Effects of microbial fermented sesame meal and enzyme supplementation on the intestinal morphology, microbiota, pH, tibia bone and blood parameters of broiler chicks

Abstract This study was conducted to compare the broiler chicks responses to raw sesame meal (RSM), either processed by microbial fermentation or enzyme supplementation. A total of 420-day-old boiler chicks (Ross 308®) were allocated to a completely randomised design experiment with 7 treatments and 5 replicates (12chicks/replicate). Treatments include: basal diet based on soybean meal (SBM), SBM substitution with 15 and 25% RSM either with phytase [5000 FTU phyzyme XP/g (PHX)] (RSM15 + PHX, and RSM25 + PHX) or without enzyme (RSM15, RSM25), and two diets in which SBM substituted with 15 and 25% fermented sesame meal (FSM15 and FSM25). The results indicated that fermentation process decreased oxalate and phytic acid (51% and 44%, respectively), and simultaneously increased in crude protein (13%), ether extract (11%) and available phosphorus (61%) (p < .05). Moreover, compared to SBM and RSM treatments, broilers fed diets containing FSM, lead to significant (p < .05) increase in Lactobacillus and decrease in coliforms count in the ileum. Furthermore, the broilers fed FSM15 diet had the lowest reduction in Escherichia coli population in the crop (p < .05). In the jejunum, the highest villus height was observed in the FSM25 diet compared to RSM diets (p < .05). Compared to SBM, inclusion of FSM25 to broiler rations resulted in blood triglycerides and cholesterol reduction by 30.7 and 23.7%, respectively (p < .05). Conclusion is that fermentation process can improve nutrient value of the RSM and could be considered as a protein source in broilers diet. HIGHLIGHTS: Fermentation process increased nutrient value of the sesame meal. Fermented sesame meal shifted intestinal microbial population more towards benefit bacteria. Fermented sesame meal increased the villus height in the jejunum and decreased blood triglycerides and cholesterol in broilers.


Introduction
Results from previous researches have shown that sesame seed (Sesamum indicum L.) is an excellent source of protein and minerals such as calcium and phosphorus (Adsule and Kadam, 1991;Ghazvinian et al. 2016). Raw sesame meal (RSM) is a by-product of the sesame seed during oil extraction process. RSM could be considered as a feed ingredient in practical diets, because its essential amino acids profile is almost equivalent to soybean meal (Mamputu and Buhr 1995), except for lower lysine and higher methionine. RSM is appeared with high level of Calcium and phosphorus, however availability of both minerals is low for birds due to phytate and oxalate contents (Rahimian et al. 2013;Sina et al. 2014;Ghazvinian et al. 2016). On the other hand, results showed, that the phytic acid has a carbohydrate binding potential, then inhibition of alpha-amylase activity, and reduction of protein digestibility in the digestive tract (Sebastian et al. 1998;Farran et al. 2000;Abbasi et al. 2019). To solve this problem, various methods of processing such as heating, soaking, phytase supplementation and fermentation have been found to affect RSM nutrient quality and availability. However, available reports indicate a large difference in results due to processing methods. Hence, the nutrient composition of RSM varies widely depending on the degree of decortication and the processing method. For example, it has been reported that the high level of oxalate and phytic acid of RSM could be appreciably eliminated when subjected to heat (Lease and Williams 1967). Also, it is well documented that phytase supplementation is an effective option in poultry diets to improve phosphorous utilisation (Ravindran et al. 2006;Abbasi et al. 2019). Administration of phytase to the diet increased the phosphorous digestibility and decomposition of phytic acid, which in turned decreased the nutrient loss in the faeces (Lei and Stahl 2001). Regarding the soaking process, Makinde and Akinoso (2013) investigated the effect of soaking process on the anti-nutritional factors of black sesame cultivars grown in Nigeria and concluded that oxalate levels in whole and dehulled black sesame seeds were decreased by 26.8 and 29.6%, respectively. On the other hand, Das and Ghosh (2015) investigated the effect of the fermented sesame meal using Bacillus subtilis subsp. Subtilis. They reported that in comparison to the diets containing RSM, fermented sesame meal appeared with the lower anti-nutritional factors (tannin, phytic acid and trypsin inhibitor). Another attempt revealed that fermented RSM by Lactic acid bacteria resulted in reduced phytic acid and tannin content (Olude et al. 2016). Furthermore, the ability of Lactobacillus acidophilus (L. acidophilus) to decompose phytic acid content from the RSM during the fermentation process has been clearly reported Ray 1999a, 1999b). Also, it has been well-documented that the fermentation of soybean, canola and camelina meals with Saccharomyces cerevisiae (S. cerevisiae) dramatically decreased phytic acid content and have an enzymatic potential to improve nutrient utilisation of these by-products (Foster and Nakata 2014;Hassaan et al. 2015;Olukomaiya et al. 2019Olukomaiya et al. , 2020Onipede et al. 2020).
To the best of our knowledge, there is no study to investigate the effect of FSM on the productivity of broiler chickens.
Summarising the above findings led to this idea that if fermented RSM by L. acidophilus and S. cerevisiae could increase nutrient utilisation and subsequently cause in improvement of broiler's chicken growth performance. Therefore, the aim of the present study was to evaluate the effects of FSM replaced with soybean meal on growth performance of broiler chickens.

