key: cord-1032797-8klmn4wh
authors: Yadav, S.; Teng, P.-Y.; Singh, A.K.; Choi, J.; Kim, W.K.
title: Influence of Brassica spp. rapeseed and canola meal, and supplementation of bioactive compound (AITC) on growth performance, intestinal-permeability, oocyst shedding, lesion score, histomorphology, and gene expression of broilers challenged with E. maxima
date: 2021-11-07
journal: Poult Sci
DOI: 10.1016/j.psj.2021.101583
sha: 4d9d3c654dc1e86d6f3d0a10c31e0e205d425e1f
doc_id: 1032797
cord_uid: 8klmn4wh

This study was performed to investigate the effect of feeding Brassica spp. including full-fat rapeseed, canola meal, and allyl isothiocyanate (AITC) to broiler chicken challenged with E. maxima. A total of 576 one-day old male broiler chicks were completely randomized to 8 treatments with 6 replicated cages and 12 birds per cage. The treatment diets consisted of nonchallenge control (NC, corn-SBM based diet), challenge control (CC), 10% rapeseed (10RS), 30% rapeseed (30RS), 20% canola (20CLM), 40% canola (40CLM), 500 ppm AITC (500AITC), and 1,000 ppm AITC (1000AITC). At d 14, all birds were challenged, except NC group, with a subclinical dose of E. maxima. Intestinal permeability was conducted on 5 d post-infection (dpi) and for oocyst shedding 5 to 6 dpi feces were pooled and collected. On 6 dpi, growth performance, lesion score, histomorphology, and gene expression were measured. The growth performance result showed that 10RS and 30RS groups had lower BW, BWG, FI, and higher FCR (P < 0.0001). During the challenge and overall periods, NC group had highest BW, BWG, and FI, and lowest FCR. The inclusion of canola meal showed lower performance during prechallenge period but was able to catch up BWG during challenge period. The AITC levels showed similar growth performance to CC group. Intestinal permeability for 20CLM, 40CLM, 500AITC and 1000AITC was similar to NC group, whereas CC, 10RS, and 30RS had higher permeability compared to NC (P < 0.0001). Oocyst shedding was significantly lower for 40CLM and NC, whereas all other treatments had higher oocyst shedding (P < 0.0001). All the challenged treatment groups had higher lesion score and microscore than NC (P < 0.0001). Histomorphology data showed that jejunum villus height (VH) for 1000AITC was similar to NC group, whereas CC group had the lowest VH (P = 0.01). The 30RS group had lower VH: crypt depth (CD) ratio in the jejunum and ileum. The gene expression at 6 dpi for claudin1, occludin, IL2, IL6, GLUT5, EAAT, B(o)AT, and LAT1 was significantly changed among the treatments. The results suggest that 30RS retards growth performance and deteriorate gut health during coccidiosis and should not be fed to chicken during the starter phase. Canola meal showed decline in growth prechallenge but maintained growth and intestinal health during the challenge period at 40% inclusion. AITC at 1,000 ppm showed similar growth as control group, but with improved gut health during the challenge period. Canola meal could be a good alternative to SBM especially during coccidiosis, whereas AITC needs to be tested vigorously in animal feeding regime.

500AITC and 1000AITC was similar to NC group, whereas CC, 10RS, and 30RS had higher permeability compared to NC (P < 0.0001). Oocyst shedding was significantly lower for 40CLM and NC, whereas all other treatments had higher oocyst shedding (P < 0.0001). All the challenged treatment groups had higher lesion score and microscore than NC (P < 0.0001). Histomorphology data showed that jejunum villus height (VH) for 1000AITC was similar to NC group, whereas CC group had the lowest VH (P = 0.01). The 30RS group had lower VH: crypt depth (CD) ratio in the jejunum and ileum. The gene expression at 6 dpi for claudin1, occludin, IL2, IL6, GLUT5, EAAT, B o AT, and LAT1 was significantly changed among the treatments. The results suggest that 30RS retards growth performance and deteriorate gut health during coccidiosis and should not be fed to chicken during the starter phase. Canola meal showed decline in growth prechallenge but maintained growth and intestinal health during the challenge period at 40% inclusion. AITC at 1,000 ppm showed similar growth as control group, but with improved gut health during the challenge period. Canola meal could be a good alternative to SBM especially during coccidiosis, whereas AITC needs to be tested vigorously in animal feeding regime.

Poultry is one of the most preferred meat, and its demand have been increasing due to its health benefit, cost per unit, availability, and acceptance by all the community (OECD-FAO, 2020). Global poultry meat consumption is expected to rise more in the developing countries which will ultimately cause rise in feed ingredient cost and demand (OECD-FAO, 2020) . Feed security, food safety and disease emergence or re-emergence are considered major hurdles to increasing strategic future of chicken production because of increasing foodborne and pathogenic diseases, antibiotic residues, cost of chicken production, limited resources of feed ingredients, and understanding interconnection of feed, health, immunity, and welfare of animals (Hafez and Attia, 2020; Hafez et al., 2021) .

