Nutrient digestibility and nutritive value
In the current study, the impact of dried OP incorporation, natural zeolite, or both in growing rabbitʼs diets on digestibility, nutritive value, N-balance, blood metabolites, antioxidant status, cecum characteristics, and microbiota was evaluated. The improvement in nutrient digestibility in the current study as a result of dried OP inclusion in rabbitʼs diets was in agreement with Suliman et al. (2019) who recorded significant increases in digestibility of DM, OM, CP, and CF when dried OP was included at 8% of the rabbits’ diet while using OP at 16% had no significant effect. Also, it was observed that replacing yellow corn in New Zealand rabbitʼs diets with 20, 40, or 60% dried OP, non-significantly improved the digestibility of DM and OM, and significantly the digestibility of CF (Ibrahim et al., 2011). In contrast, the inclusion of dried OP in rabbitʼs diets either at 5, 10, 15, 20 and 25% (Hon et al., 2009) or 1.5, 3 and 6% of the diet (Zeweil et al., 2015) had no significant effect on digestibility of DM, OM, and CP. These variations among the previous studies on nutrient digestibility as a result of dried OP incorporation may be attributed to the variability in the level of OP, diet composition, animal species, and the method of pulp processing (Bampidis and Robinson, 2006).
The improvement in nutrient digestibility may be due to the high fiber content of dried OP which decreases the passage rate and increases the digesta retention time in the rabbitʼs gut which leads to more utilization of dietary nutrients (Fraga et al., 1991). Citrus (orange) pulp is characterized by its high content of soluble dietary fibers in the form of soluble non-starch polysaccharides (NSP), pectins, and gums (Mourão et al., 2008). Furthermore, Miron et al. (2002) stated that citrus pulp is considered a source of neutral detergent fiber (NDF) and soluble carbohydrates with less lignin content. In the present study, OP diets had more NDF, ADF, hemicellulose, and cellulose and less ADL compared with the CON diet, which reflects higher CF digestibility. Hon et al. (2009) showed that dried OP contains highly digestible fibers which are very important in rabbit nutrition in comparison to poultry (Zeweil et al., 2015). A negative correlation between dietary CF and utilization of other nutrients especially CP digestibility was reported by Adegbola and Okonkwo (2002). Therefore, the improvement in CF and other nutrient digestibility with OP diets in the current study, confirms the high contents of soluble carbohydrates and fibers in dried OP. The higher fiber digestibility with OP inclusion could be explained by high NFC (non-fiber carbohydrates) content that activates the polysaccharides degrading enzymes (Zhao et al., 2013). However, the values of EE digestibility among different experimental diets were not statistically significant in the current study, there were improvements with OP diets. The antioxidant content in citrus pulp may have a role in increasing EE digestibility (Lima et al., 2014).
Regarding the effect of zeolite addition alone (Z diet) on nutrient digestibility, the current findings are in parallel to the results obtained by Balakirev et al. (2000) who noted no significant effect of 1 and 5% zeolite in rabbitʼs diets on digestibility of OM, CP, and EE, while significant increases in digestibility were observed with 3% zeolite. However digestibility of DM, OM, and CP was non-significantly improved, and the Z diet significantly increased the digestibility of CF. The positive effect of zeolite addition on digestibility especially fibers may be attributed to the high attraction between zeolite, active cations, and water which alters the passage rate of feed in the animal gut and improves fiber digestion (Johnson et al., 1988). Furthermore, the zeolite may have the ability to regulate the gut pH which promotes microbial fermentation and results in more fiber digestibility as a result of the buffering capacity of magnesium and aluminum silicate contents in a zeolite (Khachlouf et al., 2018), or due to its binding with H2 ions produced from the fermentation of dietary organic acids (Dschaak et al., 2010). Furthermore, it was indicated that zeolite administration can increase the hypertrophied function either of the intestinal villi or the epithelial cells in the ileum and duodenum, which can provide more surface area for nutrient absorption (Khambualai et al., 2009).
