MATERIALS AND METHODS

The field work of this study was carried out at the Poultry Research Unit; Kalabsho Agricultural Researches and Experiments Center, Faculty of Agriculture, Mansoura University, El-Mansoura, Egypt, during October and November, 2014. However, the blood analyses and tissue histological examinations were undertaken at the Laboratory of Poultry Physiology, Faculty of Agriculture, Ain Shams University, Egypt.

Experimental diets: Five starter and five grower experimental diets containing graded levels of CM (0.0, 10.0, 12.5, 15.0 and 20.0%, equivalent to 40, 50, 60 and 80% from soybean meal) were formulated to meet the nutrient requirements of broiler chicks, as recommended by NRC (1994).

Table 1:	Composition and calculated analysis of the experimental starter and grower diets used in the present study
¶Each 3 kg premix contains, Vit. A: 12,000,000 IU, Vit. D3: 3,000,000 IU, Vit. E: 10,000 mg, Vit. K3: 3,000 mg, Vit. B1: 200 mg, Vit. B2: 5,000 mg, Vit. B6: 3,000 mg, Vit. B12: 15 mg, Biotin: 50 mg, Folic acid: 1,000 mg, Nicotinic acid: 35,000 mg, Pantothenic acid: 10,000 mg, Mn: 80 g, Cu: 8.8 g, Zn: 70 g, Fe: 35 g, I: 1.0 g, Co: 0.15 g and Se: 0.3 g

Canola seed (Brassica napus L.) meal, evaluated herein, was purchased from the local market and added to the basal diets mainly at the expense of soybean meal with slight modifications in dietary levels of yellow corn and corn gluten meal. All the experimental diets were isocaloric and isonitrogenous and their ingredient compositions and calculated analyses are shown in Table 1.

Birds and management: One hundred and eighty, day-old Cubb-500 broiler chicks were randomly divided into five equal experimental groups, each composed of three equal replications. Chicks of each replicate group were kept at a separate compartment of wire-floored brooding battery and fed their respective starter diet until the chicks were three weeks of age; then, they were transferred to two compartments (with one feeder) of wire-floored rearing battery and fed their respective grower diet from 3-6 weeks of age. All chicks were managed similarly; had free access to feed and water and subjected to a daily photoperiod of 22 h. The chicks were vaccinated against New Castle disease, infectious bursal disease and avian influenza viruses.

Growth performance of broiler chicks: Growth performance of chicks was evaluated as Live Body Weight (LBW), Body Weight Gain (BWG), Feed Intake (FI) and Feed Conversion Ratio (FCR). Records on weekly FI and LBW of birds on a replicate group basis for both the starter and grower phases of the fattening period were maintained and thus, BWG and FCR (g feed consumed: g BWG) were calculated throughout the whole experimental period (1-42 days of age). Mortality of birds, however, was monitored and recorded daily.

Carcass traits and vital internal organs/glands: At the end of the experimental period, six birds from each dietary treatment were randomly chosen, weighed and slaughtered. Just after complete bleeding, their carcasses were scalded, feather plucked and immediately processed. Relative weights (% of LBW of bird at slaughter) of carcass dressing-out (i.e., total edible parts), abdominal fat and vital internal organs (i.e., liver, thymus, spleen and bursa of Fabricius) and thyroid glands were estimated.

Blood parameters of broiler chicks: During slaughtering, six blood samples per treatment were collected in heparinized tubes. Blood samples were immediately centrifuged for 10 min at 4000 rpm, then blood plasma was decanted and stored at -20°C until later analyses. Activity of blood plasma transaminases [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] was measured by using available commercial kits according to Reitman and Frankel (1957). Plasma concentrations of thyroxine (T4) and triiodothyronine (T3) were determined by radioimmunoassay technique using commercial radioimmunoassay kits, as reported by Akiba et al. (1982). The T3/T4 ratio was also calculated.

Histological examination of liver and thyroid gland: Representative tissue samples (from liver and thyroid gland) were taken during slaughtering and immediately fixed in 10% formalin-saline solution for 24 h before preparing the histological sections by using Paraffin method technique. All the excised sections were stained with hematoxylin and eosin stain, examined under light microscopes (X40) and then photographed by using Canon digital camera.

Experimental design and statistical analysis: A completely randomized design was used. The statistical processing of data was performed by using one-way analysis of variance of the Statistical Analysis System (SAS., 2006). The significant differences among means of the different variables were identified by Duncan’s new multiple range test (Duncan, 1955) at p≤0.05.

