Exogenous Enzymes Influenced Eimeria-Induced Changes in Cecal Fermentation Profile and Gene Expression of Nutrient Transporters in Broiler Chickens

Simple Summary Eimeria-induced coccidiosis, a common disease in the poultry industry, causes substantial economic loss globally. The developed resistance to synthetic anticoccidial drugs and increasing public and legislative pressures to decrease antibiotic utilization drive research exploring non-antibiotic alternatives to control coccidiosis. Two experiments were conducted to investigate the potential mechanisms by which enzymes may mitigate the negative effects of Eimeria on growth performance, nutrient-transporter gene expression, and cecal fermentation patterns. The results demonstrated that Eimeria changed the expression of tight junctions and nutrient transporters genes, promoted cecal protein fermentation, and inhibited cecal saccharolytic fermentation. Exogenous xylanase and protease supplementation alleviated negative effects of Eimeria effects on the above responses, and thus demonstrated benefits of enzyme supplementation beyond improvement in nutrient utilization. Abstract Two 21-day experiments were conducted to investigate the effects of exogenous enzymes on growth performance, tight junctions, and nutrient transporters, jejunal oligosaccharides and cecal short-chain fatty acids (SCFA) of broiler chickens challenged with mixed Eimeria. Two different basal diets: high fiber-adequate protein (HFAP; Expt. 1) or low fiber-low protein (LFLP; Expt. 2) were used in the two experiments. In each experiment, birds were allocated to four treatments in a 2 × 2 factorial arrangement (with or without protease and xylanase combination; with or without Eimeria challenge). In Expt. 1, with HFAP diets, Eimeria upregulated (p < 0.05) the expression of claudin-1, but downregulated (p < 0.05) glucose transporters GLUT2/GLUT5. On the contrary, enzymes downregulated (p < 0.05) claudin-1 and alleviated the Eimeria-depressed GLUT2/GLUT5 expression. In both experiments, Eimeria decreased (p < 0.05) cecal saccharolytic SCFA and increased (p < 0.05) cecal branched-chain fatty acids. The challenge × enzyme interaction (p < 0.05) showed that enzymes reversed the Eimeria effects on fermentation pattern shift. In conclusion, Eimeria altered tight junctions and nutrient transporters expression promoted cecal proteolytic fermentation and inhibited saccharolytic fermentation. Exogenous enzymes showed the potential of alleviating the Eimeria-induced intestinal gene expression changes and reversing the unfavorable cecal fermentation pattern.


Introduction
Avian coccidiosis is a worldwide disease that is caused by protozoan parasites of Eimeria spp. Seven species of Eimeria including E. acervuline, E. brunetti, E. maxima, E. mitis, E necatrix, E. praecox, and E. tenella have been identified in the broiler chicken industry [1]. The prevalence of coccidiosis usually occurs with a mixed infection instead of a single species with the mixed infection by E. acervulina, E. maxima, and E. tenella being more prevalent [2]. These three species of Eimeria spp. preferentially invade and multiply in the

