Performance Parameters, Plasma Lipid Status, and Lymphoid Tissue Fatty Acid Profile of Broiler Chicks Fed Camelina Cake

27/May/2019 Approved: 24/September/2019 ABSTRACT The effects of dietary camelina cake (CAMC) on broiler chicks’growth performance, plasma lipid status and fatty acid profile (FA) of lymphoid organs were evaluated. Six hundred broilers (3-week-old, Cobb 500) were randomly allotted for 20 days in a feeding trials into 2 groups: control (C; corn-soybean meal-canola meal basal diet) and experimental (CAMC; 80 g CAMC/kg diet replaces canola meal from C diet). Blood samples (n=12/group) were collected on day 42 for plasma profile analysis (glucose, Glu; total cholesterol, TC; high-density lipoprotein cholesterol, HDL-C; low-density lipoprotein cholesterol, LDL-C; triglycerides) and immune organs (spleen, thymus, bursa of Fabricius) for FA analysis. The FA profile of lymphoid tissue was determined by gas chromatography. Feeding the CAMC diet did not influence broilers performance parameters or relative weights of lymphoid tissue, except the weight of bursa that decreased ( p <0.05). Plasma lipids profile was affected by decreasing ( p <0.05) the concentrations of Glu, TC, HDL-C and LDL-C in broilers fed the CAMC diet. In all lymphoid tissue, the total content of n-3 polyunsaturated fatty acids (PUFA) increased (p<0.001) and the total level of n-6 PUFA decreased ( p <0.001) as the effect of the CAMC diet. The n-6:n-3 ratio decreased ( p <0.001) up to 5:1 in all lymphoid tissue. The higher ( p <0.05) concentration of arachidonic acid was found in the spleen, followed by the thymus and the bursa of the chicks fed the CAMC diet. Our results indicate that feeding 80 g/ kg camelina cakes in broiler chicks finisher diet had no negative impact on productivity, beneficially alter the plasma lipid status and fatty acids profile


INTRODUCTION
Over the last years, there has been an increased interest to modulate the n-3 polyunsaturated fatty acids (PUFA) profile of poultry meat in order to improve its nutritional value and to increase the consumption of these fatty acidsby humansdue to healthier benefits (Rymer & Givens, 2005;Wood et al., 2008;Poureslami et al., 2010;Ribeiro et al., 2013;Bhalerao et al., 2014;Nain et al., 2015).
Moreover, feeding n-3 PUFA enriched diets resulted in the deposition of these fatty acids into the lipid membrane of all tissues, including the cells of the immune system (Cinader et al., 1983;Huang & Fritsche, 1992) and could affect the immune response and reduce inflammation in different species such as chicken, mice and fish (Babu et al., 2005;Puthpongsiriporn & Scheideler, 2005;Calder, 2006;Wall et al., 2010). It is known that lymphoid tissues play an important role in the body defences against pathogens and its development can in some cases reflect immune system response and functionality (Grasman, 2002;Smith & Hunt, 2004;Akter et al., 2006). In the chickens' central lymphoid organs are the thymus and the bursa of Fabricius, while peripheral lymphoid organs include the spleen and all the lymphoid tissue associated with the intestinal mucosa. The spleen is the major site of immune responses to blood-borne antigens and is also a site of hematopoiesis (Batista & Harwood, 2009).
However, the potential to alter the nutrient composition of tissue strongly differs according to the feed ingredient considered. Recently, the development of the biofuels industry resulted in a significant increase of by-products (meals or cakes), vegetable sources rich in protein and energy which can be utilized in poultry diets.
Toour knowledge, there is no data available about the effects of CAMC, as a rich source of n-3 PUFA, on the plasma lipid status and fatty acid profile of lymphoid tissue in broiler chicks. Therefore, the aim of this study was to determine the effect of camelina cakes on performance, plasma lipid profile and lymphoid tissue fatty acid profile in broiler chicks.

MATERIAL AND METHODS
The study was performed at the experimental unit of the National Research-Development Institute for Animal Biology and Nutrition (Balotesti, Romania) based on a protocol approved by the Ethical Committee of the institute, in accordance with the EU Directive 2010/63/EU (OJEU, 2010).