In poultry feeding programs, protein is one of the most expensive nutrients with very limited sources of protein.

Plant sources includes soybean meal (SBM), cotton seed meal, canola meal, rapeseed meal, sunflower cake, groundnut cake, macadamia nut cake, and other oil-seed co-products after oil extractions (Canola, 2009; Beski et al., 2015; Yadav and Jha, 2021) . Whereas animal sources include fish meal, bone meal, blood meal, feather meal, etc. In comparison, the protein contents or amino acids balance of the animal sources are higher, but their use is limited due to availability issues, residual antibiotics, or pathogens carryover, and expensive for commercial broiler production (Hossain et al., 2015) . The plant source protein especially SBM is commonly used in commercial poultry feeding. The poultry industry is heavily dependent on SBM solely; thus, exploration of other alternative protein sources is necessary. Also, unprecedented condition such as COVID-19 increased the grain consumption by humans lowering the share for animal feed (Hafez et al., 2021) . Rapeseed and canola meal are promising alternatives to SBM to reduce the cost of production and heavy dependency on SBM.

Rapeseed meal also known as mustard seed (Brassica napus) has been used in animal feeding for long time (Hickling, 2001) . Its availability and balanced amino acid content make it suitable to be included in chicken diet. Rapeseed is the third largest oilseed crop produced worldwide, and the co-product after oil extraction is used in animal feeding as a rich source of protein (30 −45%) (Shahidi, 1990) . Rapeseed contains appropriate amino acid such as lysine and other sulfur containing amino acids which are limiting in chicken; however, its utilization is limited due to the presence of antinutritional factors (ANF) such as glucosinolate and erucic acid (Nega and Woldes, 2018) . Whole rapeseed contains additional factors such as complex carbohydrates, fiber, phytic acid, and sinapine which potentially cause reduction in feed intake (Tripathi and Mishra, 2007) , digestibility (Bellostas et al., 2007) , growth (Tripathi and Mishra, 2007) , and overall health (Chun Chang and Brian, 2004) . Previous studies used different inclusion levels of rapeseed based on the amount of glucosinolate content, animal species, age, and production level (Wondimagegne et al., 2016) . Rapeseed meal can replace up to 50% of SBM and can be included up to 10 to 15% in broiler diets (Nega and Woldes, 2018) . Another study showed that rapeseed meal can replace 25% of SBM without any negative effects on growth (Georgeta, 2009) . Whole full-fat rapeseed in ruminant feeding has been studied due to its high energy concentrate in the oil although it contains high glucosinolate level (Hristov et al., 2011) . However, feeding whole rapeseed to chicken or monogastric is not well-studied especially under disease challenged model.

Another Brassica species, canola, having low glucosinolate (<30 mmol/g) and erucic acid (<2%) was developed by plant breeding of rapeseed for oil production purpose (Bell, 1993) . With the increase in oil production, the leftover canola meal after oil extraction was utilized for the animal feeding. This variety was developed to reduce the ANF from rapeseed so that higher levels could be included in the chicken diet. Moreover, canola meal has better nutrient profiles compared to SBM in terms of essential minerals (calcium, phosphorus, and selenium), B vitamins, and sulfur containing amino acid (Wickramasuriya et al., 2015) . Canola also contains ANF in the forms of fiber, phenolics, sinapine, phytate, and glucosinolate. Among these ANF, glucosinolate is the most important one as its metabolites causes reduction in feed intake due to decreased palatability that negatively affects the growth performance of birds, (Bourdon and Aumaitre, 1990) . Although glucosinolate is inactive unless it meets an enzyme myrosinase in the presence of water that breaks it down to biologically active compounds such as thiocyanate and isothiocyanate (Tripathi and Mishra, 2007) . Although isothiocyanate is considered undesirable, it has beneficial effects as an anticarcinogenic, antioxidant, antimicrobial, and other disease therapeutic and preventive compound (Tripathi and Mishra, 2007; Maina et al., 2020) . To authors' knowledge, no study has been conducted to evaluate the effects of feeding chicken with allyl isothiocyanate (AITC) supplementation or feed ingredients rapeseed and canola meal during Eimeria challenged condition on growth performance and gut health in broilers.

