Utilization of grape (Vitis vinifera), cranberry (Vaccinium macrocarpon), wild blueberry (Vaccinium angustifolium), and apple (Malus pumila/domestica) pomaces in broiler chickens when fed without or with multi-enzyme supplement

Abstract Nutritive and functional values of fruit pomaces in poultry are unexplored. We determined apparent metabolizable energy (AME) and plasma metabolites in broiler chickens fed diets containing apple (APL), low-bush wild blueberry (LBP), cranberry (CRP), and grape (GRP) pomaces without or with multi-enzyme supplement (ENZ). A total of 360 one-day old Ross 708 male chicks were placed in 72 cages; 5 birds/cage were reared to day13 and transitioned to either cornstarch–soy protein isolate basal diet or basal with 30% of either pomace without or with ENZ. Excreta samples were collected from day17 to 20 and one bird/cage bled for plasma metabolites on day 21. Apple pomace showed a higher AME (P=0.008) than other pomaces; however, ENZ had no effect on AME. The AMEs were 3250, 2613, 2394, and 3008 kcal/kg DM for APL, LBP, CRP, and GRP, respectively. There was pomace and ENZ interaction on plasma alkaline phosphatase (P=0.04), and APL increased cholesterol levels (P<0.01). In conclusion, ENZ had no impact on energy increment in pomaces, but the AME values this study has established are nonetheless valuable for accurate poultry feed formulation. Plasma metabolites suggested pomace components are amenable to supplemental ENZ. Résumé Les valeurs nutritives et fonctionnelles des marcs de fruits sont inexplorées chez les poulets. Nous avons déterminé l’énergie métabolisable apparente (AME — «apparent metabolizable energy») et les métabolites plasmatiques chez les poulets à griller ayant reçu des diètes contenant des marcs de pomme (APL — «apple»), de bleuets sauvages à buisson bas (LB — «low bush wild blueberry»), de canneberges (CRP — «cranberry») et de raisins (GRP — «grape») avec ou sans un supplément à multiples enzymes (ENZ). Un total de 360 poussins Ross 708 mâles âgés de un jour ont été placés en 72 cages, 5 par cage, et ont été élevés jusqu’au jour 13 puis transitionnés soit à une diète de base avec isolat de protéines d’amidon de maïs/soja, soit une diète de base avec 30 % de marc avec ou sans ENZ. Les échantillons de fèces ont été collectés des jours 17 à 20 et un poulet/cage saigné pour déterminer les métabolites plasmatiques au jour 21. Le marc APL a montré une AME plus élevée (P = 0,008) que les autres marcs, mais, les ENZ n’ont pas eu d’effet sur l’AME. L’AME était de 3250, 2613, 2394 et 3008 kcal/kg de matières sèches (DM — «dry matter») pour les marcs de APL, LBP, CRP, et GRP, respectivement. Il y avait une interaction entre marc et ENZ pour la phosphatase alcaline (P = 0,04) et l’APL a augmenté les niveaux de cholestérol (P < 0,01). En conclusion, l’ENZ n’a pas eu d’impact sur l’incrément d’énergie dans les marcs, mais les valeurs d’AME établies dans cette étude ont néanmoins une valeur pour la formulation précise des aliments à poulets. Les métabolites plasmatiques suggèrent que les composantes des marcs sont susceptibles aux suppléments d’ENZ. [Traduit par la Rédaction]


Introduction
Pomaces are residues of fruit processing, composed of the fleshy pericarp, peels, seeds, stems, and other parts specific to the fruit being processed (Ross et al. 2017). Pomaces may be produced after mechanical juice extraction, while others are produced by use of chemical means. The chemical methods render the pomaces to be technically inorganically produced, as is with other feed ingredients, such as solvent treated versus mechanically extruded soybean meal (CFIA 2019; Leung and Kiarie 2020).
