Effect of Thymol Addition and Withdrawal on Some Blood Parameters, Antioxidative Defence System and Fatty Acid Profile in Rabbit Muscle

Kristina Bacova 1, Karin Zitterl-Eglseer 2, Lubica Chrastinova 3, Andrea Laukova 1, Michaela Madarova 1, Sona Gancarcikova 4, Drahomira Sopkova 5, Zuzana Andrejcakova 5 and Iveta Placha 1,\*


Received: 19 May 2020; Accepted: 20 July 2020; Published: 22 July 2020

Simple Summary: So far, the study of the bioactivity of thymol, a major constituent of Thymus vulgaris L., in the animal organism has received little attention. Our study could give us answers to questions about whether thymol accumulates in the rabbit organism after its sustained administration and if it is also able to exhibit its beneficial properties for a longer period. Thymol in powder form at the concentration 250-mg/kg feed was added to the rabbit diet for 21 days and withdrawn for the next seven days. We confirmed that thymol was sufficiently absorbed from the gastrointestinal tract and was able to express its biological activity not only during application but, also, after withdrawal. Further studies are needed to clarify the biotransformation and bioavailability of thymol in the rabbit organism with respect to the specific features of rabbit digestion.

Abstract: Thymol concentrations in rabbit plasma, intestinal wall (IW) and faeces were detected, and the effects of thymol application and withdrawal on biochemical, antioxidant parameters and fatty acids (FA) in blood (B) and muscle (M) were studied. Forty-eight rabbits were divided into two experimental groups (control, C and with thymol 250-mg/kg feed, T). Thymol was administered for 21 days (TA) and withdrawn for seven days (TW). Thymol in plasma correlated with that in the IW (Spearman's correlation coefficient (ts) = -1.000, p = 0.0167, TA) and was detected in faeces (TA and TW). In TA alkaline phosphatase (p = 0.0183), cholesterol (v = 0.0228), malondialdehyde (p = 0.003), glutathione peroxidase (p = 0.0177) in B and lactate dehydrogenase (M, p = 0.0411) decreased; monounsaturated FA (p = 0.0104) and α-linolenic acid (p = 0.0227) in M increased. In TW urea (v = 0.0079), docosapentaenoic acid (v = 0.0069) in M increased; linoleic acid (p = 0.0070), 2 n-6 (v = 0.0007) in M and triglycerides decreased (B, p = 0.0317). In TA and TW, the total protein (v = 0.0025 and 0.0079), creatinine (B; p = 0.0357 and 0.0159) and oleic acid (M; p = 0.0104 and 0.0006) increased. Thymol was efficiently absorbed from the intestine and demonstrated its biological activity in blood and the muscles.

Keywords: rabbit; thymol; bioavailability; antioxidant

#### 1. Introduction

The supplementation of human and animal diets with Thymus vulgaris (thyme) either as dried leaves or its essential oil has often demonstrated its beneficial properties. Since synthetic growth promoters were replaced with alternative herbal products in animal rearing, thyme has started receiving major attention. The most important bioactive compound contained in this plant is thymol, which exhibits antimicrobial, anticarcinogenic and anti-inflammatory activities [1-3]. According to some studies, thyme improves the performance parameters, but some other studies suggest that it has no effect. Some studies report that thyme is able to reduce levels of triglyceride and total cholesterol [4,5]. Yu et al. [6] found that thymol possesses a lipid-reducing function by altering hepatic triglyceride secretion.

The mode of action of herbs and plant extracts and the details about the accumulation of phenolic substances in animal tissues are not known or not completely understood [7,8]. In general, the bioavailability of dietary compounds depends on their digestive stability and the efficiency of their transepithelial passage [9]. Moreover, their influence on each other's intestinal absorption has to be taken into account in studies concerned with the bioavailability of essential oil compounds.

Thymol as one of the major constituents of thyme oil presents a wide range of functional possibilities in pharmacy and the food industry. Besides thymol, thyme contains high concentrations of monoterpene phenols like carvacrol, p-cymene, 1,8-cineole, linalool, borneol, camphor, β-caryophyllene, thymol methyl ether and carvacrol methyl ether, which could have influenced the thymol absorption [2,3].

Ocel'ová et al. [10] were the first who analysed the thymol concentrations in individual intestinal segments in hens. Sufficient absorption of thymol from the digestive tract and its transport by systemic circulation to tissues in broiler chickens after four weeks of thyme essential oil application were demonstrated by Ocel'ová et al. [7]. Placha et al. [2] pointed to the sparing effect of thymol against oxidative deterioration of the antioxidant defence system in poultry after sustained thyme oil dietary application at 0.05% (thymol content 248.97 mg/kg dry matter (DM)) and 0.1% (thymol content 460.22 mg/kg DM) concentrations.

Concerning the toxicity of thymol, the data are controversial. According to a recent report of the European Food Safety Authority (EFSA) [11], thymol, when administered by the oral route in a rat, mouse and guinea pig (lethal dose - LD 50, 0.98, 1.80 and 0.88 mg/kg, respectively), suggested a moderately acute toxicity. No significant alteration was observed when a thymol oil-water emulsion was administered at doses (15.39, 30.78 and 61.55 mg/kg, respectively) during 28 days [12]. Based on European Commission (EC) [13], no maximum residual limit for thymol in foodstuffs of animal origin is needed to establish when it is used as a veterinary medicinal product. These data show that further studies are required to establish the thymol appropriate concentration.

Rabbits have a unique digestive system that is represented by an original feature of rabbit feeding behaviour named caecotrophy means the excretion and immediate consumption of specific soft faeces termed "caecotrophs". This process is extremely important, because it improves feed utilization by maximizing the digestibility of nutrients [14]. Many scientific studies have tried to find the appropriate dose and form of plant extract application for improving animal health, but insights into the precise mechanism or mode of action of their components are lacking. As far as rabbits are concerned, to our knowledge, only one old study by Takada et al. [15] has been carried out regarding the metabolic outcome of thymol. Probably, the process of caecotrophy could minimize the loss of thymol bioaccessibility during the digestive processes.

The present study provides new insights for understanding the possible processes of absorption and deposition of thymol in the rabbit organism in connection with its protective role against oxidative stress. Based on our previous studies related to the thymol absorption, deposition and beneficial effects against oxidative stress in broiler chickens after a sustained administration of thyme oil, we decided to examine the thymol application into rabbit diets at the concentration 250-mg/kg feed.

#### 2. Materials and Methods

#### 2.1. Animals and Experimental Design

After weaning at 35 days of age, 48 rabbits of both sexes (meat line M9) were randomly divided into two experimental groups (control, C and with thymol addition, T), with six replicates in each (one replicate consisting of two cages, one cage/two animals). Initial live weight was 1006 ± 98 g in C and 1035 ± 107 g in T. All experimental wire-net cages (61 cm × 34 cm × 33 cm) were kept in rooms with automatic temperature control (22 ± 4 °C) and photoperiod (16 L:8 D). The rabbits could feed ad libitum and had free access to drinking water. The experiment lasted 28 days. The rabbits were fed with thymol addition for 21 days (TA), and for the next 7 days, the thymol was withdrawn (TW). Eight starved rabbits (6 male and 2 female) in each group were slaughtered in an experimental slaughterhouse at 56 (C and TA) or 63 (C and TW) days of age. Rabbits were stunned with electronarcosis (50 Hz, 0.3 A/rabbit for 5 s), immediately hung by the hind legs on the processing line and quickly bled by cutting the jugular veins and the carotid arteries.

#### 2.2. Animals Care and Use

The trial was carried out at the experimental rabbit facility of the National Agricultural and Food Centre, Research Institute for Animal Production, Nitra, Slovakia. The protocol was approved by the Institutional Ethical Committee, and the State Veterinary and Food Office of the Slovak Republic approved the experimental protocol (4047/16-221).

#### 2.3. Diets and Chemical Analyses

The basal diet (control, C) was formulated to satisfy growing rabbits' requirements [16] (Table 1) and was tested against the experimental diet (T) containing thymol (≥99.9%, Sigma Aldrich, St. Louis, MO, USA), which was added to the basal diet in white powder form at concentration 250-mg/kg feed. The diets were administered in the form of pellets with an average size of 3.5 mm. The feed was stored in darkness to protect against degradative processes and was analysed to determine the crude protein (CP), ash, ether extract, acid detergent fibre (ADF), starch and dry matter (DM) in the diets, while DM was also determined for the intestinal wall, muscle, liver and faeces according to the Association of Official Analytical Chemists (AOAC) methods [17]. Neutral detergent fibre (NDF) was analysed according to Van Soest et al. [18].



1 The vitamin-mineral premix provided per kg of complete diet: Retinyl acetate 5.16 mg, Cholecalciferol 0.03 mg, Tocopherol 0.03 mg, Thiamine 0.8 mg, Riboflavin 3.0 mg, Cyanocobalamin 0.02 mg, Niacin 38 mg, Folic acid 0.6 mg, Calcium 1.8 mg, Iron 70 mg, Zinc 66 mg, Copper 15 and Selenium 0.25 mg.

