Canola processing effects on the intestine, blood, and kidney of broiler breeder hens

Objective. Canola meal is one of the most important vegetable protein that contained the antinutritional factors. The aim of the study was to study the effect of canola processing on the intestine traits, blood metabolites, and kidney enzymes of broiler breeders. Material and methods. Four hundred fifty broiler breeder hens were used for 3 months. The completely randomized design was used with 6 treatments (unprocessed, processed by Lactobacillus Plantarum, Bacillus Subtilis, Aspergillus Oryzae, Neurospora Cytophilla, and Alkalase enzyme) and 5 replications. The collected data were analyzed by the LSmeans procedure of SAS statistical software. Results. The effects of treatments were significant on blood metabolites (p<0.05). Glucose concentration and High-density lipoprotein (HDL) were increased by processing methods. Also, the caecum, jejunum and duodenum weight were influenced(p<0.05). The weight of the duodenum and jejunum was increased and caecum weight was decreased. The processing of canola meal increased the length of the villi and decreased the depth of the crypt of the jejunum (p<0.05). Treatments effect was significant on the Alanine transaminase (ALT) and Alkaline phosphatase (ALP) (p<0.05). The effect of treatments was significant on the digestive amylase, lipase and protease activity (p<0.05). The canola process improved its digestibility. In other words, its protein quality, fatty acid profile and antimicrobial properties were improved. Conclusions. The different processing methods of canola improved the hen’s traits. It can be recommended to use the processed meal instead of the raw canola meal.


INTRODUCTION
The scientific name of canola is the Brassica Napus from the family Brassicaceae Cruciferea. In poultry nutrition, canola meal is the second most important vegetable protein after soybean meal. Canola was derived by breeding the rapeseed for reducing the glucosinolate amount (1). The erucic acid of canola oil is less than 2%, and the amount of glucosinolate in its meal is less than 30 μmol/g (2). The major anti-nutrient components of canola are phytate, glucosinolate, and tannin. These compounds reduce the palatability of the ration. Glucosinolates are not normally toxic. But, secondary products from their decomposition can have adverse effects on bird performance (3). High consumption of glucosinolates in broiler chickens reduces feed intake, decreases growth rate, hyperthyroidism, decreases thyroid hormone levels, enlarges liver, kidney, and thyroid gland, changes liver activity, and increases mortality (4,5).
In modern nutrition science, the production of new feeds through fermentation technology has been favored by fungal species (such as Rizopus Oligosporus, Aspergillus Oryza, Neurospora Cytophila and Aspergillus Niger) and bacterial species (such as Enterococcus Faecium and Bacillus Subtilis) (6,7). Fermentation technology eliminates anti-nutritional compounds and improves the structure and taste of the feed. Fermentation technology is better at processing feed than chemical methods. The use of microbial fermentation method to produce high quality protein and free of anti-nutritional compounds has been considered (8).
The inadequate use of antibiotics in the chicken farms has increased the antibiotic resistance of the bacteria. For this, the use of growthpromoting antibiotics was banned in Denmark in 1995, and then in the EU in January 2006. With this prohibition, choosing the right alternative has become a challenge for the poultry nutrition (9). The alternative ways such as fermentation technology, as a potential alternative to antibiotics, has attracted the attention of poultry nutritionists. Probiotics and prebiotics are other groups of additives introduced as alternatives to antibiotics. Probiotics are living microbial food additives that have a beneficial effect on the host by improving the gut microbial balance and enhancing the immune system (9). Prebiotics are contained non-digestible feed components and increase the growth or activity of beneficial bacterial species in the intestine and reduce the population of harmful host bacteria (10). The results of studies indicate that the addition of prebiotics to the diet not only stimulates the growth and activity of beneficial intestinal bacteria but can also increase the growth and activity of the host intestinal bacteria (10). Therefore, research has focused on other additives to address the weaknesses of probiotics and prebiotics. One of the most important and newest of these feed additives are peptides. Peptides are products obtained after incomplete hydrolysis of proteins by enzymes, acids, alkali, or fermented hydrolysis (11). Incomplete hydrolysis of proteins from plant or animal sources produces peptides of different molecular weights and high solubility in water (12). Research results show that in the process of hydrolyzing proteins by chemical methods (acid and alkaline solutions), enzymatic and fermentative, peptides are produced with beneficial feed properties such as antioxidant properties, immune system stimuli, antimicrobial, blood pressure modulation, anticancer, and anti-obesity production. (13). In the enzymatic hydrolysis of proteins, the hydrolysis process is completely controlled, resulting in peptides are produced with biologically active properties (12). Enzymatic hydrolysis of plant proteins, such as canola meal, has produced peptides that are used as natural ingredients in the production of useful feeds and can be used in animal nutrition because of their high absorption capacity from the small intestine (13).
The aim of the current experiment was to study the effects of different canola processing methods by bacteria, fungi, and enzyme on the intestine traits, blood metabolites, and kidney enzymes of broiler breeder hens.

