Contents lists available at ScienceDirect Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff Dietary organic cranberry pomace influences multiple blood biochemical parameters and cecal microbiota in pasture-raised broiler chickens Md. Rashedul Islama, Yousef I. Hassana, Quail Dasa, Dion Leppa, Marta Hernandeza, David V. Godfreyb, Steve Orbanb, Kelly Rossb, Pascal Delaquisb, Moussa S. Diarraa,⁎ aGuelph Research and Development Centre, Agriculture and Agri-Food Canada (AAFC), 93 Stone Road West Guelph, Ontario N1G 5C9, Canada b Summerland Research and Development Centre, AAFC, 4200 Highway #97 South, Summerland, British Columbia V0H 1Z0, Canada A R T I C L E I N F O Keywords: Cranberry Blood Iron Cholesterol Gut microbiota Pasture-raised chickens A B S T R A C T Cranberry processing by-products/pomace can be an excellent source of functionally bioactive molecules such as polyphenolics, complex carbohydrates, fibers, and nutritive minerals. While there are currently few applications for such processing-residues in poultry nutrition, there are many potential opportunities for the development of sustainable and value-added products. The aim of the current work was to investigate the effect(s) of four consecutive weeks of cranberry pomace (CBP) feeding on blood serum metabolic profiles and the cecal micro- biota of pasture-raised broiler chickens. Six hundred day-old Cobb 500 broiler chicks were divided into three groups: one group receiving the basal diet, and two groups each receiving the basal diet supplemented with either 1 or 2% of CBP. Blood and cecal samples were collected from the birds before, during, and after the treatment over a period of 64 days for biochemical and 16S rRNA gene sequencing analysis, respectively. The detailed analysis of CBP (and formulated final feed) indicated its content in essential amino- and fatty acids in addition to its richness in dietary fibers. Incorporating the pomace in feed induced a dichotomous response in broilers, with short-term improvements in blood-serum iron and cholesterol levels coupled with long-term modulation of cecal microbiota characterized by an increase in beneficial bacterial taxa (including Bifidobacterium, unclassified_Rikenellaceae, and Faecalibacterium) while decreasing the presence of undesirable ones (unclassified_Synergistaceae and Desulfovibrio, and unclassified_Fusobacteriaceae). Overall, the outcome of this study suggests the possibility of using organic CBP as a feed supplement with potential ability to positively influence blood metabolites and gut microbial community composition in pasture-raised broiler chickens. 1. Introduction Solid wastes, known as pomace or marc, are major by-products of many fruit/juice-processing industries (Beres et al., 2017; Hassan, Kosir, Yin, Ross, & Diarra, 2019). Substantial amounts of pomace being produced annually contain high amounts of fibers (Zhu, Du, Zheng, & Li, 2015), 70–75% moisture, and relatively adequate (when supple- mented with other sources) amounts of proteins in addition to biode- gradable but functionally-active compounds (Gassara et al., 2011; Waldbauer, McKinnon, & Kopp, 2017). Only a fraction of the several million tons of fruit pomace generated globally (Gassara et al., 2011; Shalini & Gupta, 2010) is used in a limited number of applications. The remainder is considered an under- utilized agricultural commodity with great potential for the develop- ment of sustainable and value-added products (Banerjee et al., 2017). Furthermore, waste pomace is considered an environmental burden in many jurisdictions that requires management steps and interventions before disposal (Beres et al., 2017; Gassara et al., 2011; Magyar, da Costa Sousa, Jin, Sarks, & Balan, 2016). Therefore, there is a con- siderable interest by consumers, producers, and regulatory agencies alike to find and optimize applications of this natural resource. Theoretically, the chemical composition of fruit pomaces (Fernandes, Ferreira, et al., 2019; Reissner et al., 2019) including blueberries, cranberries, grapes, apples etc.; in addition to their high content in polyphenolics (such as anthocyanins, flavonols, flavones, flavanols, flavanones, proanthocyanidins and isoflavonoids (de Souza, Willems, & Low, 2019; Fernandes, Le Bourvellec, et al., 2019)) make them good targets for incorporation as functional food ingredients (Ajila et al., 2011; Wolfe & Liu, 2003) and/or as effective im- munomodulatory and antimicrobial agents (Islam et al., 2019; Vattem, Lin, Labbe, & Shetty, 2004; Vattem & Shetty, 2002). We recently showed that berry products could promote the intestinal health of https://doi.org/10.1016/j.jff.2020.104053 Received 13 December 2019; Received in revised form 4 June 2020; Accepted 5 June 2020 ⁎ Corresponding author at: 93 Stone Road West, Guelph, Ontario N1G 5C9, Canada. E-mail address: Moussa.Diarra@canada.ca (M.S. Diarra). Journal of Functional Foods 72 (2020) 104053 Available online 23 June 2020 1756-4646/ Crown Copyright © 2020 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). T http://www.sciencedirect.com/science/journal/17564646 https://www.elsevier.com/locate/jff https://doi.org/10.1016/j.jff.2020.104053 https://doi.org/10.1016/j.jff.2020.104053 mailto:Moussa.Diarra@canada.ca https://doi.org/10.1016/j.jff.2020.104053 http://crossmark.crossref.org/dialog/?doi=10.1016/j.jff.2020.104053&domain=pdf broiler chickens by reducing the prevalence of necrotic enteritis due to Clostridium perfringens and coccidiosis due to Eimeria spp. while upre- gulating the expression of the IL4, IL5, CSF2, and HMBS genes involved in adaptive immune responses (Das et al., 2020). While many studies have demonstrated the nutritional benefits of consuming berries-based polyphenolics (including those originating from pomaces) in humans, comparatively few investigations have been directed at animals, pri- marily clinical animal models (rats and mice for example), and a very few at livestock or poultry (Hogan et al., 2010; Rodriguez Lanzi et al., 2018). Consequently, there is a gap in knowledge with regard to phy- siological aspects such as blood metabolite changes and/or gut micro- biota modulation in farm animals, particularly in response to pomace inclusion in feed. In one of the few studies that focused on the nutri- tional effects of polyphenolic-rich pomaces and their influence on farm animals and poultry; Rizal et al. (Rizal, Mahata, Andriani, & Wu, 2010) reported that wastes from carrot, apple, mango, avocado, orange, water melon and tomato mixtures can be included at 20% in diets fed to broiler chickens to effectively replace 40% corn without any negative effects on productivity. Fruit pomaces could have a nutritive value (carbohydrates, proteins, minerals and vitamins etc.) in addition to the potential health-pro- moting benefits derived from polyphenolic compounds. The above as- sumption is supported by the detailed composition-analyses of many pomaces. For example, apple pomace commonly utilized in cattle feed, contains 9.5–22.0% carbohydrates, 4.0% proteins, 6.8% cellulose, 0.42% organic acids and calcium (Shalini & Gupta, 2010). Similarly, a previous research in our laboratory highlighted the composition of cranberry pomace (CBP) with respect to carbohydrates, proteins, lipids, and