About 90% of apples (APL) in Canada are mainly produced in BC, ON, and QC, with NS trailing the three provinces. Various studies reported that dried APL pomace contained ∼43.6% dietary fibers (Sato et al. 2010), mono and disaccharides such as sucrose, arabinose, galactose, and fructose (Gabriel et al. 2013), triterpenoids (Almeida-Trasviña et al. 2014), polyphenolic compounds (Vrhovsek et al. 2004), crude protein of up to 7.1% and macro-and microelements such as sodium, potassium, calcium, phosphorus, magnesium, iron, manganese, zinc, and copper (Pieszka et al. 2015). Grape (GRP) is cultivated in Canada mainly for the wine industry and produced extensively in the Okanagan valley (Statistis Canada 2021). Characterization efforts by Makris and Kefalas (2013) demonstrated a wide array of polyphenolic phytochemicals such as catechins, stilbenes, saponins, flavanols, anthocyanins, phenylpropanoids, and derivatives of p-coumaric and benzoic acids. Studies by Silva Soares et al. (2018) demonstrated the anthelmintic, ovicidal, and antilarval properties of saponins and tannins found in GRP, up to a certain level, beyond which the tannins would then have an anti-nutritive effect due to the strong binding complexes they form with proteins and macromolecules in the body.
Wild blueberry (Vaccinium angustifolium), also called lowbush blueberry, and organic cranberries (Vaccinium macrocarpon) are grown in Canada principally in the Atlantic, QC, and BC provinces (Turner 2009;Sandra Behm 2020). Bioactive characterization of organic cranberry pomace (CRP) and blueberry pomace (LBP) by Ross et al. (2017) revealed a wide assortment of chemical constituents such as carbohydrates, including soluble and insoluble fibers, protein, minerals, and phenolics such as flavonols, anthocyanins, and tannins. Even though many tannins are considered to be anti-nutritive factors in terms of reducing iron absorption, protein digestibility, and even growth, its potential as an anti-carcinogen, antibacterial, and antioxidant as well as an immune-modulator agent among other advantages has been demonstrated (Ashok et al. 2012).
Apple (Aghili et al. 2019), GRP (Hosseini-Vashan et al. 2020;Gungor et al. 2021), CRP , and LBP (Islam et al. 2019) pomaces have been studied for their use in chicken production to different ends but with low adoption rates commercially. Considering the variation in chemical composition in pomaces, complex carbohydrates, and fibers, the digestibility and availability of nutrients of these pomaces in the chicken gut by the endogenous enzymes are unknown. Research has shown that exogenous enzymes in poultry feed break down cell wall matrices and other anti-nutritive components of feed to release starch, protein, and fats, making them available for absorption in the gastrointestinal tract (Karimi et al. 2013;Gallardo et al. 2017). Gallardo et al. (2017) further indicated that the use of multi-carbohydrase enzyme with canola meal could improve nutrient and energy utilization and fiber (non-starch polysaccharides (NSP)) solubilization for effective hindgut fermentation in broiler chicks. This breakdown is associated with degradation of the antinutritive factors such as NSP and phytic acid in feed (Kiarie et al. 2016) and leads to the release of starch, fats, proteins, minerals, and energy, according to Slominski (2011). The use of a cocktail of enzymes instead of single pure enzymes could be beneficial to improve availability of nutrients encased in structurally complex ingredients such as pomaces (Ravindran 2013;Kithama et al. 2021).
Little is known about the potential of fruit pomaces in broiler production, specifically metabolizable energy content. Such knowledge is needed to accurately formulate feed with fruit pomaces for poultry. Moreover, even though beneficial effects of pomace consumption on blood metabolites have been sporadically demonstrated in broiler models (Islam et al. 2019;Das et al. 2020;Islam et al. 2020), the benefits or impact on physiological and biochemical status of broilers has not been extensively studied. Therefore, it is paramount to evaluate the pomace effects on the plasma biochemical profile of chickens. The general objective of this study was to evaluate nutritive and functional values of APL, GRP, CRP, and LBP fruit pomaces fed without or with a multi-enzyme supplement (ENZ) in broiler chickens. The specific objectives were to evaluate the apparent metabolizable energy (AME) of pomaces and plasma biochemical profile in broiler chickens. It was hypothesized that feeding pomace with feed enzymes will lead to further release of nutrients and functional components.