Preparation of dietary treatments
L. acidophilus (PTCC:1643) was provided by Iranian research organisation for science and technology and S. cerevisiae (Saf-levure, Lesaffre Group, France) was purchased from the local market. The FSM was prepared according to the method described previously by Hassaan et al. (2015). The RSM was purchased from a local company (Arsham Co., Tehran, Iran), grounded to particle size (0.5 mm >), then diluted using distilled water at 1:1 ratio and autoclaved at 121 C for 15 minutes. After cooling in room temperature, the RSM was inoculated with combination (1:1) of L. acidophilus and S. cerevisiae as 1 Â 106 CFU/g meal. Then the mixture was packaged and sealed in sterile bag. This provided a yeast and bacteria density of 1 Â 103 cell/g substrate. Finally, inoculated RSM was incubated at 36 C for 12 days, then FSM was dried at 60 C for two days. In the beginning (day zero) and after fermentation (day 12), 20 g of FSM were sampled to chemical analysis and anti-nutritional factors content. Dry matter, crude protein, crude fibre, ether extract, ash, oxalate and phytic acid were determined according to methods of AOAC (1995).

Birds and treatments
A total of 420-day-old male broilers (Ross 308) were supplied from a commercial hatchery, individually weighed and allocated to a completely randomised design experiment with 7 treatments and 5 replicates (12 birds per pen). Feed was offered ad libitum and water was freely available. The experimental treatments were designed as follows: basal diet based on soybean meal (SBM), SBM substitution with 15 and 25% RSM either with phytase [5000 FTU phyzyme XP/ g (PHX)] (RSM 15 þ PHX, and RSM 25 þ PHX) or without enzyme (RSM 15 , RSM 25 ), and two diets in which SBM substituted with 15 and 25% fermented sesame meal (FSM 15 and FSM 25 ). The chemical compositions of the starter (1-10 days), grower (11-24 days), and finisher (25-42 days) diets are shown in Table 1. All diets were fed in mash form and experiment lasted for 42 days.

Bacteriological analysis and crop and ileum pH measurements
Two birds from each pen were randomly selected and euthanised by cervical vertebra dislocation on day 42, the crop and ileum were aseptically removed for microbial population analysis, then all samples were stored at _ 20 C until further analysis. Stored samples of crop and ileum then were homogenised and diluted in ratio of 1:10 using serial decimal dilution until 10 9 . In particular, Lactobacillus spp., total anaerobic bacteria, Escherichia coli and Coliform were counted using the deMan Rogosa Sharpe agar (Merck Co., Darmstadt, Germany), Reinforced clostridial agar (Reinforced clostridial agar, Fisher Scientific, Waltham, Massachusetts, US), Eosin methylene blue agar (Merck Co.) and violet red bile agar (Merck Co.), respectively, according to the Tuohy et al. (2002). Prepared plates were incubated at 37 C for 48 h anaerobically (Lactobacillus spp. and total anaerobic bacteria) or 37 C for 24 h aerobically (Escherichia coli and Coliform), and then colonies were counted by colony counter. Collected data from bacterial culture were transformed to logarithmic scale prior to analysis. Moreover, pH of the ileum, caecum and gizzard was measured by direct inserting of pH metre probe in three parts (proximal, middle and distal) of above organs, and average values were calculated.