Along with feed issues, another challenge in chicken production is disease outbreak. Coccidiosis is one of the most common enteric diseases caused by Eimeria species of which most common are E. tenella, E. maxima, and E. acervulina (McDougald et al., 1997; Yadav et al., 2020; Teng et al., 2020a,b) . Coccidiosis has always been an issue in chicken farms due to huge economic loss as well as long-term impacts in poultry farms as chicken is a major host for Eimeria parasites. Poultry coccidiosis has been ranked among the top 3 economically important diseases, the number one disease related issue (E. maxima), and among the top 10 impactful diseases in United Kingdom, United States, and South Asia respectively (Perry et al., 2002; Bennett and Ijpelaar, 2005; USAHA, 2019) . According to recent recalculation of global cost of coccidiosis in chicken was estimated around £ 10 billion in 2016, which includes prophylaxis, treatment, and losses cost associated with chicken production (Blake et al., 2020) . Nutrition plays a major role during susceptibility or protection, during infection or pathogenesis, and lastly during recovery or compensatory phase (Gomez-Osorio et al., 2021) . To control the economic losses or to prevent the outbreak of coccidiosis in farms, nutritional strategies for prophylaxis or treatment measures are being explored. Use of natural dietary supplements to enhance innate immunity could effectively reduce the need of treatment for the enteric infection (Lillehoj et al., 2018) .

We hypothesized that antinutritional factors present in rapeseed, canola, or AITC supplementation would affect the growth performance and gut health in broilers during Eimeria infection. Thus, the objective of the present study was to evaluate the effects of different levels of rapeseed and canola meal to partially replace SBM as well as supplementation of synthetic AITC in basal diet on growth performance and gut health parameters of broilers challenged with/without E. maxima.

The whole full-fat rapeseed was Dwarf Essex variety from Idaho and obtained locally from Athens Seed Co., GA. It was further processed by a roller grinder to split into pieces and included at desired levels to prepare a balanced diet. Solvent-extracted canola meal was obtained in pellet form which was further ground to prepare treatment diets. Synthetic AITC (Sigma-Aldrich, St. Louis, MO) was purchased and included in basal diet at levels of 500 ppm and 1,000 ppm. Control diet was based on corn-SBM. The feed ingredients were analyzed for nutrient profile and glucosinolate level before formulating feed (Table 1) .

Fresh field isolated oocyst of E. maxima was obtained from Dr Brian Jordan at Department of Poultry Science, University of Georgia. Further oocyst preparation and sporulation was performed using 2.5% Potassium dichromate following procedure used by Teng et al. 2021 .

This study was conducted at the Poultry Research Center after the approval of the Institutional Animal Care and Use Committee of University of Georgia, Athens, GA. A total of 576 one-day old male broiler chicks (Cobb 500) were randomly allocated to 8 treatments with 6 replicates cage and 12 birds per cage. The dietary treatments included nonchallenge control (NC, corn-SBM based, nonchallenged with E. maxima), challenge control (CC, corn-SBM based, challenged with E. maxima), 10% rapeseed (10RS), 30% rapeseed (30RS), 20% canola meal (20CLM), 40% canola meal (40CLM), 500 ppm AITC supplemented in basal diet (500AITC), and 1,000 ppm AITC supplemented in basal diet (1000AITC). The reason for using different levels of rapeseed and canola meal was based on previous studies that showed rapeseed can only be included in diet at 5 to 15%; thus, the present study used the average 10% as a lower level (Nega and Woldes, 2018) . Similarly, for canola meal, the range of its inclusion based on previous studies was 15 to 25%; thus, 20% CLM was used as lower level (Mushtaq et al., 2007; Min et al., 2011) . The higher levels, 30RS, and 40CLM, were used to investigate anti-Eimeria effect of their inherent high concentration of glucosinolate or its bioactive compounds. Similar dietary treatments were included in a previous study from our lab . Birds in 500AITC and 1000AITC groups received corn-SBM based control diet till d 11 of age and provided AITC treated diet from d 12. On d 14, birds in NC group were gavage with 1 mL water as sham, whereas all other treatments received 1 mL of freshly prepared E. maxima sporulated oocysts at a dose of approximately18,000 oocysts/ mL. The challenge dose was determined based on a previous study from our lab (Teng et al., 2020a) , and the current study was conducted till d 20 (6 d postinfection, dpi). All the birds had access to ad libitum feed and water and were kept under controlled temperature environment following the recommendation of Cobb management guide (Cobb, 2018).

Growth Performance The birds were weighed on d 0 (day-of-hatch) before allocation, and the body weight (BW) and feed intake (FI) were recorded during prechallenge period (d 0−d 14), challenge period (d 14−d 20), and overall period (d 0−d 20). The measured BW and FI were used to calculate body weight gain (BWG) and feed conversion rate (FCR) as shown in Table 2 . The mortality was recorded and used to calculate corrected FI.