#### 2.4. Growth Performance and Health Status

Body weight (BW) and feed intake (FI) were recorded individually once a week. The average daily FI, average daily weight gain (WG) and feed conversion ratio (FCR) were calculated at the end of the trial (on 56 and 63 d of age). Data pertaining to any animal that died during the experiment were excluded from the calculations. Mortality was recorded daily throughout the experimental periods.

#### 2.5. Thymol Antioxidant Capacity and Stability in Feed

The Trolox equivalent capacity (TEAC) was determined in thymol and in the experimental feed according with the method described by Karamać et al. [19] using the 2,2'-Azinobis-(3-Ethylbenzthiazolin-6-Sulfonic Acid (ABTS ●+) decolorization assay. The results were expressed as mmol Trolox equivalents (TE) per g. Thymol evaporation in the feed was analysed every week during thymol application using High-Performance Liquid Chromatography HPLC according to the modified method of Pisarčíková et al. [20]. Samples were analysed in triplicate and were relatively stable (0 d-151.89, 7 d-134.75 and 14 d-128.30 µg/g DM, respectively).

#### 2.6. Sampling

Blood samples for biochemical analyses were collected from the marginal ear vein (Vena auricularis) into dry nonheparinized Eppendorf tubes at experimental days 21 and 28 and were left to clot in a standing position for approximately 2 h to obtain the serum, and then, the serum was separated by centrifugation at 700× g for 15 min. Blood for analyses of antioxidant parameters was collected into heparinized tubes, and plasma was obtained after centrifugation at 1180x g for 15 min. Samples of serum, plasma, muscle (Longissimus thoracis et lumborum, LTL), small intestinal wall, liver and hard faeces (freshly voided, collected using nets mounted under the cages) were immediately frozen in liquid nitrogen and stored at -70 °C until analysis.

#### 2.7. Thymol Analyses in Plasma, Small Intestinal Wall and Facces

Detection of thymol in samples of plasma, intestinal walls and faeces was performed using headspace solid-phase microextraction followed by gas chromatography coupled with the mass spectrometry method, as described by Placha et al. [2]. Briefly, detection and quantification were carried out using a gas chromatography/mass spectrometry (GC/MS) (type HP 6890 GC) coupled with a 5972 quadrupole-mass selective detector (Agilent Technologies GmbH, Wilmington, DE, USA). Detection of thymol was confirmed by comparing its spectrum and retention time with those of the reference compound. Additionally, the Kovats index was calculated. Calibration curves were generated by plotting the peak area ratios of thymol to o-cresol used as the internal standard (Sigma-Aldrich, St. Louis, MO, USA) against the known thymol concentrations. The selective ion mode was used for the quantitative analysis of thymol. The mass fragments m/z 135 and m/z 150, as well as m/z 107 and m/z 108, were monitored as characteristic for thymol and o-cresol, respectively. Calibration curves were prepared from blank samples spiked directly with thymol (AppliChem, Darmstadt, Germany) in standard solutions with known concentrations. Each point on the calibration curve was analysed as a duplicate. The peak of thymol was detected around 19 min, and the o-cresol peak occurred around 10 min in all samples. Samples for the detection of thymol were prepared using the method described by Ocel'ová et al. [10]. Enzyme ß-Glucuronidase Helix pomatia Type HP-2 (aqueous solution, ≥100,000 units/mL, Sigma-Aldrich, St Louis, MO, USA) was added to samples to cleave thymol from its glucuronide and sulphate to obtain the total amount of thymol in the plasma.

#### 2.8. Biochemical and Antioxidant Parameters and Activity of Lactate Dehydrogenase in Blood and Tissues

Total proteins (TP; g/L), creatinine (umol/L), urea (mmol/L), triglycerides (mmol/L), total cholesterol (mmol/L), alanine aminotransferase (ALT; ukat/L), aspartate aminotransferase (AST; ukat/L) and alkaline phosphatase (ALP; ukat/L) were analysed using a DIALAB commercial kit (Prague, Czech Republic) and an ELLIPSE analyser (AMS, Guidonia, Rome, Italy).

Activity of glutathione peroxidase (GPx, EC 1.11.1.9) in blood was measured by monitoring the oxidation of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) at 340 nm in-line with Paglia and Valentine [21] using a commercial kit (Ransel, Randox, London, UK). Haemoglobin (HD) content in blood was analysed using a commercial kit from Randox, UK. The samples of LTL muscle and liver for malondialdehyde (MDA) measurement and activity of lactate dehydrogenase (LDH, EC 1.1.1.27) were washed in buffered saline to remove excess blood and connective tissue. Samples for MDA analyses were homogenised with deionized distilled water and 50 µL of 7.2% butylated hydroxytoluene and for LDH activity in cold buffer (0.05-mol/L Tris-HCl buffer, pH 7.3). The homogenates were subsequently centrifuged at 13,680x g at 4 °C for 20 min. MDA concentrations in these tissues and plasma were measured using the modified fluorometric method of Jo and Ahn [22]. The enzyme activity of LDH was measured using a commercial diagnostic kit (Crumlin, Randox, UK) with an Alize automatic biochemical analyser (Lisabio, Pouilly-en-Auxois, France) at 340 nm, as described by Andrejčáková et al. [23]. The protein concentration in the muscle and liver was quantified using the spectrophotometric method published by Bradford [24].

#### 2.9. Fatty Acids in Muscle Tissue

The fatty acid (FA) composition in the muscle tissue was determined using the method of Ouhayoun et al. [25]. Fatty acid methyl esters (FAME) were prepared by means of alcoholises in an essential nonalcoholic solution and analysed using gas chromatography on GC 6890N (Agilent Technologies, Basel, Switzerland). Results were expressed as percentages of total fatty acids.

#### 2.10. Statistical Analyses

Values of thymol, GPx, LDH, MDA and FA concentrations were tested for normal distribution with the Kolmogorov-Smirnov test. The Mann-Whitney U test was used for statistical analysis. Results were presented as the mean value ± standard deviation. Significant differences were considered at p < 0.05. Correlations of thymol concentrations between plasma and feed and plasma and intestinal wall were analysed using nonparametric Spearman's rank correlation and expressed as Spearman's correlation coefficient (rs). Statistical analyses were performed using Graph Pad Prism 5.0. (GraphPad Software, San Diego, CA, USA).

#### 3. Results

#### 3.1. Growth Performance

All broiler rabbits in the present trial were in good health, and the growth performance was normal and was not affected by the addition of thymol. Five animals (three/control and two/experimental group) died during the whole experiment. If there were no differences in the given parameters, we do not include them in the table.

#### 3.2. Thymol Antioxidative Capacity

Approximately equal TEAC value was found in the feed of the experimental group (0.76 vs. 0.74 mmol TE/g).

#### 3.3. Thymol in Feed, Plasma, Small Intestinal Wall and Facces

Thymol content in feed amounted to 148.9 ± 16.7 µg/g DM. Thymol concentration in plasma was 0.05 ± 0.02 µg/L and, in intestinal wall, 0.04 ± 0.03 µg/g DM in TA, but, in TW, it was not detected. Thymol concentration in faeces in TA was 0.89 ± 0.45 µg/g DM, and, in TW, it was 0.08 ± 0.04 µg/g DM (Table 2). Thymol concentration in plasma significantly correlated with the thymol concentration in the intestinal wall (rs = - 1.000, p = 0.0167).


Table 2. Thymol content in plasma (µg/L), feed, intestinal wall and faeces (µg/g dry matter (DM)).

ND-not detected, TA-thymol addition and TW-thymol withdrawal.

#### 3.4. Biochemical Parameters in Blood

Thymol addition significantly decreased the levels of ALP (p = 0.0183) and cholesterol (p = 0.0228). Urea significantly increased (p = 0.0079), and triglycerides decreased (p = 0.0317) in TW. TA and TW had significantly increased TP (p = 0.0025 vs. p = 0.0079) and creatinine (p = 0.0357 vs. p = 0.0159) (Table 3).

Table 3. Effects of thymol on aspartate aminotransferase (AST, ukat/L), alanine aminotransferase (ALT, µkat/L), alkaline phosphatase (ALP, µkat/L), total proteins (TP, g/L), urea (mmol/L), creatinine (µmol/L), triglycerides (mmol/L) and cholesterol (mmol/L) in rabbit blood.


a.b Values within a row with different superscript letters differed significantly (p < 0.05). Data are presented as mean ± standard deviation (SD). C-control diet, TA-thymol addition and TW-thymol withdrawal.

#### 3.5. Antioxidant Parameters in Blood and Tissues

MDA and GPx in blood (p = 0.003 and p = 0.0177) and LDH in muscle (p = 0.0411) significantly decreased in TA (Table 4).

Table 4. Effects of thymol on the antioxidant parameters and activity of lactate dehydrogenase (LDH) in rabbit blood, liver and muscle.


a.b Values within a row with different superscript letters differed significantly (p < 0.05). Data are presented as mean ± standard deviation (SD). C-control diet, TA-thymol addition, TW-thymol withdrawal, MDA-malondialdehyde and GPx-glutathione peroxidase.

