Maintaining intestinal microflora balance in heat-stressed broilers using dietary creeping wood sorrel ( Oxalis corniculata ) powder and chromium (chromium picolinate)

Aim of study: To determine the effect of dietary creeping wood sorrel powder ( Oxalis corniculata ) and chromium supplemented to broi lers (1-42 days) exposed to heat stress, on their performance and on the intestinal and caecal microbiota. Material and methods: The feeding trial was conducted on 60, day-old Cobb 500 broilers, divided equally in two groups, homogenous in terms of body weight: 46.36 ± 2.96 g (C), 46.36 ± 2.93 g (E). Each group consisted in six replicates (5 chicks/replicate). The broilers were housed in an experimental hall at 32 °C constant temperature and 23 h light regimen. Unlike the dietary control diet (C), the experimental diet (E) was supplemented with 1% creeping wood sorrel powder and 0.2 mg chromium picolinate/kg diet. Six birds (1 per each replication) were slaughtered on days 28 and 42, and samples of caecal and intestinal content were collected for bacteriological analysis. Main results: The dietary creeping wood sorrel powder and chromium supplements for heat-stressed broilers had no significant influence on their growth performance (1-42 d). Overall, the E diet had a beneficial effect on the balance of the caecal microflora ( p <0.05); however, in the intestine, the E diet had a positive influence ( p <0.05) on the balance of the intestinal microflora, only for the samples collected at 28 days. Research highlights: Dietary creeping wood sorrel powder and chromium supplements can be an efficient tool for maintaining a proper balance of intestinal microflora of heat-stressed broilers at grower stage.


Introduction
Heat stress is one of the factors that affect significantly overall animal physiology, health, and productivity. In poultry, adverse effects on the metabolic status and physiological balance (Rhoads et al., 2013), on the morphology and integrity of the intestinal barrier (Song et al., 2014) and on performance parameters (Al-Fataftah & Abdelqader, 2014;Song et al., 2014;Sarica et al., 2019) rank among the main influences of the heat stress. The gastrointestinal tract is particularly sensitive to stressors, which can cause a variety of changes, including the alteration of the normal protective microbiota (Bailey et al., 2004), and may allow pathogens like Salmonella the opportunity to bind to and colonise the intestinal epithelium (Burkholder et al., 2008). Commensal intestinal bacterial populations can protect the host from colonisation by pathogens, by competing for epithelial binding sites and nutrients, by strengthening the intestinal immune response, and by producing antimicrobial bacteriocins (Mac-Donald & Monteleone, 2005).
During the period of heat stress, lower circulating levels of vitamins and minerals have been determined in broilers, which can be associated to lower feed intake and high water intake. Beneficial effects are generated by supplemental vitamins and minerals given to broilers reared under heat stress (Khan et al., 2012), which reduce the reactions of cell oxidation. Hence, it has been reported that the negative effects of environmental stress could be prevented by the use of some phytoadditives, minerals and vitamin supplements, such as vitamin C and Cr alone or in combinations Sahin & Kuçuk, 2001;Yoo et al., 2016;Vlaicu et al., 2017;Panaite et al., 2018;Abd El-Hack et al., 2020).
Oxalis corniculata L. (Family: Oxalidaceace), commonly known as creeping wood sorrel, has a wide range of biological activities (Sharma & Kumari, 2014;Siddiqui et al., 2017). Native to Southern Europe, the creeping wood sorrel spread to the other continents as well. In Romania, it grows in the plain, and up to 1500 m altitude. A wide range of phytochemical compounds has been isolated from O. corniculata, such as flavonoids, tannins, phytosterols, polyphenols, glycosides, fatty acids (Sharma & Kumari, 2014). Siddiqui et al. (2017) showed that most of these compounds, found in the Oxalis corniculata leaves, possess antibacterial activity, the polyphenols being the most effective (Raghavendra et al., 2006), and they are involved in the growth and viability of lactic acid bacteria (Viveros et al., 2011;Gwiazdowska et al., 2015;Brenes et al., 2016). These phenolic compounds contained by O. corniculata show strong activity in the scavenging of free radicals (Sharangouda & Patil, 2007;Badwaik et al., 2011;Sharma & Kumari, 2014;Kaur et al., 2017). Given these activities, an attempt could be made to use wood sorrel as a source of antioxidant, to counteract the effects of heat stress.
