Effects of dietary extruded linseed and Lactobacillus acidophilus on growth performance, carcass traits, plasma lipoprotein response, and caecal bacterial populations in broiler chicks

Abstract The study aimed to assess the effects of dietary extruded linseed (ELS) and Lactobacillus acidophilus (LA) on performance, carcass traits, immune organ weights, plasma lipoprotein response, and caecal bacteria populations in broiler chickens. A total of 648 one-day-old chicks were divided into six groups in a 3 × 2 factorial arrangement consisting of three ELS levels (0, 6 and 12%) without or with probiotic L. acidophilus (0 and 20 g ton−1 feed). Each group had six replicates (18 birds/pen). Results showed no significant effects of ELS level nor LA supplementation on growth performance (body weight gain, feed intake, and feed conversion) of broilers during the overall period (1–42 days). The production efficiency factor increases (p = .045) with the LA addition. There were no effects of ELS level nor probiotic addition on carcase traits and immune organ weights, except for decreases in abdominal fat percentage (p = .027, respectively p = .035). The dietary ELS level significantly correlated with a decrease in the plasma total cholesterol (TC; p = .003), triglycerides, and very-low-density lipoprotein concentrations (p = .020), while the LA addition significantly correlated with a decrease (p = .015) in the plasma TC. The LA addition lowers the caecal pH (p = .007), Staphylococcus spp. and E. coli counts (p < .0001), and increase the caecal Lactobacillus spp. and lactobacilli: E. coli ratio (p < .0001). In conclusion, the use of ELS up to 12% supplemented with L. acidophilus in broiler chicks diet had a positive effect on health status, decreasing the abdominal fat deposition, plasma lipids and the caecal pathogen bacteria E. coli. Highlights Extruded linseed up to 12% in broiler chicks’ diets had no significant effects on growth performance, carcass traits or immune organs size and improve the abdominal fat deposition and plasma lipids response due to the decreased dietary n − 6: n − 3 PUFA ratio. Extruded linseed up to 12% supplemented with L. acidophilus in broiler chicks diet had a positive effect on health status improving production efficiency factor and decreasing abdominal fat deposition, the plasma lipids and the caeca pathogen bacteria E. coli.


