**Omega-3 and Omega-6 Fatty Acids in Poultry Nutrition: E**ff**ect on Production Performance and Health**

**Mahmoud Alagawany 1,\*, Shaaban S. Elnesr 2, Mayada R. Farag 3, Mohamed E. Abd El-Hack 1, Asmaa F. Khafaga 4, Ayman E. Taha 5, Ruchi Tiwari 6, Mohd. Iqbal Yatoo 7, Prakash Bhatt 8, Sandip Kumar Khurana <sup>9</sup> and Kuldeep Dhama 10,\***


Received: 17 July 2019; Accepted: 16 August 2019; Published: 18 August 2019

**Simple Summary:** In this review, we discuss previous studies, state-of-the-art technology, and the potential implications of utilizing omega-3 and omega-6 fatty acids in poultry diets, as well as the application of these fatty acids in the poultry industry for improving poultry production and health. Essential roles are played by these fatty acids in development and metabolism, growth and productive performance, immune response and anti-oxidative properties, improving meat quality, bone growth and development, and improving fertility rates and semen quality.

**Abstract:** Omega-3 (ω-3) and omega-6 (ω-6) fatty acids are important components of cell membranes. They are essential for health and normal physiological functioning of humans. Not all fatty acids can be produced endogenously owing to the absence of certain desaturases; however, they are required in a ratio that is not naturally achieved by the standard diet of industrialized nations. Poultry products have become the primary source of long-chain polyunsaturated fatty acids (LC-PUFA), with one of the most effective solutions being to increase the accretion of PUFAs in chicken products via the adjustment of fatty acids in poultry diets. Several studies have reported the favorable effects of ω-3 PUFA on bone strength, bone mineral content and density, and semen quality. However, other studies concluded negative effects of LC-PUFA on meat quality and palatability, and acceptability by consumers. The present review discussed the practical application of ω-3 and ω-6 fatty acids in poultry diets, and studied the critical effects of these fatty acids on productive performance, blood biochemistry, immunity, carcass traits, bone traits, egg and meat quality, and semen quality in poultry. Future studies are required to determine how poultry products can be produced with higher contents of PUFAs and favorable fatty acid composition, at low cost and without negative effects on palatability and quality.

**Keywords:** omega-3; omega-6; fatty acid; nutrition; performance; antioxidant; egg and meat quality; fertility; immunity; health

#### **1. Introduction**

Fatty acids, especially essential fatty acids, are gaining importance in poultry feeding systems not only for improving the health and productivity of birds, but also because of our health-conscious society that prefers properly balanced diets to minimize adverse health issues [1–3]. Among various fatty acids, omega-6 (ω-6) and omega-3 (ω-3) fatty acids are proving indispensable in a properly maintained ratio for numerous biological [4,5], physiological [2], developmental [6], reproductive [7], and beneficial health functions [3,8,9]. Adequate supplementation of poultry diets with novel and beneficial feed additives or supplements is gaining importance as it significantly improves overall poultry production and performance as well as safeguards the health of birds [10–13]. In poultry production, the advantages of using oils in diets involves a reduction of feed dust and improvement in hydrolysis and absorption of the lipoproteins that supply fatty acids [14]. In addition, oils are the main source of energy for the birds and have the highest caloric value among all dietary nutrients. They can also enhance the absorption of fat-soluble vitamins, increase diet palatability, and improve the utilization of the consumed energy. Moreover, the rate of food passage through the gastrointestinal tract can be reduced, with subsequent better absorption of all dietary nutrients [15].

In the diets of humans, ω-6 and ω-3 are essential fatty acids. However, considerable modification in dietary patterns has resulted in alterations of the consumption of such fatty acids, with subsequent elevation in the consumption of ω-6fatty acids and a marked decrease in the consumption of ω-3 fatty acids. This modification has led to an imbalance in the ω-6/ω-3 ratio, which at 20:1 now differs considerably from the original ratio (1:1). Therefore, dietary supplements of foodstuffs such as eggs and meat are a clear alternative to increase the daily consumption of ω-3 to meet the recommended doses [16]. Foods that provide ω-6 fatty acids include soybean, palm, sunflower, and rapeseed oils, whereas foods that provide ω-3 fatty acids include certain nuts, and plant and fish oils [17–20]. Omega-9 fatty acids are not essential and are found in olive oil and animal fat [21]. Fish oil (FO) includes two types of ω-3 fatty acids: docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Certain vegetables, nuts, and seed oils include alpha-linolenic acid (ALA), which can be converted to DHA and EPA in the body, with linseed oil containing more than 50% ALA [22].

High doses of omega sources in the diet may have deleterious effects on humans, such as increased bleeding risk and higher levels of low density lipoprotein (LDL) cholesterol. Omega-3 fatty acids may influence heart rates. The consumption of high rates of fatty acids (e.g., ω-6) has been linked with a higher occurrence of health problems, such as type 2 diabetes, obesity, and coronary artery diseases [23]. Maintaining a proper ratio of ω-3 and ω-6 fatty acids not only improves performance, but also prevents these health risks. Devising the correct ratios requires the addition of oils having appropriate ω-3 and ω-6 fatty acid levels [24]. Omega-3 fatty acids EPA and DHA have shown many health benefits; they are helpful in fetal development and cardiovascular function, and prevent Alzheimer's disease [25]. In addition, they play a role in modulating immunity [26,27]. The ratio of n-6:ω-3 fatty acids also plays an important role in the immune response, production performance of broilers and designing meat enriched with ω-3 polyunsaturated fatty acids (PUFAs) [26,27]. The addition of FO to the poultry diet may yield poultry products (such as eggs and meat) that are enriched with ω-3, such as EPA and DHA. Additionally, FO is more effective than other vegetable oils [28]. However, many aspects of essential fatty acids are still unknown and their diverse functions and importance in health and production should be explored. The objective of the present review is to assess the influence of dietary ω-6 and ω-3 PUFAs on the productive performance, antioxidant properties, immunity, carcass traits, bones, egg and meat quality, and semen quality of poultry, as well as their limitations in the poultry industry.

#### **2. Beneficial Applications of** ω**-3 and** ω**-6 Oils in Poultry**

Adding ω-3 and ω-6 fatty acids to the diet has become more important recently [3,29]. For at least the past three decades, studies on the beneficial activities of long-chain PUFAs (LC-PUFAs) in biological processes have been conducted. Dietary intervention with ω-3 may influence chicken immunity and lead to the production of poultry products with health benefits for the consumer [30]. Therefore, research on broiler chickens has focused on the functional action of different LC-PUFA forms and their dietary levels on the metabolism of lipids in birds. Other than the LC-PUFA source, high levels of fatty acids, particularly of the ω-3 family, lead to accelerated lipid oxidation when broiler chickens are under oxidative stress because of genetic selection [31].

Using PUFAs in poultry diets significantly reduces the cholesterol and total lipid content in the blood and egg yolk. Several studies have been conducted to minimize the harmful effects of triglycerides and total cholesterol in poultry products (edible parts). Ahmad et al. [32] reported that the cholesterol content of eggs was decreased when birds were fed a diet supplemented with ω-3 fatty acids. Moreover, increasing dietary levels of FO and milled flaxseed improved the concentration of linoleic acid (LA), EPA, and DHA in the yolk, and the fatty acid deposition from FO was found to be two times greater than that from milled flaxseed when fed at the same dietary levels [33].

Designer eggs offer balanced ratios of PUFA: SFA (1:1) or ω-6/ω-3 PUFA (1:1). Omega-3 UFAs are important nutritional factors that modulate immune functions and are of great importance for nervous system development and for lowering blood platelet aggregation and the incidence of thrombosis, hypertension, and atherosclerosis, and have anti-tumor, anti-inflammatory, and cardioprotective effects [34]. The content of ω-3 fatty acids in eggs can be increased by supplementing the diets of laying hens with certain dietary supplements, such as flaxseed, fish oil, safflower oil, linseed, fish meal, or algae. Omega-3 fatty acids can be introduced to the human consumer's body through these designer eggs; they play an important role in the maintenance of the normal functioning of the body in that they protect the body from cardiovascular problems such as heart attacks. In addition, they can replace fish products in consumer diets [35]. Ebeid et al. [36] stated that hens fed a diet containing different concentrations of ω-3 PUFA showed a linear decrease and increase (*p* < 0.05) in egg yolk content of ω-6 PUFA and ω-3 PUFA, respectively, compared to the control hen group. The levels of ALA, EPA, DHA, and docosapentaenoic acid (DPA) were higher in the egg yolks of laying hens fed linseed meal and fish oil as dietary supplements than in the un-supplemented hen group [37]. Similarly, ALA was higher in the egg yolk of hens fed diets that contained ω-3 fatty acid dietary supplements than in the control bird group [32]. It has been previously concluded that laying hens can synthesize EPA and DHA from ALA during metabolic processes if ALA is present in adequate quantities [38,39]. However, in mammals, the synthesis rates of DHA from ALA are low compared to the dietary intake and tissue demand, with the estimation of percent conversion of ALA to DHA differing widely, ranging from 0% to 9.2%, supporting the conclusion that DHA synthesis from ingested ALA is not an efficient process in humans [40]. Moreover, metabolization of ALA in vivo is not adequate to improve meat quality in ω-3 LC-PUFA, and direct supplementation of the diet with ω-3 LC-PUFA is a better alternative to modulate an increase in beneficial fatty acids of broiler meat [41]. The efficiency of ALA conversion to ω-3 LC-PUFA derivatives and deposition in peripheral tissues might not be sufficiently high to improve the nutritional value of muscle. Because there is competition among the enzymes involved in the elongation and desaturation of both LA and ALA, high amounts of LA suppress the conversion of ALA to EPA or DHA; therefore, an optimal intake of LA relative to ALA is crucial for normal metabolism [42].

Regarding egg production performances, Buitendach et al. [43] investigated the effects of dietary fatty acid saturation on the production performance of laying hens at end-of-lay (58–74 weeks of age). These authors reported no significant differences in hen-day egg production, egg weight, egg output, feed efficiency, and body weights at end-of-lay. Similar results were reported by Cachaldora et al. [44,45] who concluded that dietary fatty acid saturation had no significant effects on the egg production performance of layer hens. In contrast, Shang et al. [46] stated that body weight gain, rate of egg production, egg

weight, and feed efficiency decreased linearly with an increase in dietary fatty acid unsaturation levels during the 8-week experimental period between 40 and 48 weeks of age. Yin et al. [47] reported a decrease in egg and body weights with an increase in dietary UFAs at 50–58 weeks of age. This decrease in the performance parameters of hens as recorded by Shang et al. [46] and Yin et al. [47] is mostly attributed to the fact that these authors used conjugated linoleic acid (CLA) at higher inclusion levels (up to 7.8%) to enhance the UFA profile of their experimental diets. CLA causes weight loss in humans [47]; therefore, it appears that this specific type of unsaturated fatty acid has a similar negative effect on the body weights of laying hens and consequently on egg production and egg size.

#### **3. Improved Growth and Productive Performance**

Growth and production performance of poultry are improved by supplementation of fatty acids or their sources. The supplementation of fats and oils (as an omega source) in limited amounts leads to better utilization of feed and energy, with subsequent improvement in growth and performance [48]. The body mass and percentage body mass gain of quails was improved via dietary supplementation of sunflower and soyabean oil for a 12-week period [49]. The feed conversion ratio (FCR) and growth performance of broilers were improved via dietary supplementation with sunflower and canola oil [14]. Smith et al. [50] stated that the supplementation of animal fat, corn oil, fish oil, and a blend of vegetable and animal oils did not affect the feed intake, but positively influenced the body gain and FCR in heat-stressed broilers compared to heat-stressed un-supplemented broilers. Jalali et al. [51] found that the addition of soybean oil (high in ω-3 PUFA) significantly (*p* < 0.05) improved the FCR and body weight gain of broilers during the total and growth rearing periods. Abdulla et al. [52] found that the supplementation of soybean oil in broiler diets increased the body weight and weight gain at 6 weeks of age compared with the supplementation of LO (*p* < 0.05).

Fébel et al. [53] noticed that the difference in diets supplemented with sunflower oil (SO) or lard was not significant for the growth performance of broilers. Supplementation of fish oil in poultry diets had no influence on feed consumption, live weight, or weight gain [48] compared with the control diet (without fat). Ebeid et al. [54] reported that dietary ω-3 PUFA in Japanese quail had no adverse effects on the growth performance, such as the final body weight, feed intake, or FCR. Raj Manohar and Edwin [55] declared that dietary ω-3 PUFA in quails had a significant (*p* < 0.01) influence on body weight gain and non-significant differences in feed intake and feed efficiency. In addition, Qi et al. [56] demonstrated that decreasing dietary ω-6/ω-3 improved FCR, with the best result obtained from the diet with 5:1 ω-6/ω-3. Puthpongsiriporn and Scheideler [57] reported that four dietary ratios (17:1, 8:1, 4:1, and 2:1) of LA to ALA had no significant effect on the body weight of chickens over 16 weeks. However, Ayerza et al. [58] observed a marked reduction in the body weight and FCR of broilers fed on chia and flaxseed (rich in ALA), which may be attributed to one or more of the anti-nutritional factors present in flaxseed. Crespo and Esteve Garcia [59], Newman et al. [60], and Ferrini et al. [61] reported that the digestibility of fat increased with increasing unsaturation; therefore, the effect of the type of fat on feed efficiency could reflect the degree of unsaturation. This is consistent with the improvement of growth performance noted by Zollitsch et al. [62], Huo et al. [63], and Lopez-Ferrer et al. [48] with increasing content of UFA.

Generally, a dietary supplementation of a PUFA-enriching ingredient has an improvement effect on live weight, weight gain, and FCR of poultry. However, no adverse effects on feed intake have been reported in almost all designed studies to date.

#### **4. Improved Immune Response and Anti-Oxidative Properties**

Fatty acid supplementation affects both immune and oxidative status in poultry. It can modulate immune system through both cellular and humoral immune responses. Proliferation, maturation, function and cytokine production of lymphocytes, heterophils and splenocytes are influenced by omega fatty acids, besides antibody production like IgM and IgG (Table 1). Similarly, neutralization of oxidants and an increase of antioxidants level directly or indirectly by fatty acids minimizes the risk

of oxidative stress. The immune response and oxidative mechanisms are interlinked and affect one another, hence modulation of one can impact the other. The supplementation of natural antioxidants has become a pressing research topic [64–66]. Different studies have confirmed the many favorable effects of dietary ω-3 PUFA, including anti-oxidative properties and lipid peroxidation, as well as immune response effects [36,67]. For instance, dietary ω-3 PUFA can modulate the immune response in poultry [68]. Ebeid et al. [36] found that dietary FO supplementation below a level of 35 g/kg in the diet induced antibody titers in hens. The levels of antibodies were higher in laying hens fed oils (FO or LO) rich in ω-3 PUFAs than those in laying hens fed oil rich in ω-6 PUFAs (maize oil) [69]. Ebeid et al. [54] stated that dietary ω-3 PUFA (FO or LO) had a positive influence on humoral immunity (*p* ≤ 0.05) at 42 days of age as measured by antibody titers against Newcastle disease virus (NDV) compared to the control diet. Al-Khalifa et al. [70] declared that SO replaced by fish oil at low levels showed no evidence of adverse effects on the immune function of broilers. Jameel et al. [71] showed that chicks fed a diet supplemented with FO had significantly higher (*p* ≤ 0.05) antibody titers and percentages of spleen and bursa than that of the control group.

One of the earliest studies on the effects of fatty acid supplementation on immune tissues was by Fritsche et al. [72]. They noted that the supplementation of diets rich in ω-3 fatty acids to chicks decreased levels of arachidonic acid (AA) (C20:4n-6) in the serum and immune tissues by 50–75%. However, the levels of EPA (C20:5n-3) and DHA (C20:6n-3) increased [72], suggesting an influence on the immune system. During the early stages of life, ω-3 and ω-6 fatty acids are more important for immunity in chicks as they play a role in cellular and humoral immunity, and are regulators of inflammation [1,73]. They determine the immunoglobulin G (IgG) content of chicks produced by maternal hens, which are essential for passive immunity [74]. An inflammatory role for these fatty acids in delayed-type hypersensitivity has also been reported [75]. Further, they help in maintaining membrane integrity, thus preventing pathogen entry or infection.

Wang et al. [68] noted that the LA to ALA ratio may influence IgG-receptor activity in yolk sac membranes and thereby influence the maternal-embryo transfer of yolk IgG. Adding fish oil to broiler diets significantly induced antibody titers for the LaSota vaccine at 35 days of age after vaccination against Newcastle disease because of ω-3, which plays a role in the production of immunomodulators (leukotriene and prostaglandin). In addition, fish oil has the capacity to modulate the production of cytokines via signal transduction and lymphocytes in a population of immune cells [76]. Al-Mayah [76] showed that chickens fed a diet supplemented with fish oil at a level of 50 g/kg showed a higher production of antibodies (IgM and IgG) and globulins in the serum and maintained immune function after vaccination compared to the control group.

A moderate intake of ω-3 PUFAs enhances anti-oxidative properties, such as glutathione peroxidase (GSH-Px) activity, in laying hens [36] and decreases lipid peroxidation in abdominal fat and serum [36,67]. Ebeid et al. [54] reported that adding FO and LO at a level of 20 g/kg to Japanese quail diets significantly increased both the total antioxidant capacity and GSH-Px activity, and decreased the thiobarbituric acid reactive substances in the serum compared with the negative control. SO-enriched diets led to a reduction in the deposition of abdominal fat [77]. Additionally, adding SO and LO to the diet of birds led to a greater decrease in abdominal fat deposition than that observed after adding olive oil and tallow [67]. The addition of SO in broiler diets significantly increased (*p* < 0.05) the relative weight of the abdominal fat pad [51], whereas the abdominal fat of broilers was decreased with fish oil [64,78]. Diets high in ω-3 fatty acids increased the incorporation of these fatty acids into tissue lipids, leading to oxidative stress in cells [30]. A diet enriched in ω-3 PUFA improved the gene expression of lipin-1, which regulates triglyceride synthesis, in chicken abdominal fat [79]. Ibrahim et al. [26] stated that FO and LO supplementation had a low but significant effect (*p* ≤ 0.05) on the malondialdehyde (MDA) concentration in broilers. Reducing the ratios of ω-6:ω-3 PUFAs were found to be linked to a significant (*p* ≤ 0.05) induction of glutathione S transferase (GSH-ST). Moreover, superoxide dismutase, GSH-ST, and cardiac GSH-Px activities were augmented in the ω-3 PUFA-rich treatment, and MDA was reduced [79]. Omega-3 PUFAs have shown beneficial immune responses in infectious bursal disease

challenged broilers [80]. However, despite the noted improvements, these fatty acids must be evaluated and properly monitored for ratios to prevent adverse effects on immune status [70].

#### **5. Improving Egg Quality and Nutritional Value of Eggs**

The nutraceutical value and health benefits of eggs can be enhanced by adapting appropriate feeding strategies in poultry as well as by developing designer eggs [10,12,20]. These improve the quality and quantity of eggs [33]. Eggs are not naturally rich in ω-3 PUFA; therefore, ω-3 PUFA supplementation in poultry rations is required to obtain enriched ω-3 PUFA eggs [81,82]. Designer eggs are enriched in ω-3 fatty acids for beneficial health effects in human nutrition [12,83]. Designer eggs offer balanced ratios of PUFA: SFA (1:1) or ω-6/ω-3 PUFA (1:1) and provide more than 600 mg of ω-3 PUFA [34]. The content of ω-3 fatty acids in eggs can be increased by supplementing the diets of laying hens with certain dietary supplements, such as groundnut oil, fish oil, safflower oil, linseed, fish meal, or algae [15–18,58]. Omega-3 fatty acids include EPA, DPA, DHA, and linolenic acid (LNA), whereas AA and LA are examples of ω-6 fatty acids. Omega-3 fatty acids can be introduced to the body through designer eggs [35]. Omega 3-PUFAs serve as good fats for human health, therefore increasing PUFA contents in the egg yolk helps to decrease the bad cholesterol content [84]. The stability of ω-3 PUFAs can be improved by vitamin E and/or organic selenium, which reduces oxidation in raw eggs; thus, these confer protective effects during the marketing, storage, and cooking of ω-3 enriched eggs [85,86].

Meluzzi et al. [87] reported that the key ω-3 and ω-6 PUFAs are LNA and LA, respectively. LNA is metabolized inside the body to EPA, DHA, and DPA, whereas LA is metabolized to AA. LNA was higher in the egg yolk of hens after feeding them diets that contained ω-3 fatty acid dietary supplements than those of the control birds [88].

Ceylan et al. [89] evaluated the effect of dietary supplementation on two levels (15 g/kg and30 g/kg diet) of SO, rapeseed oil, and LO for 12 weeks in laying hens. They concluded that egg production, egg weight, feed intake, FCR, and live weight were not significantly affected by the treatments. However, hens receiving SO produced less intensively colored egg yolks than those receiving other oils in their diet (*p* < 0.01). Moreover, the composition of fatty acid in egg yolks was significantly (*p* < 0.01) affected by the treatment, whereas the cholesterol content was not influenced. There was a significant (*p* < 0.05) interaction between fat source and the level of inclusion in the diet, and LNA content was increased when hens were fed diets with linseed and rapeseed oil (30 g/kg diet). In contrast, da Silva Filardi et al. [90] studied the effects of the dietary inclusion (for 12 weeks) of different fat sources (cottonseed oil, soybean oil, lard, SO, or canola oil) on egg quality, and egg yolk lipid profiles. The different fat sources did not affect eggshell quality; however, the lipid profile of the egg yolk changed based on dietary fat sources. Optimal changes were considered to be lower levels of SFA and LA, and higher levels of ALA and DHA. Such changes were promoted by the addition of different fat sources, particularly canola oil; however, it did not enhance the egg content of PUFAs.

#### **6. Improving Meat Quality**

In human diets, there is a marked reduction in ω-3PUFA and an imbalance in the ratio of ω-6/ω-3 PUFA. Currently, the ratio of ω-6 to ω-3 fatty acids is approximately 10 to 20:1 rather than the recommended ratio (1 to 4:1). The decrease in ω-3PUFA consumption is due to the low intake of sea fish, which are the major source of ω-3 PUFA. An accepted solution for this situation could be based on the production of suitable functional foods with adjusted PUFA content, which is generally accepted to confer nutritional effects and beneficial physiological properties. The enrichment of poultry meat with ω-3 PUFA may provide an excellent alternative source for such acids in the human diet because of their relative availability [87]. Schiavone et al. [91] illustrated that the content of lipids, protein, and moisture in breast meat was not significantly affected by the addition of fish oil to the diet of the Muscovy duck. Additionally, Ebeid et al. [54] reported that adding n-3 PUFA to Japanese quail diets had no significant influence on the content of crude protein, ash, and dry matter in the meat, whereas the addition of ether extract significantly influenced these parameters. Additionally, the physical traits of the meat except for the water-holding capacity were not significantly influenced when ω-3PUFA was added to the diet. Though the fatty acid composition of meat is influenced by ω-3 PUFA supplementation or their sources in diet, meat quality parameters like meat pH, tenderness, grilling loss, toughness, and juiciness are not affected [48,54,87,91]. This can be exploited for designing functional foods with adjusted PUFA and having no differences in palatability.

In broiler diets, replacing soybean oil with LO along with the addition of pomegranate peel extract enriched muscle meat with antioxidants and ω-3 and improved broiler immunity and their serum lipid profile [92]. Also, natural antioxidants, especially those extracted from herbal plants, have greater potential for increasing the stability, palatability, and shelf-life of meat products [93,94]. The meat quality of broilers improved with fish oil supplementation in the diet [78]. Inclusion of fish oil (FO) and different fat sources [linseed oil (LO), rapeseed oil (RO), sunflower oil (SO)] for providing different PUFA (ω-3 and ω-6 PUFA) in diets and their deposition into the eggs' fat revealed that smaller proportions of FO resulted in lower values of saturated and higher values of ω-6 FA contents. Replacing FO with LO showed the lowest turn down of its derivatives by elongation and desaturation and an increase in the total ω-3 FA in the form of linolenic acid [95]. The use of LO as ground or whole flaxseed before slaughter is recommended to broiler breeders and producers as a feeding strategy to optimize ω-3 enrichment, without compromising poultry performance [96]. The fat and cholesterol content in poultry meat may decrease because of dietary supplements, such as ω-3 PUFA, and high PUFA concentrations in the diet (addition of vegetable oils) decrease the storage stability of meat [97].

A proper ratio of ω-6:ω-3 fatty acids is essential for maintaining health, oxidative balance and quality of meat. Recently, Konieczka et al. [8] found that feeding birds with a diet containing a PUFA ω-6:ω-3 ratio exceeding the recommended levels resulted in damage to the intestinal epithelial cells. Further, low PUFA ω-6:ω-3 ratio diets increased MDA in tissues including the meat. This can affect meat quality owing to peroxidative changes [8]. Hence, these authors recommended balanced supplementation to prevent oxidative damage and loss of meat quality. Similarly, Kalakuntla et al. [6] noted that the supplementation of ω-3 PUFA-rich oil sources in the broiler diet during starter and finisher phases can affect fatty acid composition, quality, and organoleptic characters of broiler chicken meat. At 2% and 3% addition levels, mustard oil, fish oil, and LO improved ω-3 PUFA levels and sensory attributes such as the appearance, flavor, juiciness, tenderness, and overall acceptability of meat; however, due to an increase of thiobarbituric acid-reacting substances, the meat quality might be compromised as it might cause oxidative damage to meat.

The content of ω-3 fatty acids in poultry meat, especially as EPA and DHA, can be readily improved by increasing the levels of ω-3 PUFA in poultry diets via the inclusion of oily fish by-products [98]. Qi et al. [56] concluded that substituting ω-3 for ω-6 in the diets of chickens resulted in a significant effect on the subcutaneous and intramuscular fat content and on meat quality (color and tenderness).

Unfortunately, although poultry meat is considered one of the main potential sources of ω-3 LC-PUFA for humans, particularly in developed countries [99,100], there are some disadvantages related to meat oxidative stability. LC-PUFAs are very susceptible to oxidation, producing off-flavors and odors in meat that are often associated with a fishy flavor [101]. This oxidative instability can influence meat quality and, consequently, reduce acceptability to consumers [41,102,103]. Oken et al. [104] concluded that the supplementation of chicken diets with fish-derived products led to unacceptable odors in the product, which has restricted the adoption of this strategy [105]. Vegetable sources such as LO may clearly increase the ω-3 PUFA content in the form of ALA, which is the precursor of the entire ω-3 family [42].

Conclusively, dietary supplementation of ω-3 fatty acids in poultry diet, particularly in the form of EPA and DHA, can improve various parameters of meat quality. However, LC-PUFAs are extremely susceptible to oxidative deterioration, resulting in off-flavors and odors, which adversely affect acceptability to consumers, especially when fish-derived products are used.

#### **7. E**ff**ects of Dietary** ω**-3 and** ω**-6 Fatty Acids on Bones**

The ω-3 and ω-6 fatty acids or their sources like fish oil, linseed oil, soybean oil and palm oil have bearing effects on mineral metabolism and hence promote bone formation, growth and development. Fat supplementation in the diet influences mineral metabolism, especially calcium, zinc, and magnesium [106], because of insoluble soap formation between these minerals and fatty acids during digestion, which makes them unavailable [107]. This can affect mineral retention, and influence bone and eggshell quality in birds. Dietary lipids play a remarkable role in the growth, development, and formation of bones [106–108]. Sun et al. [108] reported that dietary fish oil supplementation led to significantly higher bone mineral density in the proximal tibia and distal femur than supplementation with maize oil. Some studies have reported non-significant correlations between fatty acid supplementation and bone characteristics. Baird et al. [109] reported that feeding laying chickens a diet high in ω-3 PUFA did not have a significant effect on bone morphological characteristics, bone mineral content, or bone mineral density. However, there are many studies that can prove interrelation of fatty acid supplementation and bone growth and development. Ebeid et al. [36] declared that using ω-3 PUFA in Japanese quail diets improved the tibia bone and morphological characteristics and, in quails fed diets supplemented with FO and LO at a level of 20 g/kg diet, there was increased tibia bone wall thickness, tibia diameter, and the percentage of tibia ash and tibia bone breaking strength compared with that in quails fed the control diet. Abdulla et al. [52] clarified that chicks fed a diet supplemented with LO had non-significantly higher ash percentage, tibia weight, and bone-breaking strength than those fed diets supplemented with SO and palm oil. In addition, ω-3 PUFA may improve bone health by inducing calcium absorption in the gut and inducing osteoblast activity and differentiation, decreasing osteoclast activity, and stimulating the deposition of minerals in developing bones [110]. Reproducible and consistent beneficial effects of ω-3 fatty acids have been observed for bone/joint diseases and bone metabolism [111]. Recently, the importance of yolk as a mineral source for chicks and possible alterations via interventions strategies for future usage has been analyzed [112]. The amount of minerals in yolk reflects that the uptake content and enrichment can have beneficial effects [113]. In-ovo supplementation of minerals has improved bone properties and development in hatchlings and mature broilers [113–115].