Although the multiple beneficial effects of the creeping wood sorrel in traditional medicine are well established, the literature is poor in studies regarding its effect on animals. For example, in normal condition of temperature, O. corniculata has been reported to exert anxiolytic effects in mice (Gupta et al., 2012), antioxidant and hepatoprotective potential in rats (Sreejith et al., 2014). To our knowledge, there are no previous studies on broiler under heat stress.
Chromium is an essential mineral for the activation of certain oxidative enzymes and for stabilisation of proteins (Rao et al., 2012), reducing lipid peroxidation and modulating the expression of stress-related nuclear transcrip-tion factors (Orhan et al., 2012). Moreover, antioxidant minerals such as Cr can be used to prevent the effects of environmental stress (NRC, 1997;Toghyani et al., 2012). In heat stressed-broilers, chromium supplements are part of a nutritional strategy to improve growth performance (Ghazi et al., 2012), nutrient metabolism (Huang et al., 2016), the immune response (Oba et al., 2012), the antioxidant function and the response to stress measured as stress hormones level (Khan et al., 2014).
Thus, it might be highly useful for researchers in the poultry sector and in the pharmaceutical industry to widen their knowledge in promoting plants, like creeping wood sorrel, and minerals like Cr, and its antioxidant combination usage. In this context, we conducted a feeding trial to determine the effects of the dietary creeping wood sorrel (O. corniculata) and chromium (as chromium picolinate) on broilers reared under heat stress (32 °C) to maintain a proper balance of the intestinal microbiota.

Material and methods
The feeding trial was conducted in an experimental hall of the National Institute for Animal Nutrition (Ilfov, Romania) according to an experimental protocol, approved (case no. 4775/02.08.2019) by the Ethics Commission of the Institute. Sixty chicks, 1 day old (unsexed), of the Cobb 500 hybrid, purchased from a local hatchery were evaluated in a completely randomised design, with two homogenous groups in terms of bodyweight: 46.36 ± 2.96 g (control diet, C), 46.36 ± 2.93 g (experimental diet, E). Each group consisted in six replicates (5 chicks/replicate). The chicks were housed randomly in three-tiered digestibility cages (5 chicks/cage), having the following dimensions: height of front =455 mm, height of back =375 mm, total depth =550 mm, height between tiers =582 mm and tilt =14%, allowing the daily recording of the feed intake and excreta. Throughout the experimental period, the environmental temperature of the experimental hall was kept constant at 32 ºC. The light regimen was adequate to broiler age, i.e. 23 h light/ 1 h darkness. Starting from the age of 1 day, broilers received a corn and soybean meal-based (46.8% crude protein) C diet. Compared with the C (Table  1), the E diet included an additional 1% creeping wood sorrel powder (O. corniculata) and 0.2 mg Cr /kg diet. The diet formulations (Table 1) were developed by using dedicated software (Hybrimin® Futter 2008, Germany), in agreement with the feeding requirements (NRC, 1994) and the nutritional requirements of the Cobb 500 hybrid (The Management Guide of Cobb 500 Hybrid, 2015).
The creeping wood sorrel material was harvested when plants were in their late vegetative stage (44.62° N, 26.12° E). All parts of the plant (leaves, stem, flowers and roots) were dried for three weeks, under shade, at ambient temperature (20 °C), finely chopped and ground Diet for heat-stressed broilers (at about 1 mm) to obtain creeping wood sorrel powder. The drying process was made for a longer period of time at ambient temperature, as Shi (2006) showed that it ensures the maximum retention of vitamin C. The drying method used was in agreement with Hossain et al. (2010). The dose used in the study was in accordance with the European Food Safety Authority Panel on Dietetic Products, Nutrition and Allergies (EFSA, 2014). The chromium supplement was used in the premix as chromium picolinate (Cr (C 6 H 4 NO 2 ) 3 ) (Santa Cruz Biotechnology, CA, USA). Feed and water were provided for ad libitum consumption. None of the groups (C, E) had coccidiostat in the premix. All diets were fed as mash. Throughout the experimental period (1-42 days, broiler age) the following variables were monitored: body weight, BW (g); average daily feed intake, ADFI (g feed/broiler/day); average daily weight gain, ADWG (g/broiler/day) and feed conversion ratio, FCR (g feed/g gain). The individual BW was recorded on a weekly basis.