minerals, and complex phenolic (mainly anthocyanins and flavo- nols) compounds (Ross, Ehret, Godfrey, Fukumoto, & Diarra, 2017). Public interest in organically-produced chicken meat, which re- quires rearing birds under free-range conditions, is on the rise for various reasons including the prohibition of chemical pesticides, anti- biotics, preservatives and/or growth hormones, and general support for sustainable practices that eliminate the use of environment-destabi- lizing chemicals or fertilizers (Gibson & Jackson, 2017; Leslie, 2006; Siegford, Powers, & Grimes-Casey, 2008; Sorensen, Edwards, Noordhuizen, & Gunnarsson, 2006; Thamsborg, 2001). However, pas- ture-raised chickens have a higher exposure to disease-causing patho- gens, which can lead to decreased feed efficiency, reduced average weight-gain and lower bodyweight at slaughter (Gaucher, Quessy, Letellier, Arsenault, & Boulianne, 2015). These issues highlight the need for the identification of innovative, cost-effective interventions to im- prove organic-broiler production systems through treatments that po- sitively influence bird gut heath and improve or maintain product quality and safety (Jacob et al., 2012). Fruit pomaces that contain polyphenols with microbial-modulating properties have been con- sidered for this purpose (Brambillasca, Britos, Deluca, Fraga, & Cajarville, 2013; Fotschki, Juskiewicz, Sojka, Jurgonski, & Zdunczyk, 2015; Kafantaris et al., 2017; Oliveira et al., 2013). Specifically, they could be used to promote a healthy gut microbiota and support desir- able physiological, metabolic, immunological, digestive and nutrient- uptake functionalities in host birds (O'Hara & Shanahan, 2006) through different mechanisms, including the promotion of short chain fatty acids (SCFAs) production within the gut and/or favoring the growth of beneficial bacteria due to the modulating effects of polyphenolic com- pounds (Anhe et al., 2015). Research on the gut microbiome of food-producing animals has started to gain momentum due to its potential implications in food safety and public health as well as animal productivity (Borda-Molina, Seifert, & Camarinha-Silva, 2018; DuPont & DuPont, 2011; Yeoman et al., 2012). Consequently, the objective of this study was to in- vestigate the effects of a feed supplementation with CBP on blood metabolites and cecal microbiota in pasture-raised broiler chickens. 2. Materials and methods 2.1. Pomace preparation and composition analysis The CBP used in this study was prepared as described by Ross et al. (2017). Briefly, frozen organic cranberry (Vaccinium macrocarpon Ait.) purchased from Fruit d’Or (Villeroy, QC, Canada) was thawed before juicing using a hydraulic rack and cloth. The solid fraction was lyo- philized and ground using a cutting mill (SM 2000 Retsch, Haan, Ger- many) before passing through a 2-mm mesh screen (Ross et al., 2017). Pomace fiber content was analysed by fractionation according to the procedure described by Abdel-Aal, Hucl, Patterson, and Gray (2010). Briefly, the precipitate obtained after lipid extraction and protein se- paration was homogenized and treated with mQ-water (1:4 ratio) for 1 min. It was then sieved through a 210 μm screen to determine the percentage of coarse fiber retained after passage of fine solids (starch) and fine fiber (water soluble fibers). Multiple extraction and sieving steps were then carried out to determine the fine fiber percentage, which was defined as the weight of fiber retained on a 52 μm screen (Abdel-Aal et al., 2010). The amino acids profile of CBP was determined by Silliker Canada Co. (Markham, ON, Canada) using a standardized high-pressure liquid chromatography separation procedure. The chemical hydrolysis step was carried out under laboratory-controlled conditions in the absence of oxygen while the separation was achieved with an ion-exchange column coupled with post-column ninhydrin derivatization and detec- tion. The complete phenolic profile of the CBP used in this study has been previously reported (Ross et al., 2017). The analysis revealed the pre- sence of many unique abundant anthocyanins in the organic CBP, in- cluding Peonidin 3-galactoside (PubChem CID: 91810512), Cyanidin 3- galactoside (PubChem CID: 44256700), Cyanidin 3-arabinoside (Pub- Chem CID: 91810602), and Peonidin 3-arabinoside (PubChem CID: 91810651) (Fig. 1A-D). 2.2. Extraction of lipophilic fractions Lipophilic fractions of the dried CBP were extracted in triplicate from 1 to 2 g samples. Samples in 50-mL Nalgene™ Oak Ridge FEP centrifuge tubes (Thermo Scientific; Burlington, ON) were extracted with 3 × 20 mL 95% ethanol at 35 °C with magnetic stirring for 3 min, followed by 10 min shaking on a Heildoph Multi-Reax shaker (Heildoph Instruments GmbH & CO. KG, Schwabach, Germany) at approximately 2000 rpm. The mixture was spun in a centrifuge (Sorval Lynx 4000; Thermo Fisher, Mississauga, ON) at 2800g for 15 min and the super- natant was collected in round bottom flasks. The combined super- natants were then rotor-evaporated to obtain the weight of total lipids in each sample. The lipid residue was re-dissolved in 3 mL chloroform and stored at −20 °C. 2.3. Fatty acids analysis by gas chromatography The fatty acid composition of the lipophilic fractions extracted from CBP as described above was conducted according to the method de- scribed by Kramer et al. (1997). Fatty acids methyl esters (FAMEs) were prepared using base-catalyzed reactions. Briefly, a volume containing 5–10 mg of lipids was placed in a 15-ml glass test-tube equipped with Teflon-lined screw cap and dried under a nitrogen stream at room temperature before dissolution with 80 µl toluene. One millilitre of NOCH3/methanol (0.5 N) was then added to initiate the methylation reaction before heating for 30 min. at 50 °C. After cooling to room temperature, 1 mL water was added to the solution and the resulting esters were extracted with 3 mL of hexane. FAMEs were analyzed using gas chromatography system (Model 6890N; Agilent Technologies, Palo Alto, CA) equipped with a CP-Sil 88 WCOT fused silica column (100 × 0.25 mm i.d. × 0.2 um film thickness; Chrompack, Middleburg, Md. R. Islam, et al. Journal of Functional Foods 72 (2020) 104053 2 Netherlands). The column was operated at 45 °C for 4 min, heated to 175 °C at a rate of 13 °C/min, held for 27 min, heated to 215 °C at a rate of 4 °C/min and held at that temperature for 35 min. The entire run time was 86 min with sample injection volume set at 1 µl. The injection mode was split-less and a flame ionisation detector (FID) was used for detection and quantification. 