Pomaces and experimental diets
Frozen wet organic CRP and LBP pomaces were obtained from Fruit D'Or (Villeroy, QC, Canada). Apple and GRP pomaces were procured from processors (Bennett's Apples & Cider and Niagara College Teaching Winery/The Canadian Food and Wine Institute, Niagara Falls, ON). The pomaces were freeze-dried and then ground to pass through a 2 mm mesh screen using a cutting mill (SM 2000 Retsch, Haan, Germany) and stored at −20 • C until use. A basal semipurified corn starch and soybean protein isolate (CP, 87.3%) diet (Table 1) was formulated to contain 3 000 kcal/kg AME and 20% crude protein to meet or exceed minerals and vitamins specifications for a growing broiler (Aviagen 2016). The pomace diets were created by mixing basal diet and respective pomace at 7:3 (wt/wt) ratio (Woyengo et al. 2010;Kiarie et al. 2014;Ravindran et al. 2014). Each of the pomace diets was then supplemented with ENZ containing galactanase, protease, β-mannanase, β-glucanase, xylanase, α-amylase, and cellulase activities at 50, 200, 400, 600, 7 000, 2 500, and 2 800 U/g of product, respectively (Superzyme EO , Canadian Bio-systems Inc., Calgary, AB, Canada). Thus, the eight pomace diets and the control basal diet gave a total of nine dietary treatments. The basal diet was fed to facilitate calculation of AME by difference method (Woyengo et al. 2010;Kiarie et al. 2014;Ravindran et al. 2014). The diets contained 0.25% titanium dioxide indigestible marker and were prepared in mash form.

Birds and housing
The present study was conducted at the Arkell Poultry Research Station and approved by the University of

Experimental procedures
Broilers were fed a commercial starter diet for the first 13 days of life for adaptation. The commercial starter was corn and soybean meal based with concentrations of crude protein, crude fat, crude fiber, Ca, and P being 22.8%, 4.7%, 2.8%, 0.97%, and 0.62%, respectively (Floradale Feed Mill Ltd., Floradale, ON, Canada). On day 14, the 9 diets were allocated in a completely randomized design (8 replicate cages per diet) based on cage body weight. Birds had free access to feed and water throughout the study. Feed intake as well as mortality were monitored between days 14 and 21.

Sample collection
Fresh excreta samples were collected from each cage consecutively from day 17 to 20 and stored at −20 • C (Woyengo et al. 2010). On day 21, one bird per cage was bled for plasma in heparin-coated tubes; samples were placed on ice and transferred to the laboratory. The blood was spun at 4 000 g at 4 • C for 15 min to separate out the plasma layer, which was then stored at −20 • C.
Sample processing: plasma, excreta, and exogenous enzyme analyses Plasma biochemical profile, including protein, enzymes, metabolites, and minerals, was analyzed by photometric method as described by Greenacre et al. (2008) at the Animal Health Laboratory (University of Guelph, Guelph, ON). Excreta samples were pooled and oven-dried for 4 days at 60 • C. The pomaces, diets, and excreta samples were ground in a coffee grinder (Proctor Silex Fresh Grind™ Coffee Grinder) and mixed thoroughly for analyses. All samples were analyzed for the following: dry matter (DM), nitrogen (N), crude fat (CF), neutral detergent fiber (NDF), minerals, and gross energy (GE). Pomaces were further analyzed for polyphenolics, non-starch polysaccharide monosugars, and diets for amino acids. Dry matter was determined according to the method of AOAC (2005) Maness (2010). Titanium content in diets and excreta were measured on a UV spectrophotometer (Myers et al. 2004).