One bird from each replicate (6 birds per treatment) with BW within ± 10 g deviation in relation to the mean treatment weight, was slaughtered on days 28 and 42 by cervical dislocation, then immediately bled. After this, the gut was carefully excised, from the oesophagus to the cloaca. Intestinal and caecal contents (2 caeca per bird) were collected aseptically in sterilised plastic tubes and preserved at -20 ºC until the bacteriological tests (Enterobacteriaceae, Escherichia coli, staphylococci, lactobacilli, Salmonella spp.). Any digesta remaining in the two caeca was emptied by applying gentle pressure. The extracts of creeping wood sorrel and feed samples were obtained by adding 1 g of sample in 10 mL of 80% methanol; the samples were kept on a rotary shaker, in the dark, for 24 h. The extract obtained was centrifuged at 1500 × g for 10 min, and the supernatant was considered for analysis. The vitamin C content of the creeping wood sorrel was determined by the titrimetric method, as described by Kolniak-Ostek et al. (2013). In brief, 5 mL of plant extract obtained as described previously were added to 5 mL of oxalic acid (2%). The solution was titrated with 2,6-dichloroindophenol until a pink color was seen. The content of ascorbic acid (AA [mg/L]) was calculated as follows: AA= (Vi ) × 63) / Vp, where V i is the volume of 2,6-dichlorophenylindophenol used in the titration, and Vp is the volume of the sample taken for titration.
The total phenol content of creeping wood sorrel and feed samples was measured spectrophotometrically according to the Folin-Ciocalteu's method, described by Untea et al. (2018). The results were expressed as mg gallic acid equivalent (GAE)/g DW.
The total antioxidant capacity (TAC) of the creeping wood sorrel and feed samples was evaluated by the phosphate-molybdenum method of Prieto et al. (1999), based on the reduction of molybdenum(VI) to molybdenum(V) in the presence of a reducing agent (antioxidant) in the samples analytes and the further formation of a phosphate/molybdenum(V) green complex with acid pH. The results were expressed as mmol AA equivalent /kg DW and as mmol vitamin E equivalent /kg DW.
The classical medium of isolation, G.E.A.M. or Levine, was used to determine the Enterobacteriaceae and the E. coli, as described previously by Criste et al. (2017). The samples were first soaked in the medium with lauryl-sulphate (enrichment medium); then they were homogenised and left for 20-30 minutes at room temperature (23-24 °C). Decimal dilutions were made up to 10-5 in the medium with lauryl-sulphate. The dilutions of 10 -2 -10 -5 were used to seed 2 Petri dishes each per dilution, on Levine medium. The Petri dishes were incubated for 48 h at 37 °C, and the colonies were counted. E. coli formed characteristic colonies on this medium (dark violet with metallic shine). The other Enterobacteriaceae formed either dark red opaque colonies (lactic-positive species) or pale pink semi-transparent or colourless colonies (lactic-negative species). The analysis for staphylococi consists of immersing 1 g sample in 9 mL hyper-chlorinated liquid medium and innoculating in successive dilutions (10 -3 -10 -8 ) on solid hyper-chlorinated medium. The samples were incubated at 37 °C for 24-48 h, followed by the counting of the developed colonies. The lactobacilli were determined on selective mediums (MRS broth and MRS agar), characteristic for the isolation, and by the counting of these bacteria. The Salmonella spp. was determined according to SR EN ISO 6579/2003/A1:2007. The colony counter Scan 300, Interscience (France) was used to determine the colony count of Enterobacteriaceae, E. coli, staphylococci, and lactobacilli. The results were expressed as log base 10 colony-forming units (CFU) per gram of caecal contents. The complete randomised model was used to analyse the data for growth performance and intestinal and caecal microbiota. The effects of treatments were tested by analysis of variance, using the GLM procedure of the Minitab software, v. 17 (Minitab, 2015), with treatment as fixed effect according to the model Y i = T i + e i , where Y i is the dependent variable, T i is the treatment, and ei is the error. When the overall F-test was significant, the differences between means were declared significant at p<0.05 using the test of Tukey. Table 2 shows the chemical composition of the powder of creeping wood sorrel, highlighting a rather high level of crude protein (12.25%) and crude fibre (10.64%). The Diet for heat-stressed broilers product also has an important concentration of polyphenols and vitamin C. The analysis of the dietary polyphenols (Table 3), for all three growth stages, showed a higher level of total polyphenols in group E (supplemented with powder of creeping wood sorrel and Cr) than in group C (conventional diet formulation). The compound feed with creeping wood sorrel and Cr also had a higher antioxidant capacity than the conventional diet formulation.