Animals, Diets Experimental Design and Eimeria Challenge
One hundred and twenty zero-day-old Cobb 500 male broiler chicks were used in each of the two 21 d experiments. A basal diet, formulated to have a high level of fiber and adequate protein (HFAP), was used in Expt. 1, whereas the basal diet in Expt. 2 was formulated to have a low level of fiber and lower than recommended protein level (LFLP). Each of the experiments had four treatments in a 2 × 2 factorial arrangement. The factors were enzyme supplementation (with or without a combination of xylanase and protease) and Eimeria challenge (with or without). The birds in all the treatments in each experiment had the same initial body weight (day 0). Each of the four treatments had six replicate cages and five birds per replicate cage. Light was provided for 24 h on the first three days and gradually decreased to 8 h dark period on the last day. The room temperature was set at 33 • C and gradually decreased to 25 • C during the rearing period. Temperature and humidity were recorded daily.
The basal diets were corn-soybean meal-based, and wheat plus wheat bran was used as a fiber provider in the HFAP diet, producing a slightly lower AME level in Expt. 1 diet. The low-protein diets used in Expt. 2 had three percentage points lower crude protein levels compared with adequate protein diets. Five hundred FTU/kg phytase (Quantum Blue, AB Vista, Marlborough, UK; 5000 FTU/g) was supplemented in all the diets. Each basal diet was divided into two experimental diets: control diet (without enzyme supplementation), or supplemented with a combination of 0.2 g/kg mono-component protease and 0.1 g/kg of xylanase (XP). The β-(1-4)-endo-xylanase was from genetically modified Trichoderma reesei. The serine protease was produced by the sporulation-deficient Bacillus licheniformis strain. Birds receiving each of the two experimental diets were allocated equally into two groups gavaged with 1 mL distilled water or 1 mL mixed-species Eimeria oocysts solution on day 15. The mixed-species Eimeria spp. water-based solution containing 12,500 oocysts of E. maxima, 12,500 oocysts of E. tenella, and 62,500 oocysts of E. acervuline per 1 mL was prepared for a mild infection challenge. The ingredient composition of the basal diets is presented in Table 1 and the analyzed chemical composition of the experimental diets is presented in Table 2. The analyzed enzyme activities were, on average 311, 9500, and 18,700 units per kg for phytase, xylanase, and protease, respectively. The analyzed activities were 62%, 56%, and 124% of expected activities for phytase, xylanase, and protease, respectively.

Growth Performance, Intestinal Permeability, and Lesion Scoring
All animal experiment procedures were approved by the Institutional Animal Care and Use Committee at the University of Georgia, Athens, Georgia, USA (Protocol No: A2018-08-026). Birds and feed were weighed on d 0, 15, and 21. Mortality was monitored daily and used to calculate mortality-adjusted weight gain (WG), feed intake (FI), and gain: feed ratio. The intestinal permeability test was conducted on d 19, 5 d post infection (DPI). A 2.2 mg/mL fluorescein isothiocyanate dextran (FITC-d, MW 4000; Sigma-Aldrich, St. Louis, MO, USA) solution was prepared right before the test. One bird was randomly selected from each of the challenged group cages and orally gavaged with 1 mL FITC-d solution. Birds from unchallenged and no-enzyme treatment were also administrated with FITC-d as the control group. Blank blood sample from extra birds (provided with standard broiler feed) was collected to dilute FITC-d to prepare solutions for the standard curve. After 2 h of administration, birds were euthanized and blood samples were collected from the heart and pooled by treatment. Clotted blood was centrifuged at 1000× g for 12 min to separate the serum. The serum sample and standard solutions were measured by a spectrophotometer (Spectramax M5, Molecular Devices, San Jose, CA, USA) at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. All blood samples were kept in darkness during the whole procedure. The serum FITC-d concentration is positively correlated to intestinal permeability. High blood FITC-d level indicates intestinal leakage due to gut breakage caused by Eimeria spp. invasion.
At the end of the study, a 0 to 4 (no lesion to severe lesion) scale grading was used to score the coccidia lesion severity in predefined intestinal regions [18]. The upper (duodenum), middle (jejunum and ileum), and ceca sections of the intestine were scored separately.
Lesion scoring was carried out on three birds per cage.

Sample Collection
All the birds were euthanized by carbon dioxide asphyxiation on d 21. Jejunal digesta was collected from five birds per cage. The samples were oven-dried and ground for oligosaccharides composition analysis. Cecal contents were collected from two birds per cage and stored at dry ice for later SCFA analysis. Jejunal tissues were sampled from two birds per cage, snap-frozen in liquid N, and later stored at −80 • C before further analysis.

Quantitative Real-Time PCR Analysis
Quantitative real-time PCR was used to analyze gene expression related to tight junctions and intestinal nutrient transporters. Approximately 2 mm × 2 mm jejunal tissue was homogenized in QIAzol lysis reagent (QIAGEN, Hilden, Germany) and total RNA was extracted following the manufacturer's instructions. Extracted RNA was converted to cDNA in a 96-well PCR system by the use of the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) after quantity measurement and dilution. Converted cDNA was diluted and the real-time PCR reaction was performed with reaction master mix iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, U.S.) in a Step One Plus real-time PCR system (Thermo Fisher Scientific, Waltham, MA, U.S.). Samples were run in duplicate and the 2 (−∆∆Ct) method [19] was applied to analyze the results. All of the primers used in the experiments, including housekeeping and target genes, are listed in Table 3.

