3.1. Growth performance
In Europe acidifiers or preservatives such as organic acids are often used in monogastric animals’ feed production to replace antibiotics and to ensure protection from pathogens [
4]. When evaluating animals' productivity traits, feed supplementation with organic acids supports better feed conversion ratio (FCR), promotes growth, improves minerals absorption and body weight gain [
14]. Influence of dietary organic acids mixture inclusion on rabbits’ growth performance is shown in
Table 2. This kind inclusion improved rabbits’ weight at 77 d of age: compared to SCD, OAM group’s rabbits were 106.60 g heavier (
P < 0.05). Similar study with organic acids mixture (dosage 5 g to 1 kg feed) was performed by Cardinali et al. [
15]. In his study with 28-day old, weaned rabbits’ positive effect was obtained on weight gain in the second phase of fattening. Our research results after counting average daily gain (ADG) and daily feed intake (DFI) showed that feed supplemented with OAM significantly increased mentioned productivity indicators (
P < 0.05). Other scientists’ research results show that ADG can be increased even when using other kind organic acids separately or together as mixtures. For example, ADG improvements observed especially when using fumaric acid [
16], lactic and formic acids mixtures [
17], butyric acid [
18,
19]. However, not in all scientists’ studies such results were observed [
20,
21]. The positive results of growth performance are usually associated with intestinal morphological changes and improvements in feed digestibility [
19,
22]. On the contrary to ADG results, feed conversion ratio (FCR) of OAM group was slightly lower compared to SCD (
P < 0.05). Similar to our research was performed by Zeweil et al. [
23]. However, on the contrary to ours, his study showed a significantly better FCR while using dosage of 0.05% propionic acid in feed. Nevertheless, the growth rate of our study’s fattening rabbits was slightly higher in group supplemented with organic acids mixture, comparing to standard compound diet (
P < 0.05). These beneficial results on growth performance appeared to be because used organic acids mixture, which lowered gastrointestinal tract’s pH and facilitated nutrient retention. This observed positive trend could be related to more favourable microbiota in the digestive tract and improved digestion of the feed. The specific antimicrobial activity of OAM, which could have contributed to the reduction of intestinal bacteria due to low bacterial competition with the host for available nutrients and a decrease in the levels of harmful bacterial metabolites linked to reduced bacterial fermentation leading to improved feed digestibility.
3.2. Physiological properties
For an objective to investigate the state of rabbits’ health, determination of blood composition was carried out during our study. It is important parameters of animal's body physiological state, which associated with proper functioning of vital organs and, ultimately, with animal's production traits.
Table 3 shows the influence of dietary organic acids mixture inclusion on rabbits’ blood parameters at 77 d of age. However, after evaluating blood plasma results no significant effects of organic acids mixture were observed when comparing globulin, triglycerides, cholesterol, AST, and ALT contents between groups (
P > 0.05). Our results are in line with Mohmed et al. [
24] who reported that supplementation of different doses of another organic acid (humic) has no effect on AST and ALT activities in rabbits’ blood plasma. However, these results means that liver functions were normal under the treatment of OAM. Nevertheless, organic acids mixture inclusion increased total protein and albumin amounts in blood plasma (
P < 0.05). The significant total protein increase in OAM rabbits’ blood plasma could be due to the achieved significant increase in albumin. It indicates that supplemental organic acids mixture might improve protein synthesis in rabbit’s liver and digestibility of crude fibre and organic matter. However, obtained results during our study showed that OAM treatment did not have a significant effect on the most determined blood parameters, but all observed values were within the physiological limits for rabbits [
25,
26,
27]. Our present results conduct with the findings of Dorra et al. [
20] and Gorlov et al. [
1], who reported that blood parameters of rabbits were not affected by dietary organic acids inclusion. Research of Sherif [
28] found that dietary organic acids mixture at 1.0 g/kg in feed do not affect blood plasma parameters (total protein, albumin, globulin, triglycerides, cholesterol, AST, and ALT) of New Zealand white rabbits either.
Positive results of animal’s growth are usually associated with intestinal morphological changes and improvements in feed digestibility [
29], reduced intestinal pH and normal intestinal microflora [
23]. Organic acids mixture inclusion into rabbits’ diet affected most of intestine traits at 77 d of age (
Table 4). Excluding this kind of inclusion did not have any significant effects on pH and dry matter content in
duodenum and to whole intestine length (
P > 0.05). Organic acids act by reducing the intestinal pH in the gastrointestinal tract and stimulates the secretion of digestive enzymes, increase the activity of proteolytic enzymes, enhance gut growth and morphological healing in lesions. It is believed that acidifiers have an antimicrobial effect, they support the microbial balance in the intestines inhibiting the proliferation of pathogenic bacteria’s [
4]. In our study, organic acids mixture significantly reduced pH and increased dry matter (DM) in
caecum’s content (
P < 0.05). Significantly greater dry matter in OAM groups rabbits’
caecum’s content coincides with Romero et al. [
17] results, where higher quantity of dry matter content in
caecum was established while using feed supplemented with formic and citric acids mixture. More liquidly
caecum’s content may be associated with animal morbidity [
30], while our results inversely indicate increase of dry matter content in
caecum, which means less morbidity. Increased quantity of dry matter content in
caecum can be caused by feed, since fibre is good barrier to water and its mixture is pushed further into the large intestine, and then a large part of it is removed with solid faeces.