2.4. Pomace feeding regimen and bird management Six hundred 1-day old Cobb 500 broiler chicks were purchased from a local commercial hatchery (Okanagan Valley, BC, Canada) and housed in a commercial farm (Rosebank Farms, Armstrong, BC) from August 17th to slaughter on October 19th 2016 (Fig. 2). The birds were divided into three groups (2 pens/group): A group of 200 birds re- ceiving all vegetarian basal feed supplemented with 1.0% of CBP; a group of 200 birds receiving the basal feed containing 2.0% of CBP, and a group of 200 birds receiving the basal feed (no pomace supplement), which served as a control. Levels of CBP supplementation were based on previous experience and the need to balance the nutritional re- quirements of the growing birds with economical feasibility of pomace usage. The feed was prepared and mixed by Rosebank Feed mill. Starter and grower diets (Table 1) were formulated in compliance with organic broiler diet guidelines implemented by the farm to meet the Cobb broilers nutritional requirements (Islam et al., 2019). Both diets were formulated with wheat, barley, and organic peas as principal cereals, in addition to soy. Wetaskiwin-chick-macro mix (Wetaskiwin, AB, Ca- nada), distiller’s wheat, limestone, Biolys-lysine and methionine (Evonik Degussa Canada Inc., Burlington, ON) were all added to meet the Cobb broilers nutritional requirements. Analyses of dry matter (DM), total proteins, soluble-carbohydrates, fatty acids and minerals were all performed at Cumberland Valley Analytical Services Inc. (Laboratory Services for Agriculture, BC,) using Fig. 1. Structures of anthocyanins found in the organic cranberry pomace (CBP), including (A) Peonidin 3-galactoside (PubChem CID: 91810512), (B) Cyanidin 3- galactoside (PubChem CID: 44256700), (C) Cyanidin 3-arabinoside (PubChem CID:91810602), and (D) Peonidin 3-arabinoside (PubChem CID: 91810651) as re- ported in our earlier study (Ross et al., 2017). Fig. 2. Timelines for on-farm trials with pasture-raised broiler chickens: organic cranberry pomace (CBP)-feeding period, blood and ceca sampling times are in- dicated. Md. R. Islam, et al. Journal of Functional Foods 72 (2020) 104053 3 their standardized and routine laboratory analyses techniques. Water and feed were accessible ad libitum throughout the entire experiment. Pomace-supplementation treatments were applied at 8 days of age (August 24th) for four consecutive weeks, or until the birds reached 36 days of age (September 21st). The trial was continued until the birds reached 64 days of age (October 19th) during which all birds were fed a grower diet without a CBP supplementation. Cranberry pomace supplemented diet was provided during four consecutive weeks rather than the entire rearing period due to cost/labor concerns. In addition, the goal of this investigation was to track short-term effects while maximizing the beneficial effects related to gut microbiota modulation. Birds were reared using the “slow-grown broilers” style to accommodate their physiology to meet an established final targeted dress-carcass weight. From day 1 to 21, birds from all groups (treat- ments and control) were held in separate pens in the same brooder house (with the same rearing conditions). The birds were then moved to a free-range outdoor-pasture and treatment-groups were contained in their own separate rectangular areas of 160 sq. ft. each (1.6 sq. ft./bird) on the same farm until they reached 64 days of age. Each group was provided with shelter and a minimum pasture range area (protected by electrified poultry netting) consisting of a dryland forage mix under- seeded with Dutch white clover at 10%. All experimental procedures were conducted in conformity with general practices for commercial free-range broiler chicken production, using guidelines established by the Canadian Council on Animal Care (http://www.ccac.ca/en/ standards/guidelines). The protocol was approved by Rosebank Farms, a member of the North Okanagan Organic Association, which uses an exclusive “customer certified” system for accreditation (with two to three inspections by customers that take place throughout the rearing period and the night before slaughter). 2.5. Performance and mortality General health, welfare, and mortality rates were monitored three times a day (morning, afternoon, and evening). Feed consumption of each bird group was monitored daily. Feed conversion was obtained as feed intake (kg) divided by the dressed carcass weight (kg) at slaughter time (day 64 of age). Mortality was calculated weekly by dividing the number of dead birds by the number of birds placed in the pen at the start of the week multiplied by 100. 2.6. Blood serum metabolites Blood samples were collected from two birds of each replicate group (4 birds/treatment/2 pens). The birds were euthanized ethically through cervical dislocation. Sampling was carried out before applying the pomace treatment on day 7 (August 23rd) and thereafter at 14 (August 30th), 21 (September 6th), 28 (September 13th), 35 (September 20th), 42 (September 27th), and 64 (October 19th) days of age. The two blood samples were then pooled and allowed to clot at room temperature before centrifugation at 2000 x g for 10 min for serum collection. The collected sera were then transferred to sterile Eppendorf tubes and stored at −80° C. Serum mineral concentrations including calcium (Ca), iron (Fe), magnesium (Mg) and phosphorus (P) in addition to multiple lipophilic components such as cholesterol (CHO), high-density lipoproteins cholesterol (HDLC), triglycerides (TG), and non-esterified fatty acids (NEFA) were analysed by the Animal Health Laboratory (University of Guelph, Guelph, ON, Canada) using standard accredited protocols. 2.7. Cecal bacterial community structure and diversity Ceca and their contents were aseptically removed from the birds at 7, 14, 21, 28, 35, 42, and 64 days of age to determine bacterial com- munity composition and diversity. The ceca were frozen at −20 °C before extraction of genomic DNA using the PowerSoil® DNA Isolation Kit. DNA purity and yields were measured using PicoGreen (Thermo Fisher Scientific). Sequencing libraries of the 16S V3-4 region were prepared and purified amplicons were pooled in equimolar ratios for sequencing on a MiSeq instrument using the MiSeq 600-cycle v3 kit Table 1 The composition of feed with organic cranberry pomace (CBP) used in this feeding trial. Data are expressed as a percentage of dry matter (% DM) unless otherwise mentioned. Percent (%) inclusion in diet Ingredient/nutrient profile Starter (Day 0–14) Grower (Day 14–64) Control 1% CBP 2% CBP Control 1% CBP 2% CBP Ingredient Wheat 39.15 39.15 39.15 56.11 56.11 56.11 Organic peas 32.81 32.81 32.81 21.64 21.64 21.64 Barley and peas 10.67 10.67 10.67 14.07 14.07 14.07 Vitamin premix blend (Macro)a 17.37 17.37 17.37 8.18 8.18 8.18 Analysed nutrientb Dry matter (DM) 87.80 87.70 87.90 87.90 88.00 88.30 Moisture 12.20 12.30 12.10 12.10 12.00 11.70 Protein Crude protein 21.20 22.10 21.10 18.20 18.30 17.80 Adjusted protein 21.20 22.10 21.10 18.20 18.30 17.80 Soluble protein 7.10 8.40 7.60 5.40 5.40 5.10 Fiber Acid detergent fiber (ADF) 5.40 5.20 6.70 5.80 5.50 6.60 Neutral detergent fiber (NDF) 11.10 11.00 11.90 12.30 12.10 12.20 NDF/ADF 2.05 2.11 1.77 2.12 2.20 1.85 Carbohydrate Ethanol soluble fraction 3.80 4.10 3.70 1.60 2.40 3.10 Starch 46.60 44.70 44.80 56.10 55.20 54.60 Crude fat 1.91 2.30 2.28 1.81 1.58 1.33 Mineral Ash 6.29 7.16 6.87 4.27 5.45 4.23 Calcium 1.27 1.54 1.28 0.50 0.64 0.53 Phosphorus 0.76 0.81 0.76 0.63 0.68 0.62 Magnesium 0.25 0.25 0.25 0.20 0.20 0.19 Potassium 0.99 0.89 0.87 0.61 0.59 0.59 Sodium 0.19 0.24 0.20 0.11 0.13 0.10 Iron (ppm) 295.00 308.00 267.00 180.00 200.00 180.00 Manganese (ppm) 100.00 104.00 89.00 67.00 74.00 67.00 Zinc (ppm) 104.00 119.00 108.00 77.00 82.00 75.00 Copper (ppm) 23.00 26.00 25.00 12.00 17.00 13.00 Calculated energy and index Total digestible