For amino acids' (AAs) analyses, samples were prepared by acid hydrolysis according to AOAC (2005) (method 982.30). Briefly, about 100 mg of each sample was digested in 4 mL of 6 N HCl for 24 h at 110 • C, followed by neutralization with 4 mL of 25% (wt/vol) NaOH and cooled to room temperature. The mixture was then equalized to 50 mL volume with sodium citrate buffer (pH 2.2) and analyzed using the ultra performance liquid chromatography (UPLC, Waters Corporation, Millford, CA, USA). Samples for analysis of sulfur containing AAs (Met and Cys) were subjected to performic acid oxidation prior to acid hydrolysis. Tryptophan was not determined. Total phenolics, tartaric esters, anthocyanins, and flavonols in pomaces were measured using Glories method as described by Harrison et al. (2013) and Ross et al. (2017). Non-starch polysaccharide monosugars were determined by gas-liquid chromatography (component neutral sugars) and by colorimetry (uronic acids) using the procedure described by Cummings (1984, 1988) with modifications (Slominski et al. 2006). Xylanase activity in pomace diets was assayed using Xylazyme AX tablets (Megazyme International Ltd., Bray, Ireland). One unit of xylanase was defined as the quantity of the enzyme that liberated 1 μmol of xylose equivalent per minute.

Calculation and statistical analyses
The apparent retention of components (dry matter, crude protein, crude fat, NDF, and gross energy) was calculated according to Adeola (2000) using the following equation: where M feed and M excreta are the concentrations of index (TiO 2 ) in feed and excreta, respectively, while C excreta and C feed represent the concentrations of the components (energy) in faeces and feed, respectively.
The AME in APL, CRP, LBP, and GRP was calculated using the following formula by Ravindran et al. (2014): The cage was the experimental unit for all parameters measured. Data were normal (Gaussian) and analyzed as a completely randomized design using the General Linear Mixed Model (GLMM) procedure of SAS 9.4 (SAS 2016), which would account for variation in the fixed effects, interactions, and assumed correlated errors. The model had pomace, ENZ, and associated interactions as fixed factors. Multiple comparisons using Tukey's test were used to separate pomace and interaction LSmeans and independent t test for ENZ whenever the F value was significant. A P value of 0.05 or less was used to declare significance.

Chemical composition of pomaces and experimental diets
The analyzed chemical composition of the pomaces is presented in Table 2. The CRP and LBP showed comparable DM of ∼98%, whereas APL and GRP DM were 92% and 92.2%, respectively. Crude protein content was higher in LBP (11.6%), followed by CRP (7.4%), while APL and GRP showed a CP content of ∼4% for both. The gross energy for the four pomaces was higher for LBP (5 388 kcal/kg) and CRP (5 534 kcal/kg), compared to APL (4 188 kcal/kg) and GRP (4 865 kcal kg). The crude fat contents were higher in LBP (10.3%) and CRP (8.7%) than in the APL and GRP ( Table 2). The highest crude protein concentrations among the four pomaces were observed in the LBP (11.8%) and CRP (7.6%). The profiles of AAs revealed low contents of Lys, Met, and Cys in all pomaces. The most abundant AAs, specifically in CRP, LBP, and GRP, included Phe, Val, Ala, Pro, Ser, and Tyr. Among the four studied pomaces, CRP and GRP showed the highest content of Arg (Table 2). Carbohydrate composition differed greatly among the four pomaces, with GRP starch content being the highest (22.0%) compared to 2% for CRP and LBP and 8.8% for APL. The NDF fractions on the other hand stood at 72.6% and 75.0% for LBP and CRP, respectively, but GRP and APL showed NDF values of 9.6% and 34.4%, respectively. The phenolics' contents presented in Table 2 indicate marked differences among the pomaces, with CRP and LBP pomaces having the highest concentrations of total phenolics, tartaric esters, flavonols, and anthocyanins than APL and GRP.