Intestinal morphology
Morphological analysis of intestine was performed using same euthanised birds mentioned in previous section on day 42. A 3-cm midpoint segment of the duodenum, ileum and jejunum were removed, excised and flushed with cold saline and immediately placed in 70% fluid formalin, and then transferred into 70% ethanol after 72 hrs (Naderinejad et al. 2016). The intestinal segments were embedded in paraffin, and a 5-lm section of each sample was placed on a glass slide and stained with haematoxylin and eosin for light microscopy. Ten replicate measurements for each variable were taken from each sample, and average values were used in statistical analysis. The villus height (VH) was measured from the tip of the villus to the villus crypt junction, crypt depth (CD) was defined as the depth of the invagination between adjacent villi, and the epithelial thickness (ET) considered as the distance from the epithelial surface to the basement membrane of the epithelial cell (Xu et al. 2017;Abdollahi et al. 2019). Villus width (VW) was measured at the widest area of each villus, whereas the VH:CD was determined as the ratio of VH to CD. Villus surface area (VSA) was calculated using the following formula (Emami et al. 2017).
where VW ¼ villus width and VH ¼ villus height.

Tibia bone parameters
The tibia length was measured using a calliper with an accuracy of 0.001 cm. Then, both bones were weighed in the presence of air, reweighed while suspended in water to determine the wet bone volume (cm 3 ). It was assumed that water specific gravity is 1.0 g/cm 3 (measurements were made with room temperature at 22 C). To determine ash, bones were dried at 100 C for 24 h and weighed and then burned at 600 C for 24 h, cooled in a desiccator, and weighed. Bone density was calculated by dividing the bone weight to its volume (Zhang B and Coon 1997;Kim et al. 2004).

Serum biochemical analyses
On day 42, two birds per pen were randomly selected, and blood samples were collected from the wing vein before slaughter in vacutainer tubes. The blood samples were centrifuged (2500 xg for 10 min) within 30 min after sampling to obtain serum. Serum samples were divided in two aliquots and kept at À20 C until analysed. Total cholesterol, triglycerides, total protein, alanine aminotransferase (ALT), aspartate aminotransferase (AST), phosphorus and calcium values were measured using commercial kits according to the manufacturer's instructions (Pars Azmoon Kits; Pars Azmoon, Tehran, Iran). Very-low density lipoprotein cholesterol (VLDL-C) values were calculated by dividing triglyceride values to unit five.

Statistical analysis
Data analysis was conducted by one-way ANOVA using the general linear model procedure of the SAS Institute (2004). Analysis of variance and the Duncan's Multiple Range test were used to determine significant differences (p .05).

Chemical composition of RSM and FSM
The chemical composition of RSM and FSM are presented in Table 2. Fermentation process significantly decreased pH, crude fibre, oxalate and phytic acid by 30.6, 40.2, 50.8 and 43.9%, respectively, while it increased crude protein, ether extract and available phosphorus by 12.7, 11.4 and 61.1%, respectively (p < .05). However, dry matter, ash, calcium and total phosphorus remained with no significant change during fermentation process (p > .05).