MATERIALS AND METHODS
The research conditions. The research was done in 2018, using the chicken farm, facilities, and laboratory of the agriculture faculty of Islamic Azad University-Qaemshahr branch of Iran. Broiler breeder hens were rearing for 12 weeks (weeks 40 to 52). Experimental procedures of the current study were done base on the laws of the national committee for ethics in biomedical research of Iran (2018).

Hens and treatments.
In the current research, 450 hens of broiler breeder Ross strain weighing 3300±150 g (40 weeks) were used for 12 weeks. A completely randomized design was used with 6 treatments and 5 replications. Thirty pens were designed and prepared. Fifteen broiler breeder hens were included in each pen. The experimental treatments included: 1) unprocessed canola meal; 2) processed canola meal by Lactobacillus Plantarum; 3) processed canola meal by Bacillus Subtilis; 4) processed canola meal by Aspergillus Oryzae; 5) processed canola meal by Neurospora Cytophilla; and 6) processed canola meal by Alkalase enzyme Canola meal processing method. After the preparation of the canola meal, three samples (500 gr) were supplied and sent to the laboratory for chemical analysis. Five 25 kg samples were supplied and fermentation processing (fermentation with Lactobacillus Plantarum, Bacillus Subtilis, Aspergillus Oryzae, and Neurospora Cytophilla) and enzymatic hydrolysis (Alkalase) was performed on them. Then, 3 sub-samples were prepared from each sample and sent to a specialized laboratory for evaluation of quality, and traits measurement.
The ration formulation. The ration was formulated based on the nutritional requirements for the broiler breeder hen of Ross 308 (weeks 40 onwards) by corn, and soybean meal (Table 1). The studied traits.
The small intestine traits. At the end of the experiment, after the slaughtering of hens and emptying the contents of the intestine; weights and lengths of the three small intestine sections (duodenum, jejunum, and ileum) were measured. Then, 2 cm of the jejunum was washed with cold PBS solution (4°C) and placed in 10% neutral formalin buffer, and transferred to the histological laboratory (for supply transverse sections and determination of villi length and crypt depth). Histological studies were performed according to accuracy recommended protocols (14). In this study, the villi length and crypt depth were measured by Graticule.
Blood metabolites and enzymes. At the end of the experiment, two hens were randomly selected from each pen and about two ml of blood was collected through a jugular vein of the wing. Concentrations of alanine aminotransferase (ALT) aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH), total protein, albumin, uric acid, cholesterol, Low-density lipoprotein (LDL), High-density lipoprotein (HDL), calcium and phosphorus of blood samples were determined using the laboratory kits (Iran Pars-Azmoon) and spectrophotometer device (UK Jenway Genova MK3). The data was recorded for analysis.

Statistical analysis.
The data collected were analyzed by the generalized linear model (GLM) procedure of SAS statistical software (15). The statistical model used was as follows: where, y ijk is the value of each observation; µ is the mean effect; A i is the treatment effect and e ijk is the residual effect.