On 5 dpi, one bird from each cage (6 birds/ treatment) was orally gavage with 1 mL/ bird (2.2 mg/bird) of the fluorescein isothiocyanate dextran (FITC-d; 100 mg, MW 4,000; Sigma-Aldrich) to determine intestinal permeability by following the method from Teng et al. (2020a) . In brief, the birds after FITC-d gavage were kept in separate cages by treatment for 2 h. After exact 2 h, birds were euthanized and blood samples (at least 2 mL) were collected and stored in a dark container with blood vials kept in slant position at room temperature for 2 h. All the blood samples were centrifuged (Eppendorf Centrifuge 5430R, Eppendorf, Hamburg, Germany) at 1,000 £ g for 15 min to obtain serum. Blood samples were also collected from 10 extra birds (kept in the same room but not involved in experiment) whose serum was pooled, mixed with FITC-d and used to make a standard curve. The obtained serum samples (in duplicate) and standard solution (in triplicate; 100 mL per well) were kept in a dark 96-well plate (Ref. 655077, Greiner Bio-one, Monroe, NC). This plate was read in a spectrophotometer (SpectraMax ABS plus, Softmax Pro 7 software, Molecular devices, San Jose, CA) at excitation and emission wavelength of 485 and 528 nm, respectively. The values obtained were fitted to standard curve equation to obtained intestinal permeability value as shown in Figure 1 .

On 5 dpi, clean paper was kept under each cage, and fecal dropping was collected for 48 h. From each cage approximately 200 g of representative excreta was collected in sample bag and stored at 4°C until further processing following the modified procedure by Teng et al. (2020b) . Briefly, 5 g of fecal samples were diluted with 45 mL water. After proper mixing, 1 mL mixture was taken and diluted with 9 mL saturated salt solution. Homogenized mixtures were incubated for 30 s for letting oocysts float. Using a water dropper pipette (Thermo Fisher Scientific, Waltham, MA), the final samples were loaded in McMaster chambers (Jorgensen Laboratories, Loveland, CO), and the total oocysts were counted, and the data was calculated as oocysts per gram using formula as shown by Yadav et al. (2020) . Data is presented in Figure 2 .

On 6 dpi, four birds per cage were euthanized by cervical dislocation, and 15 cm of jejunum and ileum tissues with reference to Meckel's diverticulum were evaluated for gross lesion score (G-LS) by the 5-score system (Johnson and Reid, 1970) shown in Figure 3 . In addition, for microscores (M-LS), slides were prepared by mounting mucosal scraping from same location as G-LS. The slides were kept moist and studied under a microscope on the same day post sampling. Microscores were evaluated following the method by Goodwin et al. (1998) where score 0 indicates no oocyst; score 1, 1−20 oocyst; score 2, 21−50 oocyst; score 3, 51−100 oocyst; score 4, too numerous to count. Around 8 to 10 fields were observed for each slide. The result is shown in Figure 4 . On 6 dpi, 1 bird/ cage was euthanized by cervical dislocation, and 5-cm intestinal tissue samples from the duodenum loop, mid-jejunum, and mid-ileum were rinsed with phosphate buffer saline (PBS) and kept in 10% buffered formalin. Tissue samples were cut crosssectionally to 0.5 cm and loaded in tissue embedding cassettes which were sent out for further processing and slide preparation to histology-core at Poultry Diagnostic Research Center (PDRC, Athens, GA). The received slides were observed for villus height (VH) and crypt depth (CD) using a microscope (BZ-Z800, Keyence Inc., Itasca, IL) at 20X magnification. At least 3−5 representative VH, and CD were selected for measurement from each slide. Later ImageJ (version I.X.) program (Abramoff et al., 2004) was used to measure the VH, CD, and their ratios (Table 3) .

On 6 dpi, the mid-jejunum tissues were cut from the same birds for intestinal histomorphology, and digesta were flushed with sterile PBS. The cleaned tissues were collected in aluminum foil and immediately snap frozen in liquid nitrogen. The samples were stored at À80 o C until further processing. The total RNA was extracted using QiAzol lysis reagents (Qiagen, Valencia, CA) following manufacturer's instruction. After extraction, a nanodrop 2000 spectrophotometer (Thermo Fisher Scientific) was used to check quality and quantity of RNA. The cDNA was obtained from RNA by reverse transcription using high-capacity cDNA synthesis kits (Applied Biosystems, Foster City, CA). For Real-Time PCR, a Step One thermocycler (Applied Biosystems) was run for each cDNA samples kept in duplicate along with target gene primers using SYBR Green Master mix (Bio-Rad Laboratories, Hercules, CA). The gene expression of target genes was normalized with housekeeping genes and relative gene expression was determined using 2 ÀDDCt method following Teng et al. (2020a) procedure (Table 4 ) and the primers used are shown in Table 5 . 