Chemical Analysis
All the diets were analyzed for chemical profiles using standard procedures. Samples were oven-dried at 100 • C for 24 h to determine the gravimetric difference and then dry matter was calculated (AOAC Method 934.01). Nitrogen content was measured using the combustion by nitrogen analyzer (AOAC Method 968.06). The Ankom 200 Fiber Analyzer was used to measure acid and neutral detergent fibers (Ankom Technology, Macedon, NY, USA). Minerals were measured by the Central Analytical Laboratory, University of Arkansas. Matrix-assisted laser desorption ionization mass spectrometry detection was used to analyze the oligosaccharides' composition of the jejunal by the Complex Carbohydrate Research Center, University of Georgia, as previously described [17]. Gas chromatography (GC) was used to analyze the composition of cecal SCFA by a previously described method [20]. Briefly, 1 g cecal content sample was diluted in 3 mL deionized water and centrifuged at 10,000× g for 10 min. The supernatant was mixed well with 25% meta-phosphoric acid. After overnight freezing, the mixture was thawed and the supernatant was mixed with ethyl acetate at a ratio of 1:2. After vortexed and settled, the top layer of the mixture was transferred to a glass vial and analyzed on GC.

Statistical Analysis
The data for Expt.1 and 2 were analyzed separately by ANOVA using the mixed model procedure of JMP Pro 14.1.0 (SAS Institute Inc., Cary, NC, USA). The two factors were the Eimeria challenge and enzymes supplementation. Main effects refer to when there are no significant interactions, whereas simple effects refer to when there are significant interactions of factors. In cases of a significant interaction effect being detected, Tukey was used to separate the significantly different means. The Kruskal-Wallis nonparametric statistical method was used to analyze intestinal lesion scores. Significance was declared at p ≤ 0.05.

Growth Performance
In both Expt. 1 and 2, Eimeria challenge resulted in a significant reduction (p < 0.01) in WG, FI, and gain-feed ratio (Tables 4 and 5). Supplemental XP increased (p < 0.05) FI in broilers fed LFLP diets (Expt. 2) but had no effect on growth performance of broiler fed HFAP diet (Expt. 1). In both experiments, there was no significant challenge × enzyme interaction for the growth performance responses. Table 4. Growth performance of the broiler chickens challenged or unchallenged with mixed Eimeria spp. in response to feeding diets with high fiber and adequate protein levels with or without enzymes supplementation (Expt. 1).  Table 5. Growth performance of the broiler chickens challenged or unchallenged with mixed Eimeria spp. in response to feeding diets with low fiber and low protein levels with or without enzymes supplementation (Expt. 2).

Intestinal Permeability and Lesion Scores
Figures 1 and 2 show the gastrointestinal permeability response on day 19 (5 DPI). Enzyme supplementation had no effect on intestinal permeability compared with the control group (unchallenged treatment without enzymes) in both experiments. However, birds challenged with mixed Eimeria species showed higher (p < 0.01) serum FITC-d levels, indicating intestinal leakage due to gut breakage caused by Eimeria spp. invasion. The results of intestinal lesion scores are presented in Figures 3 and 4, compared to the control group (unchallenged treatment without enzyme). Eimeria challenge resulted in higher (p < 0.01) lesion scores in the upper intestine, middle intestine, and ceca which were invaded by E. acervulina, E. maxima, and E. tenella, respectively, regardless of the type of basal diets. No significant effect on lesion score was observed for XP supplementation. replicates for the simple effect; n = 12 replicates for the main effects. LF-high fiber; LP-low protein; XP-xylanase plus protease supplementation; WG-weight gain, g; FI-feed intake, g. Enzyme supplementation had no effect on intestinal permeability compared with the control group (unchallenged treatment without enzymes) in both experiments. However, birds challenged with mixed Eimeria species showed higher (p < 0.01) serum FITC-d levels, indicating intestinal leakage due to gut breakage caused by Eimeria spp. invasion. The results of intestinal lesion scores are presented in Figures 3 and 4, compared to the control group (unchallenged treatment without enzyme). Eimeria challenge resulted in higher (p < 0.01) lesion scores in the upper intestine, middle intestine, and ceca which were invaded by E. acervulina, E. maxima, and E. tenella, respectively, regardless of the type of basal diets. No significant effect on lesion score was observed for XP supplementation.