Introduction
Taking into consideration the importance of poultry meat and its quality, on the one hand, and the increasing demand of consumer for natural products, on the other hand, the use of microbial feed additives that acts as probiotic associated with n À 3 fatty acids (FA) rich feed ingredients could represent a practical way to meet these requirements.
Different kind of ingredients was evaluated over time to improve meat quality. Most of them have been shown to have high nutritional value, but the factors that limit their use is the content in antinutritional components. Among these, linseed is considered a valuable energy feed ingredient for poultry diets due to its rich content of oil (35-45%), with a favourable n À 3 polyunsaturated fatty acid (PUFA), especially a-linolenic acid (ALA; 45-52%) (Bhatty 1995;Singh et al. 2011). However, its use has received little attention due to the content in water-soluble polysaccharides (mucilage), cyanogenic glycosides, trypsin inhibitor, phytic acid (Dale and Batal 2008). These factors could negatively affect the gastrointestinal health and nutrient utilisation in poultry (Attia 2003;Apperson and Cherian 2017).
The dietary FA exerts primary physiological functions for fast-growing broilers, and it has been stated that the fat source modulates the FA composition of chicken meat, with health benefits for consumers and birds' too (Bautista-Ortega et al. 2009;Gonzalez et al. 2011;Sridhar et al. 2017). Several studies have shown that dietary linseed enhanced chicken meat with n À 3 PUFAs ( Rodriguez et al. 2001;Attia 2003;Betti et al. 2009;Mridula et al. 2015), but could affect the growth performance (Alzueta et al. 2003;Mridula et al. 2015). Furthermore, from a nutritional point of view, linseed is also a valuable source of protein (20-30%), essential amino acids, fibre, minerals, and vitamins (Anjum et al. 2013).
The extrusion technology is an appropriate method to reduce the antinutritional factors in linseed (Anjum et al. 2013;Kostadinovi c et al. 2016), to modify lipid and carbohydrate structures, and to reduce the ileal digesta viscosity (Pirmohammadi et al. 2019). Previous studies (Anjum et al. 2013;Mridula et al. 2015) reported that feeding 5-15% extruded linseed in broilers diets affects growth performance, while other research (Kostadinovi c et al. 2016;Skrivan et al. 2020) stated that 6 or 10% extruded linseed improve broilers productive performance. However, the addition of up to 10% of extruded flaxseed in the diet increased the n À 3 FA (ALA, docosahexaenoic and eicosapentaenoic FAs) and decreased the n À 6/n À 3 ratio in broilers meat, with the favourable impact of human health (Anjum et al. 2013;Mridula et al. 2015;Kostadinovi c et al. 2016;Skrivan et al. 2020). Moreover, Kostadinovi c et al. (2016) showed that 10% of extruded flaxseed had a significant effect on enzymatic and non-enzymatic antioxidative systems in the blood of broilers.
It is known that the probiotics beneficially modulate intestinal microbiota of the poultry and develop and stimulates the gut immune system with an impact on health (Mountzouris et al. 2007;Attia et al. 2017;De Cesare et al. 2020).
Lactobacillus strains have been shown to have positive effects on production performance (Smith 2014), chicken meat quality (Mountzouris et al. 2007), modulation of intestinal microflora and pathogen inhibition (Patterson and Burkholder 2003). It has been stated that mechanisms of action included competition for available nutrients, immune stimulation and the alteration of microbial and host metabolism (Patterson and Burkholder 2003;Yang et al. 2009;Huyghebaert et al. 2011;Al-Sagan et al. 2020). The major factors that affect probiotics efficacy are the bacterial strain, the dose (colony-forming unit/bird/day), the treatment period and the administration method (via feed or water) (Gallazzi et al. 2008). The L. acidophilus D2/CSL probiotic strain was isolated from the gastrointestinal tract of healthy chickens and is known for its ability to balance the intestinal microbiota (Bianchi et al. 1985). Several studies have revealed the efficiency of L. acidophilus D2/CSL used as probiotic on laying hen's productive performance (Gallazzi et al. 2008;Cesari et al. 2014), on egg production and quality, immune status, or gut microflora and health in organic laying hens (Forte et al. 2016a(Forte et al. , 2016b. A few feeding trials investigated the effects of this probiotic strain on growth performance and metabolic function in broilers (De Cesare et al. 2017), or performances and gut health in rurally reared chickens (Forte et al. 2018).
To our knowledge, there are no studies that investigated the combined effect of extruded linseed and probiotic on plasma metabolites, gastrointestinal health, and performance in broilers. Thus, we hypothesised that the probiotic addition to broiler diets including up to 12% extruded linseed could improve the productive performance and lipoprotein utilisation due to complementary effects of antimicrobial properties exert by probiotic and a rich source of bioactive compounds (e.g. ALA, lignans, antioxidants) with potential benefits on broilers gut health.
The present study aimed to assess the effects of dietary extruded linseed (ELS) and L. acidophilus (LA) addition on growth performance, carcass traits, immune organ weights, plasma lipoprotein response, and caecal bacterial populations in broiler chicks.

Materials and methods
The experimental protocol was approved by the Ethics Committee of the National Research Development Institute for Animal Biology and Nutrition, Balotesti, Romania (6283/10/2018).

Broilers and experimental design
A total of 648 one-day-old unsexed broilers (Ross 308) procured from a commercial hatchery were raised for 42 days (d) in floor pens on wood shavings litter. The lighting programme used was 23 L:1D on the first week of life, and from d 7 the light was gradually reduced until 20 L:4D, according to hybrid guide (Ross-Aviagen 2018). The floor pens were equipped with manual feeders and nipple drinkers' line. The feed was offered in mash form, and birds had free access to feed and water. The birds were vaccinated against Marek's, Newcastle and Gumboro diseases, according to standard protocol.
Broilers were assigned into six groups with six replicates each (18 birds/ pen) in a 3 Â 2 factorial arrangement consisting of three extruded linseed (ELS) levels (0, 6 and 12%) without or with probiotic L. acidophilus D2/CSL 1.0 Â 10 9 CFU g À1 (0 and 20 g ton À1 feed). The linseed (Valorex, France) used in this study was heattreated in five steps as follow: (1) seeds were grinding and mixing with water vapours; (2) maturation (80 C for 20 min) for the destruction of anti-nutritional factors; (3) preconditioning at 100 C for starch gelatinisation; (4) extrusion (140 C and 25-65 bars thermomechanical pressure) to increase the digestibility of protein and fat; (5) drying (12% moisture of final product) and cooling at 15 C.
The three-phase (starter, grower, and finisher) diets were formulated to be isonitrogenous, isocaloric and to meet the nutrients recommended by Ross 308 manual ( Table 1).
The use of ELS in broilers diets, as a rich source of PUFA, led to an increase in the ALA content of the experimental diets with 4.0-fold (ELS6), respectively 6.30-fold (ELS12) and decreased the LA: ALA ratio with 4.9-fold (ELS6), respectively 9.40-fold (ELS12), compared with the control diet (Table 1).