nutrient - TDN 77.80 77.60 77.50 79.10 77.00 82.30 Net energy lactation (mcal/ lb) 0.81 0.81 0.81 0.83 0.81 0.86 Net energy maintenance (mcal/lb) 0.85 0.85 0.85 0.87 0.85 0.91 Net energy gain (mcal/lb) 0.56 0.56 0.56 0.58 0.56 0.61 Non-fiber carbohydrates 59.50 57.00 57.80 63.40 62.50 64.40 Non-structural carbohydrates 50.40 48.80 48.50 57.70 57.60 57.7 Organic cranberry pomace (CBP) was added to the basal diet at a rate of 1% and 2%. a Vitamin premix blend for starter and grower diets contain 11.47% Soy, 4.0% Wetaskiwin-chick-macro, 0.9% Biolys, 0.85% Limestone, 0.15% Methionine, and 4.0% Wetaskiwin-chick-macro, 3.84% Distillers wheat, 0.34% Lysine, respectively. b The nutrient contents were analyzed on a dry matter (DM) basis. Md. R. Islam, et al. Journal of Functional Foods 72 (2020) 104053 4 http://www.ccac.ca/en/standards/guidelines http://www.ccac.ca/en/standards/guidelines (Illumina). Sequence processing and data analyses were performed as described in our previous study (Islam et al., 2019). The obtained fastq files were analyzed using Quantitative Insights Into Microbial Ecology (QIIME, version 1.9.1) (Caporaso et al., 2010). Paired-end reads were joined with fastq-join (Aronesty, 2011, 2013) and later quality-filtered and de-multiplexed in QIIME using default settings. The reads were clustered at 97% sequencing identity using UCLUST (Edgar, 2010) and the operational taxonomy units (OTUs) were picked against the Greengenes database (gg_otus_13_8) using an open-reference approach (DeSantis, Hugenholtz, Larsen, et al., 2006; DeSantis, Hugenholtz, Keller, et al., 2006). The taxonomic assignment of sequences was performed using the UCLUST consensus taxonomy assigner. Taxa that could not be assigned at the genus level were pre- sented as ‘unclassified’ using the highest taxonomic level that could be assigned to them (family/order/class/phylum). The sequences were aligned against the Greengenes core set with PyNast (Caporaso et al., 2009) while phylogenetic trees were constructed with FastTree (Price, Dehal, & Arkin, 2009). Alpha-diversity parameters were calculated by QIIME while the beta-diversity distance matrix was calculated based on the unweighted UniFrac metric (Lozupone & Knight, 2005) which was then used for principal co-ordinate analysis (PCoA). Heatmaps were generated according to procedures described by Babicki et al. (2016). 2.8. Statistical analyses Pomace and fatty acid composition were calculated as means ± standard deviations. Changes in cecal microbiota were tracked and visualized using either Heatmapper (Babicki et al., 2016) or as per- centages. Performance and mortality data were analyzed according to a randomized block design using the General Linear Model (GLM) pro- cedure of the Statistical Analysis System (SAS, version 9.4, SAS Institute Inc., 2016, Cary, North Carolina, United State) with treatment groups as sources of variation and the individual pens as experimental units. Data from blood parameters were analyzed by repeated measurements op- tion using treatment groups and sampling times (bird ages = source of variations) as factors. Means were estimated and the least significance difference was used to separate treatments means whenever the F value was significant. The calculated means and standard error of the means (SEM) with their respective probability values (P value) appear in the Tables. The difference between groups was considered significant at a P value of ≤ 0.05. 3. Results 3.1. Composition analysis of cranberry pomace The CBP contained on average a 46.3% and 15.5% of coarse and fine fiber, respectively (Table 2). These values were used to establish fiber levels in the starter/grower feed formulations, or a 5.2–6.7% range for acid-soluble fiber and a 11–12.3% range for water-soluble fiber (Table 1). Acid detergent fiber (ADF) is a soluble fraction that can be digested after microbial fermentation in the digestive tract of ani- mals, while neutral detergent fiber (NDF) is a coarse insoluble and/or non-digestible fibre that cannot be fermented. The incorporation of CBP in feed led to NDF/ADF ratios of 2.05 (basal), 2.11 (1% CBP), and 1.77 (2% CBP) for starter diets and 2.12 (basal), 2.20 (1% CBP) and 1.85 (2% CBP) for grower diets, respectively (Table 1). Chemical analysis of pomace indicated a moisture content of 68.37% and an ash content of 1.05% (dry matter basis), while the protein content was at 5.76% (dry matter basis) (Table 2). Hydrolysis of the proteinaceous fraction of the pomace and its chromatographic analysis showed the presence of many amino acids including histidine (0.21%), isoleucine (0.20%), leucine (0.37%), lysine (0.28%), methio- nine (0.08%), phenylalanine (0.22%), threonine (0.16%), and valine (0.24%) (Fig. 3). The most abundant amino acids (w/w) were: glutamic acid (> 0.80%), aspartic acid (> 0.50%), and arginine/leucine, each present at about 0.40% (w/w). These results suggest that the organic CBP used in this work was a good source of proteins and amino acids, notably essential amino acids needed for muscle growth in chicks at the early stages of life. Moreover, lipid and fatty acid analysis of CBP indicated a mix of mono-unsaturated (14.96%), poly-unsaturated (61.18%), and saturated (20.36%) fractions; with the most abundant fatty acids being palmitic 16:0 (10.35%), oleic acid 9c-18:1 (11.84%), linoleic acid 18:2n-6 (39.78%), α-linolenic acid 18:3n-3 (21.04%), and lignoceric acid 24:0 (3.61%) as shown in Table 3. The incorporation of CBP into the broiler feed formulations in- creased the levels of linolenic and lignoceric acids in comparison to the basal diet (Table 4). While higher linolenic acid levels are considered advantageous in poultry nutrition, supplementation also induced a second favourable change in regard to the dietary ratio of linoleic (unchanged) to linolenic acids (enhanced as mentioned earlier due to pomace incorporation). Hence the reduced ratio of linoleic to linolenic acids in feed is another empirical advantage of CBP usage noted in this study. 3.2. Performance and mortality The measured average carcass-weight of birds at processing (64 days) was 2.24 ± 0.05, 2.30 ± 0.01, and 2.07 ± 0.00 kg for control, 1%, and 2% CBP-fed birds, respectively. Cumulative mortality rates were lower in CBP-fed birds compared to control birds (con- trol = 10.79 ± 2.30, 1% CBP = 8.22 ± 1.56, and 2% CBP = 5.58 ± 2.18). Likewise, the feed conversion ratios in the treatment birds (3.33–3.64) were slightly better than that in the control group (4.03). 