Gene Expression of Nutrients Transporters and Tight Junction Proteins
In Expt. 1, Eimeria challenge upregulated (p < 0.01) the expression of CLDN1, whereas enzyme supplementation downregulated (p < 0.01) the expression of the gene. Moreover, there was a significant (p < 0.01) enzyme × Eimeria challenge interaction for the expression of glucose transporters GLUT2 and GLUT5 (Table 6). In unchallenged groups, enzyme supplementation reduced (p < 0.05) the expression, while in Eimeria-challenged groups, the expression levels were unaffected. The highest and lowest GLUT2 and GLUT5 expressions were observed in unchallenged (without enzyme) and unchallenged (with enzyme) treatments, respectively. Downregulation (p < 0.05) of glucose transporter SGLT1 was observed in enzyme-supplemented diets in Expt. 1. In addition, the Eimeria challenge downregulated (p < 0.05) the expression of CAT2 and y+LAT1. Downregulation (p < 0.05) of amino acid transporter rBAT was observed in enzyme-supplemented diets in Expt. 1. The expression of JAM2 and PepT1 were not affected by the treatments in both experiments. In Expt. 2, there was no interaction between enzyme supplementation and Eimeria challenge on gene expression. In addition, Eimeria significantly upregulated (p < 0.01) tight junction protein CLDN1 and downregulated (p < 0.01) the expression of cationic amino acid transporters CAT2 and y+LAT1 whereas enzyme supplementation had no significant effect on the gene expression (Table 7).

Gene Expression of Nutrients Transporters and Tight Junction Proteins
In Expt. 1, Eimeria challenge upregulated (p < 0.01) the expression of CLDN1, whereas enzyme supplementation downregulated (p < 0.01) the expression of the gene. Moreover, there was a significant (p < 0.01) enzyme × Eimeria challenge interaction for the expression of glucose transporters GLUT2 and GLUT5 (Table 6). In unchallenged groups, enzyme supplementation reduced (p < 0.05) the expression, while in Eimeria-challenged groups, the expression levels were unaffected. The highest and lowest GLUT2 and GLUT5 expressions were observed in unchallenged (without enzyme) and unchallenged (with enzyme) treatments, respectively. Downregulation (p < 0.05) of glucose transporter SGLT1 was observed in enzyme-supplemented diets in Expt. 1. In addition, the Eimeria challenge downregulated (p < 0.05) the expression of CAT2 and y+LAT1. Downregulation (p < 0.05) of amino acid transporter rBAT was observed in enzyme-supplemented diets in Expt. 1. The expression of JAM2 and PepT1 were not affected by the treatments in both experiments. In Expt. 2, there was no interaction between enzyme supplementation and Eimeria challenge on gene expression. In addition, Eimeria significantly upregulated (p < 0.01) tight junction protein CLDN1 and downregulated (p < 0.01) the expression of cationic amino acid transporters CAT2 and y+LAT1 whereas enzyme supplementation had no significant effect on the gene expression (Table 7).