Growth performance
Body weight (BW) and feed intake (FI) were recorded for each replicate on d 1, 10, 24, and 42. The body weight gain (BWG), and feed conversion ratio (FCR) adjusted for mortality were calculated for each period (1-10 d, 11-24 d, and 25-42 d), as well as for overall period (1-42 d). The mortality rate was registered daily. Production efficiency factor (PEF) was calculated at the end of the trial (42 d), based on the following formula: Sampling At 42 days of age, two birds per each replicate were randomly selected, weighed, and blood was collected in heparinised tubes (4 mL/bird) from the wing vein in the morning to reduce the plasma constituent's variability. After blood collection, the chicks were slaughtered by cervical dislocation. Carcases were immediately plucked, eviscerated, and head, neck, shanks, abdominal fat, organs, and intestinal tract were aseptically removed. The organs were weighed, and the major parts of cut-up carcase (breast, legs, wings with skin and bone) were dissected and weight. The relative weights of cut-up carcase parts and the immune organs were calculated as the percent of live body weight at slaughter.
The caecal contents (from the left and right caeca) were aseptically collected in tubes. After the pH of intestinal content was measure in duplicate (WTW ProfiLine pH 3310, WTW GmbH, Weilheim, Germany), the tubes were stored at À80 C until analysis.

Chemical and microbiological analyses
The proximate composition of the feeds and diets were assessed in duplicate by standardised methods (OJEU, 2009) Schiemann et al. 1972).
Amino acids profile was determined in duplicate with an HPLC Surveyor Plus Thermo Electron equipment (Massachusetts, United States) by a method described previously by V arzaru et al. (2013), which implies the acid hydrolysis for the release of amino acids from the protein molecules, preceded by oxidation with performic acid for the sulphur amino acids.
Fatty acid analyses were done in duplicate on a Perkin Elmer-Clarus 500 gas chromatograph (Massachusetts, United States) using the method described by Habeanu et al. (2014). After blood centrifugation (3000 Â g for 15 min) plasma was stored at À20˚C until analysis. A dry chemistry analyser (Spotchem EZ SP-4430, Arkray Inc., Japan) and specific solid-phase reagent strips (Spotchem, Arkray Inc., Japan) were used to determine the blood metabolites (total cholesterol, TC; triglycerides, TG; glucose, Glu; total protein, TPro; albumin, Alb; total bilirubin, TBil; creatinine, Cre; uric acid, UA). The very-low-density lipoprotein-cholesterol (VLDL) were calculated based on Tietz (1996)  for staphylococci, and Rambach-Salmonella agar (Biokar Diagnostics, Solabia, France) for Salmonella spp. Briefly, one g of caecal content samples was diluted at 1:9 (wt/ vol) in sterile saline solution. After 10-fold serial dilutions, one mL of each dilution was inoculated on Petri plates on specific agars. Plates were incubated at 37 C for 24 to 48 h, and then the bacterial units were counted (Scan 300, Interscience, France). Results were expressed as log 10 CFU/g of sample.

Statistical analysis
Data were analysed using the general linear model GLM procedures of SPSS software (v20.0, SPSS 2011) as a 3 Â 2 factorial arrangement. Two-way analysis of variance (ANOVA), followed by Tukey's multiple comparisons test, was used to assess the main effects of dietary linseed level, with or without probiotic supplementation and their interactions. The replicate pen was the experimental unit for productive performance and each sample for the other parameters. Pearson's correlation was used to assess the relationship between indicators. The results are expressed as means and standard error of the mean (SEM). Statistical differences declared at p < .05.

Chemical analyses
The proximate composition, amino acids and fatty acids profiles of extruded linseed used in this study are presented in Tables 2 and 3.