Battery cage was the experimental unit for all the measurements. Data from all the analysis were presented as mean values with standard error of mean (SEM) as shown in respective Tables. All the data except lesion score and microscore were analyzed using PROC-GLM of SAS University Edition (Version 9.4, SAS Institute Inc., Cary, NC). When significant difference was observed in one-way ANOVA, the treatments were subjected to multiple comparison using Tukey's test following the procedure by Teng et al. (2020a) . For the lesion score and microscore, the Kruskal-Wallis nonparametric analysis was performed. Statistical significances for all analyses were set at P < 0.05, and actual P values are presented in the Tables and Figures.

The growth performance of birds fed SBM, rapeseed and canola were different throughout the study (prechallenge period, challenge period, and overall period: Table 2 ) (P < 0.05). The BW and BWG were significantly lower for rapeseed and canola meal fed birds during the prechallenge period (P < 0.0001); rapeseed at the higher inclusion (30RS) had poor BW. Similarly, FI was reduced for 30RS and 40CLM (P < 0.0001), whereas 10RS and 20CLM had similar feed intake as control and both AITC fed groups. Due to changes in FI and BWG, there was difference in FCR among different treatment diets fed birds during the prechallenge period. Rapeseed at inclusion of 30% had significantly highest FCR compared to the FCR of other treatments (P < 0.0001). The data for the prechallenge period show that rapeseed at higher level in the diet for young chicks is not recommended as it caused deleterious effect on the early growth performance. For the challenge period, the BW and BWG were different between nonchallenge NC group and all other challenged groups (P < 0.0001). Within the challenge groups, there was significant decrease in BW and BWG with increasing levels of rapeseed, whereas the inclusion of canola meal caused significantly reduction in BW but not BWG compared to SBM fed CC and AITC fed challenged birds. The FI during the challenge period was reduced for 30RS, 20CLM, and 40CLM compared to NC group (P < 0.0001), whereas comparing FI among challenged birds 30RS was significantly lower than all other treatment groups except 40CLM. The FCR during the challenge period was increased for 10RS compared to NC group (P = 0.03), whereas all other treatments were in between both NC and 10RS (P > 0.05). During the overall period, the BW and BWG were highest for NC group compared to all other challenge treatment groups, and the second highest was CC, followed by 500AITC and 1000AITC group. The 10RS, 30RS, 20CLM, and 40CLM groups had significantly lower BW and BWG compared to the other groups, whereas 30RS group had the lowest BW and BWG among the treatments (P < 0.0001). Overall FI was significantly reduced for 30RS, 20CLM, and 40CLM compared to NC group. A similar pattern was observed for overall FCR where 10RS, 30RS, and 20CLM had higher FCR compared to NC group (P < 0.0001), but higher canola inclusion had very similar FCR to NC, CC, 500AITC, and 1000AITC. The rapeseed inclusion in the higher level severely caused negative impact on growth performance. Both the 500AITC and 1000AITC groups had similar growth performance to CC group during both challenge and nonchallenge periods. In the present study, birds were challenged with E. maxima and its site of infection was mainly mid gut in the region of the jejunum. As expected, no differences in duodenum histomorphology were observed for VH, CD, or their ratios (Table 3) . Jejunum VH for CC group was significantly decreased compared to NC and 1000AITC groups, whereas all other groups had values between NC and CC group (P = 0.01). There was no difference among the treatments for jejunum CD, which caused increased VH: CD ratios for NC group compared to CC and 30RS groups (P = 0.01). No difference among the treatments was observed for ileum VH (P > 0.05). The ileum CD was increased for 30RS and 1000AITC compared to NC group (P = 0.02). The ratio for VH: CD in the ileum was also decreased for 10RS, 30RS, 20CLM, 40CLM, and 1000AITC compared to NC group (P = 0.001).

The intestinal permeability result represents the levels of FITC-d that was recovered in the serum of birds; higher values mean increased permeability (lower gut integrity), whereas lower values represent decreased permeability (higher gut integrity). The result from the present study showed that NC and 1000AITC groups had lower permeability value compared to CC, 10RS, and 30RS (P < 0.0001; Figure 1 ). Interestingly, 20CLM, 40CLM, and 500AITC had significantly reduced permeability compared to 30RS, and no difference compared to NC, CC, 10RS, and 1000AITC.

The oocyst shedding was measured for 5 to 6 dpi and presented as oocyst per gram (OPG) (Figure 2 ). Usually, oocyst shedding in addition to lesion scoring and body weight gain are commonly suggested as test parameters to measure coccidiosis intervention strategies in farm (Chasser et al., 2020) ; it is also an indicator of the vaccine intake, development of anticoccidial resistance or the successful challenge by Eimeria spp. (Braunius, 1985) . The result showed that NC group had no oocyst shedding, whereas all other challenged treatments with E. maxima shed higher oocysts (P < 0.0001) except for 40CLM which was not significant to both CC and NC. The 40CLM reduced oocyst shedding by almost 15% compared to CC.