Caecum’s content viscosity levels could be used as an indicator of digestibility and nutrient absorption as well. After viscosity determination in our study, OAM samples of
caecum’s content had much greater viscosity compared to SCD group samples (
P < 0.05). Increased viscosity can affect secretion of endogenous enzymes and bile acids, which causes morphological changes in the intestine and nutrient digestibility [
31]. There are studies, which results proves that organic acids mixtures improve nutrient digestibility [
32]. Increased viscosity in our study coincides with improved growth results, however, no insufficient data on the effects of organic acids mixture on the rabbits’
caecum’s content viscosity in the scientific literature. After weighting separate intestinal parts, total weight of whole rabbit’s intestine was higher in OAM group compared to SCD (
P < 0.05). These results were in line with other studies conducted by Uddin et al. [
32] and Ghazvinian et al. [
33]. Increased intestinal weight indicates better gut development, which leads to better digestion of nutrients, and this is reflected in improved growth rates. To be more precisely, addition of organic acids mixture in feed may support nutrient digestibility and bioavailability. It is possible that it acts by lowering digestive systems acidity which lead to better utilization of nutrients. It appears that organic acids mixture used in our experiment affected the metabolism of nutrients and increased the size and morphology of the digestive system. In general, epithelial cells of the gastrointestinal tract could cause this weight gain.
Influence of dietary organic acids mixture inclusion on rabbits’ short-chain fatty acids profile and ammonium nitrogen content in
caecum’s content at 77 d of age is shown in
Table 5. Short-chain fatty acids (SCFA) is a regular source of energy for rabbits. Both, SCFA and ammonium nitrogen (N-NH
3), are absorbed into the bloodstream from
caecum. The concentration of N-NH
3 in
caecum’s content can be determined by several factors: H
2 pressure, reaction content, carbohydrate availability. As compared to ruminants, proteolytic activity is higher in the rabbit
caecum [
18]. Unfortunately, no significant effects of OAM treatment were noted on lactic acid content and N-NH
3 concentration in
caecum’s content (
P > 0.05). The profile SCFA of rabbits’
caecum’s content is specific: acetate is prevailing (65-87 mmol per 100 ml
-1), followed by butyrate (6-28 mmol per 100 ml
-1) and propionates (3-11 mmol per 100 ml
-1). The proportion depends on fibre content in the diet. For example, when increasing dietary fibre, it is usually noticeable on butyrate reduction and the proportion of acetates increasing. This proportion is reflected in our research results: amounts of acetic and propionic acids were increased by OAM and were slightly higher compared to SCD (
P < 0.05). On the contrary, OAM decreased butyric acid, which content was lower than in SCD (
P < 0.05). Rabbits are special in assessing microbial activity of their body, it should therefore be noted that butyric acid is more important SFCA than propionic, which can be critical for health assessment and prevention of enteritis. Changes in SCFA profile could lead to changes of commensal intestinal microflora composition, which cannot be assessed without a microbiological analysis.
One of the most important properties of organic acids is their influence on intestinal histomorphology. It is known that because of organic acids (particularly, fatty acids such as butyric acid), growth of the lining of the digestive tract becomes more intensive. Butyric acid is an important source of energy for intestinal epithelial cells, it stimulates the proliferation and differentiation of cells, prevents growth of cancer cells [
18]. Organic acids mixture effect on rabbits’
caecum’s villus height, crypt depth and V/C ratio at 77 d of age is shown in
Table 6. All histomorphometric results obtained in our study showed statistically significant differences between groups. Except, V/C ratios in DISTAL part of
caecum did not differ between groups (
P > 0.05). OAM group’s results showed significantly better results by increasing intestinal
villus height in all parts of
caecum (PRO; MID; DISTAL) (
P < 0.05). Longer intestinal
villus indicates absorption surface increase, and thus better approachability to nutrient absorption. Also, these results are in line with growth performance improvements achieved in OAM group. Intestinal epithelial cells, appearing in crypts, migrate by
villus surface to the summit and are squeezed into the intestinal lumen within 48-96 h [
18]. In case of shorter
villus may result in a weaker nutrient absorption, increasing secretion in the digestive tract and worse growth performance [
34]. Meanwhile, elongation of
villus and improvement of V/C ratio directly correlates with the increasing changes of epithelial cells [
35]. It is believed that the increase of
villus height indicates their functions activity [
36]. Therefore, it can be concluded that the rabbits’ intestinal
villus functions activity was improved after feed supplementation with organic acids mixture. This coincides with the studies conducted by other scientists [
15,
17,
18,
24,
37,
38]. Comparing our results to others we can agree and conclude that improved crypt depth has increased crypt cell production or an indicator of intensified mucus secretion [
39] and V/C ratio reflects the digestive capacity of the intestine [
40].