3.3. Blood metabolites In general, data showed that feed supplementation with CBP did not significantly impact blood serum calcium, magnesium, and phosphorus levels (data not shown). On the other hand, the addition of CBP at 1 and 2% significantly (P = 0.05) increased blood iron levels at day 14 to 21.67 (1%) and 16.50 µmol/L (2%), respectively (Table 5). A similar tendency was also evident for 2% CBP-treated birds at day 35. Despite Table 2 The chemical composition of the organic cranberry pomace (CBP) used in this study (adapted and expanded from Ross et al., 2017). Parameter Mean ± SD* Yielda (%) 14.47 ± 1.01 Moisture content (%) 68.37 ± 1.56 Ashb (%) 1.05 ± 0.59 Protein contentb (%) 5.76 ± 0.23 Total lipidsb (%) 4.41 ± 0.38 pH 2.74 ± 0.023 Titratable acidity (TA, %) as citric acid 0.651 ± 0.043 Titratable acidity (TA, %) as malic acid 0.623 ± 0.042 Soluble solids (SS, °Brix) 2.53 ± 0.06 Total carbohydrates and fiberc (%) 88.78 Antioxidant activity 144 µmol Trolox eq/g Total phenolics 24.87 mg gallic acid eq./g Tartaric esters 2.77 mg caffeic acid eq./g Flavonols 3.08 mg quercetin eq./g Anthocyanins 4.46 mg cyaniding-3-glucoside eq./g Starchb 17.84 ± 2.429 Total fiber contentb (%) 61.823 ± 0.348 Coarse fiber 46.305 ± 1.393 Fine fiber 15.517 ± 1.045 a Calculated as: wet pomace mass/fresh berry weight × 100%. b Dry matter basis. c Calculated by difference 100% − [Moisture content (%) + Total lipids (%) + Protein content (%) + Ash (%)] and reported on dry matter basis. * Means are presented with their respective standard deviation (SD). Md. R. Islam, et al. Journal of Functional Foods 72 (2020) 104053 5 week to week variations, the incorporation of both pomace levels (1 and 2%) led to significant (P < 0.01) increases in blood iron con- centrations near the end of the supplementation period (42 days of age), with concentrations estimated at 17.75 (1%) and 21.50 µmol/L (2%), respectively, which was higher than concentrations measured in the control group (Table 5). Hence, feed supplementation with CBP enhanced blood serum iron levels in a concentration-dependant fashion in pasture-raised broiler chickens. The influence of CBP on blood lipids was also evident from the data. A slight decrease was observed in CHO levels at the end of the CBP feeding phase (days 28–35) with levels spanning the 2.49–3.07 mmol/L range, compared with 2.78–3.59 mmol/L in the control group. This decrease was statistically significant (P < 0.01) at day 35 for pomace treatments for both supplementation levels (1 and 2%) (Table 6). Feed supplementation with pomace significantly decreased (P < 0.01) blood TG levels after the first two weeks of feeding (21 days of age), with levels ranging between 0.45 and 0.50 mmol/L in treated birds compared to 0.65 mmol/L in control birds. From days 42 to 64, TG levels in the pomace-treated birds increased gradually to reach le- vels measured in the control group as the pomace treatment was phasing out (Table 6). The opposite was true for HDLC where a sig- nificant decrease was observed at day 35 in both 1 and 2% treatments (2.29–2.51 mmol/L, 3.08 mmol/L in controls). This decrease was gra- dually replaced by increasing HDLC levels at the end of the experiment at day 64 (Table 6). 3.4. Cecal bacterial communities Interestingly, the longest lasting effect of CBP feed supplementation was observed on cecal microbiota. The 16S rRNA gene sequencing re- sults revealed that cecal microbiota composition was highly diverse, consisting of 15 known phyla (Fig. 4) with 93 different bacterial genera detected in control and treated birds throughout the entire screening period (7–64 days). The only phylum found associated with the Archaea kingdom was Euryarchaeota (less than 0.05%) which increased in re- lative abundance as birds aged from day 7 to 64. However, the increase rate was lower in birds fed CBP compared to the control birds. The predominant bacterial phyla across all sampling time-points/groups were Firmicutes, Bacteroidetes, and Proteobacteria, representing > 75% of all sequences. An increased abundance of Firmicutes, Tenericutes, Actinobacteria, and Proteobacteria associated with a visible decrease in Bacteroidetes, Lentisphaerae, and Spirochaetes phyla was apparent in CBP fed birds throughout the trial (Fig. 4). Likewise, the Fusobacteria phylum was more evident in the ceca of birds fed the control diet (especially at days 21 and 64) than in CBP-fed birds. Apparent dynamic shifts in microbial genera were primarily associated with bird age, and often with feed type. In order to focus on genuine changes introduced by the pomace treatment, we set stringent inferencing criteria that encompassed changes to all microbial taxa in the same direction (in- crease or decrease in abundance) for at least two consecutive or non- consecutive weeks during the entire trial. Furthermore, in order to address natural bird to bird variations and effects of confounding fac- tors, data from groups that exhibited standard deviation values larger than the reported means were not considered. Forty-five different bacterial genera were found at varying percen- tages in the ceca of chicks before CBP feeding. Some were barely de- tectable (< 0.001%), including Odoribacter, Synechococcus, Mucispirillum, Streptococcus, Turicibacter, Megamonas, Sutterella, unclassified_Cyanobacteria, unclassified_Actinomycetales, and unclassified_Bacteroidales. Other genera including Lactobacillus (40.5%) and unclassified_ Clostridiales (17.6%) were more abundant although bird-to-bird variation was evident, while Faecalibacterium, Oscillospira, Ruminococcus, unclassified_Lachnospiraceae, and unclassified_Ruminococcaceae were more homogenously distributed within the first week of rearing (Fig. 5). Using the screening criteria outlined above, we identified 14 out of 93 bacterial genera that were consistently correlated with pomace feed supplementation. In birds fed 1% CBP, the Bifidobacteria group increased steadily from day 14 of age (one week of feeding) until day 42 (Fig. 6). A similar trend was seen in 2% CBP-treated birds during the supple- mentation period, although to a lesser extent. The highest difference in Bifidobacteria population size was observed at the end of the pomace feeding period (days 35–42) in 1% CBP-fed birds. As expected, bacterial genera involved in plant cell-wall, fiber and Fig. 3. Amino acid profiles of organic cranberry pomace (CBP) reflecting the chemical composition of its proteinaceous fraction. The abundance of indispensable amino acids in addition to arginine, aspartic-, and glutamic acids is shown. Md. R. Islam, et al. Journal of Functional Foods 72 (2020) 104053 6 complex-starch breakdown were all more common in pomace treated birds, notably Ruminococcus and Oscillospira. Oscillospira immediately after CBP feeding accounted for 6.4 to 8.5% of genera detected in the supplemented groups, compared to 2.5% in the control (Fig. 6). This increase was maintained throughout the raising period (but at lower varying percentages) until the end of experimentation (64 days). Ru- minococcus population were similarly more common in the ceca of birds fed CBP-supplemented diet (particularly 1% CBP group), but to a lesser magnitude (0.11–3.1%) than the Oscillospira group. Genera that ferment simple sugars and plant polysaccharides into alcohols and SCFAs such as butyrate and acetate were also affected by CBP inclusion in the diet. The unclassified_Lachnospiraceae genus in- creased to 6.3% during the first week (day 14) in CBP-fed birds com- pared to 0.82% in the control birds (Fig. 6), while the percentage of unclassified Cyanobacteria displayed a similar increase at the end of supplementation, but less consistently (Fig. 6). Interestingly, several bacterial genera (including unclassified_Rikenellaceae, un- classified_Erysipelotrichaceae, and unclassified_Synergistaceae) and De- sulfovibrio showed an age-dependant presence coupled with pomace- dependent modulation. These genera were present at a very low per- centage in the early stages but began