On 6 dpi both gross lesion score and microscore were observed in the jejunum and ileum (Figures 3 and 4) . Gross lesion score results showed that NC group did not show any intestinal lesion, whereas all other E. maxima challenged treatments had significantly higher average score of 1.6, 1.9, 1.77, 1.73, 1.47, 1.7, and 1.9 for CC, 10RS, 30RS, 20CLM, 40CLM, 500AITC, and 1000AITC, respectively. Microscore results for NC, CC, 10RS, 30RS, 20CLM, 40CLM, 500AITC, and 1000AITC showed average score of 0, 3.33, 3.38, 3.53, 2.53, 2.93, 3.21, and 3.15, respectively (P < 0.0001). Comparing both the scoring techniques, microscore showed overall higher average score than gross lesion score. This could be because the presence of oocysts does not necessarily means causing damage to the intestine or having higher gross lesion. Comparison among the treatments in gross lesion score showed that although not significant to other challenged groups, 40CLM showed 8% lower score than CC group. Similarly, microscore showed numerical decrease for 20CLM by 24%, and 40CLM by 12% when compared to CC.

In this study, representative genes for tight junction proteins, immunity, and nutrient transporter were also evaluated (Table 4 ). Gene expression of claudin1 tended to be different among the treatments (P = 0.06) where 10RS and 1000AITC had higher claudin1 expression than NC group, and 1000AITC also increased compared to 40CLM. For the occludin gene, relative expression for 30RS was higher than NC and CC, and similarly 1000AITC was higher than CC group (P = 0.002). The relative gene expression of interleukin-2 (IL2) showed increased expression in 30RS, 500AITC, and 1000AITC compared to NC treatment (P = 0.05). Similarly, interleukin-6 (IL6) relative expression was upregulated for 10RS compared to 40CLM and NC group (P = 0.025). For the nutrient transporter genes, LAT1expression was increased for 10RS, 30RS, and 500AITC compared to NC group (P = 0.005). Whereas the excitatory amino acid transporter (EAAT) gene expression was significantly decreased for all the treatment groups compared to NC except for 10RS group (P = 0.003). The fructose transporter-5 (GLUT5) gene expression was also significantly altered by different dietary treatments where expression was lower for 30RS and 500AITC group compared to NC (P = 0.01). The Na + -dependent amino acid transporter (B o AT) relative expression was reduced for NC, PC, 20CLM, 40CLM, and 500AITC when compared with 30RS group (P = 0.03). No differences in relative gene expression were observed for ZO1, JAM2, MUC2, SGLT1 and PEPT1 among the treatments.

This study investigated the consequences of E. maxima challenge to broilers fed SBM in control group, 2 levels of full-fat rapeseed, 2 levels of canola meal, and supplementation with 2 levels of AITC for their potential beneficial effects in broiler chicken. Birds were challenged on d 14 with a dose between mid-low and midhigh to cause subclinical infection as recommended by Teng et al. (2020a) .

In the present study, there was difference in growth performance between non-challenged and all other treatment groups challenged with E. maxima. This is in accordance with previous studies that showed decrease in growth performance with single or mixed species of Eimeria challenge (Hamzic et al., 2015; Dos Santos et al., 2020; Yadav et al., 2020) . In a large-scale E. maxima challenge study, significant differences in BWG between challenged and nonchallenged group were found similar to the present study (Hamzic et al., 2015) . Cocci infection was also characterized by poor growth and feed conversion with high mortality even during subclinical challenge with Eimeria (Lee et al., 2013) . Although feeding canola meal showed decreased growth performance than control group, no difference was observed between 20 and 40% inclusion, except 40CLM had lowest FCR. The present study is not in agreement with a previous experiment reporting that canola meal can only be included up to 10% for starter chicks and maximum 20% during a grower phase (Canola, 2009) . Other studies had different inclusion levels of canola without negative effect on growth: 25% inclusion (Min et al. 2011 ) and below 20% inclusion (Payvastagan et al., 2012) . Throughout the current study, feeding 10RS had similar effect as 20CLM, whereas feeding 30RS had severely affected growth reduction before and after challenge. This could be solely because of the antinutritional factors present in the rapeseed. The antinutritional factors in rapeseed include GLs, erucic acid, sinapine, tannins, phytic acid, and fiber components that limit the use of rapeseed at higher inclusion (Zhu et al., 2018) . These factors also reduce nutrient digestibility by limiting nutrient availability to the birds (Zhu et al., 2018 (Zhu et al., , 2019 . The 30RS group had least feed intake compared to any other treatments because of the bitter taste of GL component which decreases the palatability along with crude fiber that decrease FI by increasing satiety (Canola, 2009; Zhu et al., 2019) . Furthermore, rapeseed used in the present study was high in fat (26% as-is basis), and the crude fat digestibility could have been lowered by the erucic acid present in rapeseed (Zhu et al., 2019) . Decrease in FI ultimately caused reduction in BW, and BWG compromising overall growth performance with rapeseed inclusion. These limitations were further accompanied by Eimeria challenge negatively affecting the birds across challenged treatments compared to NC group.