#### 3.6. Fatty Acids in Muscle

Concentrations of oleic acid (C 18:1 n-9) significantly increased in TA and TW (p = 0.0104 vs. 0.0006). MUFA and x-linolenic acid (C 18:3 n-3) significantly increased in TA (p = 0.0104 and p = 0.0227).

Docosapentaenoic acid (C 22:5 n-3) significantly increased (v = 0.0069), and linoleic acid (C 18:2 n-6) and & n-6 significantly decreased in TW (p = 0.0070 and p = 0.0007; Table 5).


Table 5. Effects of thymol on fatty acids profile (% of total FA) in rabbit muscle.

Data are presented as mean ± standard deviation (SD). C-control diet, TA-thymol addition, TW-thymol withdrawal, SFA-saturated fatty acids and MUFA-monounsaturated fatty acids.

#### 4. Discussion

#### 4.1. Growth Performance

The inclusion of thymol at the concentration used in our experiment did not show any significant effect on the animals' weight, weight gain and conversion ratio. Although thyme has been shown to improve the palatability and feed intake in growing rabbits, its beneficial effects on the live growth performance in rabbits has not yet been confirmed [26]. According to Erdelyi et al. [27], due to the specific digestive physiology of rabbits, essential oils or plant extracts have much lesser positive effects than in broilers or piglets. Windisch et al. [28] reported that the use of thyme in animal diets could be limited when applied in certain amounts, because it is highly aromatic. According to Gerencsér et al. [29], thyme leaves did not demonstrate any substantial effects on the growth performance or health status. This statement is in agreement with our study, in that the thymol did not affect the feed intake, and most animals remained in optimal condition.

#### 4.2. Thymol in Plasma, Intestinal Wall and Faeces

We found a significant correlation between thymol levels in the small intestinal wall and plasma during the period of thymol addition to the feed. This result is in agreement with Placha et al. [2], who confirmed the efficient absorption of thymol from the digestive tract into the systemic circulation in broiler chickens. According to Ocel'ová [30], after the absorption of plant compounds from the intestine, they are metabolised and eliminated from the organism.

One of the most original features of rabbit-feeding behaviour is caecotrophy. The result of this process, in which soft faeces are swallowed and then stored intact in the fundus of the stomach, is that they undergo the same digestive processes as normal feed. Some parts of the initial food intake may be recycled even up to four times in this way [31]. The detection of thymol in faeces points to its excretion from the organism in an unmetabolised form (Table 2). This unmetabolised thymol

could be recycled and again absorbed in the intestinal wall, where it is finally metabolised by the processes of biotransformation. The part of the metabolites in the intestinal wall can be transported back into the intestinal lumen by efflux transporters, converted to thymol (parental compound) and again reabsorbed by the enterocytes. Another molecule can be transported through the basolateral membrane of enterocytes directly to the blood circulation. All these processes play a crucial role in affecting the thymol concentration in blood [7].

Thymol sulphate and thymol glucuronide are the main metabolites of thymol biotransformation, and so far, little is known about their bioactivity or whether these compounds are only inactive forms [3]. Pisarčíková et al. [20] first detected thymol sulphate and thymol glucuronide in the liver and duodenal wall of broiler chickens after a sustained four-week administration of thyme essential oil, and according to Kohlert et al. [32] and Rubió et al. [9], thymol metabolites could be deconjugated to the parental compounds and, in this way, express their pharmacological properties. We have to bear in mind that, during biotransformation, plant compounds change their pharmacological properties, which usually differ from the properties of the parental compounds.

There are only a few studies concerning thymol distributions in animal tissues. Thymol conjugates have been detected in the plasma of humans and animals, and thymol after enzymatic cleavage was detected in the plasma of horses, chickens and pigs [1,10,32-35]. According to Placha et al. [2], erythrocytes can act as depots of polyphenols due to their ability to bind to the surface of red blood cells, and in this way, they can circulate in the organism. There is the question whether bound thymol is able to unbind from tissue depots if the amount of circulated thymol decreases. Since we detected thymol in rabbit faeces also after its withdrawal, this mechanism could be indicated (Table 2).

#### 4.3. Biochemical Parameters in Blood

Despite the fact that plant bioactive compounds undergo fast biotransformation and elimination, Ocel'ová et al. [10] and Placha et al. [2] confirmed the accumulation of thymol in the breast muscles, kidneys and livers of broiler chickens after four weeks of thyme essential oil diet supplementation. Moreover, they found the highest concentration of thymol in the kidneys and the lowest in liver tissues, which could point to intensive metabolism in the liver and accumulation in the kidneys. Bardal et al. [36] showed that the distribution of plant compounds from systemic circulation to the tissues is restricted, since only free drugs in plasma are able to diffuse into the target tissue.

Gumus et al. [37] reported that specific polyphenols such as thymol may reduce plasma lipids by altering the hepatic triglyceride secretion, as well as inhibiting the activity of cholesterol-synthesizing enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase. If some imbalance between the cellular free radical formation and the antioxidant defence system exists, excessive amounts of free radicals are produced, and cellular components such as the lipids are attacked. The administration of antioxidants such as thymol could combat oxidative stress by the scavenging of free radicals and, in this way, effectively reduce the serum levels of lipid parameters such as triglycerides and cholesterol [6]. Our study suggests that thymol in the rabbit organism possesses a lipid-reducing function by this mechanism (Table 3).

AST, ALT and ALP are enzymes that significantly reflect the liver function. These parameters in our study were in the range of the reference interval. We assumed that thymol in this concentration was able to express its antioxidant properties and positively affected these parameters, although only ALP was affected significantly. The values of urea and creatinine also remained within the normal ranges. This suggests that no hepatic or renal injuries occurred in this experiment. The slight increase in the urea and creatinine amounts could imply the effect of thymol and/or its metabolites on the kidney function, particularly on glomerular filtration. Ocel'ová [30] and Kohlert et al. [32] suggested the role of the kidneys in the metabolism and elimination of phenolic compounds. They assumed that thymol metabolites could be cleaved at the brush border of the renal tubule and reabsorbed as thymol back into the peritubular capillaries. As mentioned above, thymol can accumulate in the kidneys, and based on this finding, we can hypothesise that thymol concentrations in the kidneys in our experiment could

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Author Contributions: Conceptualisation: I.P.; methodology: I.P. and K.B.; validation: I.P.; formal analysis: I.P., K.Z.-E., K.B., M.M., S.G., D.S. and Z.A.; investigation: I.P. and K.B ; resources: I.P., L.C., K.Z.-E., S.G. and D.S.; data curation: I.P., L.C. and A.L .; writing-original draft preparation: I.P. and K.B.; writing-review and editing: K.Z.-E.; visualisation: I.P. and K.B.; project administration: I.P. and funding acquisition: I.P. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by the Scientific Grant Agency of the Ministry for Education, the Science, Research and Sport of the Slovak Republic and the Slovak Academy of Sciences (VEGA 2/0069/17, 2/0009/20 and 1/0204/20), as well as the Austrian Federal Ministry for Science, Research and Economics, OeAD, Ernst Mach Grant Action Austria-Slovakia.

Acknowledgments: The authors gratefully acknowledge the technical support provided by L. Ondruska, V. Parkanyi, R. Jurcik and J. Pecho from the National Agricultural and Food Centre, Research Institute for Animal Production, Nitra. The authors also thank Andrew Billingham for improving the written English of the manuscript.

Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results.

#### References


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### Article Comparison of Rabbit, Kitten and Mammal Milk Replacer Efficiencies in Early Weaning Rabbits

Panthiphaporn Chankuang 1, Achira Linlawan 1, Kawisara Junda 1, Chittikan Kuditthalerd 1, Tuksaorn Suwanprateep 1, Attawit Kovitvadhi 2,\*, Pipatpong Chundang 2, Pornchai Sanyathitiseree 3 and Chaowaphan Yinharnmingmongkol 4


Received: 19 May 2020; Accepted: 20 June 2020; Published: 23 June 2020

Simple Summary: A milk replacer must be given as the main diet to young rabbits that are separated from their mothers before they reach weaning age (31-35 days). This procedure, which is a rescue protocol, allows them to survive. Moreover, the early separation of young rabbits before weaning prevents negative consequences in lactating rabbits, which is beneficial to pet rabbit producers. Kitten (KMR®, Pet-Ag Inc., Hampshire, IL, USA: KMR) or mammal (Zoologic® Milk matrix 30/52, Pet-Ag Inc., Hampshire, IL, USA: MMR) milk replacers have generally been suggested for use in rabbits; however, rabbit milk has a unique composition. Therefore, a rabbit milk replacer (RMR) was formulated in this study for comparison with these commercial products. Early weaned rabbits at 18 days of age were fed daily with RMR, KMR or MMR until 36 days after birth, while a commercial pelleted diet and water were provided at an amount exceeding the normal intake. The results indicated that it is possible to use RMR as a milk replacer for rabbits without serious adverse consequences. However, the RMR group presented a lower trend in nutrient digestibility than the other groups, although there was no statistical significant difference. Therefore, prebiotics and/or probiotics should be added to RMR formulations to improve this parameter.