to appear in the ceca of both control and CBP-treated birds alike after the second week of rearing however, CBP feeding significantly influenced their abundance. For example, Desulfovibrio, unclassified_Rikenellaceae, and un- classified_Synergistaceae appeared on the second week of pomace feeding while the appearance of unclassified_Erysipelotrichaceae was delayed until the third week of CBP feeding. However, CBP supple- mentation induced different outcomes in these bacteria by increasing the abundance of unclassified_Rikenellaceae by up to 12% while de- creasing that of Desulfovibrio, unclassified_Erysipelotrichaceae, and un- classified_Synergistaceae (Fig. 6). Similarly, Faecalibacterium, Helico- bacter, and Sutterella displayed an overall increase (more evident with the 1% CBP treatment in some cases) while the Bacteroides demon- strated a decrease (more evident in the 1% CBP group) under the same experimental conditions. In contrast, the percentage of Lactobacilluswas unaffected by CBP (Fig. 6). The composition of the gut microbiota was also characterized by comparing the alpha-diversity parameters including phylogenetic di- versity (PD) whole tree and Shannon indices in the CBP-fed and control groups. None of the alpha diversity parameters was affected by diet, although they differed considerably from one age group to another. The unweighted UniFrac PCoA showed a clear clustering of the cecal sam- ples from control and CBP-treated bird groups at days 28 and 35 (Fig. 7). Hence, these results indicate that CBP influenced the compo- sition of cecal bacterial communities in pastured broiler chickens. Two genera (Methanocorpusculum and vadinCA11) were mapped to the Ar- chaeal phylum (Euryarchaeota), with Methanocorpusculum being sig- nificantly more abundant in the control than in the CBP-fed birds from days 27 to 42 (data not shown). 4. Discussion Free-range broiler chickens were fed with 1 and 2% CBP for four consecutive weeks. Due to its high phenolic content, CBP is believed to exert a positive influence on many blood biochemical markers in dif- ferent hosts including poultry (Aditya, Ohh, Ahammed, & Lohakare, 2018; Bajerska et al., 2018; Iannaccone et al., 2019). Several earlier studies have attributed enhanced growth performance, decreasing lipid oxidation and cholesterol levels and increasing beneficial fatty acid contents in broiler chickens to phenolic compounds including tannic and gallic acids; the latter has also been reported to significantly in- crease n-3 long-chain polyunsaturated fatty acids (Starcevic et al., 2015). The organic CBP used in this study was reported to contain ~25 mg gallic acid equivalents/g (Ross et al., 2017). Blood-serum lipids (CHO, HDLC, TG, and NEFA), and minerals were measured in the present study. Our interest in minerals was in con- sequence to the known ability of many polyphenols (such as quercetin and kaempferol) to chelate ions (Hider, Liu, & Khodr, 2001) and to reduce their bioavailability in living organisms, notably bacteria. Ear- lier studies have demonstrated the capacity of polyphenolic-rich fruit pomaces to increase mineral excretion in animal feces leading to sig- nificantly higher ash-weights (Martin-Carron, Garcia-Alonso, Goñi, & Saura-Calixto, 1997). Due to these concerns, we examined the effect of CBP-feed supplementation on the absorption of minerals by measuring blood concentrations of iron, calcium, magnesium, and phosphorous. Our data clearly demonstrated the nutritive value of CBP and its suit- ability for animal feed usage in light of its content in fibers, amino- and fatty-acids. Feed fibers play an important role in the development of broiler chickens where the coarse fiber fractions (more specifically) influence feed intake. The ratios of course/fine fiber (Mateos, Jiménez-Moreno, Serrano, & Lázaro, 2012) present in CBP led to desirable outcomes with regard to feed intake, bodyweight gain, and other production para- meters tested. A high percentage of coarse fiber could also contribute to the healthy development of the chick digestive tract (Jimenez-Moreno, Gonzalez-Alvarado, Gonzalez-Sanchez, Lazaro, & Mateos, 2010) by stimulating gizzard activity and increasing its contents. Furthermore, the fermentable digestible fraction of the CBP fiber could help modify Table 3 Fatty acid composition of the organic cranberry pomace (CBP) used in this study. Fatty acids Mean ± SD (relative %) 12:0 0.354 ± 0.0176 14:0 0.386 ± 0.0231 15:0 0.0953 ± 0.0000119 16:0 10.349 ± 0.690 9c-16:1 0.100 ± 0.00748 17:0 0.199 ± 0.0147 18:0 2.252 ± 0.0568 4t-18:1? 0.0360 ± 0.000180 5t-18:1? 0.0515 ± 0.00192 6–8t-18:1 0.242 ± 0.0126 9t-18:1 0.169 ± 0.0215 10t-18:1 0.181 ± 0.0220 11t-18:1 0.210 ± 0.0279 12t-18:1 0.201 ± 0.0494 6c-18:1 0.295 ± 0.0187 9c-18:1 11.844 ± 1.047 11c-18:1 0.940 ± 0.0612 12c-18:1 0.126 ± 0.00828 13c-18:1? 0.0428 ± 0.00684 14c-18:1? 0.152 ± 0.0261 15c-18:1? 0.0814 ± 0.00268 18:2-tt 0.0248 ± 0.00988 18:2n-6 39.780 ± 0.529 20:0 1.079 ± 0.0538 20:1(5) 0.0882 ± 0.00528 20:1(8) 0.0333 ± 0.0123 20:1(11) 0.403 ± 0.0790 18:3n-3 21.043 ± 3.254 21:0 0.109 ± 0.00143 20:2(11,14)n-6 0.208 ± 0.0224 22:0 1.217 ± 0.225 22:1n-9 0.0319 ± 0.00918 20:3n-9 0.0268 ± 0.000123 23:0 0.497 ± 0.0397 24:0 3.612 ± 1.079 22:4n-6 0.0932 ± 0.0115 26:0 0.206 ± 0.0688 Total Saturated 20.356 ± 0.861 Mono-unsaturated 14.953 ± 0.861 Poly-unsaturated 61.176 ± 2.681 n-3 21.043 ± 3.254 n-6 40.081 ± 0.563 Dietary ratio of n6:n3 1.930 ± 0.325 Md. R. Islam, et al. Journal of Functional Foods 72 (2020) 104053 7 the endogenous cecal microbiota (discussed below) by promoting the growth of beneficial bacterial genera such as Ruminococcus and Oscil- lospira. Results of the present study are in agreement with previous studies that showed the ability of bilberry pomace [with similar fiber content (Aura et al., 2015)] to increase the relative abundance of Os- cillospira and Ruminococcus in rabbit ceca (Dabbou et al., 2019). The observed increase of Bifidobacteria is also aligned with previous reports that demonstrated the ability of phenolic compounds-rich grape or wild blueberry pomace to significantly increase Bifidobacterium counts in young rats (Chacar et al., 2018) and broiler chickens (Islam et al., 2019). Monounsaturated fatty acids originating from fruit pomace li- pids (including cranberry) are becoming the focus of research due to their potential role in the structure and physiology of eukaryotic cells in addition to their functional importance in host metabolism. Linolenic acid is an essential fatty acid known to play an important nutritional role through its capacity to prevent encephalomalacia, a brain disease which induces high mortality rates in 12 days-old chickens (Rajaram, 2014). Furthermore, alpha-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) cannot be biosynthesised de novo by vertebrates and must be obtained from dietary sources. The presence of these two fatty acids in appreciable amounts (21.0 and 39.7% of total fatty acids, respectively) in CBP is nutritionally desirable and the possible accumulation of such essential acids within broiler tissues could prove advantageous for both bird health and meat quality (Kalakuntla et al., 2017; Stark, Reifen, & Crawford, 2016). Furthermore, the proportional dietary ratio of linoleic to