Jejunal Oligosaccharides and Cecal Volatile Fatty Acids Profile
No significant main or interaction effects were observed on the profile of jejunal oligosaccharides in birds receiving the HFAP diet in Expt. 1 ( Table 8). The profile of jejunal oligosaccharides in Expt. 2 (Table 9) indicates that birds challenged with mixed Eimeria spp. tended to have lower jejunal concentration of (Hex)3 (p = 0.062) and (Hex)5 (p = 0.059) but significantly lower (Hex)4 (p < 0.05), when fed LFLP diets. No significant enzyme effect nor interactions were observed.
In both Expt. 1 and Expt. 2 (Table 10 and Table 11, respectively), there were significantly lower (p < 0.05) concentrations of SCFA acetate, butyrate, and total SCFA but higher (p < 0.05) concentrations of BCFA isobutyrate, and isovalerate in birds challenged with Eimeria spp. In birds receiving HFAP diets (Expt. 1), there was significant Eimeria × enzyme interaction for isobutyrate, isovalerate, and valerate (Table 10). The concentrations of BCFA were much greater (p < 0.05) in challenged birds than in unchallenged birds. Enzyme supplementation mollified Eimeria-induced increase in cecal BCFA isobutyrate and isovalerate contents. Lower (p < 0.05) cecal valerate was observed in birds fed without enzymes in challenged compared to unchallenged birds. However, when enzymes were supplemented, challenged birds had a higher (p < 0.05) cecal valerate value compared to unchallenged birds. No significant main enzymatic effects were found. For broiler chickens in Expt. 2, there was significant Eimeria × enzymes interaction (p < 0.05) for ceca content of acetate, butyrate, and total SCFA ( Table 11). The concentrations of acetate, isobutyrate, and total VFA were highest (p < 0.05) in unchallenged-no enzyme treatments, but similar among the rest of the treatments. Table 8. Oligosaccharide profile (µg/mg) in jejunal digesta of broiler chickens challenged or unchallenged with mixed Eimeria spp. in response to feeding diets with high fiber and adequate protein levels with or without enzyme supplementation (Expt. 1).  Table 9. Oligosaccharide profile (µg/mg) in jejunal digesta of broiler chickens challenged or unchallenged with mixed Eimeria spp. in response to feeding diets with low fiber and protein levels with or without enzyme supplementation (Expt. 2).  Table 9. Cont.