RESULTS
Blood metabolites. The effect of experimental treatments was significant (p<0.05) on all blood metabolites traits (

Intestinal traits, and Jejunum morphology.
It can be seen from the results of Table 3 that, the effect of processing methods was significant on caecum, jejunum, and duodenum (p<0.05). The use of experimental treatments increased the weight of the small intestine (duodenum and jejunum) and decreased caecum weight. The highest weight gains in the small intestine (duodenum and jejunum) was observed in treatments processed with Aspergillus Oryzae fungi. The lowest caecum weight was observed in the treatment processed with Aspergillus Oryzae fungi.  The processing of canola meal increased the length of the villi and decreased the depth of the crypt of jejunum (p<0.05). The highest villi length and the lowest depth of crypt were observed in the treatment processed with Aspergillus Oryzae fungi (Table 4).  Table 5, treatments effect was significant on the ALT and ALP (p<0.05). The lowest concentration of ALT and ALP was observed in treatments processed with Lactobacillus Plantarum bacteria (6.08) and Bacillus Subtilis bacteria (2504.11), respectively. Table 5. Effect of treatments on ALT, ALP, total protein, albumin, and uric acid. The effect of experimental treatments was significant (p<0.05) on the digestive amylase, lipase and protease activity ( Table 6). The highest amylase, lipase and protease activity was observed in treatments processed with Lactobacillus Plantarum bacteria (8.98), Bacillus subtilis bacteria (21.43) and Alkalase enzyme (85.40), respectively.

DISCUSSION
By studying the results of the effects of canola processing on blood metabolites in the present study, it can be observed that the processing reduces triglyceride, cholesterol and LDL levels in the broiler breeder blood. The decrease in cholesterol may be due to the inhibition of the activity of the 3-hydroxy-3-methyl-glutaryl-coA enzyme, which subsequently decreases cholesterol production. It can also be due to the production of short-chain fatty acids such as propionic acid, which subsequently restricts the production of cholesterol (16). These changes mean improved canola quality. Improvement of canola quality was achieved by different processing methods. The results of the present study are consistent with other studies (16, 17,18). Uric acid is a marker of protein catabolism and is the most important excreted nitrogen product in birds. Changes in blood uric acid levels indicate changes that occur in protein catabolism and depend on the protein content and quality of the poultry ration (19).
Alkalase is an endo-protease of the serine-type. It has a very broad substrate specificity. On another hand, it can hydrolyze most peptide bonds within a protein molecule. Peptides and amino acids are formed which are either dissolved or dispersed in the washing water. The processed canola (by alkalase enzyme) was reported to increase villus height, decrease crypt depth, and increase the villus height to crypt depth ratio in the broiler jejunum (11). In another study, canola was processed by Bacillus Subtilis, Candida Utilis, and Enterococcus Faecalis. The use of processed canola reduced the crypt depth and increased the villus height to crypt depth ratio in the broiler jejunum. The higher this ratio, the greater the ability to digest and absorb nutrients (18). These results are consistent with the results of the present study.
The different canola processing methods increased the activity of amylase, lipase and protease enzymes. Increasing the activity of these enzymes results in the breakdown of longchain sugars, large fat and protein molecules into smaller, more digestible molecules. This way the nutritional value of canola will increase (11). These results are consistent with a similar report in this field (11).
Increased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (ALP) that enter the bloodstream of broiler chickens indicate liver damage (20). In the present study, the soybean processing by bacteria, fungi, and enzymes decreased the concentration of liver enzymes. Canola processing reduced the secretion of liver enzymes. It shows that the processing methods have reduced the antinutritional factors. As a result, the pressure on the liver was reduced. These results may be due to the favorable and protective effect of canola processing methods on the liver. The results of the present study are consistent with similar results in this field (20,21).
The results of the present study showed that the processing of canola meals by various methods (such as bacteria, fungi, and enzyme) was reduced blood undesirable metabolites of hens (such as triglycerides). On the other hand, the characteristics of the small intestine of hens that are effective in absorbing nutrients (such as crypt and willi) were improved. Also, the concentration of secreted liver enzymes decreased. All of this means improving the quality of canola meal and reducing their anti-nutritional factors through the use of various processing methods. in other words, the processed methods improved canola meal digestibility. The protein quality, fatty acid profile, and anti-nutritional properties of canola meal were improved. Therefore, can be recommended to use the processed canola instead of the raw canola meal.