linolenic acid was reduced by pomace addition. Such a reduction has been shown to enhance immunological response towards infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV) vaccines without any negative effects on bird performance (Puthpongsiriporn & Scheideler, 2005). Examination of the cecal microbiota in one-week old chickens re- vealed significant variation in bacterial taxa. Animal to animal varia- tion in the composition of the gut microbiota is expected and well Table 4 Fatty acid profiles of the starter and grower diets (relative basis) with or without organic cranberry pomace (CBP) used in the on-farm chicken trial. Fatty acid Starter: Relative basis (%) Grower: Relative basis (%) Marker Control 1% CBP 2% CBP Control 1% CBP 2% CBP Lauric C12:0 0 0 0 0.06 0 0 Myristic C14:0 0.25 0.23 0.24 0.41 0.28 0.27 Pentadecanoic C15:0 0.09 0.09 0.08 0.11 0.1 0.1 Palmitic C16:0 14.97 14.74 14.72 16.17 15.43 15.18 Palmitoleic C16:1 0.19 0.1 0.11 0.15 0.14 0.13 Heptadecanioc C17:0 0.12 0.13 0.12 0.13 0.1 0.1 Stearic C18:0 2.69 2.62 2.66 1.82 1.67 1.69 Oleic C18:1w9 20.05 20.14 20.49 20.16 20.21 20.19 Oleic C18:1w7 1.14 1.13 1.12 1.01 0.98 0.98 Linoleic C18:2w6 50.86 50.57 49.92 50.79 51.02 50.38 Linolenic C18:3w3 7.11 7.69 7.87 4.77 5.54 6.23 Arachidic C20:0 0.27 0.29 0.33 0.28 0.28 0.32 Eicosanoic C20:1w9 0.56 0.56 0.56 1.42 1.44 1.44 Eicosadienoic C20:2w6 0.06 0.05 0.00 0.1 0.1 0.09 Behenic C22:0 0.25 0.26 0.29 0.25 0.28 0.25 Erucic C22:1w9 0.23 0.21 0.20 1.32 1.24 1.31 Lignoceric C24:0 0.23 0.3 0.31 0.29 0.34 0.47 Nervonic C24:1 0 0 0.00 0.18 0.22 0.19 N/A Other 0.93 0.89 0.98 0.58 0.63 0.68 Total 100 100 100 100 100 100 Table 5 Blood-serum iron concentrations (µmol/L) in pasture-raised broiler chickens fed organic cranberry pomace (CBP)** Treatments and concentrations (µmol/L) Time Control 1% CBP 2% CBP SEM P value Day 7 14.67 14.67 16.00 0.86 0.49 Day 14 11.50 21.67 16.50 2.37 0.05* Day 21 22.50 14.00 17.25 1.76 0.02* Day 28 21.00 15.50 18.50 1.68 0.12 Day 35 20.00 17.75 22.00 0.95 0.04* Day 42 17.25 17.75 21.50 0.54 0.00* Day 64 19.25 19.50 17.25 0.89 0.20 ** Results are expressed as Mean ± SEM (Standard error of the mean). Asterisks indicate days at which iron concentrations were statistically different (P < 0.05). Table 6 Changes in blood cholesterol (CHO), high-density lipoprotein cholesterol (HDLC), and triglyceride (TG) levels in pasture-raised broiler chickens fed or- ganic cranberry pomace (CBP). Treatments Metabolite/Time Control 1% CBP 2% CBP SEM P value CHO (mmol/L): Day 7 3.47 3.46 3.43 0.18 0.98 Day 14 2.16 3.01 2.46 0.11 0.00* Day 21 3.29 2.67 2.42 0.25 0.09 Day 28 2.78 2.74 2.49 0.17 0.46 Day 35 3.59 2.75 3.07 0.09 0.00* Day 42 2.72 2.63 3.07 0.19 0.27 Day 64 3.09 3.57 3.27 0.14 0.10 HDLC (mmol/L): Day 7 2.55 2.56 2.55 0.18 1.00 Day 14 1.73 2.25 1.72 0.08 0.00* Day 21 2.41 2.12 1.97 0.12 0.09 Day 28 2.22 2.06 1.92 0.13 0.34 Day 35 3.08 2.29 2.51 0.08 0.00* Day 42 1.94 1.89 2.28 0.16 0.21 Day 64 2.39 2.62 2.68 0.13 0.28 TG (mmol/L): Day 7 1.70 1.73 1.67 0.27 0.99 Day 14 0.65 1.37 1.13 0.15 0.03* Day 21 0.65 0.45 0.50 0.02 0.00* Day 28 0.63 0.70 0.70 0.05 0.56 Day 35 0.48 0.78 0.63 0.06 0.02* Day 42 1.15 1.15 1.20 0.14 0.96 Day 64 0.48 0.68 0.48 0.03 0.00* * Results are expressed as Mean ± SEM (Standard error of the mean). Asterisks indicate days at which the studied biochemical markers’ were statis- tically different (P < 0.05). Md. R. Islam, et al. Journal of Functional Foods 72 (2020) 104053 8 documented (Alegre, 2019; Nguyen, Vieira-Silva, Liston, & Raes, 2015). Multiple causes of such variability in breeding farms or hatcheries have been identified in the past (Alegre, 2019; Nguyen et al., 2015). Despite such variability, feed supplementation could clearly modulate specific bacterial taxa. The importance of this effect needs to be understood for each bacterial taxon separately and its significance needs to be realized within the context of animal productivity and human health alike. For example, the increased abundance of Ruminococcus in response to CBP supplementation highlighted the role of this genus in plant-cell wall breakdown within the colon environment, as previously reported (Flint, 2004). Decreased abundance of this Ruminococcus genus has been ob- served in humans affected by the inflammatory bowel syndrome (Nagao-Kitamoto & Kamada, 2017) and Parkinson’s disease (Hill-Burns et al., 2017; Wood, 2015) suggesting that cranberry products may find value in the modulation of intestinal microbiota in such individuals. In the present study, the abundance of the Desulfovibrio genus (con- trol = 0.39, 1% CBP = 0. 27 and 2% CBP = 0.14%) was lower in CBP- treatment groups. Previous studies have revealed similar outcomes as- sociated with high inulin (Holscher et al., 2015) or galacto-oligo- saccharide (Vulevic, Juric, Tzortzis, & Gibson, 2013) intakes. The role of the sulfate reducing bacterium Desulfovibrio in gut health is still controversial (Karlsson et al., 2012; Rowan et al., 2010; Sawin et al., 2015). While many earlier studies have associated Bacteroides with the consumption of predominantly protein- and fat-based diets (Wu et al., 2011) or fruit pomace consumption (Islam et al., 2019), the present study showed a measurable decrease in this bacterial taxon due to CBP feeding in chickens. The role of Bacteroides in human or animal health is controversial. Some authors have reported differences in prevalence associated with lean or obese phenotypes, while others have raised Fig. 4. Taxonomic profiles of the bacterial communities (at the phylum level) in cecal samples of pasture-raised broiler chickens fed with or without organic cranberry pomace (CBP). Bacterial taxa Pr es en ce p er ce nt ag e of to ta l c om po si tio n (% ) Fig. 5. Bacterial community composition in cecal contents of one-week old pasture-raised broiler chickens. Taxa that could not be assigned a genus were presented as ‘unclassified’ using the highest taxonomic level that could be assigned to them. Md. R. Islam, et al. Journal of Functional Foods 72 (2020) 104053 9 concerns about the potential pathogenicity of bacteria belonging to the taxon (Ridaura et al., 2013; Wexler, 2007). Our data revealed an in- crease in the prevalence of unclassified_Lachnospiraceae and un- classified_Rikenellaceae in the CBP treatments. Both of these bacterial families are among the most abundant taxa reported in human/animal gut microbiome studies. Members of the anaerobic Lachnospiraceae fa- mily ferment diverse plant polysaccharides to produce beneficial SCFAs including butyrate, acetate) and alcohols (ethanol) (Boutard et al., 2014), while the Rikenellaceae family members are reported to play a role in preventing non-alcoholic fatty liver disease (Del Chierico et al., 2017). A decline in Rikenellaceae abundance has also been associated with inflammation and impairment of mucosal immune functions (Qing et al., 2019). Finally, higher abundance of Fusobacteria was evident in the control group at days 21 and 64 in CBP fed birds. Members of Fu- sobacteria, such as Fusobacterium, have been directly implicated in human colorectal cancer development and intervention strategies that could decrease their presence or transmission are eagerly sought (Huang, Peng, & Xie, 2018; Idrissi