Broilers, experimental diets, and sampling
Six hundred 3-week-old mixed-sex Cobb 500 broilers (883.26±15.04g) were used in a 20-day feeding trial (finisher phase, 23 to 42 days) conducted in a controlled experimental house. Birds were randomly allotted to 2 dietary groups, with 4 replicates of 75 broilers each and were raised in wood shaving floor pens. Each pen was equipped with manual feeders and nipple drinker lines. A lighting program of 23hL:1hD was used for the experimental period. The birds had ad libitum access to feed and water.
The broilers were fed with a control diet based on a corn-soybean meal-canola meal (C) and an experimental diet containing 80 g/kg camelina cakes (CAMC) that replaced the canola meal from C diet. The finisher diets (Table 1) were isocaloric, isonitrogenous and formulated to meet the nutrient requirements of broiler hybrid (Cobb-Vantress,2015).The camelina cake used in this study was provided from the local commercial oil processing plant and was obtained by cold-pressing oil extraction method. The chemical composition of CAMC was 93.5% dry matter, 30.4% CP, 22.5% crude fat, 8.8% crude fibre, 7.4% ash, 0.5% calcium, 0.75% phosphorus, and 12.76 MJ/ kg metabolisable energy. The protein of CAMC had a higher content of essential amino acids such a sarginine (2.85%), lysine (1.54%), threonine (1.1%), methionine (0.83%) and cysteine (0.71%).
The ingredients and analyzed composition of the experimental diets are given in Table 1.
Body weight (BW) and feed intake (FI) were measured and body weight gain (BWG), feed conversion ratio (FCR) was calculated from 23 to 42 days. Mortality rates were recorded daily, to make corrections in calculating FI and FCR during the experimental period.
At slaughter age (42 days), twelve birds per treatment (six male and six female) were randomly allocated for blood sampling and immune organs evaluation over a 12-h feed withdrawal period.
Blood samples (4 ml) were collected from wing vein in heparinized tubes, stored on ice and immediately processed. After blood sampling broilers were killed by cervical dislocation and bleeding. Carcasses were manually eviscerated and spleen, thymus and bursa of Fabricius were aseptically removed. Lymphoid organs were weighed and stored at -20°C until fatty acid analyses; their relative weights were calculated as the percent of live BW at slaughter.

Chemical and biochemical analysis
The chemical composition of the ingredients and the diets samples were analysed based on standard procedures (OJEU, 2009) (SR ISO 6490-2:1983), phosphorus (spectrophotometry method) and amino acid profile (HPLC-high performance liquid chromatography). Metabolisable energy (ME) content was cal culated based on the energy content of feed ingredients using regression equations (NRC, 1994).
Fatty acid profile of ingredients, diets and immune tissue samples were determined by the gas chromatography method (SR CEN ISO/TS 17764-2:2008) using Perkin Elmer500 chromatograph. The method was previously described by Hăbeanu et al. (2014) and consisted in transforming the sample fatty acids in methyl esters, followed by the separation of the components in the chromatography column, their identification by comparison with standard chromatograms and the quantitative determination of the fatty acids (expressed as % total fatty acid methyl esters-FAME).

Statistical analysis
For performance parameters, replication was considered as the experimental unit for the statistical analysis and other results were analyzed with every broiler as a replicate. Data were analyzed with a oneway ANOVA procedure of SPSS version 20.0 software (IBM SPSS Inc., 2014). The results were expressed as means with standard error of the mean (SEM) and value for fatty acids are expressed as the percentage of total fatty acid ester methyl (% of total FAME). Differences between means were considered statistically different at p<0.05.

RESULTS AND DISCUSSION
Fatty acid profile of camelina cake and diets. Table 2 shows that the fatty acid profile of camelina cake used in our study and obtained by cold-pressing oil extraction method, is a rich source of α-linolenic acid (ALA, 29.47%), linoleic acid (LA, 21.09%), and oleic acid (17.69%).Similar results have been reported previously by Nain et al. (2015) and Juodka et al. (2018). The fatty acid composition of diets showed that the inclusion of 80 g/kg CAMC increased the ALA content (2.87-fold) and resulted in a 3.89-fold decrease of the LA: ALA ratio, compared with the classical diet. It is stated that ALA is the precursor of long-chain n-3 PUFA such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have received considerable attention as functional nutrients for their various health-promoting effects (Rymer & Givens, 2005;Schmitz & Ecker, 2008). Thus, to obtain an efficient conversion of ALA into EPA and DHA it is important to assure an adequate dietary LA: ALA ratio (Griffin, 2008).