As presented in Table 3, mixing pomaces with the basal diet increased the levels of crude fat, while the crude protein content did not markedly change. Contents of AAs such as Arg, Ile, Pro, Phe, and Glu were higher in CRP diet than in the other diets. Xylanase activity was determined to confirm accuracy of inclusion of ENZ and feed mixing. The analyzed xylanase activities in pomace diets without ENZ were 25, 55, 119, and 29 XU/g for APL, CRP, LBP, and GRP, respectively. Corresponding xylanase activities in pomace diets treated with ENZ were 8348, 7561, 7097, and 9000 XU/kg for APL, CRP, LBP, and GRP, respectively.

Plasma metabolites
There was interaction (P = 0.04) between pomace and ENZ on plasma concentration of alkaline phosphatase and urea (Table 5). Specifically, feeding GRP with ENZ resulted in a higher alkaline phosphatase than APL with ENZ or CRP without ENZ (P ≤ 0.04). With respect to urea, birds fed GRP without ENZ had lower plasma concentration of urea than birds fed GRP with ENZ (Table 5). The main effect of pomace was noted (P < 0.01) for plasma concentration of bile acid and cholesterol. The (P < 0.01) lowest concentrations of cholesterol and bile acids were found in birds fed CRP, LBP, and GRP diets compared to the birds fed with APL pomace. There was an interaction (P = 0.008) between pomace and ENZ on plasma concentration of phosphorous and sodium (Table 5). Plasma phosphorus in birds fed APL with ENZ was higher than for birds fed LBP and GRP without ENZ, whereas birds fed GRP without ENZ exhibited higher plasma sodium than birds fed LBP and CRP with ENZ. Birds fed APL, LBP, and GRP had higher (P < 0.05) concentration of plasma CO 2 than birds fed CRP; however, APL birds had higher CO 2 (P < 0.05) concentration. Birds fed APL and GRP had higher plasma calcium than birds fed LBP and CRP. Birds fed CRP had lower plasma potassium compared with birds fed other pomaces (Table 5).

Discussion
The type, source, and nutrient composition of feed ingredients incorporated in broiler diets have profound effects on the overall growth and development of the birds. The four tested pomaces in the present study showed differences in their chemical components: significantly lower crude protein contents in APL and GRP than in LBP and CRP, higher starch in APL and GRP than in LBP and CRP, and higher crude fat and gross energy in CRP and LBP. The amino acid profiles of the pomaces did not impact the dietary crude protein levels, and as such the contribution of the amino acids is negligible. The chemical profile APL used in the present study differed from that of Aghili et al. (2019), which reported significantly high levels of crude fat and fiber; this can be attributed to the variety used and processing methods. Similar crude fat values as that in the present study were obtained by Taranu et al. (2018) for GRP, even though their NDF and ADF were much higher in tested pomace. Higher NDF and ADF contents were observed in the CRP and LBP diets compared to the APL and GRP diets, relating directly to uniqueness of pomaces. Fiber plays an important role in poultry nutrition, with implications on feed intake, gut physiology, gut motility, nutrient absorption, and gut microbiota (Kiarie et al. 2014;Tejeda and Kim 2021).
The polyphenolic compounds showed high variability, with LBP having the highest concentrations of total phenolics, tartaric esters, flavonols, and anthocyanins. Pheno-lic compounds have been extensively studied and shown to exhibit beneficial biological effects, including antimicrobial, anti-inflammatory, and antioxidant properties (Harrison et al. 2013). The differences in fruit cultivars, species, processing methods, et cetera, will produce pomaces that vary considerably in their chemical compositions, which could influence their in vivo activities in poultry. These are areas that should be explored in future studies to standardize the products and optimize them for broiler production.