Abstract: Early weaned rabbits should be fed using a milk replacer in order to survive. Therefore, a rabbit milk replacer (RMR) was developed and compared with a kitten milk replacer (KMR®) KMR) and a mammal milk replacer (Zoologic® Milk matrix 30/52: MMR). Thirty-six native crossbred rabbits aged 18 days were divided into three experimental groups (six replicates/group, two rabbits/replicate), fed RMR, KMR or MMR daily until they were 36 days old and euthanized at 38 days, while a complete pelleted diet and water were provided ad libitum. No statistically significant differences were observed in growth performance parameters, water intake, faecal weight, nutrient digestibility, internal organ weight, caecal pH, caecal cellulose activity, number of faecal pellets and amount of crude protein intake (v > 0.05). Caecal amylase activity in the KMR group and caecal protease activity in the RMR group were higher than in the MMR group (p < 0.05). The villus height and crypt depth of the MMR group were greater than in the RMR and KMR group (p < 0.05). In conclusion, it is possible to feed RMR to early weaning rabbits without serious adverse effects. However, probiotics and/or prebiotics should be supplemented in milk replacers and their benefits studied.

Keywords: digestibility; enzyme activity; gut histology; milk replacer; rabbit

#### 1. Introduction

The size of the pet market has increased sharply in recent years and was estimated to be around 131.7 billion US dollars in 2016 [1]. Moreover, the compounded annual growth rate of the global pet care market was forecast to be 4.9% between 2018 and 2025 due to changes in the new generation's lifestyle, such as living alone or child-free marriage [1]. Nevertheless, humans still need interactions with living things, which add social, medical, emotional and physical benefits to their lives [2]. Companion animals are one solution that can offer these benefits [2]. Although rabbits are not as popular as dogs and cats, they occupy third place among companion animals because they are clean, quiet, non-harmful and require little space [3].

Generally, rabbits are weaned at 31–35 days of age by rabbit producers in Thailand and other countries [4,5] and begin to consume pelleted diets around 18 days of age [6]. Milk replacer has been suggested as a means of feeding early weaned rabbits and orphaned rabbits and of solving the problem of female rabbits that do not produce milk, this being rabbits' major nutrient source for survival [7] as well as preventing gastrointestinal disease [8]. The early separation of young rabbits from their mother can prevent a negative energy balance due to lactation, which supports a higher production yield and reduces disease transmission from the mother to young rabbits, of benefit for pet rabbit producers [4,9]. In addition, the smallest rabbit breed (Netherland Dwarf) can produce around 4-6 kittens per litter; however, the milk yield of this breed is not sufficient to support their kittens, leading to a high mortality rate among young rabbits [5]. Furthermore, milk replacer can be used to rescue unweaned wild rabbits [8]. Therefore, the use of a milk replacer provides one solution to these problems.

Commercial rabbit milk replacer remains lacking or unavailable in some countries. For this reason, kitten milk replacer (KMR) has been suggested as a substitute [7]. However, although kitten milk replacer can be used in early weaning rabbits, its growth performance has been found to be inferior to rabbit milk for rabbits [8]. In addition, two milk replacer formulas using mixtures of kitten milk replacers-Fox Valley Ultraboost and/or Fox Valley 32/40 (Fox Valley Animal Nutrition, INC., Lakemoor, IL, USA)—have been used to rescue young desert cottontail (Sylvilagus audubonii) and eastern cottontail rabbits (S. floridanus), for which the mortality rate was 26-59% [8]. The high concentration of nutrients (fat, protein and energy), the near absence of lactose, the high proportion of medium-chain saturated fatty acids with bacteriostatic properties (C8:0 and C10:0) and the short milking period required constitute the unique characteristics of rabbit milk and feeding behaviour [6,10]. Therefore, milk replacer for rabbits should be formulated respecting the properties of real rabbit milk [10]. Moreover, cost-effectiveness is another problem for rabbit producers, owners and wildlife rescue center [8]. Therefore, this study aimed to compare the efficiency of a developed rabbit milk replacer with two commercial products (kitten and mammal milk replacers) based on the growth performance and health status of early weaning rabbits (18 days old).

#### 2. Materials and Methods

#### 2.1. Ethics Statement

This study was conducted following standard guidelines at the animal experimental unit, Faculty of Veterinary Medicine (Kasetsart University, Bangkok, Thailand) and was approved by the Institutional Animal Care and Use Committee of Kasetsart University, Bangkok, Thailand (ACKU62-VET-037).

#### 2.2. Animals, Diets, Milk Replacer Preparation and Experimental Design

Thirty-six 18-day-old native crossbreed rabbits with initial body weights of 134 ± 6.31 g/head (mean ± standard deviation) were taken from a local rabbit farm (Saha farm, Kanchanaburi, Thailand). Rabbits were randomly separated into three experimental groups with equal numbers of each sex (six replicates per group and two rabbits per replicate) containing: (1) rabbits fed rabbit milk replacer, which was formulated in this study (RMR); (2) rabbits fed kitten milk replacer (KMR®; Pet-Ag Inc., Hampshire, IL, USA); and (3) rabbits fed mammal milk replacer (Zoologic® Milk matrix 30/52; Pet-Ag

Inc., Hampshire, IL, USA; MMR). Rabbits were placed in a stainless cage (35 cm x 35 cm) with controlled room temperature, light and humidity at 20 ± 2 °C, 16L:8D and 75 ± 10%, respectively. The experiment was conducted for 20 days until the rabbits reached 38 days of age. At the end of the experiments, one rabbit per replicate was euthanized by intraperitoneal injection with pentobarbital sodium at 100 mg/kg (Nembutal, Ceva corporate, France) [11] and the samples were collected for further analysis, while another rabbit of each replicate was returned to the farm and reared until it reached 60 days of age.

The formulation of the RMR, including the chemical composition of the milk replacer, complete pelleted diet and rabbit milk, is illustrated in Table 1. Every day, rabbits were fed 10 mL at 38 °C of a freshly prepared mixture of milk replacer powder and clean water using a sterile syringe at 7:00 until they reached weaning age (36 days), while clean water and complete commercial rabbit diets (Lee Feed Mill, Publ. Co., Ltd., Phetchaburi, Thailand) were provided ad libitum throughout the experiment. The KMR and MMR powders were diluted with warmed water at a 7:13 ratio and homogenized. The RMR consisted of two parts: a hydrogenated palm fat part and a mixed dried powder part, containing all ingredients except hydrogenated palm fat and polyoxyethylene (80) sorbitan monooleate. For RMR preparation, hydrogenated palm fat was heated in an 800 W microwave for 60 s, changing from solid to liguid form as a result. The dried mixed powder part was then mixed with warm water. Subsequently, the two parts were homogenized and polyoxyethylene (80) sorbitan monooleate was added as an emulsifier. The ratio of mixed dried powder to hydrogenated palm fat to warm water was 24.5:17.5:78. The dilution ratio was selected based on equal dry matter content between the milk replacers and the solubility of the milk mixture.


Table 1. Ingredients and chemical components of different milk replacers, rabbit milk and diet.

RMR = Rabbit milk replacer which was performed in this study, KMR = Kitten milk replacer (KMR®, Pet-Ag Inc, Hampshire, IL, USA), MMR = Mammal milk replacer (Zoologic® Milk matrix 30/52, Pet-Ag Inc., Hampshire, IL, USA), FM = Fresh matter, DM = Dry matter, ND = Not detect; 3 A commercial pelleted diet for rabbits (Lee Feed Mill, Publ. Co., Ltd., Phetchaburi, Thailand); 9 Chemical composition of rabbit milk [10]; • Vitamin and mineral premix (Topmix-B111, Top Feed Mills Co., Ltd., Pathumthani, Thailand) were supplied per kilogram of diets at 4,800,000 UU of vitamin A; 1,200,000 IU of vitamin D3; 6000 IU of vitamin E; 600 mg of vitamin K; 600 mg of vitamin B1; 2200 mg of vitamin B2; 10,000 mg of vitamin B3; 800 mg of vitamin B12; 48 mg of vitamin B12; 48 mg of biotin; 4800 mg of Calcium pantothenate acid; 200 mg of folic acid; 24,000 mg of Zn, 16,000 mg of Fe; 32,000 mg of Mn; 32,000 mg of Cu; 200 mg of I; 40 mg of Se; 40 mg of Co; d Calculation [12]; e Calculation based on Atwater system [13].