RESULTS AND DISCUSSION

Growth performance of broiler chicks: The effects of feeding diets containing different levels of Canola Meal (CM) on growth performance of broiler chicks from 1 day-old to 6 weeks of age are given in Table 2. It was observed that Body Weight Gain (BWG) of chicks was negatively affected (p≤0.05) due to increasing dietary CM up to 20% during the starter (1-21 days old) and grower (21-42 days old) periods. Live Body Weight (LBW) responded similarly during both the starter and grower phases of the fattening period. Feeding the CM-containing diets adversely affected (p≤0.05) feed conversion ratio (FCR) only when CM level reached 20% during both the starter and the whole experimental period (1-42 days of age) but FCR was not affected by dietary CM during the grower period. Increasing dietary CM level up to 20% did not affect Feed Intake (FI) during the starter period but feeding the CM-containing diets resulted in significant increases (p≤0.05) in FI during both the grower and the whole experimental period compared with the control birds. It is important to note that no deaths occurred during the course of this study.

The above results indicate that when the dietary inclusion level of CM increased to 20% BWG and FCR were negatively affected. The reduced growth performance of broilers fed the 20% CM-containing diets, observed in the present study, may be related to the anti-nutrients or toxic substances that may be present in CM, such as glucosinolates, sinapine, non-starch polysaccharides and phytate which can interfere with digestion and bioavailability of nutrients (El-Wardany and Mohamed, 1995; Kermanshahi and Abbasi Pour, 2006; Gopinger et al., 2014).

Table 2:	Effect of different dietary levels of canola meal (CM) on growth performance of broiler chicks from day-old to 6 weeks of age
a-bFor each variable, means within the same column bearing different superscripts differ significantly (p≤0.05), NS: Not significant, SE: Standard error. *Significant at p≤0.05, **Significant at p≤0.01, LBW: Live body weight, BWG: Body weight gain, FI: Feed intake, FCR: Feed conversion ratio. Results are taken as Mean±SE

The high crude fiber content of canola meal (12%), as indicated by NRC (1994), is also involved, at least partly, in the poor growth performance of broilers fed the 20% CM-containing diets which can impair the efficiency of energy utilization in these diets (NRC., 1994).

In harmony with the present results, Payvastegan et al. (2013) investigated the effects of different dietary levels of CM (0, 10, 20%) on growth performance of broiler chicks and found that body weight gain and feed conversion ratio were significantly impaired when 20% canola meal was added in the diets between 1-21 days of age but feed intake was not affected. The present results are also in line with the findings of Min et al. (2009), who reported that body weight gain of broilers declined dramatically with increasing inclusion of CM from 0.0-25% of diet during 1-18 days of age, while FCR was not affected. In addition, Woyengo et al. (2011) studied the effect of including expeller-extracted CM in broilers’ diets on their growth performance and found that increasing dietary level of CM from 0-40% resulted in a linear decrease in feed intake by 4.8 g/21 days period for each 1% increase in CM and in body weight gain by 6.0 g/21 days period for each 1% increase in CM. However, Ahmad et al. (2007) reported that CM could be used at up to 20% of diets fed from 1-28 days of age without any adverse effects on broiler performance. Similar results were obtained by Zdunczyk et al. (2013), who observed no significant differences in the feed conversion ratio and body weight of growing turkeys fed diets containing 0, 6, 12 or 18% rapeseed meal. The contradictions in the literature on the responsiveness of animals to dietary rapeseed/canola meal is reasonable to be attributed to many factors such as genotype and age of animal, rapeseed cultivar, agronomic practices, climatic conditions, diet composition and feed processing treatments (Tripathi and Mishra, 2007).

Carcass traits and vital internal organs/glands: The effects of feeding diets containing different levels of CM on carcass traits and vital internal organs/glands of 6-week-old broiler chicks are presented in Table 3. Feeding the 20% CM-containing diets adversely affected (p≤0.05) relative weights of abdominal fat, thyroid gland and bursa of Fabricius compared with the control chicks but lower levels of CM had no effect.

Table 3:	Effect of different dietary levels of canola meal (CM) on carcass traits and vital internal organs/glands of 6-week-old broiler chicks
a,b,cMeans within the same row bearing different superscripts differ significantly (p≤0.05), NS: Not significant, SE: Standard error, *Significant at p≤0.05. Results are taken as Mean±SE

The percentages of dressing-out carcass, liver, thymus and spleen were not affected by dietary treatments.