Even though APL, GRP, LBP, and CRP pomaces have been used sporadically in horses, rabbits, chicken, and swine diets (Nicodemus et al. 2007;Brenes et al. 2016;Taranu et al. 2018;Islam et al. 2019;Islam et al. 2020), there is still a big interest in their use due to emerging feed technologies that will help in optimizing their benefits by taking advantage of their wide range of nutrients and polyphenolic compounds having antimicrobial and antioxidant properties . Fiber matrix of pomaces encapsulates polyand oligosaccharides and polyphenols that when released by fiber-degrading enzymes would be of great benefit to broiler chickens (Kithama et al. 2021). In the present study, the semipurified diets were used to minimize confounding effects of other dietary components on AME determination. The experimental period and the diets used in this study were not optimized for growth performance evaluation. APL pomace birds had less feed intake relative to birds fed basal and other pomaces. Studies by Aghili et al. (2019) showed that feeding APL pomace, with and without enzyme, decreased feed intake and reduced performance in broilers. Similar studies with APL pomace also showed reduced feed intake, postulated to be because of high fiber content (Heidarisafar et al. 2016).
Energy is a property of nutrients derived from the catabolism of carbohydrates, lipids, and protein in feed, with various feed ingredients having different energy levels that could be used in diets to meet the energy requirements for broiler chickens (Kocher et al. 2003). The available energy content of ingredients for poultry, which determines the feed intake, could be measured through different methods, including measurement of AME (Hill and Anderson 1958). The tested fruit pomaces in the present study varied in gross energy retention and subsequently AME. Higher AME contents in CRP and LBP compared to APL and GRP might be attributable to the higher content of crude fat, neutral and acid detergent fibers, and crude protein, even though APL and GRP had higher starch content. Although there are not many studies documenting the AME of fruit pomaces in poultry, one study showed that the GRP pomace in their poultry diets had an AME of 2 433 kcal/kg (Hosseini-Vashan et al. 2020). It has been reported that GRP pomaces at 0.5%, 7.5%, and 1% inclusion level had an ileal digestibility of gross energy of 81.73%, 79.42%, and 77.08%, respectively, in broiler chickens (Aditya et al. 2018). However, feeding GRP to horses (Kolláthová et al. 2020) showed improved digestibility of nutrients due to its polyphenolics aiding nutrient uptake, as was also observed in ruminants (Makkar 2003). Experiments with similar enzyme admixtures used in the present study showed higher AME values with corn and soybean meal-based diets than control diets (Kocher et al. 2003). In our study, no significant effects of the ENZ used were observed on the energy and digestibility values of studied pomaces, perhaps suggesting that the ENZ used in the present study was not optimized for pomaces. Dietary fiber in poultry research is seen from a twodimensional angle: (1) as a functional component for normal physiological functions of the gut and (2) as an anti-nutrient (Tejeda and Kim 2021); variability in assessing the digestibility of fiber in broiler diets is highly influenced by the fiber source, type, and diet formulation (Sanchez et al. 2021). NDF is composed of hemicellulose, cellulose, and lignin components of plants, and data from the present study showed NDF Table 4. Apparent retention of components and apparent metabolizable energy in fruit pomaces fed to broiler chickens without or with multi-enzymes supplement. retention was variable among the four pomaces. Among the pomaces, APL diets with or without enzyme had negative retention values for NDF, indicating increased flow of dietary and endogenous NDFs in the excreta. The mechanism for this phenomenon is unknown, but lower feed intake in birds fed APL could be a reason. In addition, the physicochemical characteristics of APL fiber and ensuing gastrointestinal effects could also have contributed. For example, broiler chickens (Thanabalan et al. 2020) fed flaxseed exhibited reduced retention of NDF, which was associated with mucilage. The avian plasma biochemical profile is an important tool used by veterinarians in the diagnosis of diseases and conditions of commercial, pet, and wild species of birds. Fruit pomaces can be a significant source of components entrapped in the fiber matrix. These include flavonoids, phenolic acids, and stilbenoids , which exert non-specific effects on living organisms and regulate the activities of enzymes and cell receptors. For example, polyphenol-rich extracts from CRP fruits displayed potential in increasing energy efficiency and insulin sensitivity and in decreasing triglyceride and cholesterol contents. In the present study, avian biochemistry was evaluated to characterize the physiological impact of APL, LBP, CRP, and GRP pomaces at an inclusion rate of 30% with or without an enzyme mixture in feed on various blood plasma metabolite levels. Measurement and assessment of chicken blood parameters does not have standards and protocols; therefore, different equipment for measurement will give varied results compared to another equipment or methodology (Brugere-Picoux et al. 2015).