Microorganism enumeration and pH measurement
Enumeration results of Lactobacillus spp., total anaerobic bacteria, Escherichia coli, and Coliform in the ileum and crop samples are shown in Table 3. In ileum, the population of Escherichia coli was not influenced (p > .05) by treatments on day 42. However, the Coliform count showed significant (p < .05) decrease in chicks fed diets containing FSM 15 (10.4%) and FSM 25 (17%) compared to RSM 15 and RSM 25 diets. Interestingly, the number of Lactobacillus was increased by RSM 15 þ PHX, FSM 15 , FSM 25 by 6.2, 7.7, and 7.5% respectively, compared to the SBM diet (p < .05), however, no significant differences were observed between SBM and other treatments. Furthermore, the treatments containing FSM 15 increased total anaerobic bacteria by 10.4, 10.6, 6.5 and 8.2% compared to RSM 15 , RSM 25 , RSM 15 þ PHX and RSM 25 þ PHX, respectively.
In the crop, the population of Lactobacillus spp. was not influenced by diets (p > .05), however, total anaerobic bacteria increased in diets containing FSM compared to other diets (p < .05). The Coliform bacteria count in the crop was higher in the RSM 25 treatment by 18.5% versus the FSM 15 (p < .05), while no significant differences were appeared between other treatments. Furthermore, compared to other diets, inclusion of different levels of FSM in diets decreased Escherichia coli population (p < .05).
The pH of the digesta in the different segments of the gastrointestinal tract across diets is summarised in Table 3. The broilers fed different levels of FSM had a lower pH in the crop than those fed other diets (p < .05), but the pH was not influenced by supplemented diets with phytase. Similarly, the pH of ileum content decreased in broilers received FSM 15 and FSM 25 diets by 6.3 and 9.2% compared to RSM 15 þ PHX and RSM 25 þ PHX diets, respectively (p < .05). Other diets did not have a significant difference compared to SBM. The lowest pH in the crop and ileum was numerically observed in diet containing FSM 25 .

Intestinal morphology
The influence of dietary treatments on the morphometry of different parts of small intestinal is shown in Table 4. In the duodenum, the measures of gut morphology (VH and CD) did not significantly differ between treatments. However, in the jejunum, the VH was increased in broilers consuming diets containing FSM 15 and FSM 25 by 13.2 and 13.9% compared to those fed RSM 15 and RSM 25 (p < .05). Also, VSA was greater in FSM diets than SBM and RSM diets in the jejunum (p < .05). The dietary treatments had no significant effect on CD, VW, VH:CD and ET in the jejunum (p > .05).
In the ileum, the birds fed FSM 25 led to significant increase in VW by 11.6, 12.3, 13.8 and 10.2% compared to the SBM, RSM 15 , RSM 25 and RSM 15 þPHX diets, respectively (p < .05). Furthermore diets with different levels of the FSM cause increased VSA when compared to other diets in the ileum (p < .05). However, VH, CD, VH:CD and ET were not influenced by diets (p > .05).

Tibia bone parameters
The effects of experimental diets on the tibia bone parameters of broilers are shown in Table 5. No Table 3. Microbial counts (log CFU/g) and pH in ileum and crop digesta of broilers at 42 days of age.  significant differences were observed in relative weight, length, density and ash percentage of tibia bone among treatments (p > .05). The body weight of the broilers fed with RSM 15 , RSM 25 and RSM15 þ PHX significantly decreased compared to other diets. Since body weight can be considered as an influential factor in tibia, therefore, the fresh weight of bone was significantly decreased in the broiler receiving RSM 15 and RSM 25 by 14.2 and 22.2%, respectively, compared to SBM diet (p < .05). In addition bone weight was numerically lowest in the diet containing RSM 25 and the broilers fed diet containing RSM 25 showed the decreased volume (cm3) of tibia compared to other treatments, except for RSM 15 (p < .05).

Serum biochemical analyses
The effects of dietary treatments on serum biochemical parameters of broilers on day 42 are shown in Table 6. No significant changes were observed in blood serum phosphorus, calcium, uric acid, total protein, ALT and AST (p > .05). However, inclusion of different levels of FSM to broiler rations significantly decreased blood triglycerides compared to birds fed SBM, RSM 25 and RSM 25 þPHX diets (p < .05). Also, the broilers fed with FSM had a lower cholesterol than those fed SBM and RSM diets (p < .05), but no significant differences were observed with those fed the combination of phytase and RSM (p > .05). As a result of cholesterol reduction, a tendency to decrease VLDL were recorded for broilers fed FSM (p ¼ .09).