The lack of significant differences in most carcass traits and internal organs, measured in the present study, in response to feeding the CM-containing diets up to a level of 15% is an indication that such level is tolerable by the broiler chicks. The increased relative weight of thyroid gland due to feeding the CM-containing diets, observed herein, may be due to the glucosinolate derivatives of CM that have a goitrogenic effect on the thyroid gland. These goitrogens have been found to depress thyroid gland activity (El-Wardany and Mohamed, 1995; Tripathi and Mishra, 2007; Khajali and Slominski, 2012). This result is in harmony with those reported by Nassar and Arscott (1986) and Slominski and Campbell (1990), who demonstrated that the high dietary levels of canola meal can cause enlargement of thyroid gland follicles. These authors attributed this thyroidal enlargement to the presence of glucosinolates in CM which leads to hypothyroidism and changes in the ratio between T3 and T4 in the blood.

In accordance with the present results, Ahmad et al. (2007) observed no significant effect of dietary CM (up to 20%) on carcass weight, breast and thigh yields or abdominal fat but gizzard weight as percentage of carcass weight increased linearly as dietary level of CM increased. In partial agreement with the present results, Payvastegan et al. (2013) found that relative weights of liver, thyroid glands, gizzard and pancreas were significantly increased when dietary CM level reached 20% but the percentage of carcass yield was not affected. Similarly, Kermanshahi and Abbasi Pour (2006) evaluated the partial replacement of soybean meal with rapeseed meal (at dietary levels of 0.0, 15 and 30%) on carcass traits of broiler chicks. They found that dietary rapeseed meal caused significant increases in the weights of liver, proventriculus and abdominal fat pad. However, Woyengo et al. (2011) reported that feeding CM-containing diets (up to 40%) did not significantly affect the relative weights (% of LBW) of kidney, heart or thyroid gland but relative weight of liver was linearly increased as dietary CM level increased.

Plasma thyroid hormones and liver enzymes: Blood plasma concentrations of thyroid hormones (T3 and T4) and the T3/T4 ratio of broiler chicks as influenced by feeding graded levels of dietary CM are presented in Table 4. The obtained results indicated that the high dietary level of CM (20%) decreased significantly (p≤0.05) the plasma thyroxin (T4) level compared with the other dietary treatments. The same trend of response was also observed for the plasma triiodothyronine (T3) level, however, the differences were not significant. The T3/T4 ratios showed also insignificant differences indicative of a slight depressive effect of CM on the rate of T4 turnover to T3.

Table 4:	Effect of different dietary levels of Canola Meal (CM) on plasma thyroid hormones concentrations and liver enzymes activity of 6 weeks old broiler chicks
a,bMeans within the same column bearing different superscripts differ significantly (p≤0.05), NS: Not significant, SE: Standard error, Results are taken as Mean±SE

The T3/T4 ratio in plasma was reported by many researchers as a good indicator of euthyroid status and hence better performance (El-Wardany and Mohamed, 1995; Kermanshahi and Abbasi Pour, 2006; Maroufyan and Kermanshahi, 2006).

Regarding the transaminases activity (AST and ALT), the present results revealed that AST activity but not ALT, was significantly increased (p≤0.05) in chicks fed the diets containing 15 and 20% CM compared with the other dietary treatments. It is well known that increased AST activity is related to degeneration of hepatic cells which was also supported by the histological observations where necrotic areas were present, especially in the 15 and 20% CM-fed groups. These results are in close agreement with the data reported in the scientific literature which support the evidence that some goitrogenic compounds and hepatotoxic agents are still present in the double-zero canola meal (Butler et al., 1982; El-Wardany and Mohamed, 1995; Maroufyan and Kermanshahi, 2006). Consequently, the dietary inclusion level of canola meal for broiler chicks should be around 15%.