#### **8. Improved Fertility Rates and Semen Quality**

Fatty acid supplementation, especially of ω-3 and ω-6, helps in improving fertility, semen quality and quantity. Kelso et al. [114] found that dietary fish oil or corn oil supplementation to chickens at a level of 50 g/kg in their diet led to significantly higher (96%) fertility rates than the rates prior to supplementation (89%). Kelso et al. [115] noticed that the supplementation of ALA in male diets led to higher fertility at 39 weeks of age because of the increased ω-3 fatty acid proportion in the phospholipids of sperm. Cerolini et al. [116] reported that dietary FA supplementation can influence spermatozoa traits. Hudson and Wilson [117] stated that the supplementation of menhaden oil at 30 g/kg in the diets of male broiler breeders improved the quality of semen and increased fertility and hatchability. Bongalhardo et al. [118] reported that supplementing cockerel diets with fish oil improved fertility, which was attributed to the lower fatty acid ratio (ω-6:ω-3) in the membrane of spermatozoa that may change the membrane resistance to peroxidative damage or its physical characteristics [119].

Fertility and quality of sperms both have been found to be affected during cryopreservation, and fatty acids act as protectants for sperms. Cryopreservation of semen affects survivability, which to a greater extent is dependent on lipid content in spermatozoa [119]. Blesbois et al. [119] noted a decrease in cholesterol/phospholipid ratio in poultry sperms following cryopreservation and a relation with fluidity, hence affecting survivability. Fatty acids can prevent damage to sperms, whether it be physical (cryopreservation) or chemical (oxidative). Zaniboni and Cerolini [120] stated that the dietary ω-3 LC-PUFA treatment of turkey prevented the negative influence of sperm storage on sperm sensitivity and quality and promoted in-vitro peroxidation and sperm death. Additionally, dietary maize oil supplementation decreased the spermatozoa number per ejaculate by 50% between 26 and 60 weeks of age. Al-Daraji et al. [121] noticed that dietary fish oil supplementation produced the best results

for sperm concentration (*p* < 0.05) based on the ejaculate volume, live sperm, total sperm count, and sperm quality factors, followed by flax oil; however, the worst results for these traits were found with treatments of corn oil and SO. Al-Daraji [122] determined that the correlation between the spermatozoa number and glucose concentration in the seminal plasma was highly significant and negative, indicating that the spermatozoa utilized glucose. Additionally, Al-Daraji [123] noticed that spermatozoa used glucose in the seminal plasma for metabolism. Al-Daraji et al. [121] also clarified that dietary SO or corn oil supplementation had a significant effect (*p* < 0.05) on the semen glucose content, alanine aminotransferase activity, and semen protein content, followed by the results for flax oil and fish oil.

A diet supplemented with a moderate ratio of ω-3:ω-6 fatty acids increased DHA and ω-3PUFAs and decreased docosatetraenoic acid and AA in rooster sperm [124]. Sperm motility, progressive motility, membrane functionality, and viability were significantly improved; the testosterone concentration increased; and a higher fertility rate was noted [125]. Feng et al. [7] reported no significant effect on the testis index; however, the spermatogonial development and germ cell layers and gonadotropin-releasing hormone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone hormone levels increased. Further, they reported that PUFAs regulate the expression of hormone receptors and steroid acute regulator protein (StAR). PUFAs significantly increased the mRNA levels of all hormone-related genes (GnRHR, FSHR, LHR, and StAR mRNA levels).

An overview of different dietary manipulations for improving nutritional quality of poultry products (egg and meat) is presented in Table 1.

#### **9. Conclusions and Future Perspectives**

The present review revealed that ω-3 and ω-6 fatty acids could be successfully utilized in poultry feeds to promote immune responses and improve the nutritional value of eggs, meat quality and growth in poultry. Omega-3 fatty acids have anti-inflammatory or inflammation-reducing properties because they can reduce the liberation of cytokines. Omega-6 fatty acids at high levels are associated with an increased prevalence of severe conditions, such as depression and heart disease. However, these fatty acids have a tremendous range of health benefits, including improved cholesterol levels and a reduced occurrence of coronary heart disease. Numerous studies have reported the favorable effects of ω-3 PUFA on bone strength, bone mineral content, and bone mineral density. Furthermore, the content of ω-3 fatty acids in eggs can be increased by supplementing the diets of laying hens with certain dietary supplements, such as groundnut oil, fish oil, safflower oil, linseed, fish meal, or algae. Dietary supplementation with different sources of ω-3 or ω-6 to cockerels improved the semen quality and increased fertility and hatchability. In the present review, we proposed that supplementing poultry diets with different sources of ω-3 and ω-6 fatty acids represented a potential strategy for poultry produced for human consumption. However, some disadvantages were related to meat oxidative stability, where LC-PUFAs were very susceptible to oxidation, resulting in off-flavors and odors in poultry meat, which negatively influence meat quality and acceptability by consumers. Therefore, future studies should investigate how we can produce poultry products with higher contents of PUFAs and favorable fatty acid composition, with low cost and without negative effects on palatability and quality, and subsequently on acceptability by consumers.

*Animals* **2019**, *9*, 573


**1.**Studiesshowingthedifferentdietarymanipulationtoimprovenutritionalqualityinpoultryproducts(egg


**Table 1.** *Cont.* buckwheat extract providing Omega-3: 0.08 g and Omega-6: 0.96 g (in each100 gm). \*\*\*\* Fish oil contain the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). \*\*\*\*\* One tablespoon of flaxseed oil is providing about 700 milligrams (mg) of EPA and DHA.

**Author Contributions:** Conceptualization, M.A. and S.S.E.; Review collection, M.A., S.S.E., M.R.F., M.E.A.E.-H., A.F.K., A.E.T., R.T., M.I.Y., P.B., S.K.K., and K.D.; validation, M.A., S.S.E. and K.D.; Writing—Original Draft preparation, M.A., S.S.E., M.R.F., M.E.A.E.-H., A.F.K. and A.E.T.; Writing—Review and Editing, M.A., R.T., M.I.Y., P.B., S.K.K., and K.D.; visualization, M.A., S.S.E., M.R.F. and K.D.; supervision, M.A. and S.S.E., M.R.F. and K.D.

**Funding:** This work did not receive any specific funding.

**Acknowledgments:** All the authors acknowledge and thank their respective Institutes and Universities for providing literature facilities.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


*Animals* **2019**, *9*, 573

144. Zhang, P.; Tang, C.; Ding, Z.; Huang, H.; Sun, Y. Effects of simultaneous supplementation of laying hens with α-linolenic acid and eicosapentaenoic acid/docosahexaenoic acid resources on egg quality and n-3 fatty acid profile. *Asian Australas. J. Anim. Sci.* **2017**, *30*, 973–978. [CrossRef]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Threonine Requirements in Dietary Low Crude Protein for Laying Hens under High-Temperature Environmental Climate**

**Mahmoud Mostafa Azzam 1,3,\*, Rashed Alhotan 1, Abdulaziz Al-Abdullatif 1, Saud Al-Mufarrej 1, Mohammed Mabkhot 1, Ibrahim Abdullah Alhidary <sup>1</sup> and Chuntian Zheng 2,\***


Received: 10 July 2019; Accepted: 6 August 2019; Published: 21 August 2019

**Simple Summary:** The threonine (Thr) requirement of laying hens in a high-temperature climate is scarcely referred in the review of literature. Therefore, our aim was to estimate the dietary Thr requirement in low CP diets in a high-temperature environmental climate. Based on our findings, the optimal dietary Thr requirements to optimize egg production, serum uric acid, and serum CuZn-SOD were 0.58%, 0.59%, and 0.56%, respectively, by regression analysis.

**Abstract:** Lohmann Brown hens (n = 420), at 28 weeks of age, were divided into five dietary treatments, and each treatment included six replicates of 14 laying hens. Dietary crude protein (14%) was presented as the control diet. Dietary L-Thr was added to the control diet for 12 weeks. Dietary Thr levels are 0.43%, 0.49%, 0.57%, 0.66%, and 0.74%, based on digestible base. From 28 to 40 weeks, hen-day egg production presented a quadratic trend to supplementing dietary Thr (R<sup>2</sup> = 0.96, *p* = 0.02), and reached a maximum level at 0.58%. Serum uric acid demonstrated a quadratic trend (R2 = 0.62, *p* = 0.02) at 0.59%. Both serum total cholesterol and 3-hydroxy-3-methylglutaryl (HMG-CoA) reductase showed lower levels (*p* < 0.05) at 0.66% Thr. Serum CuZn-SOD elevated (*p* < 0.05) at 0.49%, 0.57%, and 0.66% Thr, as compared to the control group, and showed a quadratic trend (R<sup>2</sup> = 0.87, *p* = 0.003) at 0.56%. Supplemental L-Thr decreased (*p* < 0.05) the expression of ileal HSP70 at 0.66% Thr. In summary, the optimal dietary Thr requirements to optimize egg production, serum uric acid, and serum CuZn-SOD were 0.58%, 0.59%, and 0.56%, respectively, by regression analysis.

**Keywords:** cholesterol; CuZn-SOD; HMG-CoA; HSP70; laying hens; L-Thr

#### **1. Introduction**

High temperatures negatively affect protein utilization efficiency [1]. In addition, diets that contain a high content of dietary crude protein (CP) will increase internal heat production by the elevated heat increment in a high-temperature climate [2]. Therefore, low levels of CP with supplementing limiting amino acids can overcome the bad effects of heat stress [3,4] and enhance protein utilization [5,6]. Recently, it has been reported that laying performance was equal among 14%, 15%, and 16% dietary CP [7]. Since synthetic dietary L-Thr became commercially available, it is possible to decrease CP. Thr affects protein synthesis and is the third limiting amino acid [8,9]. Recently, it has been reported that Thr is a limiting amino acid in diets containing 14% CP [10].

It has been showed that heat stress provoked lipid accumulation by elevated de novo lipogenesis, decreased lipolysis, and enhanced amino acid catabolism [11]. In addition, during stress times, the bird's body begins freeing heat shock proteins to secure itself from the harmful cellular effects of reactive oxygen species [12]. Heat stress is usually accompanied by increasing levels of 70 kilodalton heat shock proteins (HSP70) [13,14]. In addition, high temperatures disturb oxidative status [15] and increase serum total cholesterol, triglyceride, and zinc [16–20].

The present research aimed to estimate the dietary Thr requirement in low CP diets for laying hens in a high-temperature environmental climate. In addition, the effects of increasing Thr on lipid peroxidation, antioxidants enzymes activities, mineral levels, and HSP70 were investigated.

#### **2. Material and Methods**

#### *2.1. Management*

All procedures in this study were conducted according to the guide for the care and use of agricultural animals in research and teaching (American society of animal science and poultry science association, 2010), through research group (No. RG-1440-146).

Lohmann Brown hens (n = 420), at 28 weeks of age, and with almost similar live body weights (1800 g), were divided into five dietary treatments. Each treatment included six replicates of 14 laying hens (4 birds/cage; 471.5 cm2/hen). They were exposed to 16-h light. The study began during the middle of May and ended in August; it lasted 13 weeks, including one week for acclimation. The mean daily temperature and humidity are both presented in Figure 1.

**Figure 1.** Average temperature (◦C) and humidity (%) inside the experimental farm by week.

#### *2.2. Experimental Diets*

Hens were fed *adlibitum* (mash form), and water was available through nipples. Dietary CP (14%) was presented as the control diet (Table 1). Dietary Thr levels are 0.43%, 0.49%, 0.57%, 0.66%, and 0.74%, based on digestible base. Ingredient and analyzed CP and total amino acids are presented in Tables 1 and 2, respectively. L-Thr (98.5% purity) was supplied at the expense of kaolin (inert filler). Samples from each diet were analyzed for CP and amino acids according to [21]. Total amino acids in diets were analyzed using HPLC, as described by the authors of [10].


**Table 1.** The ingredients and nutrient level of the control diet.

**<sup>1</sup>** Premix per kilogram of diet: Vitamins (A, 12,000 IU; E, 20 IU; D3, 2,500 IU; K3, 1.8 mg; B1, 2.0 mg; B2, 6.0 mg; B6, 3.0 mg; B12, 0.020 mg; niacin, 25 mg; pantothenic acid, 10 mg; folic acid, 1.0 mg; biotin, 50 mg). Minerals per mg: Fe, 50; Zn, 65; Mn, 65; Co, 0.250. **<sup>2</sup>** Values of digestible amino acids were calculated according to (Rostagno et al., 2011).


**Table 2.** Amino acids (g/kg) in experimental diets.

**<sup>1</sup>** Values of digestible Thr were calculated according to (Rostagno et al., 2011).

#### *2.3. Laying Performance*

Mortalities were recorded daily. Egg numbers and egg weight were recorded daily. However, feed consumption was recorded weekly. Egg mass was calculated according to this formula (egg weight × egg production), while feed conversion ratio (FCR) was calculated according to grams of feed consumption/grams of egg mass produced.

#### *2.4. Blood, Liver, and Ileum Sampling and Laboratory Analyses*

At the end of the trial (40 weeks), 6 hens per treatment were slaughtered. The blood was collected and was centrifuged (3000× *g*) for 10 min. It was aspirated by pipette and stored in Eppendorf tubes at −70 ◦C. Serum concentrations of zinc (Zn), copper (Cu), triglyceride (TG), total cholesterol (CHO), glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT) were determined by kits from (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The levels of serum uric acid, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were analyzed by commercial kits from the previous company. The HDL-C levels in serum supernatant were determined after precipitation of lipoprotein-B using phosphotungstic acid/Mg2<sup>+</sup> (PTA/Mg2<sup>+</sup>).

Serum 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was determined following the manufacturer's protocols from Sigma (St. Louis, MO, USA).

After slaughter, the liver of each hen was collected immediately, snap-frozen with liquid nitrogen, and stored at −80 ◦C until analysis. About 0.5 g of liver of every bird was homogenized and analyzed for CHO, TG, GOT, and GPT, as described above in the serum.

#### *2.5. Oxidant and Antioxidant Status*

Serum levels malondialdehyde (MDA), superoxide dismutase (SOD), total antioxidative capability (T-AOC), and copper zinc superoxide dismutase (CuZn-SOD) were analyzed as described by [22,23]. Liver tissues were homogenized in ice-cold isotonic physiological saline to form homogenates at the concentration of 0.1 g liver/mL. The samples of liver tissues were homogenized and centrifuged, and the supernatants were collected to analyze MDA, T-AOC, and SOD.

#### *2.6. HSP70 mRNA Expression Assay*

Total RNA was isolated from 50 mg of ileum, according to the instructions (TRIzol; Invitrogen, Carlsbad, CA, USA). The quality of RNA was examined by both native RNA electrophoresis on 1.0% agarose gel and the UV absorbance at 260 nm and 280 nm. The cDNA was synthesized from 2 lg of total RNA by a reverse transcriptase at 42 ◦C for 60 min with oligo dT-adaptor primer, using the protocol of the manufacturer (M-MLV; Takara, Dalian, China). The abundance of mRNA was determined based on a Step-One-Plus Real-Time PCR (ABI 7500; Applied Biosystems, Foster, CA, USA). The PCR used a kit (SYBR Premix PCR kit; Takara, Dalian, China) as described by [10]. Average gene expression relative to the endogenous control for each sample was calculated using the 2−ΔΔCt method [24]. The calibrator for each studied gene was the average <sup>Δ</sup>Ct value of the control group (Table 3).


**Table 3.** Gene and primer sequence.

HSP70 means the 70 kilodalton heat shock proteins.

#### *2.7. Statistical Analyses*

Data (Replicate; n = 6) were statistically analyzed by one-way ANOVA (SPSS Inc., Chicago, IL, USA). Polynomial comparisons were applied to test for linear and quadratic responses of dependent variables to dietary Thr. Inflection points in response curves at increasing dietary Thr levels were calculated following [28]. To estimate the optimal Thr requirement, a quadratic regression equation based on 95% of the maximum or minimum response was used [29,30].

#### **3. Results**

#### *3.1. Laying Performance and Optimal Dietary Thr*

The results showed that both egg mass and hen-day egg production increased quadratically (*p* < 0.05) (Table 4).

From 28 to 34 weeks, hen-day egg production presented a quadratic trend to increasing dietary Thr (R2 = 0.97, *p* = 0.03) at 0.58%. In addition, hen-day egg production presented a quadratic trend (R2 = 0.96, *p* = 0.02), at 0.58% from 28–40 weeks (Table 5).


**Table 4.** Effect of graded levels of dietary Thr on laying performance of laying hens 1,2.

<sup>1</sup> Data are means of 6 replications with 14 hens/replicate; <sup>2</sup> Throughout the entire experimental period, <sup>2</sup> birds died.

**Table 5.** Estimations of the dietary Thr requirements based on quadratic regressions.


Y = Dependent variables; X = The dietary Thr level (%).

#### *3.2. Serum Biochemical Parameters*

Serum uric acid declined (*p* < 0.05) at 0.57% Thr (Figure 2) and showed a quadratic trend (R<sup>2</sup> = 0.62, *p* = 0.02) at 0.59% (Table 5).

Serum total cholesterol decreased (*p* < 0.05) at 0.66% dietary Thr. Serum HMG-CoA reductase activity decreased (*p* < 0.05) at 0.49% and 0.66% dietary Thr (Table 6). No effects were observed in the liver for total CHO, HDL-C, HDL-C, and triglyceride.


**Table 6.** Effect of graded levels of dietary Thr on the levels of lipoproteins and activities and HMG-CoA reductase of laying hens 1,2.

<sup>1</sup> n = 6 hens/treatment; <sup>2</sup> means with different superscripts; a,b differ (*p* < 0.05).

**Figure 2.** Effect of Thr levels on the levels of serum uric acid. Values are means ± standard SEM. Means on each bar with no common letter differ (*p* < 0.05).

Serum T-SOD increased (*p* < 0.05) at 0.49% dietary Thr. In addition, serum level of CuZn-SOD elevated (*p* < 0.05) from 0.49% to 0.66% dietary Thr (Table 7) and showed a quadratic trend (R<sup>2</sup> = 0.87, *p* = 0.003) at 0.56% (Table 5).

Graded levels of dietary Thr did not affect serum or liver concentration of T-AOC, MDA, Zn, Cu, GOT, and GPT (Table 7).


**Table 7.** Effect of graded levels of dietary Thr on the levels of antioxidants in the liver and serum of laying hens 1,2.

<sup>1</sup> n = 6 hens/treatment; <sup>2</sup> means with different superscripts; a,b differ (*p* < 0.05).

#### *3.3. Ileal HSP70 mRNA Expression*

The expression of ileal HSP70 decreased (*p* < 0.05) at 0.66% Thr (Figure 3).

**Figure 3.** Effect of graded levels of dietary Thr on mRNA expression of ileal HSP70. Values are means <sup>±</sup> SEM. means with different superscripts; a,b differ (*<sup>p</sup>* <sup>&</sup>lt; 0.05).

#### **4. Discussion**

It is important to formulate accurate diets to meet the requirements of laying hens because feed ingredients are expensive. In addition, laying hens have been selected for massive egg production, resulting in greater metabolic activity and reduced thermo-tolerance [31,32]. Heat stress has adverse effects on laying hens [33–35]. In addition, high temperatures increase the hens' discomfort and lead to behavioral and endocrinological changes.

In the present study, rapid panting was noticed. In addition, the expression of ileal HSP70 protein decreased (*p* < 0.05) at 0.66% Thr. It has been reported that heat shock protein protects birds from high temperatures by preventing unwanted protein aggregation and channelizing their degradation [36]. The expression of mRNA HSP70 was measured in the gut [14,37], liver [38], hypothalamus [27,39], and blood and feather [40]. Here, we focused on detecting HSP70 in ileum because it plays a vital role

in digestion and absorption, as well as immunity. In addition, the effects of Thr on intestinal function were known [41,42], and the effects of heat stress on gut function become obviously clear.

Both serum CHO and serum 3-HMG-CoA reductase decreased significantly (*p* < 0.05) at 0.66% dietary Thr. It has been reported that 3-hydroxy-3-methylglutaric acid (HMG) is a potent agent for reducing serum triglyceride and cholesterol concentrations [43,44]. It has been reported that HMG causes a 40% to 50% reduction of [1–14C] acetate incorporation into cholesterol in male rats [45]. In addition, HMG inhibited fatty acid synthesis in vivo [46]. In vitro, HMG inhibited 3-HMG-CoA reductase [mevalonate: NADP oxidoreductase (CoA-acylating), EC 1.1.1.34] and interfered with the enzymatic steps involved in the conversion of acetate to HMG-CoA [46]. Taken together, the data suggest that dietary Thr level affect CHO, especially biochemical pathways in which HMG-CoA reductase is involved. Our findings are in agreement with the results of the previous study in broiler chickens [47]. They found that plasma CHO levels decreased significantly (*p* < 0.05) when dietary Thr was sufficient [47]. Here, we did not find a decrease in total CHO levels in the liver. Recently, it has been reported that Thr supplementation did not have an effect (*p* > 0.05) on hepatic cholesterol in Pekin ducks [48]. They suggested that dietary Thr supplementation enhanced hepatic lipid metabolism by regulating lipid synthesis, transport, and oxidation. It has been reported that there is no relationship between the plasma CHO level and the level of yolk cholesterol [49–51], and, consequently, liver CHO.

The levels of GOT and GPT did not change due to treatments. The enzymatic activity of GOT and GPT are indicators of liver health. These enzymes are elevated in acute hepatotoxicity, but they are decreased with prolonged intoxication [52].

In the present study, dietary Thr at 0.49% increased serum levels of T-SOD (*p* < 0.05). In addition, dietary Thr at 0.49%, 0.57%, and 0.66% increased the levels of CuZn-superoxide dismutase (Cu-ZnSOD). The present result suggests that Thr may promote the antioxidative ability of laying hens. Previous studies [22,53] also found that supplemental amino acids (L-Thr and L-Trp) increased T-SOD in serum and the liver.

Uric acid is the metabolic product of protein metabolism and has been suggested as a dominant scavenger of free radicals [54]. We found that level of serum uric acid was declined at 0.57% Thr, which confirms that sufficient Thr increases amino acid utilization. Thr is considered a limiting amino acid in low CP diets [55,56], affecting utilization of TSAA and Lys [57]. It has been reported that the levels of plasma uric acid and excreta were higher from increasing CP than from lowering CP in the diet [58]. In addition, a decrease in the level of uric acid excretion was reported with supplementing limiting amino acids, which indicated better N utilization [59,60]. The plasma urea nitrogen and uric acid have been used to estimate amino acids requirements in swine and broilers [61–63].

It has been reported that laying performance decreased by feeding low CP diets [64]. Here, reduction CP (14%) in the control group reduced egg production quadratically. The effect of the low crude protein diet was pronounced clearly during the late cycle of laying production (43–63 weeks of age) [65]. This study was conducted during the first cycle of egg production (28–40 weeks).

Increasing dietary Thr to 0.57% improved egg production quadratically. It has been found that egg mass and hen-day egg production were reduced (*p* < 0.05) by feeding hens a Thr deficient diet [66]. This means that increasing recognition of Thr as a critical amino acid in the diet of laying hens fed a low CP diet under high-temperature environmental climate. The current results showed that 0.58% dietary Thr based on quadratic regressions guaranteed the best egg production. The present results are in agreement with [67]. They estimated that Thr requirement was 0.57% of dietary Thr from 24 to 40 weeks in Hy-Line W36.

Egg weight, feed consumption, and FCR were similar among dietary Thr levels. Previous studies reported no effect of Thr levels on the egg weight and FCR in laying hens [68–71]. It has been indicated that total Thr deficiency beyond 0.42% decreased feed intake [72].

#### **5. Conclusions**

From 28 to 40 weeks of age, the optimal dietary Thr requirements to optimize egg production, serum uric acid, and serum CuZn-SOD were 0.58%, 0.59%, and 0.56%, respectively, by regression analysis. In addition, serum total cholesterol, serum HMG-CoA reductase, and expression of ileal HSP70 decreased at 0.66% Thr.

**Author Contributions:** Data curation, M.M.A., R.A., A.A.-A., and M.M.; formal analysis, S.A.-M. and M.M.A.; investigation, M.M.A., A.A.-A., I.A.A., and C.Z.; methodology, M.M.A., R.A., I.A.A., and C.Z.; project administration, M.M.A.; writing—original draft, M.M.A.; writing—review and editing, M.M.A.

**Funding:** This research article was funded by the Deanship of Scientific Research at King Saud University, through research group No (RG-140-146).

**Acknowledgments:** The authors extend their acknowledgment to the Deanship of Scientific Research at King Saud University for funding this work through research group No (RG-1440-146).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **E**ff**ect of Supplemental Cyanocobalamin on the Growth Performance and Hematological Indicators of the White Pekin Ducks from Hatch to Day 21**

#### **Zaheer Ahmad, Ming Xie, Yongbao Wu and Shuisheng Hou \***

Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China **\*** Correspondence: houss@263.net; Tel.: +86-137-0126-1697

Received: 23 June 2019; Accepted: 25 August 2019; Published: 30 August 2019

**Simple Summary:** Vitamin B12 plays a key role in the normal functioning of the brain and nervous system as well as creation and regulation of nucleic acids (DNA and RNA). Furthermore, vitamin B12 plays a significant role in fatty acid metabolism and energy generation. Deficiency of vitamin B12 in animals may lead to weakness and anemia because it is involved in the formation of hemoglobin, which transports oxygen to body cells and red blood cells. Similarly, deficiency of vitamin B12 may also lead to hyperhomocysteinemia (increased level of homocysteine in the blood) which may depress immunity and cause cardiovascular diseases. There is no literature available regarding cyanocobalamin requirement for Pekin ducks. Therefore, the aim of our study is to determine its requirement. However, we find that cyanocobalamin has no influence on growth performance (weight gain), but it has more effect on hematological indicators (blood). On the basis of growth performance and hematological indicators we suggest that 0.02 mg cyanocobalamin/kg of feed is the dietary requirement of male Pekin ducks from hatch to day 21.

**Abstract:** The experiment was conducted to evaluate the requirement of cyanocobalamin of male Pekin ducks from hatch to 21 days of age. A total of three-hundred-eighty-four, one-day-old meat-type male Pekin ducks were randomly allocated to six treatments, i.e., dietary cyanocobalamin (vitamin B12) concentrations of 0.00, 0.02, 0.04, 0.06, 0.08 and 1.00 mg/kg, respectively in their feed. Each treatment had eight replicated pens with eight ducks for each pen. Feed and water were provided ad libitum. The experiment was conducted for 21 days. Different growth parameters including average daily weight gain (ADG), average daily feed intake (ADFI), feed conversion ratio (FCR), and hematological indicators were evaluated because, on the basis of hematological indicators, the health and nutritional status of an animal can be accessed. It is observed that supplemental cyanocobalamin has no significant effect on ADG, ADFI, and FCR but it improves hematological parameters such as white blood cells, red blood cells, and its indices and platelet counts compared to the control group (*p* < 0.05). On the basis of growth performance and hematological indicators it is concluded that 0.02 mg cyanocobalamin/kg of feed is the dietary requirement of male Pekin ducks from hatch to day 21 of age.

**Keywords:** vitamin B12; pekin ducks; weight gain; feed intake; feed conversion ratio; hematological indicator

#### **1. Introduction**

Growth of ducks is faster than all other poultry species [1]. To bias the increased growth rate, these have been genetically selected like all the other poultry species [2]. Numerous studies have suggested that vitamin B12 is necessary for chick growth and egg hatchability [3–7]. Deficiency of vitamin B12 has a critical impact on growth and existence of rats. It is also reported that the addition of high levels of protein in a diet with a deficiency of vitamin B12 causes high mortality [8]. In another

study, Dryden and Hartman (1971) observed a steady increase in growth rate with every increased unit of protein content that might not be associated with the breakdown of surplus nitrogen but with complications in the disposal of the carbon skeleton of certain or total amino acids of added protein contents in vitamin B12 deficient diets [9].

The natural vitamin contents of diets depend upon ingredients and compositions that can vary considerably between particular feedstuffs based on the season, region, and processing conditions which can induce natural variations in vitamin contents. Natural dietary contents rarely provide the intakes thought to fulfill the needs of birds to meet their normal requirements and to provide a margin of safety to meet extra metabolic demands imposed by stress and other factors [10]. Whitehead (2002) [11] demonstrated that diets supplemented with vitamins play a vital role for disease prevention and treatment. He also illustrated that biological functions are carried out by the involvement of vitamins that allow an animal to use energy and proteins for growth, health, maintenance, reproduction, and feed conversion [11]. Poultry intake B12 either by feed supplementation or ingesting feces. This vitamin plays a vital role in the nervous system and proper brain functioning, homocysteine metabolism, energy metabolism, normal blood functions, cell division, and in the immune system [12]. Similarly it is also anti-anemic in function [13]. Vitamin B12 acts as a co-factor for L-methylmalonyl-CoA mutase and methionine synthase. Methionine synthase increases the rate of homocysteine conversion to methionine which is further required for DNA and RNA synthesis while L-methylmalonyl-CoA mutase converts L-methylmalonyl-CoA to Succinyl-CoA which is a crucial biochemical reaction in fat and protein metabolism. Succinyl-CoA is also required for hemoglobin synthesis [14].