Discussion
The objective of the two experiments reported herein was to investigate the potential and mechanisms of exogenous enzymes action on mitigating the negative effects of Eimeria challenge on growth performance, gene expression of tight junctions and nutrient transporters, and cecal fermentation pattern. Eimeria-induced reduction in broiler growth performance is positively correlated with infective dose [21,22]. Bodyweight gain of birds at 1 to 6 DPI (day 14 to day 20) can be linearly reduced from 27% to 49% when challenged by increasing oocysts doses of mixed Eimeria spp. (6250 E. maxima; 6250 E.tenella; 31,250 E. acervuline to 50,000 E. maxima; 50,000 E. tenella; 250,000 E. acervulina) [21]. A medium-low dose of Eimeria was used in the current study in order to develop a mild infection resulting in a 63% reduction in body weight gain at 6 DPI, which was more severe than expected but similar to what others have reported [23,24]. There are species differences in the extent to which Eimeria impacts growth performance. The less pathogenic species such as E. praecox and E. mitis, which do not cause lesions, generally have less influence on growth performance whereas E. maxima, which mainly invades jejunum and ileum, showed a more serious impairment on feed conversion ratio (FCR) [1,25]. Exogenous enzymes do not have anticoccidial activities. However, dietary inclusion of such enzymes may reduce the negative effect of coccidia infection via the independent effect of such enzymes on promoting growth performance, nutrient utilization, the integrity of intestinal epithelial cells, or bacterial balance. For example, enzyme complexes including xylanase or protease have been shown to significantly decrease FCR in broiler chickens challenged with coccidia [14,26,27]. In Expt. 2 of the current study, enzyme supplementation increased feed intake without any effect on WG or the gain-feed ratio. The enzyme's effect during coccidia challenge is not universally observed. Enzyme supplementation had no significant mitigation effect on the impairment of the growth performance associated with coccidiosis in Parker et al. [28] or in Expt. 1 of the current study. Dietary and other factors may play a role in differences in response observed from different studies.
Eimeria challenge increases the permeability of the intestinal epithelium, and the extent of the damage can be assessed using fluorescein isothiocyanate-dextran (FITCd). Higher levels of FITC-d in serum indicate greater FITC-d leakage from the intestine, revealing the severity of impairment of tight junction barriers [29]. Upon entering the host intestine, sporulated oocysts begin several cycles of asexual multiplication before sexual multiplication. During the asexual multiplication stages, a mass of merozoites is produced and penetrates the epithelial cells of the host, resulting in severe intestinal impairment and permeability defects [3]. In the current study, the levels of FITC-d detected in bird serum were greater in challenged birds compared with non-challenged birds. However, enzyme supplementation had no effect on gut leakage. The observation was likely because enzymes cannot directly target Eimeria oocyst nor reduce its multiplication, which is the primary cause of cell destruction and gut leakage. For the same reason, there was no lesion score reduction observed in enzyme-supplemented treatments.
On the other hand, observation in the current experiments on the expression of tight junction proteins indicates that the enzymes supplementation promoted intestinal integrity. Tight junctions are multi-functional protein complexes principally consisting of three families of transmembrane proteins: claudins, occludins, and junctional adhesion molecules. These complexes act as guards to seal the paracellular space between adjacent epithelial cells, regulating nutrient absorption and restricting pathogen entry. Claudin family proteins constitute the tight junction structural framework and each protein plays a specific function [30]. In the current study, in response to the mixed Eimeria challenge, the expression of claudin-1 at 6 DPI was significantly increased. The same change of claudin-1 was reported in previous coccidia studies [21,31]. The key inflammatory cytokine TNFα led to the increase in claudin-1 in IEC-18 cells, and increased expression of claudin-1 was observed in ulcerative colitis. It is speculated that the increased claudin-1 is related to inflammatory bowel disease [32][33][34]. When attacked by inflammatory pathogens, the intestinal paracellular structure, as well as osmotic balance, are damaged, requiring a mass replacement of barrier-forming proteins including claudin-1. This intensive replacement is clinically observed as diarrhea. This replacement can not only renew intestinal barriers but also protectively exclude invasive pathogens [34]. Eimeria spp. multiplies by invading and breaking down intestinal cells intercellularly and intracellularly. All of these Eimeriainduced activities stimulate the replacement of tight junctions, consequently increasing the expression of claudin-1, which was also observed in the current study. The data in the current study demonstrated the combined effect of xylanase and protease on decreasing the claudin-1 expression. It can be reasoned that the enzymes helped to partly alleviate Eimeria-induced intestinal barrier impairment and thus helped reduce the metabolic costs associated with the disease challenge. Similar findings were reported in animals receiving xylanase supplemented diet when challenged with Clostridium perfringens [35,36].