Chemical composition of RSM and FSM
The present study was conducted to investigate the effect of feeding different levels of RSM processed by microbial fermentation and phytase enzyme on the intestinal microbiota and morphology, pH, tibia bone parameters, and blood parameters of broiler chickens.
There is growing evidence that lactic acid bacteria reduce the feed's pH during the fermentation process. The point of use the mixture of the L. acidophilus and S. cerevisiae in this study is that S. cerevisiae consumed the oxygen inside the bag (Chiang et al. 2009) and subsequently provided a better environment for L. acidophilus to produce lactic acid that resulted in reducing pH value of the FSM by 30%. In agreement to these results, it has been reported that the .288 a,b Means with no common superscripts within the column of each classification are significantly (p < .05) different, and each value represents the mean of five replicates. 1 SBM: soybean meal; FSM 25 : 25% fermented sesame meal; RSM 15 : 15% raw sesame meal; PHX: 5000 FTU phyzyme XP/g. SEM: standard error of mean. Table 6. Effects of the experimental diets on the blood parameters and digestive enzyme activity in broilers. .648 a,b Means with no common superscripts within the column of each classification are significantly (p < .05) different, and each value represents the mean of five replicates. 1 VLDL: very-low density lipoprotein; ALT: alanine amino transferase; AST: aspartate amino transferase. 2 SBM: soybean meal; FSM 25 : 25% fermented sesame meal; RSM 15 : 15% raw sesame meal; PHX: 5000 FTU phyzyme XP/g. SEM: standard error of mean. combination of lactic acid bacteria and Fungi decreased the pH of FSM during the fermentation process (Chiang et al. 2009;Ashayerizadeh et al. 2017).
In the present study, Fermentation process significantly decreased crude fibre, oxalate and phytic acid contents by 40.2, 50.8 and 43.9%, respectively, while it increased crude protein, ether extract and available phosphorus by 12.7, 11.4 and 61.1%, respectively. These findings are consistent with Olude et al. 2016 that fermentation of RSM by Lactobacillus planetarium decreased crude fibre and phytic acid contents by 48.9 and 16.7%, respectively, while it increased crude protein by 26.2%. These findings are also in agreement with Olagunju and Ifesan (2013) who reported a reduction in crude fibre, phytic acid and oxalate contents of fermented sesame seed by lactic acid bacteria by 46, 50 and 69% respectively, while the crude protein and fat contents appeared with slight increase at the end of 96 h of fermentation. Furthermore, Roy et al. (2014) reported that the amounts of crude fibre and phytic acid of FSM decreased by 28.9 and 89.9%, respectively. In agreement to present study, results from other researches showed that fermentation process using different microorganisms may increase ether extract (Campbell et al. 2017;Dai et al. 2020).
The increase in protein content can be attributed to microbial synthesis of proteins, secretion of enzymes and other biological products during fermentation by microorganisms (Elyas et al. 2002;Zhang WJ et al. 2007). The expected decrease in fibre content during fermentation could be resulted from microbial enzymes contribution in partial solubilisation of cellulose and hemicellulosic type of material (Olagunju and Ifesan 2013). The reasons for the high level of available phosphorus of FSM could be related to the enzymatic degradation of phytic acid content in this study. Enzymatic degradation of phytic acid with Lactobacillus spp. and S. cerevisiae have been previously reported (Olukomaiya et al. 2020;Onipede et al. 2020). It is widely acknowledged that decomposition of phytic acid increases the availability of many cations and thus nutritional value (West 2014;Gupta et al. 2015). Also, the enzymatic degradation of phytic acid by phytase enzyme requires an optimum pH which can be provided by natural fermentation (Gupta et al. 2015). Findings of this study revealed a reduction of oxalate which is in consistent oxalate degradation was performed using L. acidophilus (Hatch 2017;Chamberlain et al. 2019). Furthermore, the previous study has identified a S. cerevisiae acyl-activating enzyme 3 (ScAAE3) as an enzyme capable of catalysing the conversion of oxalate to oxalyl-CoA. Also provided evidence suggested a role for ScAAE3 in reducing the inhibitory effects of oxalate on growth (Foster and Nakata 2014).