Ceca of the birds synthesize vitamin B12 by using cobalt, but its production is lower than daily requirements, and suggestions were given to supplement diets with vitamin B12 [15]. In another study, researchers reported that for an increased growth rate and maximum feed utilization, a growing ration fortified with vitamin B12 had appreciable effects but it may not be optimum for achieving maximum feed efficiency [16]. Folic acid, vitamin B12, and dietary total non-structural carbohydrates have no effect on milk production and milk total solid yield. There was also a non-significant difference observed on average daily gains among the calves supplemented with these vitamins [17]. Vitamin B12 deficiency also caused health problems in many ways, from hematological manifestations to neurological disorders, varied as leucoenia, numbness in fingers, unsteadiness of gait, fasciculation, and macrocytic anemia [18]. Erythrocytes depend on vitamin B12 for their maturation and proliferation. Therefore vitamin B12 deficient erythrocytes cannot mature, resulting in hemolysis and hyperbilirubinemia [19].

Blood parameters are indices of the internal environment of the living body and they also indicate the health status of ducks [20]. To improve animals' productivity, it is important to understand their physiology and hematological characteristics. For the establishment of a diagnostic baseline for blood characteristics, hematological studies are usually undertaken on farm animals as routine management practices [21,22]. Hematological constituents serve as authentic tools for monitoring animal health because they usually reflect the physiological reactions of an animal to its external and internal environment [23,24]. Hematological indicators are very important as they designate an animal's health and nutritional status [25]. These parameters in poultry are also sensitive to stress reactions [26]. Hematological indicators are considered to be biomarkers for the immune system [27]. There is a limited literature available for hemogram parameters in vitamin B12 deficiency [28]. That is why we investigate these hematological indicators on different levels of vitamin B12 in Pekin ducks. The National Research Council (NRC) (1994) also has no data regarding the vitamin B12 requirement of meat-type Pekin ducks for growth and hematological indicators. Therefore, one of the main goals of this study is to estimate the requirement of cyanocobalamin as a supplement for the growth performance and hematology of male Pekin ducks from hatch to 21 days of age.

#### **2. Materials and Methods Estimate**

#### *2.1. Study Design*

All procedures of this study were permitted by the Animal Care and Welfare Committee of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (No. IASCAAS-AE-03, No. 20180416). A dose-response experiment with six supplemental vitamin B12 levels (0, 0.02, 0.04, 0.06, 0.08 and 1.00 mg/kg) was designed. Vitamin B12 deficient basal diet was prepared (Table 1) and it was comprised of 0.00 mg/kg total vitamin B12, according to the data of feed ingredients of the NRC (1994).


**Table 1.** Composition of basal diet (% as fed).

#### *2.2. Chicks and Diets*

The basal diet was formulated first and then six experimental diets were added with various supplemental levels of crystalline vitamin B12. A total of 384 one-day-old male White Pekin ducks with average body weights around 54 g were divided into 6 experimental groups and each group was replicated 8 times with 8 birds per pen. These ducklings were reared in steel cages with plastic floors (200 × 100 × 40 cm), from hatch to 21 days of age. They were raised and allocated randomly to an environmental control shed. The birds were offered feed and water ad libitum. Provision of water was done by using a drip-nipple, and feed was offered in pellet form. Birds were provided 24-h lighting, and temperature was maintained at 33 ◦C with relative humidity at 20% from 1 to 3 days of age, then temperature was decreased steadily to room temperature while relative humidity gradually increased to 65% until birds were 21 days of age.

<sup>1</sup> Supplied per kilogram of total diet: Cu (CuSO4·5H2O), 8 mg; Fe (FeSO4·7H2O), 60 mg; Zn (ZnO), 60 mg; Mn (MnSO4·H2O), 100 mg; Se (NaSeO3), 0.3 mg; I (KI), 0.4 mg; Mg(MgO), 200 mg; K(K2CO3), 1500; choline chloride, 1000 mg; vitamin A (retinyl acetate), 4000 IU; vitamin D3 (Cholcalciferol), 2000 IU; vitamin E (DL-α-tocopheryl acetate), 20 IU; vitamin K3 (menadione sodium bisulfate), 2 mg; thiamin (thiamin mononitrate), 2 mg; riboflavin, 10 mg; pyridoxine hydrochloride, 4mg; calcium-D-pantothenate, 20 mg; nicotinic acid, 50 mg; folic acid, 1 mg; and biotin, 0.20 mg.2 The values were calculated according to the apparent metabolizable energy of chickens.

#### *2.3. Growth Performance and Hematological Parameters*

On the 21st day, the average daily weight gain, average daily feed intake, and feed conversion ratio (FCR) of ducks from every pen were calculated using the following formula.

$$\text{Feed conversion ratio (FCR)} = \frac{\text{Feed intake}}{\text{Weight gain}}$$

Feed intake and FCR were all corrected for mortality.

#### *2.4. Sampling and Analysis*

After fasting 12 h, two ducks from each pen were selected randomly for blood collection on the basis of average body weight gain of the corresponding pen. Three to four milliliters of blood were collected in an Ethylenediaminetetraacetic Acid (EDTA)-containing tube from the jugular vein of each selected bird. These blood samples were analyzed by using an automated hematological analyzer (ABX PENTRA DX 120, Munich, Germany).

#### *2.5. Statistical Analysis*

Data were analyzed using SPSS Statistics 22.0 (Statistical Packages for the Social Sciences, released December 2013, Armonk, New York, USA). The mean comparison test was applied by using Duncan's new multiple-range test to find out the significant differences among the applied treatments at 5% the level of significance (*p* < 0.05).

#### **3. Results**

#### *3.1. Growth Performance*

The effect of dietary cobalamin on average daily weight gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) of the experiment is described in Table 2. ADG, ADFI, and FCR showed non-significant results among the vitamin B12 supplemented groups and the control group (*p* > 0.05), as presented in Table 2.

**Table 2.** Effect of dietary vitamin B12 on average daily weight gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) in meat-type Pekin ducks (mean ± SD).


<sup>1</sup> Dietary treatments where C = control (without vitamin B12), all others are vitamin B12 supplemented groups. Each mean represents values from 8 replicates (8 ducks/ replicate).

#### *3.2. Hematology*

#### 3.2.1. White Blood Cells

On day 21, ducks from the control group had significantly lower (*p* < 0.05) values for the white blood cell count (WBC) and had significantly higher (*p* < 0.05) values for the percentage of intermediate cells (MID %) than the supplemented groups. However, there were no further significant differences among the vitamin B12 supplemented groups. The absolute value of granulocytes (GRA) and percentage of granulocytes (GRA %) were lower for the control (C) group as compared to the vitamin B12 supplemented groups, although these values were not statistically significant (*p* > 0.05). There were non-significant results observed for the absolute value of lymphocytes (LYM), percentage of lymphocytes (LYM %), and absolute value of intermediate cells (MID), as presented in Table 3.


**Table 3.** Effect of dietary vitamin B12 on white blood cell count and its indices in meat-type Pekin ducks (mean ± SD).

a,b Superscript letters show significant differences (*p* ≤ 0.05). <sup>1</sup> Dietary treatments where C = control (without vitamin B12), all other are vitamin B12 supplemented groups. Each mean represents values from 8 replicates (2 ducks/ replicate). WBC: White Blood Cells; LYM: Absolute value of lymphocytes; MID: The absolute value of the intermediate cell; GRA: Absolute value of granulocytes; LYM (%): Percentage of lymphocytes; MID (%): Percentage of intermediate cells; GRA (%): Percentage of granulocytes.

#### 3.2.2. Red Blood Cells

At the end of the experiment, ducks with different levels of vitamin B12 had higher significant (*p* < 0.05) values for red blood cells (RBC), hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and standard deviation of RBC distribution width (RDW-SD) compared to the control group (Table 4). By the end of the experiment, mean corpuscular hemoglobin concentration (MCHC) and coefficient of variation in RBC width (RDW-CV) values of the control ducks had increased more significantly (*p* < 0.05) than the treated ducks. The values of RBC, Hb, HCT, MCV, MCH, and RDW-SD of the control ducks were significantly lower (1.4 to 1.8 1012/L, 82.4 to 116.9 g/L, 0.17 to 0.26 L/L, 107.5 to 148.9 fL, 54.3 to 66.6 pg, and 43.2 to 63.9%) than supplemented ducks respectively. Results also concluded that vitamin B12 plays a vital role in the synthesis of blood necessary for the different proper and optimal types of functions for the ducks and their well-being (Table 4).

**Table 4.** Effect of dietary vitamin B12 on red blood cell count and its indices in meat-type Pekin ducks (mean ± SD).


a,b,c Superscript letters show significant difference (*p* ≤ 0.05). <sup>1</sup> Dietary treatments where C = control (without vitamin B12), all others are vitamin B12 supplemented groups. Each mean represents values from 8 replicates (2 ducks/replicate).

#### 3.2.3. Platelet Count

The values of the platelets indices by day 21 are shown in Table 5. The values of the platelet count, thrombocytocrit, and mean platelet volume of the control group had significantly lower values than the supplemented groups, (41.3 to 84.6 109/L, 0.05 to 0.11 L/L and 11.4 to 13.7 fL) respectively, whereas the values of platelet distribution width of the control group were significantly higher (51.9 to 22.9%) than treated groups (Table 5).


**Table 5.** Effect of dietary vitamin B12 on platelet count and its indices in meat-type Pekin ducks (mean ± SD).

a,b,c Superscript letters show significant difference (*p* ≤ 0.05). <sup>1</sup> Dietary treatments where C = control (without vitamin B12), all other are vitamin B12 supplemented groups. Each mean represents values from 8 replicates (2 ducks/replicate).

#### **4. Discussion**

The influence of different levels of vitamin B12 on growth performance and hematological analysis was studied in Pekin ducks during the starter phase (0–21 days). There is no data available on the requirement of vitamin B12 in the Pekin duck, also the literature regarding vitamin B12 and Pekin ducks is limited.

In our study there were no significant differences found for the average daily feed intake and average daily weight gain in the group without vitamin B12 supplementation and groups supplemented with vitamin B12 in the Pekin ducks, respectively. The mean ADG, ADFI, and FCR in the present study is similar to that described in ducks earlier with vitamin B12 [12]. Our results also match the findings of other researchers who found that increasing levels of vitamin E in the diet had no significant effect on live body weight gain, body weight gain, feed intake, and FCR [29].

Without supplementation of vitamin B12 in the feed, the ducks did not show any clinical signs related to vitamin deficiency because the liver can preserve vitamin B12 for long periods of time, even feeding a vitamin B12 deficient diet. They further illustrated that about 2–5 months may be required for the elimination of B12 preserved by hens to such an extent that progeny will hatch with a low reserve of vitamin B12 [30].

Vitamin B12 had no effect (Table 2) on growth performance (*p* > 0.05). This might be due to the short period of the experiment (21 days) and its very low requirement for proper growth. In the present study, ducklings hatched from parent flocks which were feeding according to the nutrient requirements of breeder ducks. Therefore, it can be assumed that the storage of vitamin B12 in the egg yolk was stabilized, and the freshly hatched ducklings had an optimum B12 depot in the liver and in the last part of the yolk sac, and that the liver maintained the level of vitamin B12 for 21 days in the ducklings even without its supplementation in the diet [12].

Supplementation of vitamin B12 had no significant effect on the production and fertility of eggs. It was also revealed that vitamin B12 deficiency in the breeder diet had adverse effect on growth of chicks and when the chicks were collected from breeders during hatching they were supplemented with vitamin B12 but their deficiency was fulfilled up to some extent. It is therefore concluded that the supplementation of vitamin B12 in the diet of hens was more effective than the chick diet for the growth in their early stage [7].

However, limited research work has been done on hematological profiles and limited information is available on these parameters in ducks. Vitamin B12 is essential for proper red blood cell production, neurological function, and DNA formation [31–34]. The effects of vitamin B12 on the hematological indicators are shown in (Tables 3–5). The hematological indicators weremeasured in WBCs, RBCs, and platelets after both the addition and subtraction of vitamin B12. Vitamin B12 supplemented groups showed significantly higher values than the control group (*p* < 0.05).

Vitamin B12 works as a co-factor for methionine synthase and L-methylmalonyl-CoA mutase. Methionine synthase speeds up the transformation of homocysteine to methionine. Methionine is mandatory for the creation of S-adenosylmethionine, which is a universal methyl donor for nearly 100 different substrates, including DNA, RNA, hormones, proteins, and lipids. L-methylmalonyl-CoA mutase converts L-methylmalonyl-CoA to Succinyl-CoA in the degradation of propionate, which is

a critical biochemical reaction in fat and protein uptake. Succinyl-CoA is necessary for synthesis of hemoglobin [35].

Dietary supplementation with a combination of probiotics and prebiotics markedly increased the PCV, RBC, and Hb of guinea fowls [36]. Molasses also had significant effect on MCV and platelets in broiler chickens during hot dry seasons. Presence of high MCV may indicate an active erythropoiesis, as MCV is thought to be the average size of an individual erythrocyte [30]. Younger erythrocytes are larger than older ones [37,38]. Betaine had highly significant effects on hematological parameters in the meat-type ducks under stress conditions; specially values of RBC, HCT, HGB, MCV, MCHC, RDW, platelets PLT, PCT, and MPV were significantly higher (*p* < 0.05) in betaine added groups as compared to the control group but no further significant differences were found among the betaine supplemented groups [39]. Similarly the control group had significantly lower (*p* < 0.05) values for RBC, Hb, PCV, MCV, and MCH in different breeds of Omani goats from treated groups which were receiving dietary cobalt and cutaneous injections of hydroxycobalamin [40]. Hemorrhagic anemia and hemolytic anemia are caused by the decreased value of RBCs, whereas the decrease of hemoglobin causes microcythemia. It is also reported that hematocrit and packed red cells volume have an immense correlation with each other. Consequently, when the mean hematocrit decreases, the levels of hematoglobin and hemoglobin becomes lower [39]. In a study, MPV was found to be lower in patients with vitamin B12 deficiency than in the patients without vitamin B12 deficiency as a result of production of smaller platelets [18,41].

#### **5. Conclusions**

To the best of our knowledge, this is the first time that someone determined the dietary requirement of cyanocobalamin for male Pekin ducks during the starter phase on the basis of growth performance and hematological indicators. However, we found that cyanocobalamin had a non-significant effect on growth performance, but it had more significant improvements on hematological indicators. On the basis of our findings we suggest that 0.02 mg cyanocobalamin/kg of the feed may be supplemented to improve hematological variables of Pekin ducks from hatch to day 21.

**Author Contributions:** The study was designed and conducted by Z.A., S.H., M.X. and Y.W. Z.A. performed the study, analyzed the data and wrote the manuscript and S.H. reviewed the manuscript.

**Funding:** This work was supported by the earmarked fund for the China Agriculture Research System (CARS-42), the science and technology innovation project of the Chinese Academy of Agricultural Sciences (CXGC-IAS-09).

**Acknowledgments:** We acknowledge the technical support provided by Muhammad Humza, Muhammad Naeem and Sadiq Shah.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### **E**ff**ect of Chestnut Tannins and Short Chain Fatty Acids as Anti-Microbials and as Feeding Supplements in Broilers Rearing and Meat Quality**

**Federica Mannelli 1,\*,**†**, Sara Minieri 2,**†**, Giovanni Tosi 3, Giulia Secci 1, Matteo Daghio 1, Paola Massi 3, Laura Fiorentini 3, Ilaria Galigani 1, Silvano Lancini 1, Stefano Rapaccini 1, Mauro Antongiovanni 4, Simone Mancini <sup>2</sup> and Arianna Buccioni 1,5**


Received: 20 June 2019; Accepted: 2 September 2019; Published: 5 September 2019

**Simple Summary:** The poultry industry needs to replace antibiotics with natural or synthetic compounds able to overcome problems linked to the development of bacterial resistance. Tannins and short chain fatty acids are valid alternatives to contrast the growth of pathogens. However, tannins may induce detrimental effects on animal performances, especially in monogastrics, causing damage on gut villi. In contrast, short chain fatty acids are very efficient in influencing positively the morphology of small intestine wall. Hence, the aim of this trial was to develop a feeding strategy for broiler rearing, based on the use of chestnut tannins and short chain fatty acids administered as blends. No differences in animal performances or in meat quality were found among feeding groups. The results suggested that the mix of these supplements did not have negative effects on the productive performances, representing a promising alternative to antibiotics. However, further investigation is needed to better understand the effects of these supplements on animals in stress conditions.

**Abstract:** Chestnut tannins (CT) and saturated short medium chain fatty acids (SMCFA) are valid alternatives to contrast the growth of pathogens in poultry rearing, representing a valid alternative to antibiotics. However, the effect of their blends has never been tested. Two blends of CT extract and Sn1-monoglycerides of SMCFA (SN1) were tested in vitro against the proliferation of *Clostridium perfringens*, *Salmonella typhymurium*, *Escherichia coli*, *Campylobacter jejuni*. The tested concentrations were: 3.0 g/kg of CT; 3.0 g/kg of SN1; 2.0 g/kg of CT and 1.0 g/kg of SN1; 1.0 g/kg of CT and 2.0 g/kg of SN1. Furthermore, their effect on broiler performances and meat quality was evaluated in vivo: one-hundred Ross 308 male birds were fed a basal diet with no supplement (control group) or supplemented with CT or SN1 or their blends at the same concentration used in the in vitro trial. The in vitro assay confirmed the effectiveness of the CT and SN1 mixtures in reducing the growth of the tested bacteria while the in vivo trial showed that broiler performances, animal welfare and meat quality were not negatively affected by the blends, which could be a promising alternative in replacing antibiotics in poultry production.

**Keywords:** antibiotic; hydrolysable polyphenol; monoglyceride; pathogen; poultry feeding

#### **1. Introduction**

Conventional antimicrobial agents are commonly used in the poultry industry to control diseases and to prevent the mortality of birds. However, this approach conflicts with the worldwide aim to eliminate antibiotics in animal feeding. Indeed, the use of pharmaceuticals as preventing tools against pathogens has contributed to the acquisition of bacterial resistance. Moreover, problems for dejection disposal occur, due to the presence of residual antibiotics. Therefore, the poultry industry needs alternatives able to replace drugs with natural compounds or with synthetic compounds able to simulate natural molecules [1].

Polyphenols from plant kingdom are efficient antimicrobials, even if major differences can be noted. Indeed, their efficacy is affected by the solubility, which is strongly linked to the molecular structure. Among the others, chestnut tannins (CT) are hydrolysable and water-soluble compounds. Their antimicrobial activity has been previously demonstrated in poultry by Tosi et al. [2], while Redondo et al. [3] reported that *Clostridium perfringens* is unable to develop resistance against hydrolysable tannins, compared to antibiotics as avilamycin or bacitracin. However, the use of tannins in animal feeding, with particular reference to monogastrics, is discouraged for their potential anti-nutritional effects [4]. The reason is their ability in binding proteins, lowering feed intake and digestibility [4–6]. Hence, to evaluate the inclusion level of polyphenols is extremely important to avoid detrimental effects on animal welfare and performances [7,8].

Furthermore, literature shows that free saturated short-medium chain fatty acids (SMCFA; from C4:0 to C12:0) can protect the gut against several pathogenic bacteria [9–11], but their employment is limited because they are quickly absorbed in the jejunum [12–14]. Similarly, Sn2-monoglycerides are easily carried from the gut into the blood stream [6,15]. A hypothetical alternative could be represented by synthetic monoglycerides, because the industrial synthesis occurs under kinetic control of the reaction and the end-products are Sn1-substituted monoglycerides. These molecules, according to a not-natural structure, are little absorbed at the gut level, where they can exert antimicrobial effects against pathogens [16].

Several studies have been published on the efficiency of CT and synthetic monoglycerides in exercising antimicrobial activities and in ameliorating animal performances when used separately [12–14,17]. Nevertheless, no information is available in literature on the effect of blends of CT and Sn1-monoglycerides (SN1) of SMCFA as antimicrobials against the proliferation of *C. perfringens*, *Salmonella typhymurium*, *Escherichia coli*, *Campylobacter jejuni* and as dietary supplement in poultry diets. Hence, the aims of this trial were: (i) to test a possible synergic antimicrobial activity of two blends obtained from a mixture of two commercial supplements (i.e., CT extract from *Castanea sativa* Mill. and SN1) by an in vitro study, then (ii) to evaluate the effect of the same mixtures on broiler performance and meat quality.

#### **2. Materials and Methods**

#### *2.1. Chestnut Hydrolysable Tannins and Sn1-Monoglycerides Composition*

Chestnut tannins were extracted from the wood of *C. sativa* Mill. by distillation with water flow (Saviotanfeed®®, Gruppo Mauro Saviola srl Radicofani, Siena, Italy) and contained 750 g/kg (on dry matter basis, DM) of equivalent tannic acid. The chromatographic characterization of this lot of CT extract is reported in Bargiacchi et al. [18]. Sn1-monoglicerydes contained a mix of SMCFA from C4:0 to C12:0 (Silohealth®®, Silo SpA, Firenze, Italy). The glycerides and fatty acid (FA) profile of SN1 was determined according to Christie [19] and it is shown in Table 1. These supplements were the same used in the microbiological assay and during the in vivo trial.


**Table 1.** Lipid profile of Sn1-monoglycerides.

#### *2.2. Microbiological Assay*

The microbiological assay was carried out according to Elizondo et al. [20] modified as described below. The microorganisms used in this study were the following: *C. perfringens* NetB positive (strain number 191999/2014), isolated from broiler chickens affected by necrotic enteritis; *S. typhimurium* (strain number 198306/2014), isolated from viscera of egg-table layers; *E. coli* serotype O45 (strain number 184049/2014), isolated from broiler chickens affected by avian colibacillosis; *C. jejuni* (strain number 18818/2015), isolated from the skin of broiler chickens. The bacterial strains were isolated and identified using the standard procedures adopted by Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (section located in Forlì, Italy) and maintained on slants with heart infusion agar (HIA; Becton Dickinson GmbH, Germany) at +4 ◦C. To ensure culture purity, before the assays, a sample of *C. perfringens* culture was streaked on blood agar base (Oxoid Ltd., Basingstoke, UK) with 5 g/100 g of sheep blood and incubated overnight at 37 ◦C under anaerobic conditions (GENbag anaer, bioMérieux S.A., Marcy l'Etoile, France). As similar, the samples of *E. coli* and *S. typhimurium* cultures were streaked on Hektoen Enteric Agar (Becton Dickinson GmbH, Germany) and incubated overnight at 37 ◦C. The samples of *C. jejuni* cultures were streaked on modified charcoal cefoperazone deoxycholate (mCCD) agar (Oxoid Ltd., Basingstoke, UK) and incubated at 44 ◦C for 48 h in a microaerobic atmosphere (GENbag microaer, bioMérieux S.A., Marcy l'Etoile, France). Then, one colony of each strain was grown in Brain Heart Infusion (BHI) broth (Becton Dickinson GmbH, Germany) and incubated overnight at 37 ◦C (under anaerobic conditions for *C. perfringens*; microaerobic atmosphere at 44 ◦C for *C. jejuni*) and then titrated. For this purpose, 10-fold serial dilutions of each suspension were carried out in Buffered Peptone Water (Oxoid Ltd., Basingstoke, UK); each dilution was streaked on specific media and incubated overnight at 37 ◦C (under anaerobic conditions for *C. perfringens*; microaerobic atmosphere at 44 °C for *C. jejuni*). Based on the titration results, each bacterial suspension was diluted in BHI broth to obtain a concentration of 2 <sup>×</sup> <sup>10</sup><sup>3</sup> CFU/mL and then used as inoculum in the antibacterial test described below.

The antimicrobial activity in vitro assay was carried out according to Basri and Khairon [21] modified as follow. Five milliliters of each bacterial suspension (described above) was mixed with 5.0 mL of the following concentrations (in BHI broth, expressed as *w*/*v*) of the tested compounds: 6.0 g/kg of CT (T group), 6.0 g/kg of SN1 (S group), 4.0 g/kg of CT + 2.0 g/kg of SN1 (TS group) and 4.0 g/kg of SN1 + 2 g/kg of CT (ST group). Untreated tubes containing 5.0 mL of each bacterial suspension and 5.0 mL of BHI broth served as control (C group). This procedure resulted in a final concentration of the bacterial inoculum of 1 <sup>×</sup> <sup>10</sup><sup>3</sup> CFU/mL and in the following final concentrations (*w*/*v*) of the tested compounds: 3.0 g/kg of CT (T group), 3.0 g/kg of SN1 (S group), 2.0 g/kg of CT + 1.0 g/kg of SN1 (TS group) and 2.0 g/kg of SN1 + 1.0 g/kg of CT (ST group). The concentrations of supplements in T and in S groups were chosen according to the producers' guidelines. The ratio among CT and SN1 in the two blends was decided referring to preliminary studies [2,22]. The mixtures were incubated at 37 ◦C (under anaerobic conditions for *C. perfringens*; microaerobic atmosphere at 44 ◦C for *C. jejuni*). Each suspension was assayed at 0.5 h, 3 h and 24 h of incubation by making 10-fold

dilutions in Buffered Peptone Water (Oxoid Ltd., Basingstoke, UK), streaking 100 μL of each dilution on specific media and incubated as described above (adapted from Elizondo et al. [20]). The viable bacterial counts (expressed as CFU/mL) of the tested compounds for each concentration were compared with the bacterial counts obtained in the C group. The assays were repeated three times and results were expressed as the average values.

The microbial growth rate was calculated as ratio: ΔConc (CFU/mL)/ΔTime (h), where ΔConc is calculated as difference among microbial concentrations at the ranges of 0.5–3 h or 3–24 h and ΔTime is the related interval between the sampling times (0.5–3 h; 3–24 h).

#### *2.3. In Vivo Trial*

#### 2.3.1. Animals

Animal handling was in accordance with Italian Government guideline (D.lgs 26/2014, protocol number 232/2016PR). One hundred one-day-old Ross 308 male chicks were provided by a local hatchery (Incubatoio Settecrociari, Forlì-Cesena, Italy), where they were vaccinated against Marek's disease, infectious bronchitis and Newcastle disease. Birds were allotted in 20 pens (5 animals per pen singularly identified by ring) and randomly assigned to one of the 5 experimental diets (4 pens each diet). The feeding groups, summarized in Table 2, were: control group (C group), fed with a basal diet containing tannins free and SN1-monoglycerides of SMCFA free ingredients (Table 3); T group, fed with the basal diet supplemented with 3.0 g/kg on DM of CT; S group, fed with the basal diet supplemented with 3.0 g/kg on DM of SN1; TS group, fed with the basal diet supplemented with 2.0 g/kg on DM of CT and 1 g/kg on DM of SN1; ST group, fed with the basal diet supplemented with 1.0 g/kg on DM of CT and 2.0 g/kg on DM of SN1. The diets were formulated according to animal requirements (NRC, 1994) with 3 periods of growth: starter (0–12 days), grower (13–21 days) and finisher (22–35 days). The dosage of CT, SN1 and of their blends was the same used in the microbiological assay. Animals were fed *ad libitum* and had free access to water for all the 35 days of the trial. Every week, the animals from each pen were individually weighted. The individual feed intake was registered weekly for each pen and calculated dividing the total amount consumed by the number of animals present in the pen (the approved protocol did not allow the use of individual pens). Feed efficiency was calculated as estimated ratio of the individual feed intake/registered individual weight gain for each group.


**Table 2.** Experimental design.

**Table 3.** Ingredient composition (g/kg of DM) of basal diets formulated according to growing periods.



**Table 3.** *Cont.*

The litters of each group have been checked weekly for faeces compactness using an arbitrary but comparative score: 0, dry litter; 1, medium wet; 2, wet. At the 36th day, the animals were sacrificed at a slaughterhouse.

#### 2.3.2. Diet Proximate Analysis

Diets were analyzed for proximate profile as follows: crude protein (CP), ether extract (EE), crude fiber (CF) and ash were determined according to the AOAC methods 976.06, 920.39, 962.09 and 942.05, respectively (AOAC 1995). Neutral detergent fiber (NDF) was determined according to van Soest et al. [23], using heat stable amylase and sodium sulphite, and expressed inclusive of residual ash. Metabolizable Energy (ME) was estimated from feed tables according to Sauvant et al. [24]. The chemical and nutritional profile of the basal diets are reported in Table 4.

**Table 4.** Nutritional traits of basal diets according to growing periods.


<sup>1</sup> Estimated from feed tables according to Sauvant et al. (2004).

#### 2.3.3. Physical and Chemical Analysis

All carcasses were evaluated for dressing out and major traits. Breast meat from three animals of each pen was sampled for color analysis, antioxidant capacity, and oxidative status as follows.


Results were expressed as mmol of Trolox equivalent per kg of fresh meat for ABTS and DPPH methods and as mmol of Fe++ equivalent per kg of fresh meat for FRAP determination.