Janati, Karp, Sabri, & Emami, 2019; Shang & Liu, 2018). In agreement with a previous study we performed with blueberry pomace (Islam et al., 2019), unweighted PCoA (based on phylogenetic relationships) showed a clear clustering of the cecal samples from control and CBP-treated bird groups at days 28 and 35, which supports the notion that CBP supplementation affects the gut microbiota of pasture-raised broiler chickens. The current study focused on the impact of several nutritional constituents (such as amino acids, fibers, and fatty acids) besides polyphenols. Our results collectively asserted the role of such bio- functional molecules in broiler chicken health and productivity. Earlier studies concerning the use of fruit pomace in poultry feed attributed several positive effects to polyphenols, including the attenuation of Fig. 6. A heatmap showing the changes of bacterial genera presence-percentage associated without (control) or with organic cranberry pomace (CBP) feed sup- plementation. Taxa that could not be assigned a genus were presented as ‘unclassified’ using the highest taxonomic level that could be assigned to them. Fig. 7. Unweighted UniFrac principal coordinate analysis (PCoA) by microbiota in cecal samples from pastured broiler chickens fed with [green (1%) and blue (2%) circle] or without (red circle) organic cranberry pomace (CBP): (A) Samples from day 28, where the treatment (CBP) and control trend to cluster separately with CBP samples exhibit variability, and (B) Samples from day 35 show clear separation between control and CBP bird groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Md. R. Islam, et al. Journal of Functional Foods 72 (2020) 104053 10 heat-stress (Hu, He, Arowolo, Wu, & He, 2019), improved water- holding capacity of poultry meat and the mitigation of negative effects of ochratoxin A (Mazur-Kuśnirek et al., 2019), and improved fatty acid profiles and oxidative stability of turkeys meat (Juskiewicz et al., 2017). Supplementation of chicken diets with fruit by-products may also induce a significant increase in the circulation of phenolic meta- bolites and tocopherol in blood (Munoz-Gonzalez et al., 2019), in- cluding unique conjugated forms of catechins and epicatechin meta- bolites. Moreover, CBP was found to significantly increase quinic acid levels in broiler blood plasma, suggesting that it could reduce oxidative stresses in chickens by virtue of its high phytochemical content (Das et al., 2020). Finally, it seems there are several mechanisms of how the bioactive compounds (including polyphenols) of CBP influence blood parameters and cecal microbiota that need further investigations. Despite differ- ences in their chemical structure and nature, these bioactives along with their metabolites appear to act synergistically to induce not only observed effects in the present study but also to influence the immunity of chicken as reported in our recent study (Das et al., 2020). Under- standing of the close structure-function relationship between these bioactives and their in vivo effects in addition to scrutinizing how gut microbiota can metabolize pomace bioactives (two-way mutual inter- actions) to enhance their bio-accessibility and bio-functionality (Ozdal et al., 2016) could help develop efficient nutritional interventions using CBP to address the urgent needs of alternatives to antibiotics in broiler production. 5. Conclusions The present study revealed that CBP inclusion in pasture-raised broiler chicken feed induced short- and long-term beneficial effects. Some improved production parameters coupled with positive changes in blood-serum metabolites and gut microbiota were indicative of the potential of CBP as a cost-effective feed supplement for organic chicken production. Evidently, more large-scale studies are needed to establish the beneficial effects of CBP-feed supplementation in variable poultry production systems, and to further characterize mechanisms underlying observed effects on the gut microbiome and overall bird health. In general, the modulation of gut microbiota was sustained after the CBP administration period and until slaughter, whereas changes in blood metabolites were restored soon after withdrawal. Hence, the findings hinted that CBP may find value as a prebiotic in broiler chicken feed (Tabashsum et al., 2019). The SCFAs are critical compounds for bird‘s gut health and are among the products that rise from bacterial fer- mentations of carbohydrates. Given that CBP can increase the abun- dance of SCFAs-producing bacteria within bird‘s digestive system, fu- ture research efforts are warranted to characterize the associated bacterial species as well as SCFAs levels involved in such phenomena in relationship with gut’s morphology and physiology in chickens. Authors’ contributions Both M.R.I. and M.S.D. conceptualized the presented work and de- signed the experiments. M.R.I., Q.D., D.L., M.H., D.G., S.O., and M.S.D. carried out the experiments/formal analyses and participated in methodology development as well as data curation. Y.I.H. wrote the original draft besides his involvement in data visualization. K.R. and P.D. helped supervising the work in addition reviewing and editing the first draft. M.S.D. was responsible for funding acquisition as well as supervising the project. All authors have read and approved the final manuscript. Ethics statement The included animal studies have been reviewed by the appropriate ethics committees. All experimental procedures were conducted in conformity with general practices of commercial free-range broiler chicken production guidelines established by the Canadian Council on Animal Care (http://www.ccac.ca/en/standards/guidelines). The pro- tocol was approved by Rosebank Farms, a member of the North Okanagan Organic Association, which uses an exclusive “customer certified” system for accreditation (with two to three inspections by customers that take place throughout the rearing period and the night before slaughter). Sources of funding This work was supported by Agriculture and Agri-Food Canada (AAFC) under the Organic Science Cluster II Program (AIP CL-02 AGR- 10383). Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influ- ence the work reported in this paper. Acknowledgements This work was supported by Agriculture and Agri-Food Canada (AAFC) under the Organic Science Cluster II Program (AIP CL-02 AGR- 10383). We thank the Certified Organic Association of British Columbia and the participating farmers (A. and S. Gunner; Armstrong, BC) for their supports. We acknowledge X. Yin (AAFC, Guelph) technical help and assistance. References Abdel-Aal, E.-S. M., Hucl, P., Patterson, C. A., & Gray, D. (2010). Fractionation of Hairless Canary Seed (Phalaris canariensis) into Starch, Protein, and Oil. Journal of Agricultural and Food Chemistry, 58(11), 7046–7050. https://doi.org/10.1021/ jf100736m. Aditya, S., Ohh, S. J., Ahammed, M., & Lohakare, J. (2018). Supplementation of grape pomace (Vitis vinifera) in broiler diets and its effect on growth performance, ap- parent total tract digestibility of nutrients, blood profile, and meat quality. Animal Nutrition, 4(2), 210–214. https://doi.org/10.1016/j.aninu.2018.01.004. Ajila, C. M., Brar, S. 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