Microorganism enumeration and pH measurements
Data analysis in this study revealed that FSM modulated microbial balance towards increasing Lactobacillus and total anaerobic bacteria populations and decreasing Escherichia coli and coliform populations in the ileum and crop. One possible reason for the positive effects of the FSM on the intestinal microbial population, partly could be related to the low pH of the FSM which was obtained after 12 days fermentation (Table 2). Lactobacillus has been noted in the literature due to its potential for industrial mass production of fumaric acid during fermentation (Chamberlain et al. 2019). It is widely acknowledged that the Lactobacillus spp. by producing fumaric acid is known to have an antimicrobial effect, likely due to acidifying the extracellular pH and making the environment inhospitable to competing microorganisms such as Escherichia coli and Salmonella to epithelial cells adhesion (Niba et al. 2009;Lu et al. 2011;Saad et al. 2013;Elmi et al. 2020). High production of fumaric acid during the fermentation process could be the other reason behind the association between intestinal colonisation by Lactobacillus and reducing Coliform colonisation in this study. There is not any published results from study which addresses the effects of FSM on intestinal microbial of the broiler. However, fermented legumes seeds by the mixture of Lactobacillus spp., S. cerevisiae and other probiotic strains have been widely used to change the microbial population in the animal diets, of which Ashayerizadeh et al. (2018) is one. Results reported by these researchers indicate that the fermentation of rapeseed meal by the mixture of L. acidophilus, Bacillus subtilis and Aspergillus niger led to increased lactic acid bacteria in the crop, while it decreased the number of Coliform in the ileum, and decreased the pH of the ileum and crop which is in agreement to this study.
Results reported by Chiang et al. (2009), indicate that RSM fermented by the mixture of L. fermentum, Bacillus subtilis, S. cerevisiae and Enterococcus faecium led to decreased pH and subsequently increased the population of lactobacilli in broiler chickens. Similarly, Piglets fed diets supplemented with live S. cerevisiae and its superfine powders had lower pH values and decreased numbers of Escherichia coli in the ileum and caecum contents compared to the control on day 21. Moreover, the ratio of Lactobacilli to Escherichia coli in the ileum and caecum contents was increased by dietary S. cerevisiae and its superfine powders (Zhu et al. 2017). Finally, third possible reason for the positive effects of S. cerevisiae and L. acidophilus to improves microbial balance could be linked to their immunostimulatory effects that have been reported in different animal models (Koenen et al. 2004;Li et al. 2005;Ashraf and Shah 2014;Awais et al. 2019).

Intestinal morphology
In the current study, VH was higher in the jejunum of broilers fed with FSM than those fed RSM diets. Also, the VSA was greater in the jejunum and ileum of the broiler fed diets supplemented with FSM 15 and FSM 25 . No research data was found in which addressed the beneficial effects of the FSM on the intestinal morphology of broiler chickens. However, there are many studies approved positive effects of the fermented feeds on the intestinal morphology (Chu et al. 2017) because of their high Lactobacillus spp. population. For example, it has been reported that broilers fed by Lactobacillus spp. had higher VH and VH:CD ratio in different parts of small intestinal (Aliakbarpour et al. 2012;Shokryazdan et al. 2017;Elmi et al. 2020). Furthermore, Chiang et al. (2009) showed that the rapeseed meal fermented by a mixed liquid culture of L. fermentum, Enterococcus faecium, S. cerevisae and Bacillus subtilis led to increased VH and VH:CD ratio in the ileum and jejunum compared to unfermented rapeseed meal in the broiler chickens. They Also demonstrated that in the jejunum of the birds fed with fermented rapeseed meal VH:CD ratio was significantly higher than those fed soybean meal diet. Interestingly, the most parameters measured under a light microscope in this study, did not show a significant difference in various intestinal segment between SBM and RSM þ PHX. These findings are consistent with previous studies of de Souza et al. (2017) who reported the diet containing RSM with and without phytase enzyme did not have effects on the intestinal morphology of piglets. Small intestinal VH is important as it determines the functional maturity of enterocytes arriving at the villus tip. With shorter villi, enterocytes reach the villus apex earlier, when their enzyme secretory capacity is less developed, leading to reduced digestive and absorptive efficiency (Broom 2015). Because high Lactobacillus spp. in the gut are correlated with improved gut health, the FSM offer a competitive advantage over the RSM. The present findings implicated conspicuous advantage of the FSM over the RSM in improving the gut morphology.