• Oxidative status of meat. Meat samples (5.0 g) were considered for TBARS (thiobarbituric acid-reactive substances) determination. TBARS were measured to determinate malondialdehyde (MDA) levels, according to the method described by Ke et al. [31] and modified by Dal Bosco et al. [32]. Briefly, the meat samples were homogenized with a water solution of trichloroacetic acid (7.5% *w*/*v*) and diethylenetriaminepentaacetic acid (0.1% *w*/*v*). After centrifugation and filtration, the solutions were reacted with a water solution of 2-thiobarbituric acid (0.288% *w*/*v*) and heated in a water bath at 95 ◦C for 45 min. The absorbance of the samples was determined at 532 nm (V-530 Jasco International, Milan, Italy) and a calibration curve was plotted with TEP (1,1,3,3-tetraethoxypropane; 0–15 μM, final concentrations) to obtain the MDA concentration. Results were expressed as mg of MDA-equivalents per kg of fresh meat.

#### 2.3.4. Statistical Analysis

Data related to bacterial counts were expressed as log10 (CFU/mL) and normalized according to Snedecor and Cochran [33]. Data related to microbial growth rate were processed as completely randomized design with repeated measures using the MIXED procedure of SAS [34]:

$$\mathbf{Y}\_{\text{ijkl}} = \mu + \mathbf{T}\_{\text{i}} + \mathbf{D}\_{\text{j}} + \mathbf{I}\_{\text{k}}(\mathbf{D}) + (\mathbf{T} \times \mathbf{D})\_{\text{ij}} + \mathbf{e}\_{\text{ijkl}}.\tag{1}$$

where yijkl is the observation; μ is the overall mean; Dj is the fixed effect of treatment (j = 1 to 5); Ti is the fixed effect of assaying time (i = 1 to 3); Ik is the random effect of the replicate nested within the treatment (k = 1 to 3); (T × D)ij is the interaction between treatment and assaying time and eijkl is the residual error. The covariance structure was compound symmetry, which was selected based on Akaike's information criterion of the mixed model of SAS [34]. The statistical significance of the treatment effect was tested against variance of bacterial cultures nested within treatment, according to repeated measures design theory [35]. Multiple comparisons among means were performed using the Tukey's test.

One-hundred animals divided in 5 groups is the minimum number of animals in order to obtain significant differences among treatments according to the power analysis based on alpha 0.05 beta 0. 08 [33]. Data related to the feed intake, weight gain, feed efficiency of each period, were processed as completely randomized design with repeated measures using the MIXED procedure of SAS [34]:

$$\mathbf{Y}\_{\text{ijkl}} = \mu + \mathbf{T}\_{\text{i}} + \mathbf{D}\_{\text{j}} + \mathbf{I}\_{\text{k}}(\mathbf{D}) + (\mathbf{T} \times \mathbf{D})\_{\text{ij}} + \mathbf{e}\_{\text{ijkl}}.\tag{2}$$

where yijkl is the observation; μ is the overall mean; Dj is the fixed effect of treatment (i = 1 to 5); Ti is the fixed effect of assaying time (j = 1 to 5); Ik is the random effect of the replicate nested within the treatment (k = 1 to 5); (T × D)ij is the interaction between treatment and assaying time and eijkl is the residual error. The covariance structure and the statistical significance were tested as described above.

The data related to feed intake, weight gain, feed efficiency of the whole period, physical and chemical parameters of meat, dressing out and the major carcass traits of slaughtered birds were analysed by one-way ANOVA, keeping the factor "diet" as the fixed one [34]:

$$\mathbf{y}\_{\text{ij}} = \mu + \mathbf{D}\_{\text{i}} + \mathbf{e}\_{\text{ij}}.\tag{3}$$

where yij is the observation; μ is the overall mean; Di is the diet (i = 1 to 5) and eij is the residual error. Multiple comparisons among means were performed using the Tukey's test. Probability of significant effect due to experimental factors was fixed for *p* < 0.05.

#### **3. Results and Discussion**

#### *3.1. Microbiological Assay*

All the treatments were efficient in decreasing the bacterial growth of each species compared to the control (Figure 1). The T resulted the most effective treatment in controlling the growth of each bacterial species at 3 h and 24 h. For the other treatments, the behavior of the tested bacteria was different. *C. perfringens* and *S. typhymurium* resulted more sensitive to the TS than to the ST and S at 3 h and 24 h. At 3 h no significant differences were found for the S, TS and ST for *E. coli* but at 24 h the TS was more efficient in limiting the growth compared to the other two treatments (Figure 1C). No significant different growth was observed for *C. jejuni* with S and TS at 3 h. However, at the same sampling time, the growth with ST was higher than the growth with S and TS. At 24 h the growth of *C. jejuni* was lower with the TS than with the S and ST (Figure 1D).

**Figure 1.** Results of microbial in vitro assay for *Clostridium perfringens* (**A**), *Salmonella typhymurium* (**B**), *Escherichia coli* (**C**), *Campylobacter jejuni* (**D**) (data are reported as Log10 (CFU/mL)). C, control; T, 3.0 g/kg of chestnut tannin; S, 3.0 g/kg of Sn1-monoglycerides; TS, 2.0 g/kg of chestnut tannin added to 1.0 g/kg of Sn1-monoglycerides; ST, 1.0 g/kg of chestnut tannin added to 2.0 g/kg of Sn1-monoglycerides. Concentration are expressed as *w*/*v*. SEM, Standard Error Mean. The probability of significant effect due to experimental factors is reported as: α, β, γ, δ, ε for the treatments (means with different Greek superscripts are significantly different (*p* < 0.05)); A, B, C for the sampling time (means with different Latin superscripts are significantly different (*p* < 0.05)).

All the treatments lowered the growth rate of each microbial species, except for *C. jejuni* that did not show significant decreasing with the tested compounds between 3 h and 24 h, compared to the control. Furthermore, both *C. perfringens* and *C. jejuni* decreased in all the treatments between 0.5 h and 3 h, and increased between 3 h and 24 h (Table 5 and Figure 1). This observation suggested that the bactericidal effect of CT and SN1, supplied alone or in combination, is stronger at the beginning of the treatment. For *E. coli* and *S. typhymurium* only a bacteriostatic effect was observed in the treated cultures (Table 5 and Figure 1).


**Table 5.** Rate of microbial growth in the in vitro assay (data are reported as Log10 (CFU/mL)/h).

<sup>1</sup> C, control; T, 3.0 g/kg chestnut tannin; S, 3.0 g/kg of Sn1-monoglycerides; TS, 2.0 g/kg of chestnut tannin added to 1.0 g/kg of Sn1-monoglycerides; ST, 1.0 g/kg of chestnut tannin added to 2.0 g/kg of Sn1-monoglycerides. <sup>2</sup> SEM, Standard Error Mean. <sup>3</sup> Probability of significant effect due to experimental factors; a,b,c,d,e within a row, means with different Latin letters are significantly different (*p* < 0.05). <sup>4</sup> Time range considered for growth rate calculation.

Our results are in accordance with Tosi et al. [2] and Redondo et al. [3] who demonstrated that the CT can inhibit the growth of *C. perfringens*. The antimicrobial activity of tannins seems to be due to their ability to bind microbial enzymes and proteins, in ion deprivation and in inhibiting the topoisomerase, fundamental for the DNA replication [36–38]. Moreover, Ramìrez et al. [6] and Timbermont et al. [10] showed that the SN1 of SMCFA were efficient in controlling the growth of *S. typhimurium* and *C. perfringens*, respectively, consistent with our study. Their antimicrobial effect was explained with their ability to penetrate through the bacterial wall, because of their affinity with lipoteichoic acid, present in microbial membrane. Their ability to destroy the inner the membrane is probably due to their compatibility with hydrophilic and hydrophobic moieties [6,39–41]. These results suggest that the CT and SN1 alone or in combination could be useful to control the proliferation of pathogenic bacteria tested in this trial. Hence, these molecules could represent a valid alternative to antibiotics both used alone or in mixture.

#### *3.2. In Vivo Trial*

No differences among the groups were found for feed intake (Table 6), both in each single growth period and in the whole period of bird life, showing that the supplementation with CT and SN1 blends did not affect the palatability of the diets. No differences were found for weight gain and feed efficiency among groups, suggesting that the blends of CT and SN1, at the inclusion level adopted in this study, did not interfere with nutrient absorption, with respect to the single supplementation or to the control diet (Table 6). Hence, no synergic effect was found when the CT and SN1 mixtures were included in the diets. The results showed a higher feed/gain ratio during the first two weeks, compared to the growing and finisher periods. Usually, young chicks have higher feed efficiency than old birds. This trend could be due to an adaptation period of birds to the rearing condition because also the control group, fed with only the basal diet, did not show significant differences with the other feeding groups. The literature reports information on the effect of CT and SN1 when they are included alone in the diets, but few data are available on the effect of blends composed by a mixture of tannin extracts and monoglycerides. The results of this trial are in accordance with Jamroz et al. [42] and Antongiovanni et al. [13,14,43] who studied respectively the effect of CT (inclusion level of 0, 250, 500 and 1000 mg/kg on DM) and of several monoglycerides (inclusion level of 200, 350, 500 mg/kg of DM) separately, as dietary supplementation on the performance and histological characteristics of the intestine wall in chickens. No impairment of the growth performance emerged, despite a slight modification on the small intestine wall, due to the introduction in the diet of chestnut tannins

and monobutyrin (CT degrades enterocytes while monobutyrin modifies positively villi, microvilli and crypts), was observed. Moreover, previous results, reported by Schiavone et al. [17], showed that the inclusion of a natural extract of chestnut wood did not affect the apparent digestibility of CP and that this supplement had a positive effect on average daily gain and feed intake in the first two weeks of addition. For monoglycerides, especially with butyric acid, the literature confirms that they can ameliorate growth performances and health in broilers [12–14]. In contrast, for the CT, several studies reported that polyphenols reduce protein digestibility in monogastrics, decreasing the productive performances in accordance with a lower availability of this nutrient for the animal nutritional requirements [5,44–46]. In particular, tannins stimulate hypersecretion of endogenous enzymes leading to losses of sulphur aminoacids in poultry species [47–49]. The inconsistence of the results reported in many papers, including those shown here, is probably due to the kind of tannin used as dietary supplement, the animal species and the dietary dose formulation. In this study, by an accurate observation, an astringent effect of tannin has been noted in T, TS and ST groups, whose litters resulted drier than the litters of S group (at 35th day: C = 1; T = 0; TS = 0; ST = 0 and S = 1).



<sup>1</sup> C, control; T, 3.0 g/kg chestnut tannin; S, 3.0 g/kg of Sn1-monoglycerides; TS, 2.0 g/kg of chestnut tannin added to 1.0 g/kg of Sn1-monoglycerides; ST, 1.0 g/kg of chestnut tannin added to 2.0 g/kg of Sn1-monoglycerides. <sup>2</sup> SEM, Standard Error Mean. <sup>3</sup> Probability of significant effect due to experimental factors.

No significant differences in carcass quality were found among groups (Table 7), except for the liver that was smaller in the animals fed the T and S diets than the other feeding groups. This result is consistent with the findings reported by Jamroz et al. [42] and Antongiovanni et al. [13] who noted that the supplementation with polyphenols or the monoglycerides of butyric acid did not affect carcass quality, even though monoglycerides represented an energy source for animal growth and tannins are considered to be antinutritive. Unfortunately, in literature, no information is available on blends of CT and SN1, which were not able to affect carcass traits at the tested levels in our study.


**Table 7.** Major carcass traits of slaughtered birds.

<sup>1</sup> C, control; T, 3.0 g/kg chestnut tannin; S, 3.0 g/kg of Sn1-monoglycerides; TS, 2.0 g/kg of chestnut tannin added to 1.0 g/kg of Sn1-monoglycerides; ST, 1.0 g/kg of chestnut tannin added to 2.0 g/kg of Sn1-monoglycerides. Concentration are expressed as *w*/*v*. <sup>2</sup> SEM, Standard Error Mean. <sup>3</sup> Probability of significant effect due to experimental factors; a,b within a row, means with different letters are significantly different (*p* < 0.05).

Changes in the a\* and b\* values are related to changes in meat color because these parameters are markers of browning [50]. Where the SN1 was present in the diet alone an increase of the L\* and b\* values occurred (Table 8). Specifically, the S group showed the highest L\* and b\* values and it significantly differed compared to the other groups. Despite the statistical differences, ΔE calculation for the samples of chicken breast can be useful to understand how the values of color can be perceived by human eyes (Table 9). As suggested by Mokrzycki and Tatol [26], a standard observer is able to see the difference in color as follows: 0 < ΔE < 1 observer does not notice the difference, 1 < ΔE < 2 only experienced observers can notice the difference, 2 < ΔE < 3.5 unexperienced observer also notices the difference, 3.5 < ΔE < 5 clear difference in color is noticed, ΔE > 5 observer notices two different colors. Regarding the ΔE, from Table 9 it emerged that the T and TS were the most similar groups (1 < ΔE < 2), while the ST assumed a 2 < ΔE < 3.5 when compared with C, T, and TS. Interestingly, the S group was found to be the only one with a ΔE > 5 when compared to the other experimental groups, thus underlining that Sn1 monoglycerides strongly impacted meat coloration. Whether this modification could be accepted or not by consumers should be further investigated.


<sup>1</sup> C, control; T, 3.0 g/kg chestnut tannin; S, 3.0 g/kg of Sn1-monoglycerides; TS, 2.0 g/kg of chestnut tannin added to 1.0 g/kg of Sn1-monoglycerides; ST, 1.0 g/kg of chestnut tannin added to 2.0 g/kg of Sn1-monoglycerides. Concentration are expressed as *w*/*v*. <sup>2</sup> Data are reported in the L\* a\* b\* color notation system with L\* axis representing lightness, the a\* axis representing the red-green color axis (redness) and the b\* axis representing the blue-yellow (yellowness) color axis. <sup>3</sup> SEM, Standard Error Mean. <sup>4</sup> Probability of significant effect due to experimental factors; a, b within a row, means with different letters are significantly different (*p* < 0.05).

**Table 9.** Calculated ΔE2000 values for breast.


C, control; T, 3.0 g/kg chestnut tannin; S, 3.0 g/kg of Sn1-monoglycerides; TS, 2.0 g/kg of chestnut tannin added to 1.0 g/kg of Sn1-monoglycerides; ST, 1.0 g/kg of chestnut tannin added to 2.0 g/kg of Sn1-monoglycerides.

Data reported in this study showed that both the tested blends of CT and SN1 could be utilized as dietary supplements without impairing animal welfare, growing performances and meat quality, thus representing valid alternatives to antibiotics in poultry rearing. Contrariwise, the S diet deeply modified the color of breast meat which could result in a modification of consumers' acceptance.

In this trial, data related to the antioxidant status of the breast meat did not show significant differences among the groups (Table 10). Several studies demonstrated the antioxidant power of tannins and of polyunsaturated fatty acids but not for SMCFA, because of the lack of double bounds on carbon chain [51–53]. Indeed, several authors reported that ellagic tannins in humans and rats are gradually metabolized by the intestinal microbiota to produce different metabolites with antioxidant effects [54–57]. Luciano et al. [53] found that the inclusion at 8.96% (DM basis) of quebracho tannins in lamb diet produced an improvement in the antioxidant status of *Longissimus dorsi* muscle, measured as both its ferric reducing ability and its radical scavenging ability. Other authors reported similar results in beef meat [30,58,59]. In contrast, Gladine et al. [60] found no effect of polyphenols in rat muscle for the radical scavenging activity. The inability of many polyphenols to be metabolized by the gastrointestinal tract of animals is strongly linked to their molecular structure and solubility, the dose of inclusion and the animal species.


**Table 10.** Oxidative status of meat.

<sup>1</sup> C, control; T, 3.0 g/kg chestnut tannin; S, 3.0 g/kg of Sn1-monoglycerides; TS, 2.0 g/kg of chestnut tannin added to 1.0 g/kg of Sn1-monoglycerides; ST, 1.0 g/kg of chestnut tannin added to 2.0 g/kg of Sn1-monoglycerides. <sup>2</sup> ABTS and DPPH are expressed as mmol of Trolox equivalent per kg of meat; FRAP as mmol of Fe++ equivalent per kg of meat; TBARS as mg of MDA-Eq per kg of meat. <sup>3</sup> SEM, Standard Error Mean. <sup>4</sup> Probability of significant effect due to experimental factors; a, b within a row, means with different letters are significantly different (*p* < 0.05).

Nowadays, in conventional and intensive poultry production, antibiotics are used to control diseases and to prevent the mortality of birds, responsible for a huge economical loss. This approach conflicts with the sustainability of animal productions because several issues about the development of bacterial resistance, dejection disposal, food safety and human health occur. Therefore, the poultry industry needs alternatives able to replace antibiotics with natural or synthetic compounds able to simulate natural molecules. Chestnut tannins are a by-product of wood industry because they are obtained by distillation of wood used in the building industry. SN1 is obtained by recycling glycerol derived from biodiesel production. Hence, CT and SN1 are part of the concept of bio-economy. Moreover, FAO reported that livestock support the livelihoods and food supply of almost 1.3 billion people, being one of the fastest growing areas of the agricultural economy in the world. In developing countries, poultry production plays an important role in food, and pathogen proliferation represents an important public health problem that cannot be underestimated. At the same time, environmental sustainability must be ensured [61]. Data reported in this study showed that CT, SN1 or their blends could represent a valid alternative to antibiotics in poultry rearing. Although literature shows several studies in which tannins exert antinutritional effects in monogastric, in the present in vivo trial, no detrimental effects were observed on animal welfare, performance or meat quality. However, it is well known that the kind of polyphenols and the dietary inclusion level are fundamental to explain the biological and nutritional effects of dietary tannins. Besides, the literature shows unequivocal positive effects of SN1 monoglycerides in protecting gut from pathogens, by providing energy to enterocytes and by favoring the development of gut villi [12,13,16]. Hence, the blends of CT and SN1

could represent a good compromise among antimicrobial activities, animal gut protection, meat quality and production sustainability.

#### **4. Conclusions**

The in vitro study suggested that blends of CT and SN1 could be efficient against the proliferation of *C. perfringens*, *S. typhymurium*, *E. coli* and *C. jejuni*. Additionally, the in vivo trial suggested that the mixture of these supplements did not have negative effects on animal productive performances, representing a promising alternative to antibiotics. Considering all the recorded information about in vitro antimicrobial effectiveness and the broilers growing performances, meat color and the overall antioxidant capacity of meat, both the TS and ST tested blends might be good alternatives to antibiotics in the poultry sector. However, further investigation is needed to better understand the effects of these supplements on animals in stress conditions.

**Author Contributions:** Conceptualization, F.M., S.M. (Sara Minieri) and A.B.; data curation, M.D. and S.R.; formal analysis, F.M., S.M. (Sara Minieri), P.M., L.F. and S.M. (Simone Mancini); funding acquisition, M.A.; investigation, F.M., S.M. (Sara Minieri), G.T. and A.B.; methodology, G.T. and G.S.; project administration, A.B.; resources, I.G. and S.L.; Software, M.D.; supervision, A.B.; Validation, F.M., G.S. and M.D.; visualization, S.R. and M.A.; writing—original draft, F.M.; writing—review & editing, F.M., G.S. and M.D.

**Funding:** This research was funded by "Silo s.p.a., Florence, Italy" and "Gruppo Mauro Saviola, Viadana Mantova, Italy".

**Acknowledgments:** The authors acknowledge Antonio Pezzati and Doria Benvenuti for technical support, Elisabetta Bugelli and Elisabetta Guidi for administrative support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **E**ff**ect of Bamboo Leaf Extract on Antioxidant Status and Cholesterol Metabolism in Broiler Chickens**

#### **Mingming Shen, Zechen Xie, Minghui Jia, Anqi Li, Hongli Han, Tian Wang and Lili Zhang \***

College of Animal Science and Technology, Nanjing Agricultural University, No. 6, Tongwei Road, Xuanwu District, Nanjing 210095, China; 2017105056@njau.edu.cn (M.S.); 15117227@njau.edu.cn (Z.X.); jiaminghui3@163.com (M.J.); 15117409@njau.edu.cn (A.L.); hanhongli324@sina.com (H.H.); tianwangnjau@163.com (T.W.)

**\*** Correspondence: zhanglili@njau.edu.cn; Tel.: +86-25-84396483; Fax: +86-25-84395483

Received: 8 August 2019; Accepted: 16 September 2019; Published: 18 September 2019

**Simple Summary:** Cholesterol is an important lipid substance in organisms. As the precursor of bile acid, steroid hormones and vitamin D3, cholesterol plays important roles in lipid metabolism. Chicken is among the most consumed meat products worldwide; however, its cholesterol level is higher than that of other meat products. High cholesterol in a human diet will increase the risk of atherosclerosis. In addition, low-density lipoprotein cholesterol is susceptible to be oxidized, which will cause the death of broilers. Therefore, it is of great significance to enhance the antioxidant capacity and improve cholesterol metabolism in broiler chickens. Bamboo leaf extract (BLE) contains active ingredients such as flavonoids, polyphenols, and active polysaccharides, which possess anti-inflammatory, antioxidant and lipid-lowering effects. Our results show that supplementation of BLE in the basal diet improved growth and slaughter performance, antioxidant status and cholesterol metabolism in broilers. Therefore, the application of BLE as a feed additive has a certain economic value.

**Abstract:** The objective of this study was to investigate the effects of dietary bamboo leaf extract (BLE) on antioxidant status and cholesterol metabolism in broilers. One-day-old male Arbor Acres (576) broilers were randomly divided into six groups. A control group was fed a basal diet, while five experimental groups were supplemented with 1.0, 2.0, 3.0, 4.0, and 5.0g BLE per kg feed in their basal diets. The result indicated that BLE supplementation linearly improved eviscerated yield and decreased abdominal fat (*p* < 0.05). A significant decrease of serum triglyceride (TG) and low-density lipoprotein cholesterol (LDL-c) content was observed with BLE supplementation (*p* < 0.05). BLE supplementation linearly improved the total antioxidant capacity and catalase activity in both serum and liver (*p* < 0.05). Glutathione peroxidase was quadratically increased in serum and linearly increased in the liver with BLE supplementation (*p* < 0.05). The malonaldehyde content in liver showed a linear and quadratic decrease with BLE supplementation (*p* < 0.05). BLE supplementation up-regulated the mRNA expression of cholesterol 7- alpha hydroxylase and low-density lipoprotein receptor and downregulated 3-hydroxy3-methyl glutamates coenzyme A reductase mRNA expression in the liver. The antioxidant enzyme mRNA expressions were all up-regulated by BLE supplementation in the liver. In conclusion, supplemental BLE improved antioxidant status and cholesterol metabolism in broilers, which eventually led to a decrease of serum TG, LDL-c content, and abdominal fat deposition.

**Keywords:** antioxidant status; bamboo leaf extract; broiler; cholesterol metabolism

#### **1. Introduction**

In the past few decades, the proportion of animal products in the human diet has increased considerably, and the high cholesterol content in animal products has attracted great interest of researchers [1,2]. If the human diet contains high concentrations of cholesterol from the animal products, the blood cholesterol concentration will raise, and the risk of hypercholesterolemia will increase [3,4]. As a result, atherosclerosis and coronary heart disease will happen because hypercholesterolemia is one of the major factors leading to these diseases [4]. As reported previously, every 1% reduction in serum cholesterol could reduce the incidence of coronary heart disease by 2% [5]. However, chicken products are widely consumed worldwide and are also the most cholesterol-containing meat [6]. Therefore, reducing the cholesterol content in broilers is of great importance for human health. In addition, there are many oxidative stresses in poultry production, such as heat stress, immune stress and transport stress; all these stresses will increase oxidizing substances in broilers [7]. If there is a large amount of low-density lipoprotein cholesterol (LDL-c) in chicken serum, LDL-c will react with oxidizing substance, and the oxidized LDL-c will be produced [8]. While the oxidized LDL-c is highly poisonous to the cells, it will cause damage to endothelial cells and accelerate the platelet adhesion and aggregation, release growth factors, cause hyperplasia of fibroblasts and organization, and eventually speed up the development of atherosclerosis [9,10]. As a result, it will cause sudden death of broilers and lead to economic losses. For all these reasons, it becomes a hot research topic and solutions need to be further explored for enhancing antioxidant status and improving cholesterol metabolism in poultry production.

Bamboo is widely distributed around the world, and its leaves have been used for medicinal and culinary purposes in China. It is reported that bamboo leaves contain active ingredients such as flavonoids, polyphenols, and active polysaccharides [11]. Research showed that bamboo leaf extract (BLE) has multiple biological effects, especially on cardiovascular and cerebrovascular protection. As mentioned before, BLE was able to reduce the cholesterol concentration of hyperlipidemia mice and improve liver function [12]. In addition to the effect of lowering serum cholesterol, BLE is capable to increase antioxidant capacity in hyperlipidemia mice [13]. Meanwhile, the research of Sunga et al. has shown that BLE reduced the adhesion of vascular epithelial factors, regulated endothelial cells to increase vascular mobility, and reduced the risk of atherosclerosis [14]. BLE has super antioxidant ability for scavenging free radicals in vitro [15,16], and improves antioxidant enzyme activity in vivo [17]. BLE is confirmed as an ideal choice for the body to supplement exogenous antioxidants and prevent hyperlipidemia, and some Chinese functional food or medicine made with BLE, like bamboo rice, bamboo beer, bamboo toothpaste, is very popular among Chinese consumers. The existing research about BLE in poultry production mainly focuses on the growth performance, meat quality and immune organ index [18]; little research has been conducted on the antioxidant effect. Furthermore, although multiple studies showed that flavonoids are able to affect fat metabolism in animals, and BLE holds the function to improve lipid metabolism in rat or mice, the effect of BLE on cholesterol metabolism in broiler chickens is still not clear. BLE supplementation in the basal diet could enhance antioxidant status and improve cholesterol metabolism in broiler chickens, which will be beneficial for poultry production and the human diet. Therefore, this study was conducted to investigate the effect of BLE on antioxidant status and cholesterol metabolism in broiler chickens.

#### **2. Materials and Methods**

#### *2.1. Ethical Statement*

Animal feeding experiments were carried out at Jiangpu farm of Zhujiang campus of Nanjing Agricultural University, and experimental analyses were conducted at the College of Animal Science and Technology of Weigang campus of Nanjing Agricultural University. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University (GB14925, NJAU-CAST-2011-093), and the serial number of the laboratory animal use certificate issued by Science and Technology department of Jiangsu province is SYXK (Su) 2017–0007.

#### *2.2. Animals, Diets and Experimental Design*

Bamboo leaf extract (BLE) was obtained from Zhejiang XinHuang Biotechnology Co., Ltd. (Zhejiang, China), and its main components include flavonoids, polyphenols (the bamboo leaf flavonoid concentration is 70 mg per gram of BLE, and the polyphenol concentration is 50.42 mg per gram of BLE). A total of 576 one-day-old male Arbor Acres broiler chicks were obtained from a local commercial hatchery (Hewei Company, Anhui Province, China) and were randomly allotted into 6 groups with 6 replicates containing 16 birds each. Basal diets were designed for the starter phase (1–21 d) and growth phase (22–42 d) (Table 1). Chickens were supplied according to NRC (1994) recommendations for nutrition requirements. The control group (CON) was fed with a basal diet, while five experimental groups BLE1, BLE2, BLE3, BLE4, and BLE5 were fed the basal diet supplemented with 1.0, 2.0, 3.0, 4.0 and 5.0 g BLE per kg feed for 42 days. All birds were kept in three-layer pens; each replicate was divided into two pens. Temperature was maintained at 32–35 ◦C for the first five days, then gradually decreased to 22 ◦C and kept stable until the end of the experiment. During the trial period, birds had free access to feed and water.


**Table 1.** Composition and nutrient level of basal diets.

<sup>1</sup> Premix provided per kilogram of diet: VA 10 000 IU, VD3 3 000 IU, VE 30 IU, VK3 1.3 mg, thiamine 2.2 mg, riboflavin 8 mg, niacin 40 mg, choline chloride 600 mg, calcium pantothenate 10 mg, pyridoxine 4 mg, biotin 0.04 mg, folic acid 1 mg, VB12 0.013 mg, zinc 65 mg, iron 80 mg, copper 8 mg, manganese 110 mg, iodine 1.1 mg, selenium 0.3 mg; <sup>2</sup> Calculated value.

#### *2.3. Slaughter Performance*

Two birds from each replicate were weighted before slaughter and after sufficient exsanguination at 42 d. Then, the feathers, head, feet, abdominal fat and viscera were removed from the bird (except for kidney) and the carcass was reweighed. In addition, liver and abdominal fat weight were recorded separately. The right–side breast and thigh meat were weighed after removing skin and bone. The percentage weight of eviscerated yield, breast, thigh, abdominal fat and liver, compared with the live-weight was used to evaluate slaughter performance.

