Effects of dietary vinegar on performance, immune response and small intestine histomorphology in 1‐ to 28‐day broiler chickens

Abstract The purpose of this research was to evaluate the effects of apple cider vinegar in diet on the growth performance, immune response, histomorphological changes of the small intestine and some serum biochemical factors in broilers. A total of 85 broiler chicks of Ross 308 were purchased and 64 well‐conditioned chicks were selected and divided into four experimental groups with four replicates and four chicks per replicate. The diets of groups 2, 3 and 4 were mixed with 1, 2 and 3% of vinegar, respectively, and group 1 as the control group was fed by the standard diet. The results showed that body weight gain was higher in the groups fed vinegar than the control group. There were no significant changes in the feed conversion ratio between the treatment and control groups (p = .507). Vinegar intake through the diet did not change significantly the weight of Bursa of Fabricius (p = .369) and spleen (p = .122). Vinegar significantly reduced blood urea nitrogen levels in the treatment groups compared with the control group (p = .0052). There was a significant increase in the level of antibody titre against Newcastle disease virus by haemagglutination inhibition test in the groups receiving vinegar in comparison with control group (p = .0358). Compared with the control group, the villus height (p = .0022) and intestinal crypts depth (p = .0015) significantly increased in the groups receiving apple cider vinegar. In conclusion, dietary supplementation with apple cider vinegar has beneficial effects on performance, immune response and small intestine histomorphology in broilers.

basal diets were formulated to provide the nutritional requirements of the chicks. During the experiment, the chicks were given free access to water and food in mash form. The diets of chickens of groups 1, 2, 3 and 4 were mixed by spraying with 0, 1, 2 and 3% of apple vinegar, respectively. The amount of apple cider vinegar in the diets was selected on the basis of previous studies (Tasharofi et al., 2017).
The supplemented diet of each group with mentioned percentage of vinegar was freshly prepared and used from 1 to 28 days of the experiment. For this purpose 1 kg of feed was mixed with 10, 20 and 30 ml of vinegar to prepare the diets with 1%, 2% and 3% of vinegar, respectively.
To prepare vinegar, apple fruits were sliced and their seeds removed and then placed in a jar. Diluted industrial vinegar (1:4 water and vinegar) were added and the jar transferred to a warm and dark place for 3 weeks. Finally, the solid parts removed and the liquid used as apple cider vinegar.
Proton nuclear magnetic resonance ( 1 H NMR) spectroscopy was performed to confirm the existence of acetic acid in the vinegar liquid. As shown in Figure 1, 1H NMR spectrum confirmed the TA B L E 1 Feed ingredients and calculated nutrient composition of the experimental basal diets of chicks during 1-28 days of age presence of the acetic acid by the distinctive feature of the methyl signals at δ = 1.9 (as a singlet).
Feed intake of chicks was recorded and the feed conversion ratio was calculated for 28 days. All chicks were vaccinated by spraying at 1 day of age with Newcastle disease B1 vaccine and at 8 days of age with Newcastle oil emulsion vaccine (Razi Vaccine and Serum Research Institute, Iran).

| Sampling and jejunum histomorphology
On days 14 and 28, a chick from each replicate was randomly selected, weighed and followed by blood sampling, anaesthetized first and then was exposed to necropsy. After the necropsy, the organs of the Bursa of Fabricius, spleen and entire small intestine were removed and weighed with contents. The length of the small intestine from the beginning of the duodenum to the location of the blind intestines was also measured. For histomorphological changes of the small intestine, the tissue specimen was prepared with a dimension of 1 × 1 × 0.5 cm from the jejunum part and fixed in 10% neutral

| Serum Biochemical analysis, HI test
Blood samples were centrifuged at 500 g for 5 min, and sera were collected and stored at −20°C until use. Serum samples were used for the determination of cholesterol, triglyceride, albumin, total protein, blood urea nitrogen (BUN), low density lipoprotein (LDL), high density lipoprotein (HDL) and haemagglutination inhibition (HI) antibody titre. Blood serum biochemical factors were measured by an Auto Analyzer Selectra Pro M (Netherlands). HI test was used to measure serum antibody titre against Newcastle disease virus.
The HI titre was expressed as the log 2 reciprocal of the highest serum dilution producing 100% inhibition of HA activity (Rahimi & Khaksefidi, 2006).