Growth performance
The results of growth performance (Table 4) shown that during the overall period (1-42 d of age) the BWG, FI and FCR were not significantly affected by the ELS level nor LA supplementation. The production efficiency factor (PEF) was significantly increased by 5.9% (p ¼ .045) as an effect of the LA addition.
The mortality rate range in normal limits (data not shown) and was not affected by dietary treatments. No interaction between ELS level and LA addition was identified regarding productive performance.

Carcass traits and immune organ weights
There was no effect of dietary extruded linseed or probiotic addition on the carcass or cut-up carcass parts yields (Table 5), except the abdominal fat percentage that significantly decreases (11.60%; p ¼ .027), respectively (9.4%; p ¼ .035). The relative weights of immune organs were affected neither by extruded linseed level nor probiotic addition. There was no interaction between treatments.

Plasma lipoprotein response
The plasma metabolites response (Table 6) shows that the dietary ELS level significantly decreases the plasma TC (p ¼ .003), TG, and VLDL concentrations (p ¼ .020) compared to control diet. The dietary LA addition lowers the plasma TC (p ¼ .015) concentration significantly. There was no effect of dietary extruded linseed level nor probiotic addition on plasma protein response. No interaction between dietary treatments was found.
As shown in Table 7, a strongly positive correlation (p < .01) between dietary LA: ALA ratio and plasma TC, TG, and VLDL concentrations, and also abdominal fat percentage was observed.