Regarding the nutritive value, the significant (P < 0.05) increases in TDN and DE values with OP, Z, and OPZ diets may be due to the improvement in nutrient digestibility with these diets compared with the control. The highest (P < 0.05) value of DCP was recorded for the OPZ diet, which may be attributed to the highest digestibility of CP compared with other diets. Consistent with the previous results, Ibrahim et al. (2011) recorded significant increases in TDN and DE values with non-significant differences in DCP values when dried OP was included in the diets of rabbits at the levels of 3.6, 7.2, and 10.8%. However, no significant effects of OP inclusion on nutritive value either as TDN, DE, or DCP were reported with rabbits (Hon et al., 2009, Zeweil et al., 2015; Suliman et al., 2019).
Growth performance
It was observed in the current study, that the inclusion of dried OP in rabbitsʼ diet significantly (P < 0.05) improved ADG, which may be explained by the increase in nutrient digestibility with OP diets. Coincidence with that result, it was demonstrated that using orange peel extract in a rabbitsʼ diet (Hassan et al., 2021) or using dried OP in a broilersʼ diet (Abbasi et al., 2015) significantly increased ADG. It was reported by Hassan et al. (2021) that orange peel contains a significant amount of ascorbic acid (59 mg/100 g DM), which may enhance rabbit's growth performance, as it is considered a natural growth promoter due to its antioxidant effect that inhibits the cell damage (Qi et al., 2020), and prevents infection either by viruses or pathogenic bacteria (El-Desoukey et al., 2018). Furthermore, several active phenolic compounds were reported in orange peel such as flavonoids, flavones, isoflavones, coumarins, catechins, lignans, and β-carotene (Sir Elkhatim et al., 2018). These phenolic compounds were found to have a positive effect on digestive enzymes and saliva that may increase nutrient digestibility as shown in the current study, which in turn can improve animal growth (Hashemi and Davoodi, 2010). Also, pectin content in the citrus peel can treat diarrhea as a result of its high-water absorbing property (Ibrahim et al., 2011), which may be reflected in higher growth performance. On the other hand, the incorporation of dried OP (Hon et al., 2009; Ojabo et al., 2012; Suliman et al., 2019) or citrus pulp (Lu et al., 2018) in diets growing rabbits, had no significant effect on ADG.
The present results showed also a significant (P < 0.05) increase in ADG with zeolite supplementation, which could be attributed to higher TDN and DE values for the Z diet compared with the control. In agreement with the previous result, Balakirev et al. (2000) recorded higher ADG for rabbits fed diets supplemented either by 1 or 3% zeolite. However, in other studies, ADG was not significantly changed by zeolite addition either in the diets of rabbits (Fortomaris et al., 2007) or broilers (Schneider et al., 2016; Abdulrahman et al., 2022). The positive impact of zeolite supplementation on rabbits ADG may be attributed to its ability to capture harmful substances such as ammonia, mycotoxins, heavy metals, etc, and suppress their negative effect on the health and performance of the animal (Stojković et al., 2012). Furthermore, Karamanlis et al. (2008) demonstrated that zeolite can increase the utilization of dietary nutrients and improve animal performance through the following 5 mechanisms: (1) binding with ammonia produced in the intestine and eliminating its toxic effect, (2) slower the digesta transit through the intestine, (3) reducing the absorption of some toxic compounds that produced from microbial degradation in the intestine as p-cresol, (4) enhancing the activity of pancreatic enzymes and (5) elimination the inhibitory effect of mycotoxin.