Results
The BW of broilers in group E was not significantly (p >0.05) different from that of those in group C (Table 4).
Although not statistically significant, the ADFI (1-42 d) was numerically greater in the broilers from E when compared to C group (Table 4). There was no effect (p >0.05) of diet supplementation with creeping wood sorrel powder (1%) and Cr picolinate (0.2 mg/kg diet) on FCR (1-42 days). In our study, there were no mortalities in any of the two experimental groups. The dietary supplements of 1% creeping wood sorrel and Cr significantly influenced the Enterobacteriaceae, E. coli and staphylococci count in the intestinal and caecal content of the broilers (28 days). Thus, all the tested pathogenic bacteria colony forming units were lower (p<0.05) in the intestinal and caecal content of E broilers (28 days), compared to C broilers (Table 5).
At the end of the trial, the diet supplementation with 1% creeping wood sorrel and Cr had an effect against the tested pathogens only in the caecum (Table 5). Both at 28 and at 42 days, the lactobacilli populations were higher (p<0.05) in the intestinal and caecal content of E broilers than in C broilers (Table 5).

Discussion
The proximate composition of the plant (Table 2) revealed a significant content of fibre, which can be an important factor to be considered when establishing the inclusion of sorrel in the diets of monogastric animals. Regarding the mineral content, the creeping wood sorrel contained important levels of iron and zinc, according to Yang et al. (2011). These minerals are essential for broiler growth and are involved in many digestive, physiological and biosynthetic processes in the body.
The characterisation of creeping wood sorrel powder highlighted an important concentration of polyphenols and vitamin C, the results being in the same range of values reported in the scientific literature (Borah et al., 2012). The chemical composition of plants depends on the geographical area and soil and climate conditions, part of the plant, drying temperature, exposure to oxygen in the air, solvent and method of extraction and analytical method (Zechmann et al., 2011;Suzuki et al., 2014). Both polyphenols and vitamin C can react with free radicals (Grune et al., 2004;Durdun et al., 2011), thereby reducing their amounts and preventing the deleterious consequences of oxidative stress in birds. Chromium is not directly capable of preventing or reducing the formation of reactive oxygen species (ROS) (Króliczewska et al., 2004). If we consider that there is a relationship between insulin resistance and oxidative stress, in this situation, Cr act as indirect antioxidant, by decreasing the high insulin level and preventing glucose auto-oxidation, a reaction that generates ROS (Roussel et al., 2007). The same authors showed that, in the presence of Cr, much lower amounts of insulin are required, and insulin sensitivity is improved.
The antioxidant capacity of plants, measured by the TAC method, provided information about lipophilic and hydrophilic antioxidant compounds. Research has shown that there is a strong correlation between the phenolic content and the antioxidant capacity (Aryal et al., 2019). The overall antioxidant activity is not due to only one chemical compound, since the presence of non-phenolic antioxidants (vitamin C, vitamin E, carotenoids, etc) also plays an important role (Untea et al., 2018). The data reported in Table 2 confirmed the valuable concentrations of vitamin C and polyphenols; thus, wood sorrel can be considered an important source of natural antioxidants. The antioxidant potential was studied, by using a different method proposed by Kumar et al. (2012); this showed that a concentration of 30 mg/mL methanolic extract of creeping wood sorrel was necessary to inhibit 50% of DPPH radical (IC 50 ), lower than for the AA, highlighting that the extract had a higher antioxidant power than the AA. There is a positive correlation in terms of the content of polyphenols in wood sorrel and in the supplemented feed samples (E diet). The supplementation with wood sorrel improved the polyphenol content and, thus, the antioxidant capacity of the experimental feed samples. In this regard, Hu et al. (2019) acknowledged that phytochemicals with antioxidant activity offer great hope as a dietary solution for heat stress in poultry.