| Statistical analysis
Data were subjected to analysis of variance (ANOVA) procedure of SPSS Software version 25 (SPSS Inc., Chicago, IL, United States).
Replicates were considered as the experiment units. The statistical model used was: Yij = μ+Ti + Eij, where: Yij = response variables F I G U R E 1 1H NMR spectrum of the apple cider vinegar is presented from each experimental unit; μ = the overall mean; Ti = i-th treatment effect; Eij = the random terms of study. Multiple pairwise comparisons of means were performed by Duncan's post hoc test. Due to the graded level of vinegar, orthogonal polynomial contrasts were used to reveal the linear and quadratic dose response associations.
The significance level was considered to be p < .05 for the main effects and p < .1 for linear and quadratic trends. Results were reported as mean ± standard deviation.

| RE SULTS
The values measured for weight gain during the different weeks of testing in the control and treatment groups have been shown in Figure 2. As shown in Figure 2, body weight gain was higher in the groups fed by apple cider vinegar than the control group.
Results for feed conversion (FC) ratio, the internal organ weights including small intestine, the weight of Bursa of Fabricius and spleen and small intestine length in the control and treatment groups have been shown in Table 2. According to Table 2, the use of vinegar in the diet does not significantly change the FC ratio for 1-28 days (p = .507). As shown in Table 2, the vinegar intake through the diet did not cause significant changes in the weight of Bursa of Fabricius (p = .369) and spleen (p = .122), but vinegar intake increased the weight gain of the Bursa of Fabricius, although this increase is not significant.
There was no observed statistical significant difference between control and treatment groups at 14 days, regarding the weights of the internal organs (statistical results not shown).
The measured values for the biochemical factors of blood serum and HI antibody titre have been given in Table 3. As shown in Table 3, vinegar reduced blood serum BUN levels, so that 2% and 3% of it, decreases BUN significantly compared with the control group (p = .0052). There was no significant difference between control and treatment groups in the levels of cholesterol, albumin, total protein, triglyceride, HDL and LDL of blood serum. However, apple cider vinegar has reduced HDL levels in blood serum. As shown in Table 3, there was a significant difference in the antibody titre of HI between the groups receiving apple cider vinegar and the control group (p = .0358). Moreover, for BUN and HI titre the main effects and Linear trend effects were statistically significant.
The findings of histomorphologic studies of the small intestine including villus width, villus height, crypt depth and the ratio of crypt depth to the villus height have been given in Table 4. As shown in Table 4, there was a significant increase in the villi height (p = .0022) and intestinal crypt depth (p = .0015) in the groups receiving apple vinegar compared with the control group. Furthermore, the villus height and crypt depth Linear and Quadratic trends were statistically significant.

| D ISCUSS I ON
In this study, the chicks receiving apple cider vinegar had a higher weight gain than the control group containing chicks 1--28 days of age. Tasharofi et al. (2017) observed that usage of waste date's vinegar increases body weight of broiler chickens (Tasharofi et al., 2017).
Vinegar, in addition to acetic acid, contains other nutrients such as vitamins and minerals that can be effective in weight gain (Johnston & Gaas, 2006). Due to low levels of short chain fatty acids in the intestine of young chicks, they may be the best candidates for use of acidifiers (Attia et al., 2013;Panda et al., 2009). As the age increases, the production of volatile fatty acids in the gastrointestinal tract increases; this may be the main reason for the lack of effective addition of organic acids to the feed (Hernandez et al., 2006;Leeson et al., 2005).
According to the results of this research, there was an increase in the factors related to the chickens' immune system-including the weight of Bursa of Fabricius, spleen weight and antibody titre of HI in the groups receiving vinegar via feed compared with the control group. Haque et al. (2010) reported that the addition of organic acid increased the number of cells involved in the immune system in the follicles and increased the weight of the Bursa of Fabricius (Haque et al., 2010). Attia et al. (2013) observed that acetic acid in the diets of Japanese quail affected the immune system as manifested by an excess of cellular reactions in the intestine as well as lymphoid hyperplasia in the spleen tissue (Attia et al., 2013). These can be consistent with the results of our research. However, the results of this research are inconsistent with those of Brisbin et al. (2008) who reported no effect of apple vinegar on the weight of lymphoid organs (Brisbin et al., 2008). Besides, Allahdo et al. (2018) observed that vinegar had no significant effect on relative weight of lymphoid organ (Allahdo et al., 2018). and probiotics, on the histopathology of the small intestine (Paul et al., 2007). In the present study, apple cider vinegar significantly increased the height of intestinal villi in the treatment groups compared with the control group, which may be due to the improvement of the intestinal microbial flora status by the use of organic acids (Attia et al., 2013;Baurhoo et al., 2007;Xu et al., 2003).
TA B L E 2 Feed conversion, weight of internal organs including small intestine, the weight of Bursa of Fabricius and spleen, small intestine length and the ratio of the weight of internal organs to the body weight in the different groups on day 28 of the experiment