Eimeria infection causes the downregulation of nutrient transporters, which consequently contributes to possible depressed nutrient utilization and ultimately diminished growth performance [37]. In the current study, Eimeria infection significantly downregulated the expression of sugar transporters GLUT2 and GLUT5 in broilers fed HFAP diets (Expt. 1), although the decrease was rather nominal for birds receiving LFLP diets in Expt. 2. GLUT5, located on the apical side, is a facilitated-diffusion fructose transporter that facilitates the uptake of fructose into enterocytes. GLUT2, located at the basolateral side, takes over the sugar absorption transport once inside the cell and mediates the passive transport of glucose, fructose, and galactose out of enterocytes into the bloodstream [38,39]. Others have similarly observed the downregulation of intestinal GLUT2, GLUT5, and SGLT1 in Eimeriaand Campylobacter jejuni-challenged birds [7,22,37,40]. In the current study, amino acid transporters CAT2 and y+LAT1 were also downregulated in Eimeriachallenged groups regardless of the dietary fiber and protein levels. This observation is consistent with previous observations that E. maxima caused ileal and E. tenella caused jejunal CAT2/y+LAT1 downregulation [37]. CAT2 and y+LAT1 are basolateral amino acid transporters mediating the transport of cationic and neutral amino acids [41]. As important L-arginine transporters, the depression of CAT2 and y+LAT1 may contribute to the reported sharp drop of plasma arginine in E. acervulina challenged chickens [42].
The downregulation of nutrient transporter proteins caused by Eimeria challenge affects a wide range of sugar and amino acid transporter proteins such as GLUT2, GLUT5, SGLT1, PepT1, b0AT, b0+AT, EAAT3, rBAT, y+LAT2 [6,7,37,43]. The causes for the downregulation effect are not clear, but it is speculated that this response is a cell-mediated protective reaction in response to pathogenic invasion. By downregulating nutrient transporters in epithelial cells, a depleted nutritional environment is created to limit the development of parasites. In addition, the malnourished cells may trigger apoptosis and consequent epithelial renewal [6,37]. The interaction between Eimeria challenge and enzyme supplementation in HFAP diets (Expt. 1) showed that compared to the non-challenged treatments, enzyme supplementation upregulated the expression of GLUT2 and GLUT5 to possibly compensate for nutrient utilization deficit. The xylanase-mediated nutrient transporter upregulation was reported in broiler chickens challenged with Clostridium perfringens [35,44]. These observations suggest the potential of exogenous enzymes to alleviate some negative effects of Eimeria challenge in broiler chickens.
Exogenous enzymes or dietary fiber have been shown to influence the content and profile of oligosaccharides in the digestive tract [15,17,45,46]. Some of these oligosaccharides could serve prebiotic functions in birds. In a study by Lin and Olukosi [17], broiler chickens receiving diets with higher fiber content had a greater content of jejunal pentoseoligosaccharides as well as cecal SCFA. In the current study, the concentration of jejunal hexose-oligosaccharides was reduced in the challenged birds receiving LFLP diets (Expt. 2), which is likely due to the depressed feed intake in challenged birds. On the other hand, no significant difference was observed in the jejunal oligosaccharides content in the different HFAP treatments in Expt. 1. Interestingly, the Eimeria challenge increased the cecal concentration of BCFA which exclusively originates from protein fermentation, but decreased the concentration of acetate and butyrate which mainly originate from carbohydrates fermentation, regardless of the types of diet profile in the two experiments. The production of BCFA is considered a marker for estimating cecal protein fermentation [47], thus it can be speculated that Eimeria promoted cecal protein fermentation but inhibited the fermentation of carbohydrates. This is possibly due to a change in the proportion of protein and carbohydrates reaching the hindgut for fermentation or a shift in the microbial population inhabiting the hindgut of challenged, compared with non-challenged birds.
It is widely acknowledged that hindgut fermentation of carbohydrates is generally more beneficial than protein fermentation [48]. For example, butyrate resulting largely from carbohydrate fermentation is regarded as an energy source for enterocytes. Butyrate also plays a role in reducing inflammation and oxidative stress and in enhancing the colonic defense barrier. The supplementation of butyrate was reported to control coccidiosis [49]. The greater ceca BCFA content observed in Eimeria-challenged chickens may be explained by the observed downregulation in amino acid transporters. This may lead to impaired amino acids absorption in the small intestine of the host, and consequently leading to disproportionally large amounts of protein in the hindgut. Secondly, large amounts of epithelial cell debris produced by coccidiosis-induced intestinal damage ultimately flow to the ceca as potentially fermentable protein substrates. In addition, an Eimeria-induced cecal microflora shift has been indicated in previous studies [49], leading to an increase in Firmicutes (mucins and amino acids fermentation) and Proteobacteria (amino acids fermentation) and a decrease in Bacteroidetes (carbohydrates fermentation) [50][51][52]. Therefore, protein fermentation is promoted at the expense of carbohydrates fermentation. Instructively though, in the current study, enzyme supplementation mitigated the Eimeria-induced enhancement of cecal BCFA in HFAP diets (Expt. 1). These observations demonstrated a potential positive effect of enzyme supplementation in alleviating the shift in microbial fermentation patterns caused by Eimeria challenge.

Conclusions
In conclusion, Eimeria challenge triggered changes to the expression of claudin 1 and nutrient transporters irrespective of the diet types. In addition, Eimeria infection resulted in the promotion of cecal protein fermentation and inhibited carbohydrates fermentation. Exogenous enzymes showed the potential of alleviating Eimeria-induced intestinal gene expression changes and mitigating the unfavorable cecal fermentation pattern, and thus demonstrated that enzyme supplementation may benefit beyond nutrient improvement.