Histological observations: The histological structure of livers from different experimental groups fed the CM-containing diets is illustrated in Fig. 1a-e. It is clear from Fig. 1a that the hepatocytes of the control chicks are normal with some dark-stained eosinophilic cells surrounding the central vein. This structural appearance of hepatocytes was also observed in hepatic sections of birds fed the diets containing 10 and 12.5% CM and to a little extent, in those fed the 15% CM-diets (Fig. 1b-d, respectively). However, there are some congested areas, moderate hypertrophy of hepatocytes, some necrotic areas and infilterable fluids could be seen in all sections. The severity of these observations is dependent on the dietary inclusion level of CM. Progressive changes in the liver architecture could be seen in Fig. 1e, where chicks were fed the 20% CM diets. There are many focal sinusoids in/between many compressed hepatic cords accompanied with necrotic areas, infilterable fluids and dilated central vein engorged with blood. These observations are supported by the microscopic examination of livers, where they were greatly enlarged in birds fed the higher levels of CM (15 and 20%). At autopsy (6 weeks of age), some livers were enlarged, friable and variable in color from tan to pale yellow in the 20% CM-fed group. This observation coincided with the significant increase in plasma activity of AST of birds fed the 15 and 20% CM diets (Table 4), indicative of some damage in liver cells. The enlarged livers were also observed as for their relative weights in these treatment groups (15 and 20% CM) which recorded insignificantly higher values than other experimental groups (Table 3). The previously mentioned changes in the liver histology may be due, in part, to the increase in the metabolic rate associated with the high growth rate of broiler chicks, in general. Since liver is the main metabolic organ in the body, some histological and anatomical lesions should be expected.

Fig. 1(a-e):	Cross section in the liver tissue of broiler chicks received different dietary levels of canola meal, T.S in the liver of broiler chicks receiving (a) Control diet, (b) 10, (c) 12.5, (d) 15 and (e) 20% CM (H and Ex40), respectively cv: Central vein, h: Hepatocytes, s: Blood sinusoids, if: Infilterable fluids, nc: Necrotic area and b: Bile duct

These changes did not affect the performance of broilers in the present study but some growth depression was noticed in the 20% CM-fed group.

Concerning the effect of feeding various levels of CM on thyroid gland histology, the microscopic examination showed an obvious increase in the number of thyroid follicles (per microscopic field), especially in Fig. 2a-b. This is concomitant with the larger amount of colloid within the follicular lumen. The epithelial lining of the follicles tended to be high cuboidal or columnar, indicative of a possible hyperactivity of the gland. On the other hand, the thyroid gland sections from chicks that were fed the diets containing 12.5 and 15% CM (Fig. 2c-d) exhibited moderate changes including low amounts of colloid and an enlargement in some follicles, especially in the 15% CM-fed chicks. There are also dense connective tissue septa that are separating the follicles and giving them a polyhedral appearance in the 20% CM-fed chicks. It is also of great interest to observe that the epithelial lining of thyroid follicles (Fig. 2e) became low cuboidal or squamous in its appearance with little colloidal material in the follicles, indicative of hypothyroid activity.

The previous results indicate that the negative effect of dietary CM was primarily observed beyond the inclusion level of 15%.

Fig. 2(a-e):	Cross section in the thyroid glands of broiler chicks received different dietary levels of canola meal, T.S in the thyroid gland of broiler chicks receiving (a) Control diet, (b) 10, (c) 12.5, (d) 15 and (e) 20% CM (H and Ex40), respectively. T: Thyroid follicles, P: Parafollicular cells, C: Colloid and S: Septa

Although, the effects of feeding CM-diets up to 15% were not severe enough to depress the growth performance of broiler chicks but there is no doubt that some goitrogenic substances still present in the canola meal. The presence of antinutritional factors in CM, particularly glucosinolates and erucic acid, has been reported to be less than 2% erucic acid of the total fatty acids in the oil and less than 30 μmol of alkenyl glucosinolates per gram of oil-free dry matter of the seed (Bell, 1993); however, they still exert a detrimental effect on thyroid gland function, especially when high level of CM is included in broiler diets. It is evident that the birds can compensate for the goitrogenic effect by increasing thyroidal mass to supply extra-thyroid hormones. This is accompanied by enlargement of follicles as a result of the excessive secretion of the thyroid stimulating hormone from the pituitary gland which causes thyroidal hypertrophy and enlarged follicles.

The present observations are in close agreement with the earlier findings by Van Etten (1969) and Nassar and Arscott (1986), who found that the thyroid gland enlargement is due to rapeseed glucosinolates which are hydrolyzed by the enzyme myrosinase and yield some compounds such as thiocyanates, isothiocyanates, goitrin and nitriles that interfere with the function of thyroid gland and adversely affect the growth performance of monogastric animals (Fenwick, 1982; McCurdy, 1990; Tripathi and Mishra, 2007). These compounds block the iodine uptake by the thyroid gland, causing hypothyroidism without any damage to the follicles. It appears that the extraction and refining processes of canola seed oil can be effective to isolate most but not all, goitrogens present in canola meal.

CONCLUSION

Summarizing, taking the previous histological characterization of liver and thyroid gland, plasma levels of thyroid hormones and growth performance of broilers into account, it could be concluded that canola meal can safely be included at up to 15% in broiler chicks’ diets.