The total protein content provides some information regarding general status; however, more clinically useful data are indicated by plasma albumin and globulin ratio (Brugere-Picoux et al. 2015;Harr 2002). The total protein or albumin and globulin were not affected by dietary pomaces; however, a trend was observed for enzyme to increase total protein and globulin and subsequently reduce the albumin to globulin ratio. These proteins are primarily synthesized in the liver and play important roles in fat metabolism (Harr 2002;Greenacre et al. 2008). The primary factors affecting albumin and globulin synthesis include protein and amino acid nutritions, colloidal osmotic pressure, the action of certain hormones, and disease status (Harr 2002). Thus, the tendency for higher circulating plasma globulin observed in birds fed enzyme could be linked to increased release of nutrients in the gut. Circulating plasma enzymes reflects damage to an organ or tissue, particularly the liver and kidney (Rozenboim et al. 2016). Alkaline phosphatase (ALP) is found in bile ducts, bone, liver, intestine, placenta, and tumors (Sharma et al. 2014). Elevations of the blood ALP level occur with hepatobiliary disease but also during healing fractures, vitamin D deficiency, bone disease, and malignancy (Sharma et al. 2014;Brugere-Picoux et al. 2015). Enzyme-treated GRP pomace had a significantly higher plasma ALP compared to the rest of the enzyme-treated and non-enzyme-treated pomaces. Increase in plasma ALP has also been noted in pigs fed diets supplemented with phytase enzyme linked with increased bone mineralization (Kiarie et al. 2022). Additionally, there were non-significant differences in ALP levels between the enzymetreated and non-enzyme-treated pomaces. High cholesterol levels in blood are indicative of obesity with liver steatosis, high lipids in diet, or fasting (Brugere-Picoux et al. 2015).
With consideration of these underlying factors, the low feed intake of the birds on APL-supplemented diet may be the attributable factor to the high cholesterol, even though more studies are warranted regarding impact of APL diets on lipid metabolism. Plasma sodium, potassium, and phosphorus levels were within normal range compared to the baseline, and there was an interaction between pomace and enzyme for sodium and phosphorus levels (Brugere-Picoux et al. 2015;Harr 2002). Decrease in plasma potassium could be an indication of diuretic inefficiencies in birds, while an increase in its levels would indicate a case of kidney disease or dehydration (Brugere-Picoux et al. 2015). Similar kidney disease symptoms would be observed in the case of increased phosphorus levels in plasma, while decreased quantities would lead to bone disorders (Sharma et al. 2014;Brugere-Picoux et al. 2015). Calcium concentration in plasma also plays a major role in bone modeling, kidney conditions, and metabolism of vitamin D. In the present study, APL pomace-fed birds showed the highest level of plasma calcium compared to the other four pomaces. These observations in conjunction with ALP responses suggested that feeding pomace with enzyme influenced mineral metabolism, perhaps linked to increased release of bioactives related to mineral utilization in broilers. A study on horses fed GRP pomace showed similar effects on plasma mineral levels (Kolláthová et al. 2020).
In conclusion, present results showed that considering AME, the four fruit pomaces tested can be used in poultry rations. These high inclusion rates (30%) in the present study did not show adverse effects based on plasma metabolites; however, we hypothesize that the high fiber fraction may have contributed to the low feed intake of the birds. It should be noted that lower LBP and CRP inclusion rates (1%-2%) have been applied in broiler chicken feeding programs for a longer period (30 days) without notable detrimental effects . Based on high fiber and other phytochemical contents, cranberry and blueberry pomaces could be explored for efficient use in poultry. In the present study, the diets were semipurified, and so future studies should aim at including the pomaces into standard corn-soybean-based or wheat-based diets and their assessment at lower inclusion levels to optimize their benefits and animal performance.