The non-significant differences recorded in the current study in DMI among the different experimental groups are in parallel with those obtained by Hon et al. (2009), Ojabo et al. (2012), Zeweil et al. (2015) and Lu et al. (2018) when either OP, orange peel or citrus pulp were incorporated in rabbitsʼ diet. In the same context, DMI was not significantly altered with zeolite addition to broilersʼ diets (Schneider et al., 2016). Contrarily, significant increases in DMI were observed with orange pulp or peel inclusion in the diets of rabbits (Hassan et al., 2021) or broilers (Abbasi et al., 2015; Abdulameer, 2018). Also, Fortomaris et al. (2007) reported significant increases in DMI when zeolite was added to rabbit diets at the level of 1.25 and 2.5%.
In concordance with the present data, non-significant differences were found in FCR values when rabbit's diet contained OP (Hon et al., 2009), orange peel extract (Hassan et al., 2021), or citrus pulp (Lu et al., 2018). Suliman et al. (2019) recorded significantly higher values of FCR when 8 and 16% of dried OP were included in the rabbitsʼ diet. Furthermore, no significant effect on FCR was conducted by Fortomaris et al. (2007) when zeolite was added at 1.25% of the rabbit's diet. While Balakirev et al. (2000) mentioned that FCR was decreased with 1 and 3% zeolite addition to rabbit's diet. The best FCR value in the current study that was recorded with the OPZ diet (by 7.56%) could be attributed to the highest ADG for rabbits fed the OPZ diet with no significant difference in DMI between OPZ and CON diets.
Nitrogen balance
In the present study, all the experimental groups showed significantly similar values of N-intake, which may be due to that all diets were formulated to be iso-nitrogenous. Meanwhile, significant (P < 0.05) decreases in the values of total N-excreted and significant increases (P < 0.05) either in the values of N-retention or N-balance were observed with OP, Z, and OPZ groups. In matching with the previous findings, Ferrer et al. (2021) reported significant decreases in NH3 emission and NH3 content in Pigsʼ slurry when OP was included in the diets in two forms (dried and sun-dried silage). Similarly, Burmańczuk et al. (2015) found that adding zeolite at 5% of broilersʼ diet significantly decreased ammonia volatilization by about 33%. Also, N in pigs’ manure was reduced by 15 and 22% when zeolite was added to diets in 2 different particle sizes (Leung et al., 2007).
It was indicated that the inclusion of indigested dietary fiber ingredients in the form of non-starch polysaccharides (NSP) such as OP (Mourão et al., 2008; Hon et al., 2009), which includes hemicellulose, cellulose, fructans, galactomannans, glucomannans and pectins (Grieshop et al., 2001), is effective in altering the excretion pattern of nutrients and reducing the loss of odor compounds in feces (Mroz et al., 2000). Furthermore, Noblet and Goff (2001) illustrated another mechanism that may be involved in reducing NH3 emission from pigʼs slurry with OP diets, as it increases the microorganismsʼ fermentation of the total dietary fibers in the hindgut which leads to higher VFA production and decreases the slurry pH.
Although urinary-N: fecal-N ratios in the current study were not significantly affected by the inclusion of dried OP or zeolite in the diets, their values were relatively decreased with OP, Z, and OPZ diets compared with control. Ferrer et al. (2021) suggested that the decrease in NH3 emission with diets containing OP may be due to not only the decrease in total N excretion but also a result of N partition between urine and feces. Several studies confirmed that total dietary fibers; especially soluble fibers; may have the ability to alter N partitioning from urine to feces, which leads to increased N excretion in feces and a decrease in its content in urine as urea (Ferrer et al., 2018). Higher NDF intake with OP diets is another possible explanation for N partitioning (Ferrer et al., 2021). Similar findings were reported in N partitioning when different fibrous by-products were included in pigsʼ diets (Antezana et al., 2015). Furthermore, the obtained results in the current study, suggest that natural zeolite can be used as a feed additive in rabbitsʼ diets to reduce NH3 volatilization. That result is probably attributed to the ability of zeolite to bind ammonia that occurs in the digestive tract or due to its positive effect on CP digestibility (Karamanlis et al., 2008).