#### 2.3. Perfomance, Digestibility and Faecal Evaluation

The animals were weighed at 18, 24, 30 and 36 days of age, whereas average daily feed intake (ADFI), average daily weight gain (ADG), feed conversion ratio (FCR), water intake and weight of facces output were evaluated at 19-24, 25-30 and 31-36 days of age. The apparent digestibility of dry matter, organic matter, ether extract and crude protein was conducted at 23-27 and 31-35 days of age and contained six replicates/groups. The procedures for feeding, faecal collection, chemical analysis and calculation were in accordance with [14]. Briefly, feed intake was measured during the period of the digestibility trial. Facces were removed from the cage at 9:00 on the first day of the digestibility trial. Subsequently, all faeces on a net under the cage were collected at 9:00 for four days. The faeces were weighted immediately after collection, put in a sterile plastic bag and kept at -20 °C for further chemical composition analysis following the procedure of [14]. Another study was conducted, where the amount of daily faecal pellet excretion was measured by counting the dried faecal pellets between 19 and 36 days old from photos.

#### 2.4. Internal Organs, Gut Histology and Caecal pH

The internal organ weight and the body weight of the euthanized rabbits were determined. The duodenal part of the small intestine was fixed in 10% buffered formalin for further villus morphometric evaluation. Briefly, small pieces of middle duodenum after fixation were processed, embedded in paraffin, sectioned at 7-um thicknesses by means of a rotary microtome (Leica RM2155; Leica Instruments GmbH, Nussloch, Germany) and stained by haematoxylin and eosin method. Villi height and crypt depth were evaluated under a microscope using an image analysis programme (Image Pro Plus; Media Cybernetics, Bethesda, MD, USA). Caecal pH was measured directly using a Crison MicropH 2001 pH meter (Crison Instruments, Barcelona, Spain). The caecal content was immediately placed in sterile plastic tubes under ice for enzyme preservation and kept at -20 °C for further analysis of caecal enzyme activity.

#### 2.5. Caecal Enzyme Activity

The crude enzyme extracted from the caecal content was extracted by homogenized caecal content with phosphate buffer solution (pH 7) at a 1:5 ratio (w/o). The homogenates were centrifuged at 18,000× g for 30 min at 4 °C to obtain the supernatant used to evaluate amylase, protease and cellulase activity. Amylase and cellulase activity were assayed according to [15,16] using 5% soluble starch and 1% carboxyl-methyl cellulose (CMC; medium viscosity) as the substrate, respectively. One hundred microlitres of crude enzyme extract were added to activate the digestion of the substrates. The products of the carbohydrate-digestive enzymes were stained using 1% dinitrosalicylic acid (DNS) and measured using a spectrophotometer at 540 nm against a linear range of maltose standards for amylase and glucose standards for cellulase. Protease activity was assayed according to the method described by [17] using 0.6% casein as the substrate. The product of the protein-digesting enzyme was measured spectrophotometrically at 660 nm against a linear range of tyrosine. The activity of the observed digestive enzymes was expressed as U.

#### 2.6. Crude Protein Assessment

Each rabbit's feed intake between 19-24, 25-30 and 31-36 days old and the amount of crude protein in the milk replacer and diet were used as information to calculate the amount of crude protein intake.

#### 2.7. Statistical Analysis

The results of this study are represented as the mean and pooled standard error of the mean. A completely randomized design was employed in this study. Therefore, one-way analysis of variance (ANOVA) was used to compare the different types of milk replacers (fixed factors) for internal organ characteristics, caecal pH, caecal digestive enzyme activities and duodenal histology, whereas the growth performances, water intake, faeces excretion, apparent digestibility, number of faecal pellets and amount of protein intake were analyzed by two-way mixed analysis of variance, with treatment groups or age serving as the between-subjects or the within-subjects factor, respectively. Duncan's multiple range test was used for post hoc analysis. Differences were considered statistically significant at p < 0.05. All statistical analyses in this study were performed with R-statistic software using the Rcmdr package [18].

#### 3. Results

The effects on performance, apparent digestibility, amount of faeces excretion and crude protein intake from rabbits fed the different milk replacers are shown in Table 2. No statistically significant differences between the groups and the interactions between the studied factors (groups and age) for all parameters in Table 2 were apparent (y > 0.05). The age increment was correlated with increased body weight, ADFI, ADG, FCR, water intake, faeces excretion and crude protein intake (p < 0.05), whereas apparent digestibility did not affect dry matter, organic matter or ether extract (v > 0.05). However, the crude protein digestibility of rabbits at 31-35 days old was lower than rabbits at 23-27 days old (v < 0.05). The rabbits fed KMR and MMR displayed higher nutrient digestibility in both age ranges compared with rabbits fed RMR, but there was no statistically significant difference (v > 0.05). Rabbits in RMR, KMR and MMR were received the crude protein from milk daily at 2.23, 2.61 and 1.62 g dry matter/head. No deaths, morbidities or clinical signs were observed in rabbits during the experimental period. Moreover, a rabbit in each replicate was not euthanized at the end of the experiment and remained alive until it reached two months of age. In addition, there were no problems of milk perception and palatability in any group in this experiment, because the rabbits sucked milk directly from the syringe without any force feeding.

Table 2. Effect of different milk replacers on rabbit performances, apparent digestibility and crude protein intake.


RMR = Rabbit milk replacer, which was used in this study, KMR = Kitten milk replacer (KMR®, Pet-Ag Inc, Hampshire, IL, USA), MMR = Mammal milk replacer (Zoologic® Milk matrix 30/52, Pet-Ag Inc., Hampshire, IL, USA), SEM = pooled standard error of mean, BW = Body weight, ADFI = Average daily feed intake, ADG = Average daily weight gain, FCR = Feed conversion ratio; 3,6, d The differences in superscript letter in the same row represented statistical significant differences (p < 0.05); 1 The rabbits in the RMR, KMR and MMR groups received crude protein from milk at 2.23, 2.61 and 1.62 g/head/day, respectively.

The consequences for internal organ weight, caecal pH, duodenal wall histology and digestive enzyme activities between the groups are compared in Table 3. The weight of each internal organ was not statistically significantly different between the groups (y > 0.05). Caecal pH was not influenced by the differences in milk replacers (v > 0.05). Caecal amylase activity in the MMR group was lower than in the KMR group (p < 0.05), whereas greater caecal protease activity was observed in the RMR group compared to the MMR group (p < 0.05). Cellulase activity was not affected by the treatments (p > 0.05). Respectively, the shortest villus and the shallowest crypt depth were found in the RMR and the KMR groups compared to the MMR group (p < 0.05).


Table 3. Effect of different milk replacers on internal organ weight, caecal pH, intestinal villi morphology and digestive enzyme activity.

RMR = Rabbit milk replacer, which was used in this study, KMR = Kitten milk replacer (KMR®, Pet-Ag Inc, Hampshire, IL, USA), MMR = Mammal milk replacer (Zoologic® Milk matrix 30/52, Pet-Ag Inc., Hampshire, IL, USA), SEM = pooled standard error of mean, BW = Body weight, FI = Feed intake, ADG = Average daily weight gain, FCR = Feed conversion ratio; 4 % The differences in superscript letter in the same row represented statistical significant differences (y < 0.05); 1 Thoracic organs includes lungs and heart; - Intestinal organs includes stomach, small intestine, large intestine, caecum and rectum with content.

The average number of faeces pellets from two rabbits of each replicate in the experiments is illustrated in Figure 1 and Table A1. The graph shows a steady increase in the number of faecal pellets with increasing age (v < 0.001); however, a sharp drop occurred in all study groups at 35 days of age, followed by another increase. The largest number of faecal pellets existed in the MMR group compared to the RMR group (p < 0.05; Appendix A Table A1), with the KMR group between them (p > 0.05). A significant interaction between the fixed factors (age and treatment group) was identified (p < 0.05). Generally, the same increasing trend was observed in all groups, except for the sharp rise in the number of faecal pellets in the MMR, RMR and KMR groups at 22-23, 24-26 and 34-36 days of age, respectively.

Figure 1. Effect of different milk replacers on number of faecal pellets (RMR, rabbit milk replacer in this study; KMR, kitten milk replacer, KMR®, Pet-Ag Inc., Hampshire, IL, USA; and MMR, mammal milk replacer, Zoologic® Milk matrix 30/52, Pet-Ag Inc., Hampshire, IL, USA).