#### *2.4. Sample Collection*

At the end of the experiment, two birds (near the average body weight of each replicate) from each replicate were selected, and non-anticoagulant sterile blood vessels were used to collect blood samples from the jugular vein. The blood was left for 2 hours at 4 ◦C, then centrifuged at 3500 rpm for 10 min, and the supernatant was stored at −20 ◦C for analysis. A sample was cut from liver tissue from the middle of the left lobule in anatomical location and stored at −80 ◦C for antioxidant enzyme and mRNA expression analysis.

#### *2.5. Cholesterol Metabolism Parameter Analysis*

The total cholesterol (TC, kit number: A111-1-1), total triglyceride (TG, kit number: A110-1-1), high density lipoprotein cholesterol (HDL-c, kit number: A112-1-1), low density lipoprotein cholesterol (LDL-c, kit number: A113-1-1) and blood glucose (GLU, kit number: F006-1-1) in serum were measured by different commercial kits purchased from Nanjing Jiancheng Institute of Bioengineering (Nanjing, China).

#### *2.6. Serum and Liver Homogenate Antioxidant Enzyme Analysis*

One gram of liver tissue from a sample preserved at -80◦C was homogenized with 4.5 mL of 0.9% sodium chloride buffer with tube embed in ice by using an Ultra-Turrax homogenizer (Tekmar Co., Cincinnati, OH, USA), and the homogenates were centrifuged at 3500 rpm for 10 min. The supernatant was used to measure superoxide dismutase (SOD, kit number: A001-1-1), glutathione peroxidase (GSH-Px, kit number: A005-1-1), catalase (CAT, kit number: A007-1-1), total antioxidant capacity (T-AOC, kit number: A015-1-1) activities, and Malondialdehyde (MDA, kit number: A003-1-1) content by different commercial kits purchased from Nanjing Jiancheng Institute of Bioengineering (Nanjing, China) according to its instruction.

#### *2.7. RNA Extraction and Quantitative Real-Time PCR*

Trizol Reagent (Vazyme, NanJing, China) was used to extract total RNA from liver tissue, which was then treated by deoxyribonuclease I to remove the contaminant DNA. RNA was quantified based on the absorption of light by a Nanodrop ND-2000c spectrophotometer (Thermo Scientific, Camden, UK) at 260 nm (A260) and 280 nm. From each sample, 1 μg of RNA was used to synthesize cDNA in a 20 μL reaction mixture using the Primer-ScriptTM reagent kit (TaKaRa, Dalian, China) according to the manufacturers' instructions. The real-time quantitative polymerase chain reaction was carried out by using the SYBR Premix Ex Taq II kit (TaKaRa) in an ABI 7300 fluorescence quantitative PCR instrument (Applied Biosystems, Foster City, CA, USA). The 20 μL reaction system included 10 μL of SYBR Premix Ex Taq buffer, 0.4 μL each of forward and reverse primers and dye, 2 μL of cDNA template, and 6.8 μL of distilled water. The real-time PCR cycling conditions were as follows: 95 ◦C for 30 s, 40 cycles of 95 ◦C for 5 s, and 60 ◦C for 30 s. The relative mRNA expression was determined using β-actin as an internal reference gene. The significance and correlation of quantitative results were analyzed by using 2-ΔΔct as per Livak and Schmittgen [19]. Primer sequences are shown in Table 2.


**Table 2.** Primer sequences used for Real-time PCR.

<sup>1</sup> CYP7A1: cholesterol 7- alpha hydroxylase; LDLR: low-density lipoprotein receptor; HMGCR: 3-hydroxy 3-methyl glutamates coenzyme A reductase; SREBP-2: sterol regulatory element binding transcription factor 2; SOD: Superoxide dismutase; CAT: Catalase; GSH-Px: Glutathione peroxidase.

#### *2.8. Statistical Analysis*

All data were preliminarily processed by using Excel 2016 and analyzed through one-way analysis of variance (ANOVA) using SPSS statistical software (Ver. 20.0 for Windows, SPSS, Inc., Chicago, IL, USA). The data were analyzed as a completely randomized design with a replicate as an experimental unit. Duncan's multiple range test was performed to determine differences between treatments. The effect of BLE supplementation at various levels was evaluated using an orthogonal polynomial contrast test for linear and quadratic effects. Differences were regarded as significant at *p* < 0.05.

#### **3. Results**

#### *3.1. Growth Performance*

In the starter phase, compared to the CON group, average daily feed intake was significantly higher in the BLE2 and BLE5 groups (*p* < 0.05), and average body weight in the BLE5 group increased significantly (*p* < 0.05). In the growth phase, compared with the CON group, average daily feed intake and average daily gain in the BLE2 and BLE5 groups were significantly higher than that of the CON group (*p* < 0.05). However, the BLE1 and BLE2 groups showed a significant decrease in feed: gain ratio (*p* < 0.05). Average daily gain and feed: gain ratio showed a quadratic improvement with increasing BLE dosage, and there was a linear and quadratic enhancement on average body weight when the BLE levels increased. During the whole rearing period, average daily gain and feed: gain ratio improved significantly in the BLE2 group over the CON group (*p* < 0.05).

#### *3.2. Slaughter Performance*

As shown in Table 3, compared with the CON group, the percentage of eviscerated yield in BLE supplementation groups was significantly (*p* < 0.05) increased (except for birds in BLE3 group), the percentage of abdominal fat was decreased significantly in BLE supplementation groups (*p* < 0.05). In addition, the percentage of eviscerated yield showed a linear improvement (*p* = 0.007), and abdominal fat percentage showed a linear (*p* = 0.027) decrease with increasing level of BLE supplementation. Moreover, there was a quadratic decrease in the percentage of liver weight as BLE supplementation increased. No difference was observed in breast and thigh meat percentage among BLE groups (*p* > 0.05).


**Table 3.** Effect of dietary BLE on slaughter performance of broilers.

Note: a.b.c means within the same row with no common superscript differ significantly (*p* < 0.05); <sup>1</sup> standard error of the means; <sup>2</sup> Orthogonal polynomials were used to evaluate linear and quadratic responses to the levels of BLE treatment; <sup>3</sup> CON: basal diet, BLE1, BLE2, BLE3, BLE4 and BLE 5 group, basal diet adding 1.0, 2.0, 3.0, 4.0 and 5.0g/kg BLE, respectively.

#### *3.3. Serum Cholesterol Metabolism Parameters*

Compared with the CON group (Table 4), the serum content of TG in BLE2, BLE3 and BLE4 groups were significantly decreased (*p* < 0.05), except for BLE1, the LDL-c content in serum was significantly decreased with increasing levels of BLE supplementation (*p* < 0.05). In addition, there was a quadratic (*p* = 0.002) decrease in the TG content and a linear (*p* <0.001) decrease in the LDL-c content with the increasing inclusion of BLE in the diet. No significant difference was observed in TC, HDL-C and GLU contents among groups (*p* > 0.05).

**Table 4.** Effect of dietary BLE on serum cholesterol metabolism parameters of broilers.


Note: a.b.c means within the same row with no common superscript differ significantly (*p* < 0.05). TC: total cholesterol; TG: triglyceride; HDL-c: high-density lipoprotein cholesterol; LDL-c: low-density lipoprotein cholesterol; GLU: glucose; <sup>1</sup> standard error of the means; <sup>2</sup> Orthogonal polynomials were used to evaluate linear and quadratic responses to the levels of BLE treatment; <sup>3</sup> CON: basal diet, BLE1, BLE2, BLE3, BLE4 and BLE 5 group, basal diet adding 1.0, 2.0, 3.0, 4.0 and 5.0g/kg BLE, respectively.

#### *3.4. Antioxidant Index of Serum*

Birds in BLE4 and BLE5 groups showed higher T-AOC activity in serum (Table 5) than other groups (*p* < 0.05). The CAT activity in serum of BLE2, BLE3 and BLE5 groups was significantly higher than that in the CON group (*p* < 0.05). Supplementation with BLE significantly increased GSH-Px activity in the serum of broilers (*p* < 0.05). In addition, BLE linearly increased T-AOC and CAT activity (*p* < 0.001, and *p* = 0.003), and quadratically increased GSH-Px activity in the serum as the addition level increased (*p* < 0.001), and GSH-Px activity in BLE2 and BLE3 groups was significantly higher than that in other BLE supplementation groups (*p* < 0.05). SOD activity and MDA concentration were not affected by BLE supplementation (*p* > 0.05). Birds in the BLE2 group showed a numerical minimum serum MDA concentration among groups.


**Table 5.** Effect of dietary BLE on serum antioxidant index of broilers.

Note: a.b.c means within the same row with no common superscript differ significantly (*p* < 0.05). T-AOC: total antioxidant capacity; CAT: catalase; SOD: Superoxide dismutase; GSH-Px: Glutathione peroxidase; MDA: malondialdehyde; <sup>1</sup> standard error of the means; <sup>2</sup> Orthogonal polynomials were used to evaluate linear and quadratic responses to the levels of BLE treatment; <sup>3</sup> CON: basal diet, BLE1, BLE2, BLE3, BLE4 and BLE 5 group, basal diet adding 1.0, 2.0, 3.0, 4.0 and 5.0g/kg BLE, respectively.

#### *3.5. Antioxidant Index of Liver*

The effect of dietary BLE on the liver antioxidant index is shown in Table 6. Except for SOD in BLE4 group, the T-AOC and SOD activities in BLE supplementation groups were significantly higher than in the CON group (*p* < 0.05). Compared with the CON group, the CAT activity in BLE4 and BLE5 groups, and GSH-Px in the BLE5 group significantly improved (*p* < 0.05). In addition, linear (*p* = 0.028, and *p* <0.001) and quadratic (*p* = 0.009, and *p* = 0.007) increasing relationships between BLE level and T-AOC and CAT activities were observed, and there was a linear (*p* = 0.010, and *p* = 0.011) increase in SOD and GSH-Px activities as BLE supplementation increased, and SOD activity in BLE2 and BLE5 groups was significantly higher than in the BLE4 group (*p* < 0.05). Except for the BLE1 group, the MDA concentration was significantly decreased by BLE supplementation (*p* < 0.05); a linear (*p* = 0.014) and quadratic (*p* = 0.018) decrease effect was presented with increasing BLE. Moreover, a numerical minimum MDA concentration of liver was observed in the BLE2 group.



Note: a.b.c.d means within the same row with no common superscript differ significantly (*p* < 0.05). T-AOC: total antioxidant capacity; CAT: catalase; SOD: Superoxide dismutase; GSH-Px: Glutathione peroxidase; MDA: malondialdehyde; <sup>1</sup> standard error of the means; <sup>2</sup> Orthogonal polynomials were used to evaluate linear and quadratic responses to the levels of BLE treatment; <sup>3</sup> CON: basal diet, BLE1, BLE2, BLE3, BLE4 and BLE 5 group, basal diet adding 1.0, 2.0, 3.0, 4.0 and 5.0g/kg BLE, respectively.

#### *3.6. Antioxidant Enzyme Gene Expression in the Liver*

As shown in Figure 1, the SOD, GSH-Px and CAT mRNA expressions were all up-regulated with BLE supplementation as compared to the CON group (*p* < 0.05). In addition, the GSH-Px mRNA expression in BLE5 was significantly higher than that in BLE1 and BLE2 groups (*p* < 0.05), and CAT mRNA expression in BLE3, BLE4 and BLE5 groups was significantly higher than that in BLE1 and BLE2 groups (*p* < 0.05).

**Figure 1.** Effects of dietary BLE on antioxidant enzymes mRNA expression in liver of broilers. GSH-Px: Glutathione peroxidase; SOD: Superoxide dismutase; CAT: Catalase. Note: a.b.c means within the same gene of the histogram with no common superscript differ significantly (<0.05); CON: basal diet; BLE1, BLE2, BLE3, BLE4 and BLE 5 group, basal diet adding 1.0, 2.0, 3.0, 4.0 and 5.0g/kg BLE, respectively.

#### *3.7. Cholesterol Metabolism Related Gene Expression of Liver*

Compared with the CON group, the cholesterol 7- alpha hydroxylase (CYP7A1) and low-density lipoprotein receptor (LDLR) mRNA expressions were significantly up-regulated by BLE supplementation (*p* < 0.05), and the CYP7A1 mRNA expression in BLE3 was significantly higher than in other BLE supplementation groups (*p* < 0.05). The highest LDLR mRNA expression was observed in the BLE4 group, and BLE2, BLE3 and BLE5 groups also showed favorable mRNA expression of LDLR compared with the BLE1 group (*p* < 0.05). In addition, the 3-hydroxy3-methyl glutamate coenzyme A reductase (HMGCR) mRNA expression was downregulated significantly except in the BLE1 group (Figure 2). Also, HMGCR mRNA expression in the BLE5 group was significantly lower than that in BLE2 and BLE3 groups (*p* < 0.05).

**Figure 2.** Effects of dietary BLE on cholesterol metabolism-related gene expression in liver of broilers. CYP7A1: cholesterol 7- alpha hydroxylase; HMGCR: 3-hydroxy3-methyl glutamates coenzyme A reductase; LDLR: low-density lipoprotein receptor; SREBP-2: sterol regulatory element binding transcription factor 2. Note: a.b.c.d means within the same gene of the histogram with no common superscript differ significantly (*p* < 0.05); CON: basal diet; BLE1, BLE2, BLE3, BLE4 and BLE 5 group, basal diet adding 1.0, 2.0, 3.0, 4.0 and 5.0g/kg BLE, respectively.

#### **4. Discussion**

Muscle and fat are the main traits of carcass yield, while excessive fat deposition is a problem in the current poultry industry. It will not only affect broiler processing and feed conversion but also decrease carcass quality and the acceptance of consumers [20]. The present study indicated that BLE supplementation linearly improved eviscerated yield. Yang [21] reported that diet supplemented with 1.6 g bamboo leaf flavonoids per kg feed could improve carcass yield in broilers. As reported in the literature, flavonoids have a mild estrogen-like effect [22], which may contribute to muscle deposition of broilers. Bamboo leaf flavonoids are the major active components of BLE. It is reasonable to suggest that the linear improvement of eviscerated yield was attributed to increasing concentration of BLE. In addition, abdominal fat deposition was linearly decreased with BLE inclusion in the present study. It is well established that flavonoids regulate fat deposition and metabolism in animals. Li [23] reported that hawthorn leaf flavonoids reduced fat deposition in broilers in a dose-dependent manner. Most phytogenic flavonoids have a similar structure and functions [24]. Furthermore, Yang [25] demonstrated that BLE is confirmed to possess adipocyte differentiation properties. According to the present results, it is suggested that the flavonoids in BLE may contribute to reduce abdominal fat deposition. Although flavonoids in BLE play a major role in improving growth and fat metabolism, the lipid-lowering effect of polysaccharide in BLE cannot be ignored as it is reported that bamboo leaf polysaccharide could significantly decrease the liver fat content in mice [5], and the quadratic decline percentage of liver weight rate may result from the decrease of liver fat content in the present study.

Oxidative stress is a key factor leading to cardiovascular diseases, such as atherosclerosis, hyperlipidemia, inflammation and other chronic diseases [26]. When the redox state of the body is out of balance, the accumulation of reactive oxygen species will cause damage to vascular endothelial cells. On the other hand, the elevated TC and LDL-c in serum are the arch-criminal cause of cardiovascular diseases [27]. Thus, there are important links between antioxidant status, cholesterol metabolism and cardiovascular disease.

The antioxidant parameters in liver and serum indicate the antioxidant capacity of the organisms. Antioxidant enzymes like SOD, CAT and GSH-Px cooperate to eliminate excess free radicals and maintain homeostasis. The T-AOC refers to total antioxidant capacity, while MDA is one of the lipid peroxidation metabolites, reflecting the degree of oxidative stress. It is worth mentioning that CAT and GSH-Px activities in both serum and liver presented a dose-dependent enhancement as BLE increased. Zhang [28] reported that BLE could enhance GSH-Px enzyme activity in serum of aged rats. In addition, Zhang [29] demonstrated that liver-injured mice supplemented with bamboo leaf flavonoids showed improved GSH-Px enzyme activity and alleviated liver injury. As reported in the literature, the antioxidant effects of flavonoids in living systems are ascribed to their capacity to transfer electron free radicals, chelate metals catalysts, activate antioxidant enzymes, and inhibit oxidases [30]. It is reasonable to suspect that the flavonoids in BLE play a major part in improving antioxidant enzyme activities. However, the GSH-Px enzyme of serum in BLE2 and BLE3 groups was significantly higher than in other BLE groups. According to the research, BLE has peroxide scavenging capacity [31,32], so we speculated that with the increasing inclusion of BLE in the diet, the antioxidant capacity was mainly attributed to BLE itself. Our results showed that the MDA in liver tissue was linearly and quadratically decreased as BLE increased. Flavonoids and polyphenols play a role in inhibiting the formation of thiobarbituric acid-reactive substances [33]. In addition, studies showed that broilers supplemented with plant extracts rich in flavonoids, polyphenols and polysaccharide, such as *Ginkgo biloba* leave extract [34], and *Artemisia annua* extract [35], could enhance free radical scavenging capacity. In some animal models, like hyperlipemia [36], gastric mucosal damage [37] and myocardial ischemia reperfusion [38] rats, when supplemented with BLE remedy, the oxidative damage caused by these interventions was alleviated, accompanied by a low MDA concentration in serum or liver. According to our present results, it is suggested that BLE supplementation in broiler chickens improved antioxidant capacity and alleviate oxidative stress. Elisabeth [39] explained that a variety of electrophilic compounds including polyphenol and plant-derived constituents trigger the nuclear factor erythroid 2-related factor 2 pathway response. The SOD, CAT and GSH-Px are downstream genes of this pathway. Our results showed that mRNA expression in the liver was significantly up-regulated. It is reported that BLE can activate hepatic phase II enzymes [40] or the AKT pathway [41] to improve inflammation and oxidative stress, all related to antioxidant effects. With the strictly demonstrated antioxidant effect of BLE in vitro studies [31,42], it is well-founded to speculate that BLE could both increase antioxidant enzymes and decrease chain breaking in broiler chickens, and these effects explain the linear improvement of T-AOC as BLE increased.

Cholesterol homeostasis is very important for broilers, as its metabolism dysfunction will lead to atherosclerosis, bile duct blockage or gallstones and other diseases [43,44]. The TC, TG, HDL-c, and LDL-c contents in serum are important indicators of cholesterol metabolism. In the present study, the TC and HDL-c concentrations in plasma were not affected by BLE supplementation. As reported in the literature, HDL-c is mainly secreted by the liver and small intestine, which plays a major role in transporting cholesterol and maintains a relative stable concentration itself [45]. Ding [12] and Liu [36] also found that BLE did not affect HDL-c concentration in hyperlipemia rats. These findings are similar to the present results. In addition, we speculated that the non-affected TC concentration with BLE supplementation in the present study may be attributed to the high content of TC in broilers [46]. The TG content in serum influences the accumulation of fat deposition. Our results present a quadratic decrease in TG and LDL-c contents in serum with BLE supplementation. According to the research, the TG, and LDL-c contents in serum were reduced significantly with BLE supplementation [13] in some hyperlipemia rat models. Furthermore, Yang [41] reported that BLE possesses an anti-inflammatory function in macrophages and inhibits adipogenic differentiation. A large number of studies have shown that flavonoids and polyphenols have regulatory effects on fat deposition and cholesterol metabolism in animals. Genistein [47], hawthorn leaf flavonoids [23], and soy isoflavone [48] reduce blood lipid and improve cholesterol metabolism in broilers. It is reasonable to speculate that bamboo leaf flavonoid possesses the same function, and supplementation of BLE could decrease the TG and LDL-c contents in serum of broilers and improve cholesterol metabolism. LDL-c is susceptible to free radicals, and high free radical scavenging capacity could reduce the oxidized LDL-c content [49]. It is speculated that the reduction of LDL-c concentration with dose may result from the antioxidant capacity improvement.

Although the lipid-lowering effects of BLE were strictly demonstrated, little research has been conducted on the effects of BLE on cholesterol metabolism and related mRNA expression in broiler chickens. The absorption, transformation, and synthesis of cholesterol metabolism are involved with some key regulators. For further investigating the effect of BLE on cholesterol metabolism, Quantitative Real-Time PCR was performed for these regulators. HMGCR is a rate-limiting enzyme in the whole process of cholesterol synthesis; increasing HMGCR activity will promote endogenous biosynthesis of cholesterol in the liver [50]. CYP7A1 catalyzes the conversion of cholesterol to bile acid; up-regulating CYP7A1 activity could decrease cholesterol levels [51]. LDLR mediates plasma LDL-c, which is ingested into cells for metabolism and degradation, and approximately 75% of plasma LDL-c was cleared in the liver [52]. Chen [47] reported that 50 mg/kg genistein significantly decreased HMGCR and CYP7A1 mRNA expression levels and increased LDLR mRNA expression in broiler liver. In addition, dietary of soy isoflavone showed the same outcomes in high-cholesterol diets rat [48]. Similarly, genistein exhibited the same effect of inhibiting HMGCR activity in cells [50]. Bamboo leaf flavonoid is the main compound of BLE, with a similar structure to genistein and soy isoflavone [30,53], and the present results are consistent with abovementioned studies in terms of HMGCR and LDLR mRNA expression. Furthermore, BLE showed an excellent inhibitory effect on HMGCR mRNA expression, indicating that BLE may reduce cholesterol synthesis and promote the clearance of LDL-c in serum. Studies on CYP7A1 activity regulation are not identical; both exogenous and endogenous cholesterol affect the expression of CYP7A1, and bile acids also have a negative feedback effect on CYP7A1. Our results showed significant up-regulated mRNA expression of CYP7A1, and higher CYP7A1 activity may contribute to the conversion of absorbed LDL-c to bile acid in the liver. By combining serum lipid parameters with the expression of liver cholesterol metabolism genes, it is deduced that BLE improved cholesterol metabolism by up-regulating LDLR and CYP7A1 mRNA expression to promote the conversion of LDL-c into bile acid, and down-regulated HMGCR expression to reduce cholesterol synthesis. However, due to the complicated mechanism of cholesterol metabolism, our study only

presented a basic result of the effect of BLE on cholesterol metabolism, and the mechanism still needs to be further studied.

#### **5. Conclusions**

In conclusion, broiler chickens supplemented with BLE presented a linear improvement of eviscerated yield and reduced abdominal fat deposition. BLE supplementation improved antioxidant capacity by enhancing SOD, GSH-Px and CAT mRNA expression and reducing lipid oxidation, and a dosage of 2.0 to 3.0 g/kg presented the best outcome. Supplemental BLE decreased LDL-c concentration in serum, up-regulated mRNA expression of CYP7A1 and LDLR, and down-regulated mRNA expression of HGGCR. Supplementation of BLE improved cholesterol metabolism of broilers to some extent, but the specific mechanism needs further investigation.

**Author Contributions:** Conceptualization, M.S.; Formal analysis, M.S., Z.X., M.J., A.L. and H.H.; Funding acquisition, L.Z.; Supervision, L.Z.; Writing—original draft, M.S.; Writing—review & editing, T.W. and L.Z.

**Funding:** This research was funded by The National Natural Science Foundation of China (NO.31601973), National innovation and entrepreneurship training program for college student in Nanjing Agriculture University (NO.2019103070267), College student innovation and entrepreneurship training program in Nanjing Agriculture University (NO. S20190014).

**Conflicts of Interest:** All authors declare no conflict of interest in this work.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **Denatonium Benzoate-Induces Oxidative Stress in the Heart and Kidney of Chinese Fast Yellow Chickens by Regulating Apoptosis, Autophagy, Antioxidative Activities and Bitter Taste Receptor Gene Expressions**

**Enayatullah Hamdard 1,**†**, Zhicheng Shi 1,**†**, Zengpeng Lv 1, Ahmadullah Zahir 2, Quanwei Wei 1, Mohammad Malyar Rahmani <sup>1</sup> and Fangxiong Shi 1,\***


Received: 5 August 2019; Accepted: 9 September 2019; Published: 19 September 2019

**Simple Summary:** Denatonium benzoate is a strong bitter taste receptor agonist, extensively used for its activation of different cell pathways. Taste signals have been associated to food recognition and avoidance, and bitter taste provokes an aversive reaction and is assumed to protect chickens from consuming poisons and harmful toxic substances. The results of the study revealed that dietary supplementation with medium and high doses of denatonium benzoate damaged the epithelial cells of the heart and kidneys by inducing apoptosis and autophagy and reduced the growth of chickens, respectively. However, mRNA expressions of bitter taste receptors, downstream signaling effector genes, apoptosis-, autophagy- and antioxidant-related genes were higher on day 7, while these expressions were subsequently decreased on day-28 in the heart and kidney of Chinese Fast Yellow chickens in a dose-response manner.

**Abstract:** The sense of taste which tells us which prospective foods are nutritious, poisonous and harmful is essential for the life of the organisms. Denatonium benzoate (DB) is a bitter taste agonist known for its activation of bitter taste receptors in different cells. The aim of the current study was to investigate the mRNA expressions of bitter taste, downstream signaling effectors, apoptosis-, autophagy- and antioxidant-related genes and effector signaling pathways in the heart/kidney of chickens after DB dietary exposure. We randomly assigned 240, 1-day-old Chinese Fast Yellow chicks into four groups with five replicates of 12 chicks and studied them for 28 consecutive days. The dietary treatments consisted of basal diet and feed containing DB (5, 20 and 100 mg/kg). The results revealed that dietary DB impaired (*p* < 0.05) the growth performance of the chickens. Haemotoxylin and eosin staining and TUNEL assays confirmed that medium and high doses of DB damaged the epithelial cells of heart/kidney and induced apoptosis and autophagy. Remarkably, the results of RT-PCR and qRT-PCR indicated that different doses of DB gradually increased (*p* < 0.05) mRNA expressions of bitter taste, signaling effectors, apoptosis-, autophagy- and antioxidant- related genes on day 7 in a dose-response manner, while, these expressions were decreased (*p* < 0.05) subsequently by day-28 but exceptional higher (*P* < 0.05) expressions were observed in the high-dose DB groups of chickens. In conclusion, DB exerts adverse effects on the heart/kidney of chickens in a dose-response manner via damaging the epithelium of the heart/kidney by inducing apoptosis, autophagy associated with bitter taste and effector gene expressions. Correlation analyses for apoptosis/autophagy showed agonistic relationships. Our data provide a novel perspective for understanding the interaction of bitter taste, apoptosis, autophagy and antioxidative genes with bitter taste strong activators in the

heart/kidney of chicken. These insights might help the feed industries and pave the way toward innovative directions in chicken husbandry.

**Keywords:** denatonium benzoate; bitter taste receptors; apoptosis; autophagy; heart; kidney; chicken

#### **1. Introduction**

Taste is well-known biological descriptor for sweet, bitter, sour, salty receptors and ion channels, which plays a critical role in the life and nutritional status of chickens and other organisms. Bitter taste perception provides animals with critical protection against the ingestion of poisonous and harmful toxic compounds [1]. Taste signals have been associated to food avoidance and recognition, as well as feed or liquid intake in different species of animals [2–4]. With the simultaneous inflation in the cost of animal feed and higher standards of livestock products, people endeavor to discover novel feed additives and effective alternatives to traditional antibiotics [5,6]. Efforts have been made to extract incredible numbers of potential additives from natural plants, and they often display bitter taste. However, bitter taste receptors are part of a superfamily which includes more than twenty members. This makes very difficult to do functional studies for each bitter taste receptor. In addition, bitter taste receptors expressions are not limited to taste buds but also exist in extra-gustatory organs, so it is critical to determine their extra-gustatory functions. Several studies have already revealed that bitter taste receptors exert a variety of functions in different cells and tissues [7,8]. Chickens have only three bitter taste receptors: ggTas2r1, ggTas2r2 and ggTas2r7 [9]. Due to their low number of bitter taste receptors, chickens are a good minimalistic model for understanding the functions of bitter taste receptors in non-gustatory tissues [9]. Denatonium benzoate as a bitter taste receptor stimulus can bind to the bitter taste receptors to activate bitter taste signaling. Furthermore, it has been reported that this receptor family plays a critical role in the heart [10], thyroid [11], and gastrointestinal muscle [12]. However, their roles in the kidney has never been determined. Therefore, our present project aimed to examine the roles and underlying mechanisms of bitter taste transduction signaling associated with mRNA expression patterns of bitter taste receptors, apoptosis-, autophagy- and antioxidant-related genes in the hearts and kidneys of chickens.

Cysteine aspartate-specific proteases (CASPs or caspases) serve as intrinsic initiators of apoptosis by cleaving substrates at aspartate residues [13]. In mammals and humans the caspase protein family currently consists of 13 and 11 isoenzymes, respectively [14]. The function of caspases is closely associated to the initiation and execution of apoptosis, with caspases categorized as either initiator or effector caspases. CASP2, CASP8, CASP9 and CASP10 are initiator caspases, and CASP3, CASP6 and CASP7 are effector caspases [15]. Known as interleukin 1β-converting enzyme, CASP1 plays an important role in both apoptosis and inflammation [16]. Apoptosis is regulated by stepwise activation of caspases for the processing or cleaving of other caspases [17].