Because Eimeria spp. cause severe intestinal damage, the proper tools/ biomarkers that could identify intestinal entities related to infection would be great help to evaluate mitigation strategy and nutritional manipulation during infection by Eimeria or prophylaxis to control before exposure (De Meyer et al., 2019; Criado-Mesas et al., 2021) . Some of the most studied parameters are gut permeability, intestinal histomorphology, intestinal lesion score, gene expression related to intestinal barrier proteins, nutrient transporters, and immune genes (Teng et al., 2020a; Yadav et al., 2020) .

A single layer of epithelial cells joined together by tight junctions helps maintain gut selective permeability in healthy birds. The gut permeability significantly increases with the infection by Eimeria (Teng et al., 2020a; Yadav et al., 2020) . The pathological changes in the gut such as altered intestinal histomorphology, lesion score, tight junction proteins, as well as systemic changes all start with the increase in gut permeability caused by the disintegration of mucosal barriers by the presence of Eimeria (Belote et al., 2019) . In the present study, the gut permeability increased for the CC, 10RS, and 30RS groups compared to NC; this is an indication of reduced tight junction integrity and barrier function. Moreover, reduction of jejunum VH and VH: CD ratio in these treatments also reflects gut health and integrity reduction by E. maxima infection.

Although oocyst shedding was not significant among the challenged groups, there was no oocyst detected in the NC group, suggesting that there was no cross contamination between challenged and nonchallenged cages throughout the experiment. Hamzic et al. (2015) found the correlation between BWG and oocyst count where higher BWG has higher oocyst count, and the present study showed numerical decrease in the oocyst shedding of 40CLM compared to CC group. Previous studies also found huge variation between treatments in the challenged groups especially for oocyst shedding and lesion score (Pinard-van der Laan et al., 2009; Hamzic et al., 2015) Lesion score was observed in the jejunum and ileum of challenged group as mid-gut is the site of action for E. maxima. A study by Hamzic et al. (2015) showed that intestinal lesion score is a good indicator of health status in the Eimeria infected birds. However, Belote et al. (2019) claimed that the "I See Inside" (ISI) methodology is better tool to diagnose even mild alteration caused by subclinical coccidiosis. NC group in the present study did not show any lesion score in both methods of observation (gross lesion scoring and in microscoring). Although not significant within challenged birds there was numerical decrease in lesion for canola fed birds.

Coccidiosis causes damage to the intestinal mucosa, altering their absorptive surface during severe infection, decreasing the digestibility of nutrients, and leading to reduced growth performance (Dalloul and Lillehoj, 2006; Dos Santos et al., 2020; Hafez and Attia, 2020) . No difference in histomorphology of the duodenum was noticed when only E. maxima was used because E. maxima specifically attach and infect jejunum and proximal ilium. The 1000AITC group had similar jejunum VH as NC group although the cell turnover rate was high in the ileum, causing significantly higher CD leading to decreased VH: CD ratio in the ileum for 1000AITC fed birds. According to Criado-Mesas et al. (2021) , the higher cell turnover in Eimeria challenged birds is due to activation of mTOR complex 1 pathway to reduce the intestinal mucosal disturbance. Previous studies showed no difference in VH and VH: CD ratio when canola was included up to 40% (Figueiredo et al., 2003; Chiang et al., 2009 ). This agrees with the present study although there was reduced VH: CD ratio in the ileum, which was supported by a study from Gopinger et al. (2014) showing that 20% canola inclusion decreased VH: CD ratio. This effect on histology of the ileum could also be due to challenge with E. maxima which increased the metabolic cost of epithelial cell turnover in the ileum. Teng et al. (2020a) reported that increasing Eimeria challenge doses caused linear negative effect on gut histology. The present study had decrease in jejunum VH and VH: CD ratio for challenge control and VH: CD ratio for 30RS, whereas other treatments were able to maintain intestinal morphology in between NC and CC group. The disturbance in intestinal parameters during Eimeria infection also causes dysbiosis leading to increase in opportunistic pathogens like C. perfringens, Enterococcus, Streptococcus and decrease in non-pathogenic Lactobacillus and Faecalibacterium (De Meyer et al., 2019; Chen et al., 2020) . This dysbiosis further deteriorates the situation in intestinal parameters due to production in toxic materials with the rise in harmful bacteria.