Performance parameters
The effects of dietary CAMC on production parameters and lymphoid tissue weights of broiler chicken are shown in Table 3. There was no significant difference (p>0.05) in BW, WG, FI and FCR between the dietary treatments at the end of the finisher phase (42 days). Similarly, previous studies feeding diets up to 10% camelina meal reported no significant differences in turkey broiler performance (Frame et al., 2007) or in broiler final body weight, feed efficiency and carcass weight at 42 days of age (Aziza et al., 2010). Other studies also indicated that the addition of camelina oil in broiler chicken's diets had no significant effects on performance and carcass quality parameters (Pietras & Orczewska-Dudek, 2013;Jaskiewicz et al., 2014). On the contrary, Thacker & Widyaratne (2012) stated that dietary camelina meal inclusion up to15% decreased BW gain and impaired the feed conversion of broiler chickens. Pekel et al. (2015) fed broilers with 10 and 20% camelina meal from 21-28 days of age and reported detrimental effects on performance and attributed this negative impact to the presence of glucosinolates in camelina by-product.
In our study, the relative weights of lymphoidorgans at 42 days (Table 3) were not affected (p>0.05) by the CAMC addition in the diet, except the weight of the bursa of Fabricius that significantly decreased (p=0.039). Previous studies have shown that lymphoid tissue development can reflect the immune status (Grasman, 2002;Smith & Hunt, 2004;Akter et al., 2006). Wang et al. (2000) reported that feeding laying hens with diets rich in n-3 PUFA stimulates the growth of immune tissue (thymus, spleen, and bursa) up to 4 weeks of age. The authors suggested that after 4 weeks of age the immune tissue weights began to decrease, and the bursa degenerated between the 4 th and the 8 th weeks of age. Al-Khalifa et al. (2012) have shown that feeding n-3 PUFA rich diets (30, 50 and 60 g/kg fish oil) had no effect on the relative weight of the spleen, but significantly higher the thymus weights in broilers fed diet with 50 g/kg fish oil and significantly lower the bursa weights in broilers fed diets containing 50 and 60 g/kg fish oil than those of broilers fed the control diet or 30 g/kg fish oil.

Performance Parameters, Plasma Lipid Status, and Lymphoid Tissue Fatty Acid Profile of Broiler Chicks Fed Camelina Cake
eRBCA-2019-1053 of the diet (r= -0.58, p=0.002). The values of very lowdensity lipoprotein cholesterol (VLDL-C) and triglycerides were lower compared to the C group with no significant difference (p>0.05). It is considered that VLDL-C concentrations are good indicators of fat deposition in the bird (Whitehead & Griffin, 1984;Grunder et al. 1987). This confirms the results of our study regarding the abdominal fat content, expressed as % of BW at slaughter, which was lower (1.86% vs. 1.90%; p>0.05; Table 3) compared to the C group as the effect of CAMC addition.
Our results are in line with what was previously reported by Taranu et al. (2014) who found that feeding camelina cake in finishing pigs improves the blood biochemistry profile by decreasing plasma glucose and increasing plasma antioxidant capacity. Fébel et al. (2008) have shown that feeding broilers with different sources rich in PUFA decrease plasma total cholesterol, and the decrease could be attributed to a suppression of hepatic cholesterol production by the high PUFA levels. VLDL-Cholesterol=Triglycerides divided by 5 (Tietz, 1996).
Means of 12 broilers per treatment, at 42 days of age.
ab Means within a row with no common superscript are significantly different (p<0.05).