#### 4. Discussion

The RMR was formulated according to the profile of rabbit milk; therefore, its chemical composition was the most similar to the composition of rabbit milk [10]. KMR contained a higher proportion of GVYHI TVSXIMR ERH [EW PS[IV MR JEX ERH IRIVK] XLER 616 ERH VEFFMX QMPO 3R XLI SXLIV LERH 116 [EW PS[IV MR GVYHI TVSXIMR ERH LMKLIV MR JEX ERH IRIVK] XLER 616 ERH VEFFMX QMPO % LMKL HIRWMX] SJ RYXVMIRXW ERH IRIVK] [EW XLI YRMUYI GLEVEGXIVMWXMG SJ VEFFMX QMPO [LMGL GSRXEMRIH VIWTIGXMZIP] EVSYRH JSYV ERH XLVII XMQIW LMKLIV TVSTSVXMSRW SJ TVSXIMR ERH PMTMHW XLER GS[W QMPO ?A % WLSVX QMPOMRK XMQI MW E GSQQSR RYVWMRK FILEZMSYV I\TPEMRMRK XLI LMKL HIRWMX] SJ VEFFMX QMPO ?A 8LI EQSYRX SJ QMPO VITPEGIV JIH XS ]SYRK VEFFMXW [EW GEPGYPEXIH SR XLI FEWMW SJ WXSQEGL GETEGMX] 1MPO VITPEGIV MR TS[HIV JSVQ [EW YWIH JSV EPP JSVQYPEXMSRW MR XLMW WXYH] FIGEYWI E LMKLP] GSRGIRXVEXIH QMPO QM\XYVI GER FI JSVQYPEXIH JVSQ HVMIH TS[HIV FYX RSX MR PMUYMH JSVQ ?A 6EFFMX QMPO TVSXIMR GSQTVMWIW EVSYRH ERH GEWIMR ERH [LI] TVSXIMR VIWTIGXMZIP] ?A 8LIVIJSVI GEWIMR WIVZIH EW E QENSV TVSXIMR MRKVIHMIRX MR XLI QMPO VITPEGIV JSVQYPEXMSR JSV 616 ERH XLI X[S SXLIV GSQQIVGMEP QMPO VITPEGIVW [LIVIEW HVMIH WOMQQIH QMPO TS[HIV VITVIWIRXIH ERSXLIV TVSXIMR WSYVGI [LMGL [EW YWIH MR E PS[IV TVSTSVXMSR XLER GEWIMR 6IWTIGXMZIP] IMXLIV [LI] SV QMPO TVSXIMR GSRGIRXVEXI [EW WYTTPIQIRXIH MR /16 ERH 116 [LIVIEW MR 616 XLI] [IVI RSX % PS[ PIZIP SJ PEGXSWI MW TVIWIRX MR VEFFMX QMPO XLIVIJSVI GS[W SV KSEXW QMPO MW PMQMXIH EW E QMPO VITPEGIV JSVQYPEXMSR ?A 1SVISZIV PEGXEWI EGXMZMX] MR VEFFMXW HIGVIEWIW [MXL EKI ERH HSIW RSX VIWTSRH XS XLI PEGXSWI GSRGIRXVEXMSR MR XLI HMIX 8LIVIJSVI I\GIWWMZI PEGXSWI MRXEOI GER PIEH XS E HMKIWXMZI HMWSVHIV ?A (M IVIRGIW MR XLI X]TI SJ VE[ TVSXIMR [EW RSX IZEPYEXIH MR XLMW WXYH] XLI MRXEOI SJ EQMRS EGMHW [SYPH LEZI FIIR WY GMIRX JSV VEFFMXW FIGEYWI GEWIMR GSRWMHIVIH ER MHIEP TVSXIMR [EW YWIH EW XLI QEMR MRKVIHMIRX ERH XLI TIVGIRXEKI SJ GVYHI TVSXIMR MR EPP QMPO VITPEGIVW [EW LMKLIV XLER RYXVMIRX VIUYMVIQIRXW ?A

\*EX MR VEFFMX QMPO VITVIWIRXW XLI QENSV IRIVK] WSYVGIW JSV VEFFMXW ?A [LIVIEW I\GIWWMZI WXEVGL MRXEOI TVSQSXIW HMKIWXMZI TVSFPIQW ERH MRGVIEWIW XLI QSVXEPMX] VEXI SJ ]SYRK VEFFMXW ?A 8LI LMKLIWX RMXVSKIRJVII I\XVEGX [EW TVIWIRX MR XLI /16 JSVQYPEXMSR LS[IZIV RS EHZIVWI I IGXW [IVI SFWIVZIH MR XLMW KVSYT 1IHMYQGLEMR JEXX] EGMHW QEMRP] GETV]PMG '
 ERH GETVMG EGMH '
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z SJ HEMP] IRIVK] VIUYMVIQIRXW 8LI GEIGEP IRZMVSRQIRX KVIEXP] E IGXW XLI QMGVSFMEP GSQQYRMX] MR VEFFMXW IWTIGMEPP] MR XIVQW SJ RYXVMIRX JIVQIRXEXMSR I GMIRG] GER VIWYPX MR E LMKLIV ZEPYI SJ GEIGEP T, MRGVIEWMRK XLI VMWO SJ E HMKIWXMZI HMWSVHIV ?A 8LI GEIGEP T, SJ XLI 616 KVSYT WIIQIH XS FI LMKLIV XLER XLEX SJ XLI SXLIV KVSYTW ,S[IZIV XLI GEIGEP T, SJ XLI 616 KVSYT [EW PS[IV XLER MRWY GMIRX XS TVSQSXI E KYX LIEPXL TVSFPIQ ? IVIRGI MR GEIGEP T, KVS[XL TIVJSVQERGI HMKIWXMFMPMX] QSVFMHMX] ERH QSVXEPMX] HYVMRK XLI I\TIVMQIRXW WYTTSVXMRK XLI TSWWMFMPMX] SJ YWMRK 616 MR VEFFMXW

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 MR XLI /16 JSVQYPEXMSR GSRXVMFYXIH XS LMKLIV IR^]QI EGXMZMX] ERSXLIV VIWIEVGL WXYH] ?A % LMKLIV WYVZMZEP VEXI [EW SFWIVZIH MR XLI QMPO VITPEGIV [MXL TVSFMSXMGW ERH TVIFMSXMGW  
 GSQTEVIH XS [MXLSYX XLIWI EHHMXMSRW  
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 QE] VITVIWIRX XLI FIWX TVSGIHYVI XS EGLMIZI KSSH HIZIPSTQIRX SJ XLI QMGVSFMEP GSQQYRMX] MR IEVP] [IERIH VEFFMXW ,S[IZIV E QMGVSFMEP

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energy density, which bacteria can use as energy sources to produce butyrate. Intestinal epithelial cells can utilize such short-chain fatty acids as an energy source, promoting proliferation, differentiation and gut immunity [21]. Thereby, the greatest villus height and villus crypt depth were seen in the MMR group, enabling better digestion and absorption and providing higher nutrient digestibility RMR as a result [22]. Furthermore, the amount and characteristics of hard faeces can be used to indicate digestive health [7]. The total weight of faecal excretion did not differ between groups, but the largest number of faecal pellets was found in MMR, followed by KMR and RMR. Moreover, no soft faeces were found under the rabbit cage, indicating that crude protein intake did not exceed their requirement [19].

Lower growth performance and survival rates were reported in the group fed with KMR compared with the group fed with rabbit milk from lactating does |8]. Unfortunately, this study did not compare the milk replacers with rabbit milk. Early separation at 14 days of age may have been the cause of the negative consequences found in the study of [8], whereas separation at 18 days of age did not result in any serious adverse outcomes here. In addition, a high mortality rate was observed in the study of [8] because the rabbits were too young, were injured and were experiencing high levels of stress due to being wild rabbits.

#### 5. Conclusions

Differences in efficiency between the RMR developed in this study and commercial milk replacers (KMR and MMR) used in 18-day-old rabbits were revealed in the current study. Based on the results, it was possible to use RMR as a milk replacer for 18-day-old rabbits and to wean at 36 days of age, this not providing any adverse consequences for final body weight, ADG, FCR, ADFI, nutrient digestibility, internal organ characteristics, caecal pH, amount of faeces excretion and crude protein intake. Lower nutrient digestibility was observed in the RMR group without statistically significant differences. Therefore, probiotics and/or prebiotics can be supplemented in formulations to promote a suitable microbial community and to provide benefits in terms of growth performance and nutrient digestibility.

Author Contributions: Conceptualization, A.K.; investigation, P.C. (Panthiphaporn Chankuang), A.L., K.J., C.K., T.S., P.C. (Pipatpong Chundang) and A.K.; resources, P.S. and C. Y.; data curation, A.K.; writing-original draft preparation, A.K.; writing-review and editing, P.C. (Panthiphaporn Chankuang), A.L., K.J., C.K., T.S., P.C. (Pipatpong Chundang), A.K., P.S. and C.Y.; project administration, A.K.; funding acquisition, A.K. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by Student Development Fund, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand.

Acknowledgments: The authors would like to thank Faculty of Veterinary Medicine, Veterinary Teaching Hospital Kamphaengsaen campus (Kasetsart University, Nakhon Pathom, Thailand) and Mr.Saha Bairak (Saha farm, Kanchanaburi, Thailand) for providing the rabbit milk replacer and rabbits, respectively. Moreover, we would like to acknowledge Thanaporn Sriprathardtrakul, Jiraphat Wangka and Sirapoom Narktap for the support in rabbit rearing and feeding.

Conflicts of Interest: The authors declare no conflict of interest.

#### Appendix A

Table A1. Effect of different milk replacers on number of faecal pellets.



Table A1. Cont.