Blood biochemical parameters and antioxidative status
Regarding blood biochemical, the current data indicate non-significant differences among the experimental groups in plasma total protein, albumin, globulin, AST, ALT, and total lipid concentrations, which are in harmony with the findings obtained by Lu et al. (2018) and Hassan et al. (2021) with rabbits when OP was included in diets. Moreover, the addition of zeolite in broilersʼ diet had no significant effect on blood total protein, albumin, globulin, AST, and ALT levels (Abdulrahman et al., 2022).
The non-significant improvement in the concentration of blood globulin in the present study with zeolite diets may be considered an indicator of improving animal immunity (Pavelić et al., 2018). Several mechanisms were reported to explain that positive effect, either by reducing the possibility of some metabolic diseases in animals such as diarrhea and hypocalcemia (Stojković et al., 2005) or due to the detoxification property of zeolite as a result of its ability to bind several harmful substances as mycotoxins, radioactive elements and poisons (Katsoulos et al., 2015). Also, the improvement in blood globulin levels by incorporating OP in the diets may be attributed to the steroidal flavonoids content in OP that stimulates cortisone secretion, as 80–90% of cortisone was found to bind with globulin (Gardill et al., 2012).
In agreement with the current results, significant decreases in concentrations of blood cholesterol, triglycerides (Abbasi et al., 2015), and LDL (Hassan et al., 2021) either with dried OP or OP extract inclusion in broiler or rabbitsʼ diets, in the same order. However, OP had no significant effect on cholesterol and triglyceride concentrations in other studies (Abdulameer, 2018; Suliman et al., 2019). The ascorbic acid content in OP was found to have a role in preventing lipids peroxidation (Padayatty and Levine, 2016). Also, its antioxidant effect which reduces cholesterol and triglycerides levels in blood and tissues may be attributed to its role in cholesterol hydroxylation in bile acid, because it acts as a cofactor of the 7-alpha-hydroxylase enzyme (Samman et al., 1997). Polidori et al. (2004) declared the inhibitory effect of ascorbic acid on LDL oxidation due to the reduction in levels of lipid peroxide in blood. Moreover, Njus et al. (2020) showed the ability of ascorbic acid to reduce the development of hypercholesterolemia in rabbits. Furthermore, it was indicated that the isoflavones content in orange peel could affect lipid metabolism as a result of reducing cortisol levels (El-Shazly et al., 2017), regulating cholesterol homeostasis (Medjakovic et al., 2010), upgrading LDL receptors (Fukuchi et al., 2008) and stimulating gene expression of essential protein and enzymes in lipid metabolism (Ezekwesili-Ofili and Gwacham, 2015). Another explanation for reducing cholesterol and triglycerides with OP in its pectin content prevents bile acid reabsorption and increases lipase activity as a result (Bok et al., 1999). Moreover, it was evident that citrus flavonoids such as naringin and hesperidin play a role in the reduction of cholesterol and triglycerides due to the inhibition of hepatic enzymes (Lee et al., 2003) or regulation of glucose metabolism and lipogenesis (Evans et al., 2004). On the other hand, it was suggested that zeolite can reduce the total cholesterol level in the blood by adsorbing bile salts in the digestive tract, which subsequently increases the bile salts synthesis (Prvulović et al., 2007). Also, the hypocholesterolemic effect of the zeolite may be explained by the alteration in the rate of dietary cholesterol absorption (Alexopoulos et al., 2007) or due to short-chain fatty acid absorption by zeolite (Ly et al., 2007).
