similarly, there was a quadratic depression in body weight, ADG and FCR in response to dietary VALM supplementation.Such growth depression could be attributed to the lower digestibility of VALM, since no significant effect of supplementation on feed intake was observed.The current study showed that VALM supplementation in broiler feed, not exceeding 3 g/kg, did not negatively affect performance.The effects of different levels of VALM incorporation in the diet were evaluated by quadratic regression analysis, and a reliable equation was obtained for the FCR, whereby: y = 0.00132x 2 -0.00361x + 1.72382 (P<0.05,R 2 =0.5269).
The optimal inclusion of VALM in the diets was calculated to be 2.42 g/kg of feed.
V. amygdalina is known to contain terpenes, sesquiterpene lactones, vitamins A and C, steroidal glycosides, phenols, flavonoids, alkaloids and triterpenoids (Alara et al., 2019;Odukoya et al., 2019).These compounds are involved in the improvement of digestive enzyme activities and, consequently, intestinal absorption.According to Fiesel et al. (2014), supplementation with plant products rich in polyphenols improved FCR in growing pigs.Such effects are likely to be due to increased efficiency in feed utilisation, resulting in improved growth.There is evidence to support the claims that herbs, spices, and other plant extracts have qualities that stimulate the appetite and digestion, as well as having antibacterial benefits (Kamel, 2001).Different compounds found in plant extracts have intrinsic bioactivities that can affect animal metabolism and physiology.The weight gain observed in this study may have been due to an improvement of the histological structures of the intestine, in particular the intestinal villi, as increasing the surface area of absorption leads to better absorption of nutrients, although this was not measured in the current trial.
Supplementation with VALM had no effect on the relative weights of the liver, pancreas, proventriculus, gizzard, small intestine or large intestine.These results concurred with those of Hernandez et al. (2004), who showed no changes in the weights of the gizzard, liver or pancreas of broiler fed diets containing essential oil extracts from oregano, cinnamon and pepper or a labiatae extract from sage, thyme and rosemary.In addition, Jamroz et al. (2005), who investigated the effect of essential oils in broiler diets based on maize and locally produced grains, reported similar findings.According to Visek (1978), the consumption of antibiotics as a growth promoter decreased intestinal weight by weakening the gut wall and shortening its overall length; however, this impact was not observed in the present study.All organs had larger relative weights at d 21 than those at d 42.All of the tested organs' relative weights declined   with age.These results are in agreement with the paper by Iji et al. (2001).
According to Platel and Srinivasan (2000), when administered in the diet at concentrations of 150, 200, and 5,000 mg/kg, respectively, the active ingredients of certain spices -capsaicin, piperine and curcumin -stimulated pancreatic enzymes in rats without changing feed intake or body weights.According to the findings of Engberg et al. (2000), dietary antibiotics seem to boost the activity of chymotrypsin and amylase in pancreatic tissue from broilers given wheat-and soybean-based feed.The activity of the digestive enzymes in intestinal digesta was not all affected similarly by the dietary supplements evaluated in this experiment.V. amygdalina significantly increased amylase and trypsin activities in digesta from the VA-3 group compared with the control birds.These findings implied that the expression of digestive enzymes was stimulated by VALM administration.An earlier study found that the intestinal digestive enzymes, amylase, lipase, and protease, in broilers were enhanced by polysaccharides from Astragalus membranaceus (Wu, 2018).The fundamental processes by which this occurred, particularly in the small intestine, are currently poorly understood.
Techniques for assessing intestine absorption performance are the D-xylose and GSH absorption tests.The most significant intracellular non-protein thiol molecule, GSH, is essential for maintaining a healthy redox environment in vivo.The current study revealed that the level of absorption of D-xylose and GSH increased in the VALM-treated groups compared to the control.A significant difference was noted, especially for the VA-3 groups compared to the control, for D-xylose on d 12 and 15 and GSH on d 9.This phenomenon can could explained by the development of intestinal villi in the intestines of the birds that consumed V. amygdalina, thus favouring a better absorption of D-xylose and GSH, although this was not measured.
Animals have immunity that is linked with the gut lumen via the mucosal surface channel.The key mechanism through which mucosal immunity responds to viral infections through SIgA activity.As the initial line of defence against viral infection, SIgA is produced in large quantities and binds to viruses during invasion.This prevents viruses from infecting epithelial cells and causing disease (Yang et al., 2011).As one of the key components of the intestinal mechanical barrier, intestinal epithelial cells are the first to interact with external antigens and pathogens.Intestinal epithelial lymphocytes can provide insight into the strength and effectiveness of the mucosa's local immune defence system (Lillehoj and Lee, 2012).In chickens, there are three primary types of immunoglobulins that are involved in immunity maintenance; SIgA, IgM, and IgG. (Ulmer-Franco, 2012).Intestinal mucosal epithelia that separate the body's interior from the external environment are protected and maintained in a homeostatic state by SIgA (Corthésy, 2013).The main purpose of SIgA is to prevent bacteria and mucosal antigens from accessing the delicate and susceptible mucosal barrier.The IgG released by B cells directly supports an immunological response, including viral and toxin neutralisation.
IgG and SIgA concentrations in the jejunum mucosa were increased in birds receiving VALM compared to the control in this study.Increased immunoglobulin levels cause complement proteins to be stimulated, which in turn helps birds' specialised immune systems function more efficiently to ward off diseases.Furthermore, a complicated interaction among numerous cytokines regulates the immunological response.According to reports, V. amygdalina has antiinflammatory and antioxidant properties that suppress the production of prostaglandin, which is an anti-immune molecule, and, hence, improves humoral response (Wang et al., 2020).In the present study, V. amygdalina regulated IgA and IgG contents by lowering pro-inflammatory cytokines, which helps to maintain a healthy cytokine environment and protect the integrity of the intestinal epithelium.

Conclusions
Incorporating 3 g/kg of VALM into the diet had a positive impact on broiler growth performance, digestive enzyme, absorption function, and immunological status.All these beneficial effects make VALM a suitable alternative to replace antibiotics as growth promoters for the poultry industry.

Table 2 . Effects of different treatments on growth performance of broilers.
Mortality rate and mean daily feed consumption during the growth phase (d 22 to 42) were not linearly and quadratically affected by the inclusion of VALM in the diet (P>0.05).