Lymphoid tissue fatty acid profile
The effects of dietary CAMC on the fatty acid profile of the spleen, the thymus, and the bursa of Fabricius in broiler chicks are presented in Table 5. As response to feeding CAMC in broilers the total saturated fatty acids (SFA) was increased (p=0.029) by 1.08-fold in the thymus and 1.06-fold in the bursa, while the predominant SFA, palmitic acid (C16:0) increased in all immune tissues (1.06-fold in the bursa, 1.07-fold in the spleen and 1.09-fold in the thymus; p=0.002). The content of palmitoleic acid (C16:1) increased in all lymphoid tissues (1.33-fold in the spleen and in the bursa, 1.40-fold in the thymus; p<0.0001). However, the content of oleic acid (C18:1cis-9) decreased in the thymus and in the bursa (1.11-fold; p=0.009). Villaverde et al. (2006) reported that feeding broilers with rich PUFA diets could decrease the oleic acid due to an inhibition effect of increased dietary n-3 PUFAs on the Δ9-desaturase enzyme, lowering de novo synthesis of monounsaturated fatty acids (MUFAs).
Our results have shown that the MUFAs concentration significantly decreased (p<0.0001) in the thymus (1.04-fold) and in the bursa (1.07-fold) tissues as an effect of dietary CAMC addition. It is stated that the increase of PUFAs concentration led to decreases in MUFAs in tissues due to their inhibitory role of the desaturase enzyme needed for synthesis of MUFAs (Lefevre et al., 2001).
As expected, the composition of the CAMC diet, especially the essential FA content is reflected in the PUFA profile of lymphoid organs. The LA (C18:2n-6) content as predominant n-6 PUFA significantly decreased in all tissues (1.58-fold in the bursa, 1.61-fold in the thymus and 1.30-fold in the spleen; p<0.0001) of broilers fed CAMC diet and the total PUFAs content also decreased (p<0.0001) compared with C diet. However, the increase in ALA (C18:3n-3) content (4.33-fold in the bursa, 4.29-fold in the thymus and 6.06-fold in the spleen; p<0.0001) led to an increase of the total n-3 PUFAs in all the immune tissues (4.0-fold in the spleen, 3.24-fold in the thymus and 3.29-fold in the bursa; p<0.0001) as effect of feeding CAMC diet.

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Eicosapentaenoic acid (EPA; C20:5n-3) content increased (p<0.0001) in the immune organs of broilers fed CAMC diet compared with C (3.62-fold in the thymus, 4.8-fold in the bursa and 6.88-fold in the spleen). It was noticed that the spleen, that is the major immune organ, contained more EPA than other immune tissues and this fact could be related with a higher capacity for eicosanoid production, which is predominated by arachidonic acid (Al-Khalifa et al., 2012). The AA and EPA are metabolites of LA and ALA (Schmitz & Ecker, 2008) and usually AA decrease when n-3 PUFA increases (Komprda et al. 2005).
The sum of n-6 PUFAs decreased (p<0.0001) in the bursa (1.51-fold), thymus (1.45-fold), and spleen (1.28fold) compared with the C diet. These results are in line with previous studies that stated that the LA and ALA compete for the same desaturase enzymes (Crespo & Esteve-Garcia, 2001;Lopez-Ferrer et al., 2001).
As results of dietary CAMC inclusion, the n-6:n-3 ratio decreased in all immune tissues (4.87% in the thymus, 3.81% in the bursa and 3.61% in the spleen; p<0.0001) compared with C diet. It is stated that a lower ratio of n-6:n-3 PUFAs in poultry diets decreases the competition of ALA with LA for enzymes involved in bioconversion to long-chain (LC) n-3 PUFAs, resulting in increased tissue content (Riediger et al., 2009;Nain et al., 2012). In our study, the higher deposition of sum LCn-3 PUFAs was noticed in all immune tissue, especially in the thymus and the spleen (p=0.047) as an effect of dietary CAMC addition.
To our knowledge, there are no published reports evaluating the effect of dietary camelinacakes on the lymphoid tissue fatty acid profile. However, Al-Khalifa et al. (2012) studied the effect of feeding increasing levels of fish oil (30, 50 and 60g/kg) on immune function in Ross 308 broilers from 21 to 47days. These authors reported no significant effect of fish oil enriched diets on the proportions of ALA in the bursa; a significant Means within rows with no common superscript are significantly different (p<0.05) for tissue effect.
In conclusion, the results of the present study indicate that feeding 80 g/kg camelina cake in broiler chicks, as a rich source of n-3 PUFA, had no negative impact on productivity, beneficially alter the plasma lipid status and fatty acids profile of lymphoid tissue.