RMR = Rabbit milk replacer in this study, KMR = Kitten milk replacer (KMR®; Pet-Ag Inc., Hampshire, IL, USA), ithir = Nammal milk replace (Zoologic® Milk matrix 3052; Pet-Ag Inc., Hampshire, II, Ord,
MMR = Mammal milk replace (Zoologic® Mille martix 3052; Pet-Ag Inc., Hampshire, II,

#### References


Animals 2020, 10, 1087

22. Makovicky, P.; Tumova, E.; Volek, Z.; Makovicky, P.; Vodicka, P. Histological aspects of the small intestine under variable feed restriction: The effects of short and intense restriction on a growing rabbit model. Exp. Med. 2014, 8, 1623–1627. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution ー(CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## Review Autochtonous Strain Enterococcus faecium EF2019(CCM7420), Its Bacteriocin and Their Beneficial Effects in Broiler Rabbits-A Review

#### Monika Pogány Simonová 17\*, Ľubica Chrastinová 2 and Andrea Lauková 1×


Received: 22 June 2020; Accepted: 10 July 2020; Published: 14 July 2020

Simple Summary: Weaning is the most important and critical period in rabbits breeding; the cecal digestion is very complex and only small dietary and/or environmental changes can disturb the stable microbial population/fermentation and gut health, leading to digestive dysbiosis and increased morbidity, often with fatal outcome and big economic losses. Control of the microbiota, prevention of digestive disturbances and improving gut health and immunity can be achieved through the natural substances application in rabbit nutrition. While probiotics are frequently used in rabbit farms, the in vivo administration of bacteriocins (antimicrobial substances produced by bacteria, which usually also possess probiotic properties) in these animals is often limited and has become an area of research activity. Moreover, the most of probiotic strains used in rabbits are non-autochthonous (have a different origin than the rabbits ecosystem). Therefore, our study focused on improving rabbits' health using the autochthonous strain Enterococcus faecium EF2019 (CCM7420) and its enterocin (Ent7420) in broiler rabbits. The antibacterial and anticoccidial effect of additives was observed, with good colonization ability of the CCM7420 strain. Both additives showed a tendency to modulate the serum biochemistry parameters and to improve the immunity, jejunal morphology, weight gains, feed conversion ratio and meat quality (physicochemical traits and mineral content).

Abstract: The present review evaluates and compares the effects achieved after application of rabbit-derived bacteriocing strain Enterococcus faccium CCM7420 with probiotic properties and its bacteriocin Ent7420. The experiments included varying duration of application (14 and 21 days), form of application (fresh culture and lyophilized form), combination with herbal extract and application of the partially purified enterocin-Ent7420, produced by this strain. Results from these studies showed that E. faccium CCM7420 strain was able to colonize the gastrointestinal tract (caecum) of rabbits (in the range < 1.0-6.7 log cycle, respectively 3.66 log cycle on average), to change the composition of intestinal microbiota (increased lactic acid bacteria, reduced counts of coliforms, clostridia and staphylococci), to modulate the immunity (significant increase of phagocytic activity), morphometry (enlargement absorption surface in jejunum, higher villi height:crypt depth (VH:CD) ratio), physiological (serum biochemistry; altered total proteins, glucose and triglycerides levels) and parasitological (Eimeria sp. oocysts) parameters and to improve weight gains (in the range 4.8-22.0%, respectively 11.2% on average), feed conversion ratio and meat quality (physicochemical traits and mineral content).

Keywords: rabbit; Enterococcus faecium; enterocin; microbiota; intestinal morphology; phagocytic activity; serum biochemistry; meat quality; weight gain

#### 1. Introduction

Rabbit breeding has a great potential because of the small body size, short generation interval, rapid growth rate, high productive capacity and healthy, easily digestible meat of rabbits [1,2]. Moreover, rabbits can convert a higher amount (20%) of the protein they eat into edible meat, compared with pigs (16-18%) and cattle (8-12%; [3]). In several European countries, in which the rabbit breeding has a long history and high production efficiency, presently is regressing, whereas in the developing countries of the world rabbit farming has become to an important emerging enterprise. In growing rabbits, the most critical period is the weaning, when the kits are separated from mother and the milk is substituted with solid feed [4]. During these environmental and physiological changes/stresses, the rabbits are very sensitive to digestive disturbances, also called non specific enteritis (usually caused by dietary stresses, parasites-Coccidia and bacteria-Clostridia sp. and enteropathogenic Escherichia coli) and gastrointestinal infections (epizootic rabbit enteropathy-ERE, a multifactorial gastrointestinal syndrome, [5]). These dietary and bacterial changes are the main reason of morbidity and mortality and have negative effects on feed consumption, growth performance and health status of animals in this period and also on the economic aspects of rabbit farming.

To overcome this period, to reduce economic losses and to improve and stabilize the health status and gastrointestinal tract development, antibiotic growth promoters (AGPs) have been widely used for years. Although, these synthetic drugs showed good effects on production indicators, on the other hand, there was a risk of increasing resistance to antibiotics and transferring of antibiotic resistance genes from animal to human, which also threatened the human health and quality of meat and food [6]. For this reason, AGPs have been banned by the European Union (began in 1986 in Sweden and completely banned in January 2006, when the last four antibiotics have been permitted as feed additives was no longer allowed to be marketed or used from this date; IP/03/1058; [7,8]). As a result of the ban, researchers had to substitute AGPs and to find new feed additives that were supposed to be safer, without leaving residues and spreading resistance to themselves, but also improving health and productivity of rabbits. Therefore, the antibiotics have been replaced with new, naturally based supplements: probiotics, prebiotics, synbiotics, enzymes, bacteriocins, organic acids, herbs and their extracts, which are well-tried tools for disease prevention and therapy in various animal species, including rabbits [9].

The last two decades have seen a substantial increase in the use natural supplements and/or additives in animal nutrition, in which their antimicrobial activity has been highlighted many times. The EU in its Regulation EC 1831/2003 defined the terms "feed additives" as "substances, microorganisms or preparations, other than feed material and premixtures, which are intentionally added to feed or water" and the "antimicrobials" as "substances produced either synthetically or naturally, used to kill or inhibit the growth of microorganisms, including bacteria, viruses or fungi, or of parasites, in particular protozoa" [10]. According to the World Health Organization (WHO) probiotics are defined as "live microorganisms which, when administered in adequate amount, confer a health benefit on the host". Although this definition is widely accepted, a 2007 guidance document from the European Commission on Regulation EC 1924/2006 on nutrition and health claims (NHCR) categorizes the term 'probiotic' as a health claim on the basis that it implies a health benefit. For this reason, the term "beneficial microbes" is used more often instead of a "probiotic microorganism" [1].

The use of several natural feed additives has already been reviewed in rabbit breeding [10,12–15]. The positive effects of probiotics and their antibacterial products-bacteriocins on health, growth performance, nutrient utilization and metabolism changes, microbial composition [14,16-35], blood serum biochemistry, oxidative stress, immune response, intestinal morphology [21,24,28-31,34,36-38] and meat quality of rabbits [32,34,39-43] was described. However, in spite of the achieved results, there are still few declared probiotic preparations based (Lactina, Toyocerin; [44,45]; Prorabit-declared in Slovakia; [25]) and detailed studies of microorganisms with beneficial properties that have also the ability to produce antimicrobial substances, enterocins.

The aim of this review was to summarize all achieved properties and physiological effects of the bacteriocin-producing strain with probiotic properties Enterococcus faccium CCM7420 (EF2019 previous working labeling, [46]) isolated in 2003 from rabbit feces in the Laboratory of Animal Microbiology of the Institute of Animal Physiology, Centre of Biosciences of the Slovak Academy of Sciences (Košice, Slovakia) and tested to date in 180 rabbits. These experiments included varying duration of application (2 and 3 weeks), form of application (fresh culture in water; the concentration of cells was ×10° CFU/mL in a dose 500 µL/animal/day; lyophilized (freeze-dried) form rehydrated in water (x10) CFU/mL; dose 500 µL/animal/day) as well as mixed in feed and pelleted (15 g/100 kg feed), application of its partially purified bacteriocin (PPB)-enterocin (Ent) EF2019 (applied into water) and fresh culture in combination with natural substance (Eleutherococcus senticosus).