Programmed cell death or apoptosis has a vital role in various biological events. This is a process of single cell death controlled by the activation of specific genes, the elimination of unwanted or damaged cells by apoptosis is an indispensable action that occurs via several mechanisms which maintain cellular homeostasis, and normal regulation of the immune system [18]. An earlier study showed that denatonium benzoate enhanced intracellular Ca2+, damaged mitochondria and induced apoptosis in airway epithelial cells, respectively [19]. Programmed cell death is activated by intracellular stresses and developmental cues. Well-known representative intrinsic regulators, the extended BCL2 family proteins, play crucial roles in cell death regulation and are able to regulate several cell death mechanisms, including apoptosis, necrosis and autophagy [20–22]. Caspase-independent mechanisms lead to the release of apoptosis-inducing factor (AIF) from the mitochondria, inducing large-scale DNA fragmentation in several cell types including heart and kidney cells to induce apoptosis caused by AIF [22]. BCL2 family proteins contain at least one of four BCL2 homology (BH) domains (i.e., BH1, BH2, BH3 or BH4), and the number of BH domains included in proteins is associated with their apoptotic functions [23].

Autophagy is the pathway involved in forming an organelle called an autophagosome. This pathway moves something from the cytoplasm of the cell into the lysosome for degradation. The term, which derives from the Greek words 'auto' meaning self and 'phagos' meaning to eat, is defined as a catabolic pathway involving the degradation of cellular components via the lysosomal machinery [24,25]. Autophagy is the natural process by which the cells can clear out damaged mitochondria, recycle proteins, and get rid of intracellular pathogens. However, all organisms need a balance of autophagy with anabolic processes. There are about 30+ different proteins involved in the formation of the autophagosome. Researchers are still actively figuring out how all of the bits and pieces of the process go together, but recent genetic studies have shed a lot of light on the pathway. A family of genes known as the autophagy-related genes, whose abbreviations start with A/TG, code for several of the proteins integral to autophagy. Several of these genes have variants that have been studied in reference to pathogen susceptibility, autoimmune diseases, cancer, and sepsis [26–28].

There is a complex system containing natural enzymatic and non-enzymatic antioxidants that protect the body from oxidative damage. Briefly, the antioxidant enzymes (SOD, MnSOD, CAT, GSH, and GSH-Px) appear to be the first line of defense during oxidative stress, and exert beneficial effects preventing oxidative damage in poultry raising [29–31]. Catalase (CAT) participates in defense mechanisms against oxidative stress by converting H2O2 into water and molecular oxygen [32]. The antioxidant glutathione peroxidase enzymes (GPX) are implicated in the protection of cells against oxidative damage by reducing H2O2 and other organic peroxidases to water with reduced glutathione [32].

Denatonium benzoate (DB), one of the most bitter-tasting substances known, is described as extremely unpleasant at a lower amount and can cause perceptible bitterness [33]. DB has been demonstrated extensively as a bitter taste agonist and used to activate bitter taste receptors in many cell types, including taste cells [34]. DB is intensely bitter and non-toxic, and can be detected by human taste receptors [35]. For these reasons DB has been broadly added to liquid detergents, cosmetics, plastic toys and personal care products to avoid the consumption of harmful substances [36]. Aside from its strong bitter taste, DB also exerts biologic effects on various physiological systems in different organisms. Exposure to DB quickly suppressed ongoing intake and delayed gastric emptying in rodents [37,38].

However, there is limited knowledge about the biologic effects of DB and its potential molecular mechanisms in chicken heart and kidneys and no studies have been conducted to date to investigate the relationship between bitter receptors, signaling effectors, and apoptosis-, autophagy- and antioxidantrelated genes in the chicken heart and kidney. Therefore, the objective of this study was to investigate the mRNA expressions of bitter taste and its downstream signaling effectors, apoptosis-, autophagy-, and antioxidant-related genes and transduction signaling pathways in chicken heart and kidney to DB dietary exposure in a dose-response manner. The results were confirmed by RT-PCR, qRT-PCR, haemotoxylin and eosin, TUNEL assay, correlation of apoptosis, autophagy and bitter taste receptors and its associated downstream signaling pathway, complete amino acid sequence alignments of related genes, selected gene heat mapping and the potential molecular mechanisms of action of dietary exposure to denatonium benzoate on the heart and kidney cells of Chinese Fast Yellow chickens were determined.

#### **2. Materials and Methods**

#### *2.1. Chemicals*

Denatonium benzoate (98%) was purchased from Adamas Reagent Co. Ltd. (Nanjing, China) and then stored at room temperature until the end of the experiments. RNA0se (phenol 38%), isoprophyl alcohol, trichloromethane, 100% absolute alcohol, DNA/RNAase-free water all were purchased collectively from RNA detection from (Takara Bio Inc., Shiga, Japan), Cat. # RR047A v

201810Da PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) and Cat. # RR420A v201810Da, TB Green™ Premix Ex Taq ™ (Tli RNase H Plus), were both purchased from TaKaRa (Dalian, China).

#### *2.2. Birds Management and Treatments*

A total of 260 1-day-old local Chinese Fast Yellow chicks were housed individually in the Animal Room of Nanjing Agricultural University under standard conditions, and were randomly divided into four (4) treatment groups: Control group (Control), low-dose DB-treated group (5 mg/kg, Low-Den), Medium-dose DB-treated group (20 mg/kg, Medium-Den) and high-dose DB-treated group (100 mg/kg, High-Den), respectively. The control group was fed with basic corn-soybean diet according to NRC (1994) (Table 1). The Low-Den, Medium-Den and High-Den treatment groups were fed the same commercial diet with denatonium added at 5, 20 and 100 mg/kg, respectively. Each treatment includes five cages, and each cage consisted of 12 chicks. Chicks were reared in a ventilated chicken house in which the lighting regime was 16-h light:8-h dark, the relative humidity was approximately 50 ± 5% and chickens were offered formulated feed (Table 1) ad libitum with freely available tap water for 28 consecutive days. Average body weight was calculated on day 1, day 07, day 14 and day 28, respectively. The experimental protocol and procedures were designed and approved in accordance with the Guidelines for the Care and Use of Animals prepared by the Institutional Animal Care and Ethical Committee for Nanjing Agricultural University (Permit number: SYXK (Su) 2019-0047), Nanjing, China.

#### *2.3. Feed Mixing and Formulation*

A basic corn-soybean diet with the ingredients listed in Table 1 was purchased from ADM (Nanjing, China).


**Table 1.** Feed formulation for the entire period of experiment (d 1–d 28).

The complete diet provided the following contents (% per kilogram): vitamin A, 12,500 IU; vitamin D3, 2,500 IU; vitamin E, 30 IU; vitamin K3, 2.65 mg; vitamin B1, 2 mg; vitamin B2, 6 mg; nicotinic acid, 50 mg; pantothenic acid, 12 mg; vitamin B6, 4 mg; folic acid, 1.25 mg; vitamin B12, 0.025 mg; biotin, 0.25 mg; Fe, 50 mg; Zn, 75 mg; Mn, 100 mg; Cu, 8 mg; I, 0.35 mg; Co, 0.2 mg; and Se, 0.15 mg.

The feed was mixed with a manual electric mixing machine available in the Nanjing Agricultural University animal house, according the experimental design.

#### *2.4. Sample Collection*

On days 7 and 28 of age (starter and grower stages), 10 chickens with body weights near the average of their group were slaughtered via exsanguination. The heart and kidneys were gingerly separated, and immediately cut up into two sections. One section was promptly fixed in 4% paraformaldehyde for histological analyses, whereas the second section was stored at −80 ◦C for the analysis of gene expression.

#### *2.5. TUNEL Assay*

The terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate nick-end labeling (TUNEL) assay was carried out referring to the kit manufacturer's instructions. Heart and kidney apoptosis were determined using a TUNEL Bright Green Apoptosis Detection Kit (A112, Vazyme Biotech, Nanjing, China). According to the protocol, the paraffin sections of heart and kidney were deparaffinized, rehydrated and then incubated with Proteinase K (20) at room temperature for 20 min. Later, the sections were incubated with TdT enzyme buffer containing double distilled H2O, eecombinant TdT enzyme, equilibration buffer and Bright Green Labeling Mix at 37 ◦C for 60 min under dark conditions. Finally, after washing three times in PBS, the sections were stained with 4 ,6-diamidino-2-phenylindole (DAPI) staining solution (C1005, Beyotime Biotechnology, Shanghai, China) for 5 min under dark conditions. The negative control was prepared as above without incubation of the TdT enzyme buffer to ensure no non-specific reaction occurred during the experiments. Images were taken through a LSM 700 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany). The total numbers of apoptotic cells (green color) and total cells (blue color) were counted using the Image-Pro Plus software 6.0. The apoptotic index was defined as the ratio of apoptotic cells to total cells.

#### *2.6. Body and Organ Weight Measurements*

All chicken live body weights were measured at four different stages on days, 1, 7, 14 and 28 of the experiment and a total 10 chicken in each group (two chicken/replicate) were used in each stage of killing to collect and measure heart and kidney weights at day 07 (starter stage) and at day 28 (grower stage), respectively (Figure 1).

**Figure 1.** Effects of DB supplementation on the body and organ weight gain (gr) of chickens. (**A**) Body weight of chickens at 0, 07, 14, 28 days of age (n = 10). (**B**) Organ weights of chickens at day 7 and day 28 of age (n = 10). Data are presented as mean value ± SEM. Values without the same mark (a, b, c) represent statistically significant differences (*p* < 0.05). Subfigure A uses line graphs with experimental days on the *X*-axis for body weights, and subfigure B separates the data into two bar graphs for heart and kidney, respectively.

#### *2.7. Haemotoxylin and in Eosin Staining (Histological Observations)*

To observe histological changes, heart and kidney samples were fixed in 4% paraformaldehyde for 12 h, then dehydrated through a graded ethanol series, cleared with xylene, embedded in paraffin wax, and serially sectioned at 4-μm thickness. The sections were stained with H&E, and histopathological changes were then viewed under a YS100 microscope (Nikon, Tokyo, Japan). Four (4) sections from each stage were observed for determination of apoptotic and autophagic cells in the tissues as reported previously [39].

#### *2.8. Total RNA Extraction and mRNA Quantification*

The total RNA from heart and kidney for RT-PCR and real-time PCR was extracted and purified from frozen collected tissues using RNAose (Takara Bio Inc,), which includes gDNA Eraser (Perfect Real Time) for elimination of genomic (g) DNA according to the manufacturer's protocols. The concentration and quality of total RNA was identified by a micro-spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The samples with the 260/280 ratios of 1.8–2.0 and 260/230 ratios of 2.0–2.2 were chosen for further RT and qRT-PCR reactions. Afterward, mRNA was reverse transcribed into complementary DNA (cDNA) using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time, Cat. # RR047A v201810Da) in accordance to the manufacturer's instructions. Gene-specific primers for bitter taste receptors (ggTas2Rs), apoptosis related genes, autophagy genes and antioxidant genes were generated with aid of the nucleotide database of The National Center for Biotechnology Information (NCBI) [40]. according to their published cDNA sequences (Table 2). The target genes and the housekeeping gene were synthesized by Sangon (city, country) and applied for real-time PCR and their primer sequence is shown in Table 2. Amplicon lengths for real-time PCR were between 92 and 200 bp. The PCR final reaction mixture of 20 μL included 10 μL of TB Green Premix Ex Taq (Tli RNase H Plus) (2×), 0.4 μL of ROX Reference Dye 1 or Dye 2 (50×), 2 μL of cDNA template diluted in ratio of 1:3, 0.4 μL of each primer (10 μM) and 6.8 μL of double distilled H2O (TaKaRa). PCR reactions were performed in 96 well reaction plates on a 7500 Real-time PCR instrument (Applied Biosystems, ABI, Beverly, MA, USA), all genes were assayed three times. under the following conditions: hold stage (95 ◦C for 30 s), PCR stage (40 cycles of 95 ◦C/2 min, 60 ◦C/34 s) apparently, to verify the amplification of a single product, a stage with a temperature increment (Melt Curve Stage) was conducted to generate a melting curve under the following conditions: (95 ◦C/15 s, 60 ◦C/1 min), followed by a temperature increment of 95 ◦C/15 s. Relative gene expression levels were analyzed by the 2\*\*\*\*\* method after normalization against β-actin.

#### *2.9. Statistical Analysis*

#### Body and Organs Weights Measurements

Body weight and two (2) selected organs weight measurements were described previously [41]. Significant differences between treatment groups and control group were analyzed by one-way analysis of variance (ANOVA) followed by a Duncan post hoc test. A *p* < 0.05 value was considered as statistically significant, marked with (a–d). Value =MEAN ± SEM, weight unit (g).

#### *2.10. Gene Expression Profile*

For relative gene expression analysis apoptosis, autophagy, antioxidant, downstream signaling effectors and bitter taste receptor genes for each organ at different growing stages were compared to he chosen control gene (β-actin) in two tissues (heart and kidney) using one-way ANOVA. In addition, multiple comparison among means of mentioned target genes and β-actin gene levels in each group were calculated using Dunnett's test (marked with a–e) as shown in the respective figures. An alpha level of 0.05 was set for all tests. Results were described as the mean ± standard error of the mean (SEM). *p* < 0.05 was considered to be statistically significant. These statistical analyses were conducted

with GraphPad Prism 6 (IBMP Crop, Unites States), and IBM SPSS Statistics, version 20 software (SPSS Inc., Chicago, IL, USA).

#### **3. Results**

#### *3.1. Body and Organ Weights Measurements*

After continuous feeding for 28 consecutive days with DB-containing diets or control, the body weights of Chinese Fast Yellow chickens were significantly decreased in the medium-dose (20 mg/kg) and high-dose (100 mg/kg) groups (*p* < 0.05), however, no differences were observed between control and the low-dose (5 mg/kg) groups (Figure 1). After 28 days of dietary exposure, all DB-treated groups showed significantly reduced body weights (*p* < 0.05; Figure 1A,B). However, the average body weight of control and low-dose treated groups were almost similar and no major changes were observed during the experiment. The heart and kidney weight gains were recorded twice during the time of experiment, the heart weight gains on day 07 in the control, low-dose and medium-dose of DB-treated groups were significantly (*p* < 0.05) similar, but reduced in the high-dose DB group, respectively, whereas, the heart weight gain on day-28 in all DB-treated groups were significantly (*P* < 0.05) reduced compared to the control group. In addition, the kidney weight gain on day-07 was similar in all DB-treated groups, interestingly the kidney weight gaining in comparison to heart weight gaining was higher in all groups. Moreover, the kidney weight gain on day-28 was equally reduced in all three DB-treated groups (low-dose, medium-dose and high-dose) but significantly (*p* < 0.05) increased in the control group, respectively (Figure 1A,B).

#### *3.2. Histological Observations*

#### 3.2.1. Histological Changes in the Heart of Chicken

To evaluate whether dietary DB exposure caused pathological changes in the hearts of chickens, we examined the heart tissue cellular morphology on days 07 and 28 via H&E staining and observed apoptotic changes. Compared to the control group, there was moderate necrosis, myocardial inflammatory infiltration and pyknotic cells, but no morphological distortion of cardiac cells in the low-dose of DB group (Figure 2A,B). In the medium-dose DB groups, there were greater changes compared to the low-dose ones, while severe changes were observed in the high DB dose groups. We also observed cell necrosis, apoptotic cells (cell damage/death), pyknotic cells, and distortion of the normal morphological characteristics of the cells due to the toxic effects of DB for all respective doses. When the DB dietary treatment was continued for 28 consecutive days these effects were increased, with karyorrhexis and karyolysis due to severe necrosis which caused autophagy, shrinkage of fibroblasts leading to condensation, and the number of apoptotic cells in the hearts of chickens was increased (Figure 2A,B).

The results indicate that a high dose of DB can cause severe necrosis of the cells, shrinkage, distortion and changes in the characteristics of the cells which finally lead to apoptosis and autophagy, respectively.

**Figure 2.** Effects of Denatonium benzoate supplementation on chicken heart histomorphology on days 07 and 28 (**A**,**B**). On day-07 necrosis (red arrow), apoptotic cells (white arrow), pyknotic cells (yellow arrow) and distortion of the morphological characteristics of the cells (green arrow) due to the toxic effect of DB were seen. On day-28, karyolysis due to severe necrosis which caused autophagy (black arrow), apoptotic cells (white arrow) shrinkage of fibroblasts which led to condensation (green arrow) indicate the effect of DB.

#### 3.2.2. Histological Changes in the Kidney of Chicken

Histological changes are a direct indication of kidney damage. In our study, microstructure studies were performed on chicken kidneys. The chicken kidney microstructure is shown in Figure 3A,B.

By comparing the control group results with the groups treated with three different doses of DB, we found several histological changes in the kidney of chickens on days 07 and 28 (Figure 3A,B). Major changes were observed in the high-dose DB group on day-07 of dietary exposure to DB. These changes included Proximal Convoluted Tubules (PCTs) with swelling due to hydrophiid degeneration and influencing the smaller in size collecting tubes. Inflammed infiltrated cells were also observed and separation of basement membrane of Distal Convoluted Tubules (DCTs) which led to karyolysis, apoptosis and autophagy. No live cells were found in the damamged areas of kidneys due to the toxic effects of medium and high doses of DB (Figure 3A,B). In addition, the chickens were more sensitive

during the initial days of DB dietary exposure, therefore the major histological effects were visible on day-07 of exposure rather than day-28 and there were no major differences in terms of histopathological observations on day-07 and day-28 (Figure 3A,B). The results indicated that the chicken kidney is a vital organ for the filtration and excretion of hydrophilic substances inside the body. Therefore, swelling of nephrotic cells were observed which can lead to nephrosis and dysfunction of nephrons, the glomerulus is sensitive to toxic compounds and this can easily lead to inflammation and damage of nephrotic cells.

**Figure 3.** Effects of DB supplementation on the histomorphology of chicken kidney on day-07 and day-28 (**A**,**B**). On day-07, the black arrow shows that PCTs became swollen (hydrophiid degeneration). The white arrow indicates inflamed infiltrated cells and the green arrow indicates separation of the basement membrane of DCTs; Karyolysis occurred where no cells were found due to the toxic effects of DB. The effects on day 28 showed no major differences.

#### *3.3. Confirmation of Apoptosis in the Heart and Kidney of Chicken by TUNEL Assay*

The results after staining samples with a TUNEL assay kit are shown in Figure 4A, BTUNEL-positive cells with green colored nuclei represent apoptotic cells.

**Figure 4.** Tunnel assay of heart and kidneys at 07 days (**A**) and at 28 days (**B**) of age by immunofluorescence. The blue color represents the total cells in the heart and kidneys, and the green color represents the apoptosis cells in the heart and kidneys with three different doses of DB (low-dose, medium-dose and high-dose).

These apoptotic cells were observed on day-07 and day-28 in the hearts and kidneys of chicken. However, the numbers of apoptotic cells in both hearts and kidneys were higher on both day-07 and day-28 in the medium- and high-dose DB-treatment groups compared to control group (*p* < 0.05; Figure 4A,B). In comparison with the medium-dose group, the high-dose group exhibited a greater percentage of apoptotic cells on day-28 in both hearts and kidneys of the chickens (*p* < 0.05). No significant pathological differences were observed in apoptotic cells in the low-dose group in contrast with the control group on days 07 and 28, respectively.

#### *3.4. Determination of Real Time (RT) and Quantitative Real-Time (qRT- PCR) Expressions*

#### 3.4.1. The mRNA Expressions of Bitter Taste and Downstream Signaling Effectors in the Heart

RT and qRT-PCRs results showed that after dietary exposure to DB the expressions of ggTas2R1, ggTas2R2 and ggTas2R7 in the hearts of chickens were significantly (*p* < 0.05) close on days 07 and 28 for all three different DB doses, with some slight subsequently higher expressions in the BD high and medium dose groups, respectively (Figure 5A,B). We observed however correlated expressions of bitter taste receptor genes, which were gradually up-regulated in a dose-dependent manner, with exception in ggTas2R7 on day 28, while the rest of the genes were similarly up-regulated (Figure 5A,B).

The expressions of downstream signaling effectors (α-gustducin, PLCβ2, IP3R3, TRPM5) in the heart of chickens on day 07 were significantly (*p* < 0.05) higher in most of the DB-treated groups than control (Figure 5A,B). whereas, the low- and high-dose DB groups on day-07 displayed significantly (*p* < 0.05) enhanced expressions of α-gustducin, PLCβ2 and IP3R3, while only the low-dose group displayed higher expressions of TRPM5 (Figure 5A,B), respectively.

The expressions of downstream signaling effectors on day-28 were only significantly (*p* < 0.05) up-regulated for α-gustducin, in the medium and high dose DB groups, while PLCβ2, IP3R3 and TRPM5 were significantly (*p* < 0.05) down-regulated in all DB-treated groups compared to control (Figure 5A,B). Nevertheless, the high DB (100 mg/kg) groups still exhibited significantly (*p* < 0.05) increased mRNA expressions of ggTas2R1, ggTas2R2, ggTas2R7, α-gustducin, PLCβ2, IP3R3, TRPM5 in contrast with the control group (*p* < 0.05; Figure 5A,B).

**Figure 5.** *Cont*.

**Figure 5.** RT and qRT-PCR showed effects of DB supplementation on heart mRNA expressions of bitter taste receptors and downstream effectors at 07 days (**A**) and 28 days (**B**) of age. Data are presented as mean value ± SEM (n = 6). Values without the same mark (a–d) represent statistically significant differences (*p* < 0.05). gg, Gallus gallus; PLCβ2, phospholipase Cβ2; IP3R3, type 3 inositol-1,4,5-trisphosphate receptor; Denatonium benzoate- Low Dose treated group, Denatonium benzoate- Medium Dose treated group, Denatonium benzoate- High dose treated group.

#### 3.4.2. Correlation Analysis

Correlation analyses (Tables 2 and 3), exhibited a highly positive correlation among bitter taste receptors and downstream signaling gene sets separately, which may be due to their similar biological function. Moreover, a weak negative correlation among bitter taste genes-and signaling effectors-related genes was observed, which suggests that bitter receptors and signaling effectors may function frequently antagonistically.

**Table 2.** Correlation analysis of bitter taste and downstream signaling effectors related genes in the heart of chicken.


\*\* Correlation is highly significant at the 0.01 level (2-tailed), while \* correlation is less significant.


**Table 3.** Correlation analysis of bitter taste and downstream signaling effectors related-genes in the kidney of chicken.

\*\* Correlation is highly significant at the 0.01 level (2-tailed), while \* correlation is less significant

#### 3.4.3. The mRNA Expressions of Bitter Taste and Downstream Signaling Effectors in the Kidney

ggTas2Rs showed significantly (*p* < 0.05) higher expressions in the kidney of chickens compared to the medium dose chickens, contrary to ggTas2R7, which was expressed higher in the DB high dose group on day-07 and then gradually lower expressed in day-28 with slight differences in ggTas2R7 (Figure 6A,B), respectively.

The results indicate that ggTas2R1, ggTas2R2, ggTas2R7 have contrary expressions in kidney on day-07 and day-28 (Figure 6A,B). It was found that the expressions of ggTas2Rs were dose-dependent on DB different doses on day-07, while ggTas2R1 expressions were highly dose-dependent in the DB medium dose group among all treated groups in day-07 (Figure 6A,B). However, on day-28 the expressions of bitter taste-related genes (ggTas2Rs) were significantly (*p* < 0.05) down-regulated and there were dose-independent correlations with expressions (Figure 6B).

On day-07 the expressions of two downstream signaling effectors genes (IP3R3, TRPM5) were significantly higher in the low, medium and high DB dose groups compared to α-gustducin and PLCβ2 (Figure 7A). However, on day-07 the high dose (100 mg/kg) DB group showed significantly higher expressions among all treated groups, exhibiting a high dose-dependent correlation (Figure 6A). In addition, on day-28 the downstream signaling effectors individually up-regulated the expression of α-gustducin in the medium and high dose DB groups, while PLCβ2, IP3R3 and TRPM5 were significantly (p < 0.05) down-regulated in all DB- treated groups compared to control (Figure 7B). Therefore, we concluded that the downstream signaling effector gene expressions were quite similar on day-07 and day-28 and no major differences were observed between them (Figure 6A,B)

**Figure 6.** Effects of DB supplementation on kidney mRNA expressions of bitter taste receptors and downstream effectors at 07 days (**A**) and 28 days (**B**) of age. Data are presented as mean value ± SEM (n = 6). Values without the same mark (a–d) represent statistically significant differences (*p* < 0.05). gg, Gallus gallus; PLCβ2, phospholipase Cβ2; IP3R3, type 3 inositol-1,4,5-trisphosphate receptor; Denatonium benzoate- Low Dose treated group, Denatonium benzoate- Medium Dose treated group, Denatonium benzoate- High dose treated group.

**Figure 7.** Effects of DB supplementation on kidney mRNA expressions of apoptosis-related genes at 07 days (**A**) and 28 days (**B**) of age. Data are presented as mean value ± SEM (n = 6). Values without the same mark (a–d) represent statistically significant differences (*p* < 0.05). CALPN1, Calpain 1; CALPN2, Calpain 2; CASP-2, Caspase 2; CASP-3, Caspase 3; CASP-7, Caspase 7; CASP9, Caspase 9; BCL2, B-cell CLL/lymphoma 2; BCL2L1, BCL2 like 1; MCL1, myeloid cell leukemia sequence 1; BID, BH3 interacting domain death agonist; NOXA, similar to ATL-derived PMA-responsive peptide; Denatonium benzoate-Low Dose treated group, Denatonium benzoate- Medium Dose treated group, Denatonium benzoate-High dose treated group.

#### *3.5. Determination of Real Time (RT) and qRT-PCR Expressions of Apoptosis Related Genes*

3.5.1. The mRNA Expressions of Apoptosis Related Genes in the Heart of Chicken

The qRT-PCR results showed significant (*p* < 0.05) changes in the expressions of apoptosisrelated genes in both day-07 and day-28, whereas lower expressions were observed in the heart of chicken on day-28, respectively (Figure 8A,B). Comparing the CALPN1 and CALPN2 caspase family genes (CASP2, CASP3, CASP7 and CASP9) and BCL2, BCL2L1, MCL, BID and NOXA on day-07,

a significantly (*p* < 0.05) higher expression pattern was observed in CALPN1 among the selected genes, that gradually reduced the CASP family-related genes and consequently the BCL2 family (Figure 8A). Lower expressions were observed in BCL2 and NOXA genes among others (Figure 8A). Therefore, the expression levels of apoptosis-related genes in day-07 were dose-dependent in the heart of chicken (Figure 8A).

On day-28 of the experiments, the apoptosis-related gene expression results revealed higher expressions of both CALPN1 and CASP9 among the genes (Figure 8B), while other genes were also significantly (*p* < 0.05) expressed, but the expressions were still lower in contrast with day-07 (Figure 8A,B). Remarkably, lower expressions on day-28 in the heart of chicken were observed in NOXA among apoptosis-related genes in the low-dose DB group (Figure 8B). To conclude, the expressions in both stages in the heart of chicken were dose- and time-dependent, hence, we found a dose-dependent relation but a time-independent relation in expressions was exhibited. Therefore, higher expressions were observed in CALPN and CASP family genes against a high dose (100 mg/kg) of DB but lower in the low-dose chickens. Thus, these genes might have similar sensitivity against DB in the heart of chicken.

**Figure 8.** Effects of BD supplementation on heart mRNA expressions of apoptosis-related genes at 07 days (**A**) and 28 days (**B**) of age. Data are presented as mean value ± SEM (n = 6). Values without the same mark (a–d) represent statistically significant differences (*p* < 0.05). CALPN1, Calpain 1; CALPN2, Calpain 2; CASP-2, Caspase 2; CASP-3, Caspase 3; CASP-7, Caspase 7; CASP9, Caspase 9; BCL2, B-cell CLL/lymphoma 2; BCL2L1, BCL2 like 1; MCL1, myeloid cell leukemia sequence 1; BID, BH3 interacting domain death agonist; NOXA, similar to ATL-derived PMA-responsive peptide; Denatonium benzoate-Low Dose treated group, Denatonium benzoate- Medium Dose treated group, Denatonium benzoate-High dose treated group.

#### 3.5.2. Correlation Analysis

Correlation analyses (Tables 4–7), exhibited a highly positive correlation among apoptosis and autophagy-related gene sets in the heart and kidney of chicken, separately, which may be due to their similar biological function in selected organs. Moreover, a strong positive correlation among different apoptosis and autophagy-related genes was observed, which suggests that different apoptosis genes (CASP and BCL2 families) may function agonistically, while a very weak correlation was also observed among some genes, which suggests that they may function anti-agonistically, as shown in Tables 4–7, respectively.

**Table 4.** Correlation analysis of apoptosis related-genes in the heart of chicken.


\*\* Correlation is significant at the 0.01 level (2-tailed), while \* correlation is less significant.