With the damage to gut barrier and epithelium during subclinical doses of E. maxima infection, malabsorption of nutrients has been associated with altered gene expression especially nutrient transporter genes, tight junction genes, and immune genes Teng et al., 2021) . The damage of absorption sites and presence of diarrhea ultimately lead to loss of dietary nutrients in excreta. This is one of the pathways leading to decreased growth performance during challenge. Although coccidiosis may damage mucosa, triggering impaired absorption of nutrients (Criado-Mesas et al., 2021) , gene expression of MUC2 (component of mucus layer) was numerically reduced in the present study. As AITC are known candidates to reduce oxidative stress, the challenged birds should have coped up with the inflammation associated with oxidative stress which is responsible for changes in gene expression (Criado-Mesas et al., 2021) . The gene expression in the intestine also depends on the severity as in case of necrotic enteritis genes are more profoundly expressed compared to mild-coccidiosis (Criado-Mesas et al., 2021) . The expression of tight junctions also depends on pathogen as some pathogens utilize these proteins to attach whereas for other pathogens tight junctions act as barrier to enter the underlying cells; thus, the expression of tight junctions should be destroyed or modified for the pathogens to invade intestinal epithelial cells (Awad et al., 2017) . Criado-Mesas et al. (2021) found down-regulation of claudin1 and occludin following Eimeria challenge; however, it was up-regulated in the present study. The present study also found downregulation of nutrient transport genes, which elucidate the changes to gut histomorphology. There was downregulation of brush border membrane, amino acid transporters (B o AT) except for rapeseed and higher AITC levels which indicate reduction in uptake of neutral amino acids such as alanine, serine, cysteine, and threonine from gut lumen to epithelial cells which also disrupt other cationic amino acids transport across membrane as explained by Teng et al. (2021) .Along with other nutrient transport genes EAAT expression decreased which is responsible for internalization of glutamate and aspartate across membrane (Kanai et al., 2013; Teng et al., 2021) . Similar results were obtained by Su et al. (2014) where brush border membrane amino acids transporters are downregulated, whereas the basolateral membrane amino acid transporter like LAT1 was upregulated especially for rapeseed fed birds and low level of AITC in present study. During the acute infection by Eimeria, birds respond by decreasing the amino acid pool in the enterocytes limiting the nutrient for cell causing cell death that also respond in decreasing E. maxima replication and increase the shedding from the cells as witnessed in the fecal oocyst shedding count increase.

In response to Eimeria presence in the intestine, the IL2 gene was significantly upregulated for 30RS, 500AITC and 1000AITC in the present study. Previous study by Li et al. (2002) suggests that duodenal IL2 was upregulated in Eimeria resistant birds when compared for susceptibility between different bird lines. IL2 as a proinflammatory cytokines is able to activate T cells and other immune cells as mode for rapid inflammatory response against Eimeria (Broom and Kogut, 2019) . IL6 acts as both proinflammatory as well as anti-inflammatory at the same time and is upregulated during Eimeria infection. In the present study, gene expression of IL6 in 40CLM was very similar to NC group which means that birds were able to resist with the inflammation caused by Eimeria whereas upregulation of IL6 in the 10RS group suggest that bird immune cells were fighting against inflammation due to exposure to E. maxima in the gut. Thus, immune gene expression of IL2 and IL6 in the jejunum of Eimeria infected birds showed more resistance to infection and higher fighting capability of rapeseed and AITC fed birds.

In summary SBM, rapeseed, and canola meal reacted differently to the Eimeria infection. Rapeseed at the 30% inclusion had severe negative impact on growth and gut health, whereas a synthetic compound of GL metabolites (AITC) was able to maintain gut integrity and bird performance. This was the first study to evaluate AITC in feed as a supplement, and further in-depth knowledge about this compound will help explore the beneficial role of the natural plantbased compound to control poultry diseases. Canola meal inclusion although decreased body weight of birds, both 20 and 40% were able to maintain feed efficiency and better gut health compared to the other treatments in Eimeria challenge groups. The gut parameters including gut permeability, histomorphology, tight junction protein gene expression, lesion scoring, as well as oocyst shedding were positively influenced by canola inclusion in feed even during the acute E. maxima infection period. Thus, 40% canola meal and 1,000 ppm of AITC supplementation could be good supplements during coccidiosis outbreak. Further investigation is essential to determine optimum doses of inclusion and elucidate mechanism to alleviate negative effects by Eimeria infection.

Authors want to thank Kim's Lab members for helping in sample collection, processing, and suggestions on this manuscript. Special thanks to Yuguo Hou Tompkins and Hanyi Shi for help and generous support throughout the study.

There is no conflict of interest.

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