However, T-AOC did not significantly alter either with OP or zeolite diets, the antioxidant status of rabbits was improved as a result of significant (P < 0.05) reduction in MDA by 22.38, 29.94, and 38.66% in OP, Z, and OPZ groups, respectively. Similarly, Hassan et al. (2021) reported a non-significant effect of OP on T-AOC. On the other side, T-AOC was significantly increased with zeolite addition only when broilersʼ diet was contaminated with aflatoxin B1 (Abdulrahman et al., 2022). It was confirmed that citrus contains more than 60 flavonoids (Benavente-Garcia and Castillo, 2008). The digestion of these compounds in the small intestine can result in increasing their level in the blood (Walsh et al., 2009). These flavonoids showed anti-inflammatory, antioxidant, and immune stimulation effects (Harborne and Williams, 2000). A relationship between these phenolic compounds and T-AOC was demonstrated by Lu et al. (2018). Moreover, Anticona et al. (2020) reported high antioxidant activity in orange peel extract. The higher plasma T-AOC with dried OP diets may be ascribed to the content of phenolic compounds in OP that may inhibit the free radical activity and protect the animal tissues from lipid peroxidation (Zheng et al., 2009), or show a metal chelation activity (Andjelković et al., 2006).
Malondialdehyde (MDA) the oxidative stress marker, is produced as a result of lipids (polyunsaturated fatty acids) peroxidation (Kasperska-zajac et al., 2008). Qu et al. (2019) indicated the role of zeolite as an antioxidant feed additive in the reduction of MDA concentration in broilers. The reduction effect on lipid peroxidation may be attributed to the zeolite properties as ion exchange, adhesion, adsorption, and cation binding such as binding with oxygen, the one of lipid peroxidation substrates (Pavelic et al., 2002). Consistently, Wu et al. (2013a) demonstrated a significant decrease in MDA with 2% zeolite supplementation in broilers' diets. Also, supplementation of turkey diets either with 1 or 2% zeolite significantly reduced MDA concentrations in both meat and liver (Hcini et al., 2018). The same authors explained the detoxification effect of zeolite which decreases lipid peroxidation as a result of free radical scavenging.
Cecum characteristics
In accordance with the current results, Ferrer et al. (2021) reported a reduction in the pH of pigsʼ slurry when OP was included in diets either dehydrated or sun-dried silage. Higher fermentation of dietary fibers with OP incorporation, which subsequently increases VFA production (Noblet and Goff, 2001), may be the reason for lower pH values. Also, Wu et al. (2013b) reported lower cecal pH values with synthetic zeolite, while no significant difference was observed in pH value between the natural zeolite-supplemented group and control. The high affinity of zeolite for active cations and water was suggested to improve osmotic activity and fermentation process in the gut, which may regulate pH by acting as a buffer for hydrogen ions produced from organic acids fermentation (Dschaak et al., 2010). Furthermore, a relationship between the viscosity of ingesta, its density, and pH value was observed. As viscosity increases, as a result of zeolite administration, the ingesta density and intestinal pH decrease (Ismail et al., 2011).
Consistent with the current data of cecum microbiota, Alefzadeh et al. (2016) recorded significant decreases in E. coli with 2 or 4% orange peel powder included in broilersʼ diets. In the same context, Abbasi et al. (2015) reported significant decreases in cecal E. coli when broilersʼ diets contained 0.5% dried OP. However, the same authors showed that using dried OP at 1, 1.5, or 2% of the diet had no significant effect on E. coli count. That reduction effect of OP could be related to the inhibitory effect of OP essential oils on E. coli count (Lee et al., 2014). Moreover, the saponins, tannins, essential oils, phenolic compounds, and flavonoids contents in both citrus pulp and peel were found to have antibacterial activity (El-Desoukey et al., 2018), which can inhibit the synthesis of cell protein (Shetty et al., 2016). Consistently, zeolite addition exhibited a reduction in E. coli counts either in the pigsʼ diet (Wang et al., 2021) or in the broilersʼ diet (Wu et al., 2013b). The lower count of E. coli with zeolite may be a result of good microbial environmental balance, or due to the catalytic property of zeolite that can enhance the activity of enzymes, alter the pH of the digestive tract, and improve lumen ionic composition (Wang et al., 2021).