Although there was a slight improvement in the performance variables (final BW, ADFI, ADWG) of broilers fed with the E diet, compared with those fed with the C diet, these results were not statistically assured (p>0.05). Thus, diet supplementation with Cr and wood sorrel did not affect (p>0.05) the performance of heat-stressed broilers in the trial period (1-42 days).
In contrast, Toghyani et al. (2012) reported 4% higher FCR in broilers exposed to high ambient temperatures (33 °C) to which diets supplemented with 1.50 mg/kg Cr-nicotinic were fed, when compared with those on diets without Cr supplementation. Subhani et al. (2018) conducted a study on unsexed Hubbard broilers (7-42 days), by using a dietary ethanolic extract of O. corniculata (250 mg/kg and 500 mg/kg respectively), alone or combined with aflatoxin B1 (350 ppb). In contrast with our study, Subhani et al. (2018) showed that the use of creeping wood sorrel extract (250 mg/kg and 500 mg/kg, respectively) given to broilers reared in normal environmental conditions resulted in a BW (42 days) and cumulative feed consumption (42 days) that were significantly lower than those of the broilers treated with the conventional diet. These differences can be due to the dietary form of inclusion, the inclusion level, the broiler hybrid, the rearing conditions, and to the fact that our study used a combination of creeping wood sorrel and chromium. Some studies support the synergistic action of Cr and other antioxidants in stress conditions; by reciprocally amplifying their actions, they lead to enhanced performance in poultry Haq et al., 2017;Al-Sultan et al., 2019). Except for performance, a positive effect of the dietary combination of vitamin C and Cr was related to their potent antioxidant property against oxidative stress (Al-Sultan et al., 2019). There is a relation between Cr supplementation and AA intracellular availability. It has been claimed that a higher level of glucose decreases the intracellular AA content (Seaborn et al., 1994), and that Cr supplementation reduces plasma glucose concentrations in broilers. Consequently, Cr plays indirectly a role in the increase of the intracellular availability of vitamin C, by intensifying the action of insulin on the cellular uptake of glucose (Sahin et al., 2002).
The data obtained in this study revealed that dietary creeping wood sorrel powder and Cr had an effect against the tested pathogens (Enterobacteriaceae, E. coli, staphylococci) in the caecum of heat-stressed broilers (Table 5). This fact is important, as heat stress is known to favour the increase of harmful bacteria over beneficial ones, with bacterial density reaching a maximum in a different section of gut intestinal tract within the first week of age (Yadav & Jha, 2019). Interestingly, only in the early stage of growth (28 days) did the supplemented diet (E) decrease significantly the CFU of Enterobacteriaceae, E. coli, staphylococci in both caecum and intestinal segment, while no effect was recorded in the intestine at 42 days (Table 5). Yadav & Jha (2019) stressed the fact that the microbiota becomes more diverse and tends to be relatively stable in older age. The early growth period is important for microbial balance and consequently for broiler health because gut intestinal health problems in broilers typically occur between the ages of 20 to 30 days (Teirlynck et al., 2011). It is likely that the dose of 1% creeping wood sorrel powder was insufficient, or maybe the polyphenols did not possess bioavailability in the broilers' intestines to cause susceptibility to pathogenic bacteria at 42 days. Even though the compound shows strong antioxidant or other biological activities in vitro, it would have little biological activity in vivo if little or none of the compound gets to the target tissues (D'Archivio et al., 2010). Caecum is the part of large intestine where the final stages of nutrient and water absorption occur, with the synthesising of certain vitamins, accumulation of residues in order to eliminate. Probably, in our case, polyphenols resulting from creeping wood sorrel had a stronger bioavailability in the caecum than in the intestine, thus reflecting in its antimicrobial effect, reducing the pathogens populations in caecum. An explanation in this regard may be that, in the small intestine, the ingested polyphenols are absorbed only in a share of 5%-10%, while the remaining unmodified polyphenols (~90%-95%) cross the intestinal tract and accumulate in the large intestine (Santhakumar et al., 2018). Thus, in the present study, the different results on microflora at 42 days between caecum and intestine might be due to the degree of bioavailability of polyphenol in several segments of the gastrointestinal tract and to broiler growth stage.
