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 di-saccharides 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 low-bush 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 (Islam et al. 2020), 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 anti-nutritive 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.

Materials and Methods

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 2mm mesh screen using a cutting mill (SM 2000 Retsch, Haan, Germany) and stored at −20°C until use. A basal semi-purified corn starch and soybean protein isolate (CP, 87.3%) diet (Table1) was formulated to contain 3000 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, 7000, 2500, and 2800U/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.

Table 1.

Composition of basal diet, as fed basis.

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 (TiO₂) 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.

Results

Chemical composition of pomaces and experimental diets

The analyzed chemical composition of the pomaces is presented in Table2. 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 (5388kcal/kg) and CRP (5534kcal/kg), compared to APL (4188kcal/kg) and GRP (4865kcalkg). The crude fat contents were higher in LBP (10.3%) and CRP (8.7%) than in the APL and GRP (Table2). 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 (Table2). 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 Table2 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.

Table 2.

Analyzed chemical composition of pomaces, as fed basis.

As presented in Table3, 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 29XU/g for APL, CRP, LBP, and GRP, respectively. Corresponding xylanase activities in pomace diets treated with ENZ were 8348, 7561, 7097, and 9000XU/kg for APL, CRP, LBP, and GRP, respectively.

Table 3.

Analyzed composition of experimental diets, as fed basis.

Apparent metabolizable energy and apparent retention of nutrients

The birds readily consumed experimental diets, and there was no mortality throughout the experimental period. The average feed intakes for the 7 experimental periods were 249, 146, 280, 298, and 251g/bird for the basal, APL, LBP, CRP, and GRP diets, respectively. Data in Table4 show that there was no interaction between ENZ and pomace for apparent retention of DM (P=0.369), N (P=0.068), CF (P=0.784), GE (P=0.878), and AME (P=0.852) but there was for NDF (P=0.045). The interaction was such that the apparent retention of NDF for APL fed with ENZ was lower than that for non-ENZ group. The main effects of pomace were significant for apparent retention of DM (P=0.002), N (P<0.001), NDF(P<0.001), GE (P<0.001), and AME (P=0.008). The APL showed higher apparent retention of GE (76.2%) than CRP (64.6%), LBP (66.2%), and GRP pomace (70.4%). Similarly, the highest AME (3349kcal/kg) was observed with APL compared to CRP (2394kcal/kg), LBP (2612kcal/kg), and GRP (3008kcal/kg). Supplemental ENZ had no effect on AME (P=0.109).

Table 4.

Apparent retention of components and apparent metabolizable energy in fruit pomaces fed to broiler chickens without or with multi-enzymes supplement.

Plasma metabolites

There was interaction (P=0.04) between pomace and ENZ on plasma concentration of alkaline phosphatase and urea (Table5). 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 (Table5). 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 (Table5). 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 CO₂ than birds fed CRP; however, APL birds had higher CO₂ (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 (Table5).

Table 5.

Plasma metabolites in 21-day old broiler chickens fed diets with fruit pomaces without or with fiber degrading enzymes.

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. Phenolic 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 (Islam et al. 2020). Fiber matrix of pomaces encapsulates poly- and 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 semi-purified 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 2433kcal/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 two-dimensional 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 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 (Islam et al. 2020), 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 enzyme-treated 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 (30days) without notable detrimental effects (Das et al. 2020). 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 semi-purified, 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.