**Table 5.** Correlation analysis of apoptosis related-genes in the kidney of chicken.

\*\* Correlation is highly significant at the 0.01 level (2-tailed), while \* correlation is less significant

**Table 6.** Correlation analysis of autophagy-related-genes in the heart of chicken.


\*\* Correlation is highly significant at the 0.01 level (2-tailed), while \* correlation is less significant

**Table 7.** Correlation analysis of autophagy-related-genes in the kidney of chicken.


\*\* Correlation is highly significant at the 0.01 level (2-tailed), while \* correlation is less significant

#### 3.5.3. The mRNA Expressions of Apoptosis-Related Genes in the Kidney of Chicken

After 28-days of continuous DB dietary exposure of the chickens, the results of both PCRs confirmed the expressions of apoptosis related-genes in the kidney of chickens (Figure 7A,B). On day-07, the expressions of CALPN2, CALPN1, CASP2, CASP3, CASP6, CAPS9 were significantly much higher than other apoptosis genes, respectively (Figure 8A). However, on day-07, the lowest expressions were found in NOXA among the genes in the kidney of chicken (Figure 7A). On day-28 of exposure, the highest significant expressions of apoptosis related-genes in the kidney of chicken were observed in CALPN1 and CAPS7 among the selected genes, but lower expressions were confirmed for BID, respectively (Figure 7B). The results indicate that the apoptosis-related gene expressions in the heart and kidney of chicken at the two stages of exposure were dose-dependent while we found an independent correlation with time (Figures 7A,B and 8A,B).

#### *3.6. Determination of Real Time (RT) and qRT-PCR Expressions of Autophagy-Related Genes*

#### 3.6.1. The mRNA Expressions of Autophagy-Related Genes in the Heart of Chicken

To determine autophagy-related genes expression in the heart of chicken, we performed RT and qRT-PCR to examine their expressions. On day-07, we observed that the lowest expressions for all DB-treated groups were for ATG5 and Beclin-1 among six selected autophagy-related genes (ATG5, Beclin-1, Dyanin, LC3-I, LC3-II, mTOR) respectively (Figure 9A,B). Meanwhile, the highest expressions of autophagy-related genes were found in LC3-II, Dyanin and LC3-I among the genes gradually (Figure 9A). Furthermore, significantly higher expressions for autophagy-related genes on day-07 was observed in the high dose (100 mg/kg) DB groups as shown in Figure 9A, whereas, the lowest expressions were exhibited in the Low-dose (5 mg/kg) DB-treated group and then in the medium-dose (20 mg/kg) DB groups (Figure 9A). We concluded from the above results that the expression levels of autophagy-related genes are directly correlated with the DB dose, therefore the expressions were dose-dependent, which lead to their higher and lower expressions in a dose-dependent manner. To evaluate the effects of DB on the autophagy-related gene expression profile in the heart of chicken we detected the expressions of six autophagy-related genes (ATG5, Beclin-1, Dyanin, LC3-I, LC3-II, mTOR) using RT and qRT-PCR techniques to confirm the expression levels. The results of both PCRs on day-28 showed that higher expressions were found in Dyanin and the lowest expression was found for Beclin-1 among all selected genes, respectively (Figure 9B). however, the expressions in ATG5, LC3-I, LC3-II and mTOR were almost similar and no major differences were observed among them (Figure 9 A,B). Notably, there was no significant differences in the expressions of ATG5, Beclin-1, LC3-I, LC3-II and mTOR and the expressions level was dose-dependent for those genes, with the only exception of mTOR, where we found a lower expression in the high dose DB group (Figure 9B). Remarkably, the expression of Dyanin was significantly higher at both stages (day-07 and day-28) in the heart of chicken among the six autophagy-related genes (Figure 9A,B). These results indicated that DB benzoate exposure aggravated ER stress and increased autophagy and the autophagic effects are dose dependent.

**Figure 9.** Effects of DB supplementation on heart mRNA expressions of autophagy- related genes at 07 days (**A**) and 28 days (**B**) of age. Data are presented as mean value ± SEM (n = 6). Values without the same mark (a–d) represent statistically significant differences (*p* < 0.05). ATG5; Beclin 1; Dynein; LC3-I; LC3-II, mTOR; Denatonium benzoate- Low Dose treated group, Denatonium benzoate- Medium Dose treated group, Denatonium benzoate- High dose treated group.

#### 3.6.2. The mRNA Expressions of Autophagy-Related Genes in the Kidney of Chicken

The autophagy-related gene expression figures indicated that among six examined autophagy-related genes in the kidney of chicken, the expression level of mTOR gene on day-07 is dramatically increased (Figure 10A), while the rest of the genes were significantly almost equally expressed in the kidney of Chinese Fast Yellow chickens, but the higher expressions were observed in the DB high dose group for all six autophagy-related genes of the experiment with the exception of ATG5 (Figure 10A). These findings indicate that, there is direct relationship between dose and autophagy-related gene expressions in the kidney of chickens.

On day-28, higher expressions were found in Dyanin like on day-07, but lower expressions were displayed for Beclin-1 among autophagy-related genes, respectively (Figure 10B). However, the expression profile of autophagy-related genes in the kidney of chicken was dose-dependent and showed significantly gradually changing expressions (Figure 10B). Therefore, the results suggest that, autophagy-related gene expressions are dose-dependent on both day-07 and day-28 in the kidney of chicken and high dose (100 mg/kg) of DB was found to be the dose with the highest effects among the treatments.

**Figure 10.** Effects of DB supplementation on kidney mRNA expressions of autophagy- related genes at 07 days (**A**) and 28 days (**B**) of age. Data are presented as mean value ± SEM (n = 6). Values without the same mark (a–d) represent statistically significant differences (*p* < 0.05). ATG5; Beclin 1; Dynein; LC3-I; LC3-II, mTOR; Denatonium benzoate- Low Dose treated group, Denatonium benzoate- Medium Dose treated group, Denatonium benzoate- High dose treated group.

#### *3.7. Determination of Real Time (RT) and qRT-PCR Expressions of Antioxidant Genes*

#### 3.7.1. The mRNA Expressions of Antioxidant-Related Genes in the Heart of Chicken

Chicken heart is one of the organs susceptible to oxidative processes, and its oxidation state can be reflected by the levels of antioxidant gene expression, for example, glutathione peroxidase (GPx1) and catalase (CAT), which are responsible for the clearance of hydroxyl radicals [42]. Our results showed that the expression profile of oxidative stress-related genes in the heart of chicken on day-07 was significantly (*p* > 0.05) increased in a dose-dependent manner and we found high level expressions in the DB high dose (100 mg/kg) group among the three selected doses (low-dose, medium-dose and high-dose), however the expression pattern of GPX1 gene was significantly higher compared to other antioxidant genes (CAT, SOD1) (Figure 11A). On day-28, the expression level of three antioxidant-related genes were significantly (*p* > 0.05) increased consequently and higher-level expressions were detected in the DB high dose chickens, the gene expressions for GPX1, SOD1 and CAT were dose-dependent

and CAT showed higher expression among them (Figure 11B). The results suggest that the heart is sensitive to oxidative stress and shows significant (*p* > 0.05) elevated expressions of antioxidative stress genes in a dose-dependent manner on both day 07 and day 28, while low-dose chickens displayed inconspicuous decreased expressions, respectively (Figure 11A,B).

**Figure 11.** Effects of DB supplementation on heart mRNA expressions of antioxidant-related genes at 07 days (**A**) and 28 days (**B**) of age. Data are presented as mean value ± SEM (n = 6). Values without the same mark (a–d) represent statistically significant differences (*p* < 0.05). GPX1, glutathione peroxidase 1; SOD, superoxide dismutase 1; CAT, catalase; Denatonium benzoate- Low Dose treated group, Denatonium benzoate- Medium Dose treated group, Denatonium benzoate- High dose treated group.

#### 3.7.2. The mRNA Expressions of Antioxidant-Related Genes in the Kidney of Chicken

Because of the high sensitivity of chicken kidney to oxidative stress, the oxidation state could be reflected by the level of antioxidant genes expression profile. Most enzymatic components of this antioxidant defense system are commonly known as "antioxidant enzymes" (e.g., catalase, superoxide dismutase, glutathione peroxidase). We evaluated the expressions of such antioxidant enzymes at two different ages of chicken as a parameter to assess oxidative stress in selected organs (heart and kidney). The experimental data revealed that the expressions of three antioxidant genes (GPX1, SOD1, CAT) were expressed with approximately equally significance (*p* > 0.05) and there were slight differences in the expression level among the genes, which indicates that the expressions and sensitivity of genes related to antioxidative activity in the kidney of chicken are correlated with the dose of DB, the same as was shown in the heart of chicken, respectively (Figures 11 and 12A,B).

**Figure 12.** Effects of DB supplementation on kidney mRNA expressions of antioxidant- related genes at 07 days (**A**) and 28 days (**B**) of age. Data are presented as mean value ± SEM (n = 6). Values without the same mark (a–d) represent statistically significant differences (*p* < 0.05). GPX1, glutathione peroxidase 1; SOD, superoxide dismutase 1; CAT, catalase; Denatonium benzoate- Low Dose treated group, Denatonium benzoate- Medium Dose treated group, Denatonium benzoate- High dose treated group.

#### *3.8. Amino Acid Sequences Complete Alignment*

We performed complete amino acid sequence alignment for the experiment selected genes. First, we searched for the amino acid complete sequence through exploring the NCBI database, then we did a complete alignment using two bioinformatic tools (ClustalX and Gene-Doc). The amino acid sequence alignment showed us that those genes which are identical have black color while those which are similar with each other have gray color for the alignment indications. The empty area means there is no similarity and identity among the genes, respectively. Interestingly, we found more identical and similar genes in bitter taste receptors family (ggTas2Rs), downstream signaling effectors genes and antioxidant genes, while there was little similarity among apoptosis- and autophagy- related genes. The illustrated alignment figures for bitter taste receptors genes, downstream signaling effectors genes, apoptosis related genes, autophagy related genes and antioxidant genes are shown in the Supplementary Materials.

#### *3.9. Heat Map of Selected Genes*

We performed a heat map analysis for all selected genes of the experiment (in Figure 13). In this heat map analysis, we precisely showed all gene expressions levels and confirmed the same results as described earlier in the Results section.

**Figure 13.** Heat map of expression profiles (bitter taste, downstream signaling effectors, apoptosis, autophagy and antioxidant genes) in supplementation with three different doses of DB in two stages (day-07 and day-28) in the heart and kidney of chicken.

#### **4. Discussion**

In the present study, we investigated the biological effects of denatonium benzoate (DB) on growth performance, mRNA expressions of bitter taste receptors, its downstream signaling effectors genes and related pathway, apoptosis, autophagy, antioxidant related genes, histological changes and correlations among genes expressions in the heart and kidney of Chinese Fast Yellow chickens on both day-07 and day-28 of the experiments using RT, qRT-PCR, Hematoxylin and Eosin and TUNNEL assays. We found that DB induced apoptosis, autophagy and increased the expressions of antioxidant-related genes in the heart and kidney of the chickens. However, the expressions of bitter taste receptors genes and its downstream signaling effectors were significantly higher on day-07 compared to day-28 for different DB doses, but the High-dose DB had more potential effects on apoptosis, autophagy, antioxidant, bitter taste receptors and its downstream signaling effectors gene expression than other doses, which significantly induced apoptosis and autophagy in the heart and kidney of chicken on both day-07 and day-28. Remarkably, we also found that bitter taste receptors and the associated signaling effectors, apoptosis, autophagy and antioxidant gene expressions were dependent in a dose-response manner. These findings suggest that the bitter taste receptors have a potential role among the extra-gustatory organs of the chicken, and high-dose DB causes severe necrosis via apoptosis and may result in autophagy in chicken heart and kidneys, while these symptoms were obviously observed on day-07, which proves that the chicken were more sensitive to DB exposure at the beginning of the experiments and later they adapted accordingly.

DB (485–740 mg/kg) exhibited a low toxicity rate in acute oral LD50 tests in rats and rabbits, while chronic toxicity studies have indicated that gavage of 16 mg/kg/day resulted in no compound-related toxicity in monkeys and rats [43,44]. In our current study, the average exposure amount of DB in the Low-dose (5 mg/kg), Medium-dose (20 mg/kg) and High-dose (100 mg/kg) groups was calculated on a daily basis for the feed and was less than the doses above. Therefore, DB dietary exposure for 28 days significantly reduced the growth performance and organ (heart and kidney) weights of the chickens. In agreement with our results, four weeks of treatment with bitter agonists like DB or quinine resulted in decreases in body weight gain associated with decreased feed intake [45]. Moreover, DB has been shown to influence ongoing interdigestive behavior, food intake and gut peptide secretion in healthy volunteers and DB may be able to suppress the contraction of smooth muscles, which inevitably affects the nutrient integration, palatability, digestibility and impairs body weight gain [33,46]. Interestingly, our results indicated that at day-07 and day-28 the ggTas2r2 expressions were higher among three tested bitter taste receptors, and separately, the higher expressions were found in the DB High-dose (100 mg/kg) group, which indicate that the expressions were dose-dependent. Overall, the expressions were significantly decreased on day-28 in contrast do day-07. This finding indicates that chicken sensitivity to DB decreased consequently.

The avian circulatory system is the main transport system of the body. It is the means by which nutrients, enzymes and other important needs for the proper functioning of body systems, organs, tissues and cells as well as body defense components are transported to where they are required. The heart is the most significant and vital organ of the avian circulatory system and its main function is blood supply/pumping of the blood [47]. To our knowledge this is the first time the potential mechanism underlying the heart pathological changes caused by different doses of DB has been determined. We performed haemotoxylin and eosin staining as well as TUNNEL assay examinations on day-07. In the haemotoxylin and eosin staining expreiments, we found particular pathological changes which alter cell necrosis, apoptosis, pyknotic cells, and distortion of morphological characteristics of the cell due to toxic effect of denatonium on the heart of chicken. Interestingly, these changes were more due to medium and high dose DB exposure. However, on day-28, we observed severe necrosis which caused apoptosis, autophagy and some shrinkage of the fibroblasts which can lead to condensation due to the effect of a high dose of BD (Figure 2A,B).

The urinary system is very complex because of its function. The kidneys maintain the water balance by removing excess water from the blood stream. Additionally, the kidneys maintain the electrolyte balance, and eliminate metabolic wastes, particularly nitrogen products. In addition, they need their own supply of nutrients for the maintenance of their own tissues and cells. When the kidneys are diseased or damaged and unable to carry out their functions efficiently, the animal becomes debilitated and death often occurs quickly [48]. The present study showed that, on day-07 and day-28, after exposure to different doses to DB the PCTs swell due to hydrophiid degeneration, the basement membrane of DCTs becomes separated and karyolysis occurred in the kidney of chickens due to the toxic effects of medium and high dose DB, respectively (Figure 3A,B). These findings were obviously more severe on day-28 compared to day-07 and suggest that after long term treatment with DB, the chicken kidney may suffer dysfunction.

Apoptosis is involved in cellular growth and development, and is important for the turnover of heart and kidney epithelial cells and tissue homeostasis [49]. Severe apoptosis is harmful for the heart and kidney, and can lead to cellular dysfunction [49,50]. It is reported that BD inhibits airway epithelial cell proliferation, decreases the number of cells and promotes cell apoptosis in a dose-dependent manner via a mitochondrial signaling pathway [19]. We performed TUNNEL assays to confirm pathological changes caused by denatonium benzoate in the heart and kidneys of chicken. As described previously, the apoptosis-related genes showed higher expressions in the heart and kidney of chicken, pathological changes that were also confirmed by haemotoxylin and eosin staining. In the present study, we detected more serious apoptosis in the heart and kidney epithelial cells of the medium and high dose DB groups. In addition, we found greater number of apoptotic cells in the high dose DB

group than in the medium-dose group at 28 days, these findings revealed that denatonium benzoate amplified apoptosis in a dose-effect manner. Consistent with our results, a previous study indicated that DB inhibited airway epithelial cell proliferation, and increased cell apoptosis in a dose-effect manner [19]. Other studies also demonstrated that bitter-tasting compounds induced apoptosis in cancer cells [51]. In our study, high-dose DB exerted seriously negative effects on the heart and kidney of Chinese Fast Yellow chickens. Interestingly, low-dose DB reduce the body weight without affecting the heart and kidney epithelium after long-term adaptation. We hypothesize that low-dose DB could be added into the feed for obese layers to control the body weight due to obesity-induced dysfunctions in layers [52]. This hypothesis requires further investigations to evaluate.

The downstream signaling effectors genes (α-gustducin, PLCβ2, IP3R3 and TRPM5) of bitter taste receptors displayed similar expression patterns as the bitter taste receptors. However, these expressions were higher in day-07, while the age of chicken increased the amplified genes' expressions (bitter and downstream signaling) in the heart and kidney of chicken were attenuated in both low-dose and medium-dose groups apparently. The results indicate that heart and kidney have a better tolerance to bitter stimuli after long-term of exposure to low and medium doses of DB. Taste transduction gene mRNA expression showed variations in the heart and kidney, through administration of DB, which suggests possible extra-gustatory effects for these genes on heart and kidney cell function of the chicken which require further investigations.

The transduction of taste is a fundamental process that allows animals to discriminate nutritious from noxious substances. Three taste modalities, bitter, sweet, and amino acid, are mediated by G protein-coupled receptors that signal through a common transduction cascade: activation of phospholipase Cβ2, leading to a breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol and inositol 1,4,5-trisphosphate, which causes release of Ca2+ from intracellular stores. The ion channel, TRPM5, is an essential component of this cascade; however, the mechanism by which it is activated is unknown. The bitter taste signaling transduction requires the involvement of Ca2<sup>+</sup> influx [53,54]. It is clarified that increased cytosolic [Ca2+] is reversed by Ca\*\*-ATPase [55,56]. Ca2+-ATPase is responsible for actively maintaining the balance of Ca2<sup>+</sup> concentration within the cytoplasm and cellular organelles [57]. In the present study, the reduced activity of Ca2<sup>+</sup>-ATPase revealed that the function of the Ca2<sup>+</sup> pump was affected in the heart and kidney. In addition, in humans and rodents, mitochondrial dysfunction and oxidative damage could cause Ca2+-ATPase damage [58,59]. Hence, in order to further understand the exact mechanism of Ca2+-ATPase damage in chicken further studies are required. Likewise, in agreement with our results in this experiment, the Ca2<sup>+</sup>-ATPase activity in low-dose DB and medium-dose DB groups were recovered with an adaptation to denatonium after 28 consecutive days of exposure. Moreover, excessive Ca2<sup>+</sup> concentration is able to activate the Ca2+-dependent cysteine proteases (calpain family) [60]. The major calpain isoforms are calpain 1 and calpain 2, which are expressed in different tissues including heart and kidney of the chicken [61]. Calpain is demonstrated to be capable of inducing the activation of caspase family, which results in apoptosis [56,62]. The activation of calpain could cause tissue damage, apoptosis and autophagy [62,63]. Calpain 1 (u-calpain) and calpain 2 (m-calpain) require micromolar [Ca2<sup>+</sup>] and millimolar [Ca2<sup>+</sup>] to activate, respectively [60]. We speculate that long-term of bitter taste receptor agonist caused [Ca\*\*]c to increase in heart and kidney epithelial cells in micromolar degree according to the result of enhanced CAPN1 expression and invariant expression of CAPN2. Elevated gene expressions of CAPN1 and apoptosis executioners (BCL2, BCL2L1, Caspase 2, Caspase 3, Caspase 7, Caspase 9, MCL1, BID, NOXA) in high-dose group indicated that high-dose DB induced more apoptosis in the heart and kidney of chicken. The apoptosis result was validated by a TUNEL assays. These data increase the possibility that after administration of DB, bitter taste receptors expressed in the heart and kidney of chicken are involved in the process of apoptosis via a calpain/caspase-dependent mechanism.

Autophagy is an evolutionary conserved catabolic process that includes different forms of digestive pathways, namely macro-autophagy, micro-autophagy, chaperone-mediated autophagy and non-canonical autophagy, regulating the degradation of a cell's own components through the lysosomal machinery [64]. Dramatically it plays a key homeostatic role in every cell type to maintain the balance between the synthesis, degradation, and consequent recycling of cellular components [65]. Currently, more than thirty different autophagy-related genes have been identified by genetic screening in yeast, and many of these genes are conserved in plants, flies and mammals, respectively [21,66]. Particularly, Bcl-2, a major apoptosis inhibitor, binds Beclin-1 to prevent its interaction with AGT5, thus resulting in the inhibition of autophagic initiation [67]. Conversely, when cleaved by caspase-3, Beclin-1 loses its ability to promote autophagy but renders cells sensitive to apoptosis [68]. Some reports indicate that autophagic degradation prevents apoptosis by eliminating harmful cellular wreckages [69,70], whereas others suggest that boosted autophagy results in increased apoptotic vulnerability [68,71]. However, data in birds are rare. Here, we report our results for the first time to indicate the autophagy-related genes expressions in the heart and kidney of chicken exposed to dietary DB treatment for 28 consecutive days. All selected autophagy-related genes in this experiment (ATG5, Beclin-1, Dyanin, LC3-I, LC3-II and mTOR) had high basal expression levels in the two examined tissues from chicken both at day-07 and day-28, respectively. However, the expressions of autophagy related genes were confirmed by RT and qRT-PCR analysis. Moreover, the expressions level of ATG5, Beclin-1 and mTOR were significantly lower in day-07 and day-28, while, we visualized higher significant expressions for Dyanin, LC3-I and LC3-II in both experimental stages in the heart of chicken. Interestingly, these expressions were in contradiction with the kidney of chicken data, where we observed higher significant expressions of Beclin-1, Dyanin and mTOR at both day-07 and day-28 among other selected genes, respectively. These results suggest that autophagy may play a crucial role in regulating many toxicity- and apoptotic-related complications which may be due to exposute to BD. On the other hand, limited knowledge is available on the role of the effect of the modulation of the autophagy process in the DB exposure context in chickens.

The endogenous cellular defense system consists of a number of antioxidant enzymes and proteins that maintain the cellular redox status, which is critical for various biological processes and functions. Most enzymatic components of this antioxidant defense system are commonly known as "antioxidant enzymes" (e.g., catalase, superoxide dismutase, glutathione peroxidase). Additionally, several experimental works evaluate the activity and expression of such antioxidant enzymes in different physiological conditions as a parameter to assess oxidative stress in a given system. As reported by Yuzhalin and Kutikhin [72]. Long-term accumulation of ROS and high levels of reactive oxygen species (ROS) may enhance oxidative damage at the DNA level. This process may affect several genes responsible for the regulation of proliferation, growth, survival, apoptosis, autophagy, invasion, leading to genomic instability and deregulation of several pathways [72]. Several enzymes, such as super oxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), nitric oxide synthase (NOS), and paraoxon's (PON), function to prevent damage caused by ROS [73].

Therefore, in the present study, our results indicated different expressions level in RT and qRT-PCR analysis for the confirmation of oxidative stress in the heart and kidney of chicken due to exposure to different doses of denatonium benzoate for 28 consecutive days. However, the expressions of GPX1, SOD and CAT were almost similar on both day-07 and day-28 in the heart of chicken but we observed higher significant (*p* < 0.005) expressions among them in the BD high-dose treatment groups. This indicates that the oxidative genes expressions are dose-responsive, and it confirms our previous apoptosis and autophagy results, while similar expression patterns were observed in the kidney of chicken in day-07 and day-28 of the experiment. Remarkably, GPX1, SOD and CAT expressions were significantly (*p* < 0.005) similar, while there were slightly higher expressions in the DB high-dose treatment groups, respectively. These results suggest that oxidative stress damage is correlated with apoptotic and autophagic changes in the heart and kidney of chicken in a dose-responsive manner.

#### **5. Conclusions**

In summary, exposure to DB for 28 consecutive days impaired the growth performance of chickens. The present study demonstrates that dietary DB has adverse effects on the heart and kidney epithelial cells of chickens in a dose-response manner via apoptosis, autophagy and antioxidative status involving bitter taste transduction. DB can increase the oxidative stress and promote the mitochondrial apoptotic pathway via regulating ATP synthesis and mitochondrial apoptosis. Our data provide a novel perspective for understanding the interaction of heart and kidney with strong bitter taste receptor agonist. This might open a new window and maybe helpful for deeper studies of the roles and underlying mechanisms of bitter taste receptors in chickens and could have a great contribution for the improvement of chicken feedstuffs.

**Supplementary Materials:** The following are available at http://www.mdpi.com/2076-2615/9/9/701/s1.

**Author Contributions:** Conceived and design the experiments: the work was conceived by E.H. Performed the experiment: E.H., Z.S. & Z.L. Analyzed the data: Data was analyzed by E.H., A.Z. and M.M.R. Contributed reagents/materials/analysis tools: Z.S., Z.L., Q.W. Manuscript writing: Manuscript writing was performed by E.H. All authors reviewed and approved the manuscript.

**Funding:** This study was supported by the Agricultural Independent Innovation Project in Jiangsu Province, China CX (18)2002. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing Jiangsu China 210095.

**Acknowledgments:** We thank to the Agricultural Independent Innovation Project of Jiangsu Province for providing the fund for successful implementation of the project.

**Conflicts of Interest:** The author declares no conflict of interest is present in this manuscript

#### **Abbreviations**


#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **The E**ff**ect of Dietary** *Camelina sativa* **Oil or Cake in the Diets of Broiler Chickens on Growth Performance, Fatty Acid Profile, and Sensory Quality of Meat**

#### **Sylwia Orczewska-Dudek \* and Mariusz Pietras**

Department of Animal Nutrition and Feed Science, National Research Institute of Animal Production, 32-083 Balice, Poland; mariusz.pietras@izoo.krakow.pl

**\*** Correspondence: sylwia.orczewska@izoo.krakow.pl

Received: 14 August 2019; Accepted: 24 September 2019; Published: 27 September 2019

**Simple Summary:** Feeding broiler chickens components rich in polyunsaturated fatty acid (PUFA), especially n-3 family fatty acid (*Camelina* oil or expeller) can be an effective way to improve both animal health and meat quality. The rate of mortality was the lowest in the group fed *Camelina* oil or expeller. Broiler chicken meat enriched with bioactive PUFA n-3 can be an alternative source of these fatty acids in the human diet. Introduction to the broiler diet of 40 g/kg *Camelina* oil, as well as 100 g/kg *camelina* expeller cake, significantly increased PUFA n-3 fatty acid and lowered PUFA n-6/PUFA n-3 fatty acid ratio. Furthermore, meat of chickens fed with *Camelina* oil was characterized by better juiciness.

**Abstract:** The aim of the present study was to determine the effect of supplementing the diets of broiler chickens with *Camelina sativa* oil or cake as a source of polyunsaturated fatty acids (PUFAs) on their growth performance, fatty acid profile, and sensory quality of meat. The 456 Ross 308 broilers aged 21–42 days were divided into 3 groups with 4 replicates of 38 birds in each. Chickens in the control group I (CTR) were fed a standard grower–finisher feed mixture containing 60 g/kg rapeseed oil. The experimental components, *C. sativa* oil—CSO (group II) or cake—CSC (group III), were included in a diet based on wheat and soybean at 40 and 100 g/kg, respectively. The use of *Camelina* oil and cake as feed components did not have a significant effect on the growth performance of the chickens. Analysis of the fatty acid profile in the lipids of the breast muscles showed that *Camelina* oil and cake reduced the content of monounsaturated fatty acids (*p* < 0.05) but increased the content of n-3 polyunsaturated fatty acids, especially α-linolenic acid (C18:3) (*p* < 0.01). Furthermore, both components reduced the ratio of n-6/n-3 PUFAs in the breast muscles (*p* < 0.01). Sensory analysis revealed that *Camelina* oil had a beneficial effect on meat juiciness, whereas *Camelina* cake slightly worsened the flavor and tastiness of the meat. In conclusion, supplementing the diet of broiler chickens with *Camelina* oil or cake can be an efficient method for modifying the fatty acid profile of the meat lipids in a beneficial way, without any negative impact on the growth performance of the chickens. According to the dietetic recommendations for humans, broiler chicken meat with a higher level of PUFA n-3 can be a good alternative source of these fatty acids in the human diet. Furthermore, *Camelina* oil improved the juiciness of breast meat.

**Keywords:** *Camelina* oil; *Camelina* cake; polyunsaturated fatty acids; growth performance; broiler chicken

#### **1. Introduction**

The ratio of n-6/n-3 polyunsaturated fatty acids (PUFAs) in the feed mixtures used for fast-growing broiler chickens plays a significant role in the prevention of metabolic disturbances [1] and heart failure, which is a cause of sudden cardiac death [2–4]. Numerous studies have indicated that the main cause of the sudden death of birds is the high content of n-6 PUFA in the feed mixtures. The results showed that the serum and heart muscle of these birds contained increased amounts of arachidonic acid (AA; C20:4) and a reduced total level of n-3 PUFA, especially eicosapentaenoic acid (EPA; C20:5n-3) [3,5,6]. Furthermore, the fatty acid profile of meat lipids is a significant factor determining meat quality. The ratio of n-6/n-3 PUFAs in chicken meat ranges from 7:1 to 15:1, and the breast muscles (*Pectoralis major*) are characterized by the most beneficial proportion [7,8]. The wide variation of this ratio is the result of using feed mixtures based on cereal seeds (corn, barley, wheat, and triticale) and plant oils (sunflower, corn, and soybean), as well as oilseeds characterized by a high content of n-6 linolenic acid [9].