Tibia bone parameters
There were no published results concern to RSM effects on the tibia bone characters. However, since body weight had a significant covariate effect on bone weight (Yalc¸in et al. 2001), the reason for decrease in bone weight of the broilers fed diet supplemented with RSM may be correlated with reduction in their body weights, and consequent effects on the bone volume (Table 5). Furthermore, the lower bone weight and volume might be related to the high level of oxalate and phytic acid in RSM that result to decrease in availability of calcium and phosphorous (Rahimian et al. 2013;Sina et al. 2014;Ghazvinian et al. 2016).

Serum biochemical analyses
Serum biochemical parameters are often used as bioindicator to monitor the health and nutritional status of chickens. Obtained results in this study indicated that the serum triglycerides and cholesterol contents were lower in FSM-treated chickens than those treated with diets containing SBM, RSM 15 and RSM 25 . In agreement with results of present study, Ashayerizadeh et al. (2018) showed that serum cholesterol, triglycerides and VLDL-C contents of broiler chicken fed fermented rapeseed meal diets were lower than other treatments. Similar results that showed a decrease in triglyceride level using fermented products (fermented cottonseed Meal) in the broiler chicken diets have been reported by Nie et al. (2015). The reduction mechanisms of cholesterol and triglycerides that were previously reported have a close relation with the population of the lactic acid bacteria. In this study the Lactobacillus spp. and total anaerobic bacteria count were increased in broiler fed with FSM. Three reasons to explain the decline in blood cholesterol concentration of FSM-treated chickens have been suggested. First, lactic acid bacteria by inhibiting the enzyme activity of 3-hydroxy-3-methyl-glutaryl-coA, decreases the amount of cholesterol synthesis (Chen et al. 2013). Second, the production of short-chain fatty acids, especially propionate by lactic acid bacteria limits the hepatic cholesterol synthesis (Homayouni et al. 2012). Third, antihyperlipidemic activities of sesame could be credited to the induction of cholesterol turnover by increasing faecal excretion of steroid and production of hepatic bile acid (Mushtaq et al. 2020).
In this study, the RSM with and without phytase did not have significant effects on the blood parameters, which agrees with previous findings on the Japanese quail (Ghazvinian et al. 2016;Rezaeipour et al. 2016) and broiler chickens (Al Harthi and El Deek 2009). In contrast, Shanti et al. (2012) reported the dietary RSM at the level of 25% increased the plasma triglycerides and calcium contents, whereas it had no significant effect on the AST and ALT activities, cholesterol, total protein, and phosphorus concentration.

Conclusions
Based on the in vitro results, the fermentation of RSM by L. acidophilus and S. cerevisiae for 12 days not only sufficiently increased availability of phosphorus and calcium by enzymatic degradation of phytic acid and oxalate, but also increased the crude protein of FSM. The low pH and high lactic acid bacteria in FSM, made it the best trigger to modulate microbial balance towards benefit bacterial and consequently improving intestinal morphology of broilers. The in vivo results indicated that there is not any differences between broilers fed FSM and SBM diets in tibia bone quality. In contrast, those fed diet containing RSM 25 showed the lowest body weight, fresh tibia weight and volume. Furthermore, the serum triglycerides and cholesterol contents of the broilers reared with two levels of FSM were lower than those fed SBM and RSM 25 . All in all, the fermented RSM by L. acidophilus and S. cerevisiae not only showed some probiotic properties to change microbial balance and improve intestinal morphology but also could be considered as a beneficial source of protein for SBM substitution up to 25%.

Ethical approval
Farm experimental procedures were conducted in accordance with animal ethics committee guidlines of Guilan university.

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

Data availability statement
The data that support the findings of this study are available from the corresponding author, Majid Mottaghitalab, upon reasonable request.