Caecal bacterial populations
There was no significant effect of dietary ELS level on caecal pH and microflora at 42 days of age (Table 8).
valuable protein source (182.6 g kg À1 DM) with a balanced essential amino acids profile (7.6 Lys and 10.34 TSAA g kg À1 DM). The results obtained are in line with the previous reports (Kostadinovi c et al. 2016;Ahmad et al. 2017;Skrivan et al. 2020).
In the current study, the results on the broilers productive performance shown that the extruded linseed level nor LA supplementation has no significant effects on overall BWG, FI and FCR (Table 4). Anjum et al. (2013) stated that BWG and FI decrease, and FCR increase in broilers fed with different level of extruded flaxseed (5, 10, 15%). Mridula et al. (2015) reported that fed extruded flaxseed between 5% and 10% impaired growth performances of broiler chicks. The growth impaired was attributed to the presence in flaxseed of antinutritional compounds (e.g. cyanogenic glucosides, mucilage) and vitamin B6 antagonism (Bond et al. 1997;Attia 2003). Contrary, Kostadinovi c et al. (2016) indicated that 10% extruded flaxseed improve broilers productive performance vs 2.5, 5% and control diets, and Skrivan et al. (2020) stated that 6% extruded flaxseed increase BW of Ross 308 cockerels at 35 d compared to the control or hemp seed diets. Lactic acid bacteria have been proved to act positively by inhibiting or diminishing other contaminants into the host body. A possible explanation consists of delivering enzyme and other substance when these bacteria are passing by the intestinal tract. The present study showed that L. acidophilus addition slightly increased the BWG of broilers and resulted in significantly increases (by 5.9%) on the PEF. Previous research has shown that Lactobacillus addition as a probiotic in poultry diets positively affects growth performance (Loh et al. 2010;Shim et al. 2012;Askelson et al. 2014;Al-Sagan et al. 2020), digestive status , and stimulates an immune response (Brisbin et al. 2011). It is stated that factors such as nutrition, environment (hygienic condition), rearing management, probiotic dose, bird's age, and administration method (via feed or water) could influence the probiotics efficiency (Yang et al. 2009).
Our results showed no significant effect of ELS level nor probiotic addition on carcase traits and relative immune organ weights (Table 5), except the abdominal fat percentage that significantly decreases with 11.60% (p ¼ .027), respectively 9.4% (p ¼ .035). Similarly, some studies have shown that dietary flaxseed did not significantly affect the carcase traits or the relative organ weights (Pekel et al. 2009;Mridula et al. 2015). Naseem et al. (2012) found that probiotics did not affect lymphoid organs. Anjum et al. (2005) and Mehr et al. (2007) revealed that probiotic addition decreases abdominal fat weight, while other research reported a concomitant decrease in blood triglyceride concentration (Kalavathy et al. 2003;Mansoub 2010). Abdominal fat could be linked by decrease activity of acetyl-CoA carboxylase, the rate-limiting enzyme in fatty acid synthesis, as the effect of probiotic addition that may also explain the blood triglycerides decreased (Santoso et al. 1995).
Plasma lipid metabolites are considered markers of fat metabolism in poultry organisms, and the age, sex, genetic type, environmental and feeding conditions are the major factors of influence (Krasnode Rbska-Depta and Koncicki 2000; Attia et al. 2020). In our study, plasma lipid metabolites were positively influenced by dietary treatments (Table 6). The dietary ELS level significantly correlated with a decrease in the  plasma TC, TG, and VLDL concentrations, while the LA addition significantly correlated with a decrease in the plasma TC. An explanation is that linseed contains a higher level of n À 3 PUFA, which is known to suppress lipoprotein lipase activity and regulate adipose tissue growth in broilers (Chan and Cho 2009). This explanation is strengthened by the significant correlation that we found in our study between dietary LA: ALA ratio and plasma lipid profile (Table 7). Our previous research stated that 80 g/kg camelina cake in broiler chicks' diet, as a rich source of n À 3 PUFA, beneficially affects plasma lipids decreasing the glucose, TC, HDL-C, and LDL-C levels, and alter fatty acids profile of lymphoid tissue (Gheorghe et al. 2019). Star cevi c et al. (2014) observed that 5% of linseed oil in Ross 308 broiler diets lowered the serum cholesterol level, with no significant effect of the other lipids fraction. Present results show a significant decrease in plasma TC concentration and tend to decrease plasma TG and VLDL level, as the effect of LA addition. It has been stated that probiotic supplemented diets in broilers lower blood cholesterol (Anjum et al. 2005;Naseem et al. 2012). According to Kalavathy et al. (2003), some bacterial probiotic strains can absorb cholesterol into the cells, hydrolyse bile salts or inhibit hydroxyl methylglutaryl-CoA, the rate-limiting enzyme of cholesterogenesis, and lower the cholesterol in the organism.
Modulation of gut microbiota by probiotic addition such as Lactobacillus spp. could improve health and prevent certain diseases (Mai and Draganov 2009) by inhibiting the proliferation of pathogenic bacteria (Van Winsen et al. 2002). In the present study, the L. acidophilus decreased pH and inhibited the growth of Staphylococcus spp. and E. coli populations in the caeca, concomitant with a significant increase in the Lactobacillus spp. and Lactobacillus: E. coli ratio, which is beneficial for intestinal health (Table 8). The results obtained on intestinal microflora are supported by the positive effects noticed in the productive performance. To our knowledge, there are fewer studies regarding the use of L. acidophilus as a monostrain probiotic in broiler chicks. The present results are in line with other studies that noticed that supplementation with L. acidophilus via feed efficiently colonises the intestinal tract and exerts a competitive exclusion effect on pathogenic bacteria because it is part of the chickens' healthy gut microflora (Lin et al. 2008;Li et al. 2014;Mookiah et al. 2014). De Cesare et al. (2020) studied the effects of L. acidophilus on caecal microbiome and performance in broilers and reported similar L. acidophilus amounts in the caecal contents of broilers fed control or supplemented diet. The authors noticed a positive effect of L. acidophilus supplementation on the metabolic functions, increasing the b-glucosidase and promoting health. Forte et al. (2018) observed that supplementing the Kabir chicks' diet with L. acidophilus had no significant effect on enterococci, staphylococci, and E. coli populations. The authors reported a Lactobacillus population increased and a tendency to lower coliforms compared with the control diet. Forte et al. (2016b) have shown that Lactobacillus positively modify the gastrointestinal balance of microbiota in laying hens, increasing the presence of beneficial bacteria such as Bifidobacterium spp., and reducing pathogenic bacteria such as E. coli, clostridia, and staphylococci. Contrary, other studies on the L. acidophilus use in broilers reported no significant effect on the lactobacilli population or partial effects on coliforms count (Jin et al. 1998;Salarmoini and Fooladi 2011).

Conclusions
The results indicated that the use of extruded linseed up to 12% in broiler chicks' diets had no adverse effects on growth performance, carcass traits or immune organs size and improve the plasma lipids response due to the decreased dietary n-6: n-3 PUFA ratio. In addition, the association between extruded linseed and probiotic (L. acidophilus) positively affects the production efficiency factor, abdominal fat deposition, plasma lipid metabolites, and beneficially alters the caeca bacterial population E. coli.

Disclosure statement
No potential conflict of interest was reported by the author(s).