Studies have revealed that the meat of chickens, similar to other monogastric organisms, can be efficiently enriched with n-3 PUFA by using an appropriate diet [10,11]. It was found that the use of feed mixtures supplemented with oils as a source of n-3 PUFA during the second growth phase of broiler chickens modified the fatty acid profile of meat lipids in a beneficial way [12,13]. The introduction of oil rich in α-linolenic acid (ALA) in the feed mixture used for broiler chickens increased the concentration of this acid and its long-chain derivatives, including EPA, DPA, and docosahexaenoic acid (DHA) in the meat lipids, which resulted in a decreased proportion of n-6/n-3 PUFAs [8,12]. Compared to mammals, broiler chickens have a greater ability to convert ALA to long-chain derivatives due to higher activity and wider substrate specificity of elongases responsible for the conversion of DPA to C24:5n-3 and then to DHA [14,15].

According to Haug et al. [8], the inclusion of poultry meat as a potential source of n-3 PUFA in the diet of contemporary populations can contribute to reducing the risk of cardiovascular diseases. In their experiment, the authors modified the fatty acid profile of the leg and breast muscles in broiler chickens by using a feed supplemented with a mixture of rapeseed and flax oil as a source of ALA. People consuming such enriched meat for 4 weeks showed an increased concentration of EPA in the serum. The results of other authors have also indicated that the meat of broiler chickens can be included as a potential source of n-3 PUFA in the human diet [11,16,17].

The oil of *Camelina sativa* is one of the richest known plant sources of ALA of the n-3 group [18,19]. *Camelina sativa* is an oil plant that attracts renewed interest of industry and agriculture after it was replaced in the post-war period by higher yielding rapeseed. The renewed interest in *Camelina sativa* results from a higher demand for fat raw materials necessary for production of biofuels. *Camelina* belongs to the oldest crop plants of the *Brassicacea* family. Soil requirements of this plant are modest; it can grow on poor soils and is resistant to drought and frost [20]. Moreover, it requires a lower fertilization rate than rapeseed and is resistant to insect pests [19,20]. *Camelina* oil is considered valuable mostly due to its nutritional values and chemical composition [19]. In particular, the cold-pressed oil is characterized by a high content of PUFAs and natural antioxidants such as tocopherols (791 mg/kg) [20] that make it exceptionally durable and fit for human consumption for 6 months [18]. It is also distinguished from other oils by a special taste and pleasant clear flavor of medium intensity. *Camelina* oil of a domestic variety has been shown to have high efficacy in modifying the fatty acid profile of meat lipids in broiler chickens [21–23]. In addition, it was observed to have a beneficial effect in reducing the ratio of n-6/n-3 PUFAs [22,23]. On the other hand, *Camelina* cake is characterized by high protein content (up to 45%) with a beneficial composition of amino acids [24] and fat content with a high proportion of ALA [19,25]. The energy value of *Camelina* cake in poultry, pig, and cattle was estimated at 8.0, 14.0, and 15.0 MJ ME/kg DW, respectively [24]. However, due to the presence of non-starch polysaccharides and glucosinolates, adding a high percentage of *Camelina* cake in the feed mixture can adversely affect the growth performance of broiler chickens [26,27].

Therefore, the aim of the present study was to determine the effect of *C. sativa* oil or expeller cake included as components in the diets of broiler chickens on the growth performance, fatty acid profile of lipids in the breast muscle (*Pectoralis major*), and sensory quality of meat.

#### **2. Materials and Methods**

#### *2.1. Birds, Housing, and Feeding*

The experiment was carried out according to the guidelines of the Ethics Committee for the Use of Animals in Research. No explicit approval of the committee was needed because the birds were only fed different diets (none of them were toxic—the regulation 68/2013 (16 January 2013) of the European Union Commission allowed the use of *C. sativa* seeds and products obtained by their processing, including oil and *Camelina* meal, as a feed component in animal diets) and no invasive procedures were performed on them. A total of 456 Ross 308 broiler chickens (hens and cockerels) were raised in group pens on litter from 1 to 42 days of age under standard housing conditions with free access to feed and water. During the first growth phase (1–21 days of age), the chickens were fed a starter feed mixture which did not contain the tested additives. In the second period of rearing (22–42 days of age), chickens were randomly divided into 3 groups with 4 replicates of 38 birds each. The chickens in the control group CTR (group I) were fed a standard grower diet containing 60 g/kg of rapeseed oil, whereas birds in the experimental groups were fed a feed mixture containing 40 g/kg of cold-pressed oil obtained from the seeds of spring *C. sativa* var. Borowska—CSO (group II) and 20 g/kg of rapeseed oil, or 100 g/kg of expeller cake—CSC obtained from *Camelina* of the same variety and 50 g/kg of rapeseed oil (group III). The composition and nutritional value of the grower feed mixtures used for the broiler chickens are presented in Table 1. Diets were formulated to provide nutrients according to the Polish recommendations for broilers [28] and to contain the same amount of metabolizable energy and crude protein within each set.


**Table 1.** Composition and the calculated nutrient contents of grower–finisher feed mixtures (g/kg).

\* Vitamin–mineral premix provided the following (per kg of feed mixture): Retinyl acetate—10,000 IU; cholecalciferol—2000 IU; tocopherol—20 mg; menadione sodium bisulfite—2.0 mg; thiamine—1.5 mg; riboflavin—5 mg; pyridoxine—3 mg; cyanocobalamin—0.02 mg; Ca-pantothenate—12 mg; folic acid—1 mg; biotin—1 mg; niacin—25 mg; choline chloride—400 mg; manganese—100 mg; iodine—0.8 mg; zinc—65 mg; selenium—0.2 mg; and copper—8 mg. Abbreviations: CTR, control group; CSO, *C. sativa* oil group; CSC, *C. sativa* cake group.

#### *2.2. Data Collection and Chemical Analyses*

The individual body weight of the chickens was determined at 1, 21, and 42 days of age, and the number of dead birds was noted throughout the experiment. Feed intake by the groups was determined for each pen. On the basis of the experimental data collected, the following basic parameters of production were calculated: Body weight gain (BWG), feed conversion ratio (FCR) per kilogram BWG, and mortality of birds. At the end of the experiment, at 42 days of age, 8 birds from each group (4 cocks and 4 cockerels) were slaughtered using a method adapted to their age, species, and body weight. The procedure was carried out in accordance with the Annex IV of European Parliament and Council Directive 2010/63/EU of 22 September 2010 on the protection of animals used for scientific purposes. If no pain or suffering was inflicted during the trial, the regulations allowed sacrificing the experimental birds before sampling. Accordingly, the birds were electrically stunned and then decapitated. The mass of fresh carcasses (after slaughter), as well as mass of cold carcasses (after cooling it for 24 h in temperature of +4 ◦C for 24 h), were determined. The simplified slaughter analysis of the carcasses was performed after cooling them at +4 ◦C for 24 h [29]. Samples of the breast muscles were collected for further analysis.

The diets were analyzed for the profile of higher fatty acids (HFA) and the content of tocopherols and tocotrienols. The content of HFA was determined using the modified method by Loor and Herebain [30] based on ISO 12966–2:2011. The fatty acids were separated in the form of methyl esters and determined using a VARIAN 3400 gas chromatograph with a flame ionization detector (250 ◦C, range = 11; carrier gas: helium, 3 mL/min; gas injection: 0.7 μL) and an RTX™-2330 capillary column (105 m × 0.32 mm, 0.2 μm). Tocopherols and tocotrienols were determined using liquid chromatography, according to the method described by Manz and Philip [31], with a Merck-Hitachi HPLC system equipped with a LiChroCART® 250–4 Superspher® 100 RP-18 cartridge on a 4-μm column and an FL detector (Ex. 295 nm and Em. 350 nm).

The collected samples of the breast muscles (*musculus Pectoralis major*) were analyzed for basic chemical composition, fatty acid profile, sensory parameters, and malonaldehyde content after 90 days of frozen storage (−20 ◦C). The basic chemical composition of the breast meat was determined according to the AOAC method [32]. The content of fatty acids in meat lipids was determined in the form of methyl esters using gas chromatography according to the procedures validated by the Central Laboratory of the National Research Institute of Animal Production in Aleksandrowice, Poland. Fat was extracted from the samples with a mixture of chloroform and methanol (2:1) according to the modified method of Floch et al. [33], and the extract was evaporated at 65 ◦C under nitrogen. The residue was saponified with 0.5 NaOH in methanol (80 ◦C, 20 min), and then esterified with BF in methanol [34] at 80 ◦C for 10 min, followed by the addition of hexane. After salting out with a saturated NaCl solution, the hexane layer was collected into a chromatographic vial and subjected to gas chromatography using a VARIAN 3400 system with an RTX-2330 capillary column (105 m × 0.32 mm, 0.2 μm; detector range = 11, 250 ◦C; carrier gas: helium, 3 mL/min), a Varian 8200 CX Autosampler, and Varian Star 4.5 software package for data analysis.

The TBA values in the breast meat were expressed as milligrams of malonaldehyde (MDA) per kilogram of meat. To determine TBA, meat samples were prepared according to a modified version of the method of Salih [35], as modified and described by Pikul [36]. TBA values were measured by the colometric method at the presence of 2-thiobarbituric acid.

Breast meat samples held for sensory analysis were frozen at –20 ◦C until evaluation. Breast meat was analyzed after cooking to determine the sensory impact of the tested components used on flavor and tastiness quality. The sensory evaluation of meat samples was conducted by eight internal panelists. The day before the analysis, 200 g of each sample was thawed at 4 ◦C, and cooked individually in a covered container in 400 mL of 0.6% saline, until the temperature inside the meat reached 70 ◦C. The temperature was measured using a special thermometer. After cooling, the meat samples were evaluated within 10 min. The flavor, juiciness, tenderness, and tastiness of the meat were evaluated based on a 5-point scale, where 5 meant strong appreciation and 1 an extreme dislike, on the basis of the method described earlier by Matuszewska and Baryłko-Pikielna [37].

#### *2.3. Statistical Analysis*

For evaluating the growth performance during the experimental period of 21–42 days, a total of 162 birds per treatment with 4 replications each were considered. As the other results were analyzed by including 8 replications per treatment, statistical analysis of the obtained indices was performed using a one-way analysis of variance. The significance of differences between the experimental groups was evaluated using the multiple-range Duncan test. The differences were deemed statistically significant at a confidence level of *p* < 0.05. The procedures were carried out using the SAS statistical package (version 9.2), procedure GLM.

#### **3. Results**

The addition of *Camelina* seed oil or expeller to the grower feed mixture used for broiler chickens influenced its PUFA profile (Table 2).


**Table 2.** Fatty acid profile of the feed mixtures used for broiler chickens (% of the sum of fatty acids).

The use of *C. sativa* oil or expeller cake reduced the content of saturated fatty acids (SFAs) from 19% (found in the feed mixture used for control group) to 16.1% and 11.7%, respectively, in the feed mixture used for experimental groups, and increased the content of n-3 PUFA (from 1.5% to approximately 12%), especially ALA. The supplemented feed mixtures were characterized by a narrow ratio of n-6/n-3 PUFAs amounting from 2.1 to 2.5 compared with the control feed mixture (6.8). The contents of αtocopherol in the feed were similar in all the groups. Supplementing the diet with *Camelina* oil, and especially cake, increased the content of γ-tocopherol by 20.4% and 110%, respectively, and slightly decreased the level of β-tocopherol (Table 3).


**Table 3.** Contents of natural antioxidants in the feed mixtures used for broiler chickens (mg/kg of feed).

The level of gamma-tocopherol in the feed mixture used for experimental groups was 36.6 (CSO group) and 63.9 mg/kg (CSC group), while that in the feed used for the control group was 30.4 mg/kg. The content of delta-tocopherol in the feed containing *Camelina* oil (4.01 mg/kg) or cake (5.7 mg/kg) was higher compared with the feed used for the control group (3.26 mg/kg) by approximately 23% and 75%, respectively.

Rearing results of broiler chickens are shown in Table 4. At the first period of rearing, there were no significant differences in the body weight of chickens and feed consumption, as well as in feed consumption ratio between the groups. Mortality rate in each group was similar and ranged from 0.91 to 1.29%. The addition of *Camelina* oil or cake to feed mixtures during the second growth phase did not significantly affect the BWG of the experimental groups compared with the control group. Moreover, the feed intake and conversion per kilogram of BWG remained at a similar level in all of the groups. Most birds that died during the second growth phase and the whole experimental period belonged to the CTR group (Table 4).


**Table 4.** Production parameters of broiler chickens. Abbreviations: BWG, body weight gain.

The addition of *Camelina* oil or cake to the diet did not affect the carcass weight and slaughter yield (Table 5). However, a reduction in the percentage of abdominal fat was noted in the carcasses in the CSO and CSC groups. The carcasses of chickens fed *Camelina* oil (CSO) were characterized by the largest weight and the greatest percentage of breast muscles (*p* < 0.05) and the lowest fat content. The carcasses of chickens in the CSO and CSC groups also showed the lowest percentage of the liver compared with the CTR group. A significant (*p* < *0.05*) reduction in proportion of skin with subcutaneous fat was observed in the carcasses of chickens from the CSO group.


**Table 5.** Slaughter analysis of the carcasses of broiler chickens.

a, b—the mean values in a row marked with different letters differ statistically significantly at *p* < 0.05.

The use of *Camelina* oil and cake as grower diet components for broiler chickens did not significantly affect the dry mass and the content of total protein and crude fat in the breast muscles (*Pectoralis major*) (Table 6).

**Table 6.** Results of the chemical analysis (%) of the breast muscles (*Pectoralis major*).


The results of the analysis of PUFAs in the lipids of the breast muscles (*Pectoralis major*) demonstrated that *Camelina* oil and cake caused a highly statistically significant (*p* < 0.01) increase in the content of n-3 PUFA, especially ALA (Table 7).

In addition, a significant increase (*p* < 0.01) in EPA was noted in the CSO group, which was fed with the diet supplemented with 4% *Camelina* oil. In the CSO group, as well as in the CSC group, the content of AA (C20:4) belonging to n-6 PUFA was significantly reduced (*p* < 0.05). The ratio of PUFA/SFA was significantly higher and the ratio of n-6/n-3 PUFAs was significantly reduced (*p* < 0.01) in groups II and III, compared with the control group. In the CSO and CSC groups, the content of monounsaturated fatty acids (MUFAs) was statistically significantly reduced (*p* < 0.01) compared with the CTR group, while the level of erucic acid (C22:1) was significantly increased (*p* < 0.01). In the CSC group receiving the *Camelina* oil-supplemented diet, the SFA content was significantly increased compared to the CTR and CSC groups (*p* < 0.05), mostly due to the increased levels of palmitic acid (C16:0) (*p* < 0.01) and stearic acid (C18:0) (*p* < 0.05). Among the acids of this group, significant increases were observed in AA (C20:0) (*p* < 0.01) and behenic acid (C22:0) (*p* < 0.05). The addition of 4% *Camelina* oil to the diet also significantly increased the content of conjugated linoleic acid (CLA) in the CSO group (*p* < 0.01).


**Table 7.** Fatty acid profile of the lipids of the breast muscles (*Pectoralis major*) (% of the sum of acids).

a, b—the mean values in a row marked with different letters differ statistically significantly at *p* < 0.05; A, B, C—the mean values in a row marked with different letters differ statistically significantly at *p* < 0.01.

After 3 months of frozen storage (−20 ◦C), the content of malonaldehyde was found to be reduced (Figure 1) by 19% and 20%, respectively, in the meat of the broiler chickens in the CSO and CSC groups compared with the CTR control group. However, statistical analysis did not confirm the significance of these differences.

**Figure 1.** Malonaldehyde content (mg/kg of sample) in the breast muscles (*Pectoralis major*) of broiler chickens fed with rapeseed oil—CTR group, *Camelina* oil—CSO group, and *Camelina* expeller cake—CSC group.

Supplementation of the diet of broiler chicken with *Camelina* oil (CSO group) significantly (*p* < 0.05) influenced the juiciness of the cooked meat compared with the control group (Table 8). On the other hand, the meat of the CSC group chickens fed *Camelina* cake-supplemented diet was characterized by an inferior tastiness and flavor.


**Table 8.** Results of the sensory analysis of the breast muscles (*Pectoralis major*) of broiler chickens (according to a 5-point scale: 5—the highest score; 1—the lowest score).

a, b—the mean values in a row marked with different letters differ statistically significantly at *p* < 0.05.

This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.

#### **4. Discussion**

The quality and nutritional value of oilseeds, oil pressed from them, and the by-products of oil production depend on their chemical compositions, fatty acid profile, and especially, the contents of antinutrients [26,27,38]. Recent studies have indicated that the oil and cake of *C. sativa* can be used as a source of PUFAs, mostly of the n-3 group, and natural antioxidants such as tocopherols without any impairment of the sensory quality of the poultry products [39–42]. Antinutrients contained in *Camelina* seeds include mostly glucosinolates, the content of which mainly depends on the variety and environmental conditions that prevail during the plant vegetation [38]. Moreover, the seeds contain crude fiber which can also have a negative impact on the production performance of chickens. Thus, the efficiency of production depends on the choice of *Camelina* variety and the percentage of seeds or by-products of oil pressing in the feed mixtures.

In the present study, the addition of 4% *Camelina* oil to the diet of broiler chickens did not significantly affect their growth performance. Moreover, *Camelina* oil had no effect on the final body weight and FCR. Similar results were shown by Pietras and Orczewska-Dudek [23], who investigated the effect of the addition of 3% and 6% dietary *Camelina* oil to broiler diet. In addition, Ja´skiewicz et al. [21] demonstrated that the addition of *Camelina* oil to broiler diet both during the first (1.43%) and second phase of growth (2.16%) did not adversely affect the production parameters. Analogous results were reported by Ja´skiewicz et al. [22] who used 6.91% *Camelina* oil in the starter diet and 4.07% *Camelina* oil in the grower diet.

The addition of *Camelina* cake to feed mixtures caused a slight reduction in growth rate, and thus led to a lower BWG in chickens. Feed conversion per kilogram of BWG was also found to be slightly increased in this group; however, the differences were not confirmed as significant by statistical analysis. Similar results were obtained by Aziza et al. [42,43] who supplemented the feed with 2.5, 5, and 10% *Camelina* cake. These authors indicated that the addition of *Camelina* cake to the diet of broiler chicken at 10% did not impair the production performance. On the other hand, Pekel et al. [44] demonstrated a negative effect of *Camelina* cake supplement on the production performance of chickens. They found that the addition of 10% *Camelina* cake in the feed mixture used for broiler chickens diminished their growth between 15 and 37 days of age, reducing feed intake, which resulted in a significant reduction of the final body weight. This could probably have resulted from a higher content of glucosinolates in *Camelina* cake used in their experiment, a higher level of fiber, and a limitation of nutrient availability [38].

Valkonen et al. [45] showed that increasing the content of *Camelina* cake (from 0 to 25%) in the feed mixtures used for broiler chickens produced a linear negative effect on the feed consumption, body weight, and feed conversion, but a beneficial effect in lowering the mortality. The authors indicated that the best growth performance was obtained using 5% and 10% *Camelina* cake in the feed. Moreover, the study by Widyaratne [46] demonstrated that when the percentage of *Camelina* cake in the feed mixtures used for broiler chickens was increased from 3 to 15%, the BWG of chickens and feed intake decreased in direct proportion to the increase. *Camelina* cake also had a negative effect on feed conversion (kg/kg BWG). Furthermore, when *Camelina* seeds were added to feed mixture at 30%, a slower growth rate and a low final body weight were observed. It was observed [41] that the addition of 5% and 10% *Camelina* cake to the diets of turkeys led to both growth depression and reduction of feed intake in the birds. These discrepancies between the present study and the above-mentioned studies could probably be due to the use of different varieties of *Camelina* grown under different climatic conditions. This was confirmed by the study of Waraich et al. [20], who indicated that the *Camelina* variety, climatic conditions during vegetation, and fertilization program influenced the contents of fatty acids, vitamins, and glucosinolates in seeds.

The lower percentage of dead birds noted during the second growth phase in groups fed with the diet containing *Camelina* oil or cake was in agreement with the results obtained by Aronen et al. [47]. It can be expected that the beneficial effect of *Camelina* oil is associated with its high content of ALA, which contributes to a reduction in the formation of pro-inflammatory eicosanoids when chickens are fed a diet deficient in ALA but containing high levels of phytosterols. Moreover, it was found that both *Camelina* oil and cake are a rich source of ALA of n-3 group and caused a reduction in the ratio of n-6/n-3 PUFAs in the feed mixtures used for broiler chickens which, according to Chen et al. [48], propitiously increased the level of antioxidant enzymes in the heart muscle and improved the immunological function of the thymus. In addition, Swi ˛ ´ atkiewicz et al. [49] suggested that n-3 PUFA improved the immunological functions in animals; in particular, they reduced the prevalence of acute and chronic inflammatory response generated towards harmful factors.

The present study demonstrated that the addition of 10% *Camelina* cake significantly reduced the proportion of the breast muscle in the carcasses by 7.5% compared with the control group. However, Aziza et al. [42,43] did not notice such a relationship. These authors concluded that irrespective of the percentage, *Camelina* cake did not have any negative effect on the quality and tissue composition of the chicken carcasses. In our study, it was observed a reduction in the content of abdominal fat in the carcasses of chickens that received the feed mixture supplemented with 4% *Camelina* oil or 10% cake. These results are in accordance with the data obtained earlier in the study by Pietras and Orczewska-Dudek [23], who also observed a reduction in the percentage of abdominal fat and weight of skin with subcutaneous fat in the carcasses of chickens fed with diet containing 6% *Camelina* oil.

On the other hand, Ja´skiewicz et al. [22] did not notice any significant effect of 2.04% *Camelina* oil added in the feed mixture used for chickens on the content of abdominal fat in the carcasses. According to Valkonen et al. [45], the amount of abdominal fat in the carcasses of chickens that were fed the diets supplemented with *Camelina* cake linearly decreased with an increase in the content of *Camelina* cake in the feed (from 5 to 10%). Similar results were reported by Crespo and Esteve-Gracia [13] and Ferrini et al. [50], who revealed that PUFAs reduced the accumulation of abdominal fat in contrast to SFAs and MUFAs. According to Takeuchi et al. [51] and Sanza et al. [52], PUFAs inhibited the synthesis of lipids in the liver and enhanced the processes of thermogenesis. This mechanism explains why PUFAs reduce fat in the abdomen and other parts, and as a consequence, decrease the total content of fat in a carcass [52,53]. In our study, both *Camelina* oil and cake did not affect the content of crude fat in the breast muscles of the broiler chickens. This is in line with the reports of other authors [13,54], who concluded that the source of fat in feed mixtures and their fatty acid profile did not influence the content of crude fat in the meat samples of broiler chickens.

The results of the analysis of HFA in the lipids of the breast muscles (*Pectoralis major*) indicate that both *Camelina* oil and cake significantly reduced the percentage of MUFAs, especially oleic acid, and increased the percentage of PUFAs, mostly of the n-3 group. According to the dietetic recommendations for humans, reducing the ratio of n-6/n-3 PUFAs is desirable because a narrow ratio of n-6/n-3 PUFAs is beneficial for maintaining a proper balance between eicosanoids formed from both groups of fatty acids [55,56]. The obtained results confirmed the data reported by other authors [22,23,42,43]. According to Thacker and Widyaratne [46], the addition of 15% *Camelina* cake induced a statistically significant increase in the content of n-3 PUFA and beneficially narrowed the ratio of n-6/n-3 PUFAs. In addition, Nain et al. [57] indicated that feeding broiler chickens with both a mixture enriched with 24% *Camelina* cake for 28 days and 16% *Camelina* cake for 42 days significantly increased the content of n-3 PUFA in the lipids of the breast and leg muscles, exceeding the content of 300 mg/100 g of meat.

In the present investigation, the lowest level of MUFAs was observed in the breast muscles from chickens that were fed with the mixture supplemented with 4% *Camelina* oil. In addition, the desaturation index SCD-1 (C18:1/C18:0) was significantly lower in this group, which can indicate a reduction in the activity of stearoyl-CoA desaturase which catalyzes the synthesis of MUFAs in the liver [58]. Moreover, Paton and Natambi [59] and Green et al. [60] confirmed the role of this enzyme as an inhibitor of the synthesis of MUFAs from SFAs, especially of the transformation of stearic acid into oleic acid and palmitic acid into palmitoleic acid. In the present study, the content of EPA was found to be statistically significantly increased in the experimental groups. The increase in EPA resulted from the elevated content of ALA, a precursor of long-chain fatty acids, which was confirmed by the studies Azcon et al. [61] and Jiang et al. [62]. It was found from the experiment that the addition of *Camelina* oil or cake to broiler diet significantly modified the fatty acid profile of the lipids of the breast muscle (*Pectoralis major*), leading to a significant increase in ALA (C18:3n-3).

Other authors [11] also observed that an oil rich in PUFAs influenced the lipid metabolism, leading to a greater accumulation of ALA in tissue lipids and reduction of SFA. As explained earlier, the increase of SFAs in the lipids of breast muscles (*Pectoralis major*) of broiler chickens fed with a mixture enriched with 4% *Camelina* oil could be caused by the suppression of the transformation of SFAs to MUFAs due to the diminished activity of an enzyme participating in these reactions. It is supposed that the highest increase in CLA also observed in this group additionally contributed to the reduction in the activity of SCD-1, which significantly decreased the content of SFAs in the lipids of the breast muscle. The group that received the feed mixture supplemented with *Camelina* cake showed a significantly increased content of linoleic acid (C18:2 n-6). LA is biologically converted into AA, the level of which was found declined in the lipids of the breast muscle (*Pectoralis major*) of chickens fed with *Camelina* oil or cake. Betti et al. [63] also noted in their study that the increase in n-3 PUFA resulted in a reduction of AA in the phospholipids of the breast muscle.

The results of several studies have demonstrated that the meat of animals containing more PUFAs is more susceptible to oxidative processes [63], which has a negative impact on its organoleptic characteristics and shelf life [11]. Supplementation of α-tocopherol to the chicken diet increases its content in body tissues and limits the oxidation of fat in the breast muscles (*Pectoralis major*) [42,43,64]. It was also confirmed that malondialdehyde (MDA) content in the breast muscles (*Pectoralis major*) of broiler chickens in the experimental groups, measured after 3-month frozen storage, was lower by 6% compared with the control group. The high content of natural antioxidants such as tocopherols and tocotrienols in *Camelina* oil and cake increased tocopherol content in the feed, which resulted in its increase in cell membranes, thus slowing down the oxidation of the lipids in the breast muscle (*Pectoralis major*). Moreover, *Camelina* oil was found to contain high levels of phytosterols and phenolic compounds that also contribute to the limitation of PUFA oxidation.

The obtained results agree with those reported by Aziza et al. [42] who discovered that *Camelina* cake added to feed mixture at 10% efficiently restricted the oxidation of fatty acids and improved the oxidative stability of meat lipids. In the present study, supplementation of the feed mixture with 4% *Camelina* oil did not impair the organoleptic quality of the cooked meat, and even significantly improved its juiciness. The obtained results were consistent with the earlier observations of Pietrsa and Orczewska-Dudek [23]. However, these authors did not note the effect of 3% and 6% *Camelina*

oil on the juiciness of breast muscle (*Pectoralis major*) in chicken. The current study showed that the addition of 10% *Camelina* cake to the feed mixture used for broiler chickens had a less favorable effect on the sensory quality by worsening the flavor and tastiness of the meat. In contrast, a beneficial effect of 5% *Camelina* cake added as a supplement to the feed mixture used for broiler chickens on the tenderness and juiciness of the meat was documented [41]. Such an effect was not observed when the content of *Camelina* cake was increased to 10%, which was also noticed in presented study. In addition, Valkonen et al. [45] indicated that *Camelina* cake had a favorable effect on the sensory properties of the breast muscles.

#### **5. Conclusions**

On the basis of the obtained results, it can be concluded that the addition of *Camelina* oil or expeller to the diet of broiler chickens can be an efficient method for modifying the fatty acid profile of the meat lipids in a way that is beneficial according to the dietetic recommendations for humans, without compromising the growth performance of the birds.

A high content of tocopherols in *Camelina* oil and *Camelina* meal slows down the oxidative processes of breast meat lipids, which is reflected in a lower content of malondialdehyde.

Additionally, *Camelina* oil had a beneficial effect on meat juiciness, whereas *Camelina* cake slightly worsened the flavor and tastiness of the meat.

*Camelina* expeller cake can be a cheaper alternative source of polyunsaturated fatty acids, as well as natural antioxidants, but the level of *Camelina* expeller used in broiler chicken diet should be more thoroughly investigated in future.

**Author Contributions:** Conceptualization, S.O.-D. and M.P.; Investigation, S.O.-D. and M.P.; Methodology, S.O.-D. and M.P.; Project administration, M.P.; Supervision, M.P.; Writing—original draft, S.O.-D.; Writing—review & editing, S.O.-D. and M.P.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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