Furthermore, in our study, this antibacterial effect could be enhanced by the combination of the creeping wood sorrel with Cr, which is involved in the antioxidant mechanism. Chromium as an insulin cofactor, it acts as a secondary antioxidant (Króliczewska et al., 2004;Farag et al., 2017;Krol et al., 2017), and it also enhances the immune system of heat stressed-broilers (Dalólio et al., 2018). A strong immune system leads to broiler gut health because the gastrointestinal tract is not only a site for digestion and absorption of nutrients but it also acts as a metabolic and immunological organ, maintaining the intestinal integrity and thus, preventing bacterial adherence to the mucosa (Adedokun & Olojede, 2019). In vitro studies showed that the extracts of different parts of O. corniculata are a good source of secondary metabolites demonstrating natural antioxidant and antibacterial activities (Kaur et al., 2017). Other investigations related to the antimicrobial effects of creeping wood sorrel (Mohan & Pandey, 2016;Panda et al., 2016;Kaur et al., 2017). This is also supported by the results reported by Meghla et al. (2016), who revealed that the ethanolic, methanolic, and aqueous extracts of O. corniculata showed to be effective against the tested Gram-positive and Gram-negative bacteria. On the other hand, Criste et al. (2017) noticed that the use of phytoadditives known for their antioxidant and antibacterial properties (1% oregano powder or 1% rosehip powder) given to broiler chicks (14-35 d) exposed to heat stress (32 °C) depressed the pathogen bacteria from the ileum and supported the replication of the lactic acid releasing bacteria. Stress has an important role in determining the extent and type of bacteria colonisation (Hamidi et al., 2019). They also showed that some of the bacteria can modulate the expression of genes in host epithelial cells, thus creating a favourable habitat for themselves, and can prevent the growth of other bacteria introduced later. This is the reason why one must make sure of gut population with beneficial bacteria, such as Lactobacillus species, much more so under conditions of heat stress.
Both at 28 and at 42 days, the lactobacilli populations were higher (p<0.05) in the intestinal and caecal content of E broilers than in those of C broilers (Table 5). Some reports stated that phenolic compounds can also selectively stimulate the growth of beneficial bacteria, such as Lactobacillus and Bifidobacterium and may, therefore, modulate gut microbiota (Tzonuis et al., 2008;Gwiazdowska et al., 2015;Lipiński et al., 2017). However, the explanation of phenolic compounds selectivity for beneficial bacteria growth is still unclear. A possible explanation for the stimulatory effect of polyphenolic compounds have on bacterial growth is that some microorganisms are able to use these compounds as nutritional substrates (Ghorbani et al., 2014). In the particular case of lactobacilli, these bacteria can metabolise phenolic compounds supplying energy to cells and positively affecting the bacterial metabolism (Garcia-Ruiz et al., 2008). The present results showed that creeping wood sorrel had an effect mostly on lactobacilli as gram-positive bacteria rather than gram-negative bacteria. Papuc et al. (2017) reported that gram-positive bacteria are more resistant to the action of polyphenol than gram-negative bacteria. This is due to differences in cell wall composition; the outer hydrophilic membrane of Gram-negative bacteria is mainly composed of lipopolysaccharides (Nohynek et al., 2006) and it hinders polyphenol connections to the peptidoglycan layers of these microorganisms (Cui et al., 2012). Moreover, Cr can interact with gut microbiota. Feng et al. (2019) showed that the use of Cr as a nutritional supplement and micronutrient may provide significant protection to the gut microflora, particularly Lactobacillus, against some of the commonly used antibiotics. The data revealed that a combination of 1% creeping wood sorrel and Cr had a beneficial effect on the lactobacilli populations in the intestine and caecum of chickens during heat stress condition, maintaining the body's homeostasis.
In conclusion, dietary creeping wood sorrel (O. corniculata) and chromium supplements for broilers reared under heat stress (32 °C) had no significant influence on their growth performance (1-42 d). The inclusion of 1% creeping wood sorrel and 0.2 mg Cr picolinate/kg diet in broiler diet had a positive effect on the preservation of a proper balance of bacteria species (in caecum and intestine) only at the grower stage of the heat-stressed broiler. However, no effect was recorded on the pathogenic bacteria tested in the intestinal microflora of broiler at finisher stage.