#### 2. Enterococcus faecium CCM7420 (EF2019) and Its Bacteriocin-Enterocin (Ent7420)

Enterococcus facium EF2019 (CCM7420) is a bacteriocin-producing strain [47], which was isolated from the rabbit feces and genetically confirmed by the PCR method and subsequently by MALDI-TOF mass spectrophotometry as well as the sequencing procedure of this strain was provided (Dr. Kopćáková, IAP CBs SAS). This strain produces lactic acid, tolerates low pH (3.0; 63% surviving of cells) and is able to grow even in 5% oxgall-bile (80% surviving of cells), shows sensitivity to antibiotics, including vancomycin [25,48] and possess lipolytic activity [49]. Other unpublished data suggests that the CCM7420 does not produce biogenic amines and enzymes such as ß-glucuronidase, ß-galactosidase or N-acetyl-B-glucosaminidase (enzymes produced by unfriendly gut bacteria; their increased levels are usually the indicators of colon cancer), and it does not show any gelatinase (absence of the gelE gene) or hemolytic activities with low ability to form biofilm (0.092). The strain was deponed into Czech Collection of Microorganisms in Brno, Czech Republic to have number CCM7420. This strain showed the broadest inhibitory activity from all tested rabbits enterococcal strains against the indicators E. avium EA5, Listeria innocua LMG13568 and L. monocytogenes CCM4699 and against other tested enterococci and staphylococci tested such as clostridia, pseudomonads, enterobacteria and coliform bacteria [48]. The presence of the structural genes for enterocins (ent) A, P and L50B was detected; however, the CCM7420 did not possessed gene for ent B [47]. The molecular mass of its bacteriocin-like substance ranged from 3 to 10 kDa. Proteinaceous substance produced by CCM7420 strain was partially purified (partially purified bacteriocin (Ent) 2019 =7420). It is thermostable substance as well as stable at pH 4.0, 7.0 and 9.0. Its production starts in early logarithmic growth phase and it culminates in the late logarithmic phase of CCM7420 strain growth. By its properties, it can probably be included in the II. classification group of bacteriocins. Ent2019 or Ent7420 added to the growing strain L. innocua LMG13568 (after 4 h) inhibited its growth already at 1 h after enterocin addition with a difference of 1.5 log cycles (5 h of cultivation). This effect was prolonged up to 24 h. The Ent7420 was tested against more than 300 strains of enterococci, staphylococci, clostridia, pseudomonads, enterobacteria and coliforms [48]. The inhibitory activity of this substance was preserved after 24 months of storage at -20 °C (6400 AU/mL; [48]) and also after lyophilization (freeze dried) and redissolution in PBS buffer (25600 AU/mL; not published data). The CCM7420 strain is currently available in the ProRabbit, probiotic product for rabbits and other rodents, made by the International Probiotic Company Košice (Slovakia).

#### 3. Application Effects of E. faecium CCM7420 and Its Enterocin Ent7420 Observed in Experiments

#### 3.1. Effect on Growth Performance

The results from all these experiments indicated that the CCM7420 strain could improve the average daily weight gain (ADWG; between 4.8 and 22.0%; Table 1) regardless of the form of bacteriocin-producing strain (fresh, p < 0.001 compared to control data; or freeze dried-lyophilized-numerical increase) and its application time (2 or 3 weeks). The feed conversion ratio (FCR) was influenced only through the Ent7420 administration and the combinative application of the CCM7420 strain with Eleutherococcus senticosus extract. The effect of probiotics, including registered probiotic preparations and new beneficial microorganisms on the growth performance of rabbits have been already described/reviewed; they usually confirmed the increased body weight [14,15,17,19,24,25,31,32]. The most of these studies described faster growth and higher weight gain of rabbits using probiotic preparations and feed additives based on the following bacterial strains and yeasts alone or in their combinations: Saccharomyces cerevisiae, S. boulardii, Bacillus licheniformis, B. cereus var. toyoi, Pediococcus acidilactici, Lactococcus lactis, Lactobacillus acidophilus, L. plantarum, L. helveticus, L. delbrueckii and L. sporogenes [14]. Up to now, only several commercial products recommended especially for rabbits contain especies strain Enterococcus facium as a component of the probiotic bacterial mix (Lactina-E. faccium NBIMCC 8270 [44]; Prorabbit—E. facium CCM7420 [25]) or for companion animals with diarrhea (Pro-enteric Triplex-E. faccium DSM 10663/NCIMB 10415 [50]). In all experiments, higher ADWG was noted during CCM7420 administration. Surprisingly, the highest increase of ADWG (by 22%, Table 1) was noted after 2 weeks addition (model experiment, only seven animals were used in the group). The three weeks long CCM7420 dietary inclusion also improved the growth performance; however, only by 6.7% on average (from results of fresh culture application in two experiments; [25,26]). Similarly to our results, Lauková et al. [21] and Szabóová et al. [35] also reported higher ADWG through bacteriocinogenic and probiotic E. faccium AL41 (CCM8558) and CCM4231 strains application in rabbits. Improved body weight in rabbits was also noted after probiotics administration by Bovera et al. [30], Bhatt et al. [32] and Kalma et al. [51]. On the other hand, after PPB CCM7420 addition increased the body weight only slightly (by 2.2%, Table 2), but in this group, better FCR was achieved, compared to strain application. The bacteriocin dietary inclusion showed better feed conversion also in the case of other bacteriocins: EntM, EntCCM4231, EntEF55 and gallidermin [21,52-54], applied in rabbit husbandry.


Table 1. The effect of Enterococcus faecium CCM7420 and its enterocin Ent7420 on the growth performance of rabbits.

The fresh culture of the CCM7420 strain was applied into water (at concentration of cells x10° CFU/mL; dose 500 uL/animal/day); lyophilized (freeze-dried) form rehydrated in water (x10° CFU/mL; dose 500 uL/animal/day) as well as mixed in feed and pelleted (15 g/100 kg feed).

Table 2. The effect of Enterococcus facium CCM7420 and its enterocin Ent7420 on the fecal microbiota of rabbits.


The fresh culture of the CCM7420 strain was applied into water (at concentration of cells x10° CFU/mL; dose 500 µL/animal/day); lyophilized (freeze-dried) form rehydrated in water (x10° CFU/mL; dose 500 µL/animal/day) as well as mixed in feed and pelleted (15 g/100 kg feed). Statistical analysis was performed using one-way analysis of variance (ANOVA) with the post hoc Tukey test with the level of significance set at (p < 0.05), within experimental groups during each individual experiment.

#### 3.2. Effect on Fecal Microbiota

In all experiments, administration of E. faccium CCM7420 was associated with increased enterococci (v < 0.01) and lactic acid bacteria (LAB) counts during the treatment period by a 1.3 log cycle on average (Table 2), while the application form or time length had no impact on the bacterial counts increase. The numerical increase of enterococci and LAB was recorded also in previous experiments with non-autochthonous E. faecium probiotic strains inclusion in rabbits [21,29,56]. Other authors also noted abundance of microflora in caecum and higher lactobacilli counts after Lactobacillus strains application [57]. Outgoing from results of several studies focusing on the molecular profiling of rabbits gut [58-60] and colonization ability of applied enterococcal probiotic strains [33], enterococci were found as predominant microbiota from the phyla Firmicutes and they were able also to colonize the caecum and the small intestine (ileum and jejunum) in sufficient counts. The microbiological examinations in fecal samples confirmed the presence of E. faccium CCM7420 strain. This strain was able to colonize the digestive tract of rabbits, reaching counts in the range 2.8-6.7 log cycle during the 2 or 3 weeks treatment. These numbers are comparable with the level of autochthonous probiotic strains of several Enteroccus spp. after their application in rabbits [35]. Obviously, decreased CCM7420 counts (1.0-3.3 log cycle) were noted in the post-treatment period (3 weeks after strain cessation), but it was still able to persist in the rabbit's intestine. However, the lowest counts in fecal samples were achieved during the experimental application of the lyophilized CCM7420 strain [60], this level is comparable to that one achieved through fresh culture addition of non-autochthonous, bacteriocinogenic and the probiotic E. faccium AL41 (CCM8558) strain in rabbits [21]. Important results concerning the spoilage microbiota, including clostridia, coliforms and staphylococci were described in our experiments. Significant reduction of coagulase-positive staphylococci was noted in fecal samples of rabbits, administering fresh culture of CCM7420 (v < 0.01; [24]); the S. aureus counts were also reduced by 0.5-1.2 log; p < 0.001 [25,26]. The CCM7420 strain seems to be useful in rabbits suffering from diarrhea disturbances involving coliforms (reduced by 1.6 log; p < 0.001 [26]) and/or clostridia (reduced by 0.5-1.5 log; p < 0.05 [25,26]). Similarly to our results, decreased counts of coliforms, S. aureus and clostridia were presented in probiotic-treated rabbits [21,23,29]. In rabbits administering the enterocin Ent7420, reduction in coliforms, coagulase-positive staphylococci, including S. aureus

was noted [25]. Our results are in accordance to those presented by Lauková et al. [21], who tested the effect of nisin on the rabbits gut microbiota and noted reduced counts of the most bacterial species. The gut microbiota is an important constituent in the intestine's mucosal barrier; the increase of the host defense had been already demonstrated by the application of potentially beneficial microorganisms and other natural antimicrobials (bacteriocins, organic acids and plant extracts; [61]).

Changes in bacterial composition-decrease of fecal coliforms, Pseudomonas-like sp., Clostridium-like sp. and S. aureus-during the additives supplementation confirm the antibacterial effect of CCM7420 strain. The inhibitory effect of the bacteriocin-producing and probiotic enterococci and their enterocins on rabbits' intestinal microbiota was already reported in our previous studies [24,25,28,29,52]. Kritas et al. [23] also described the lower frequency of E. coli and C. perfringens in rabbits treated by probiotic. The dominancy in antimicrobial activity of CCM7420 strain combined with E. senticosus can be confirmed by the fact that the E. senticosus extract possesses slight or no antimicrobial activity [26]. We supposed that the dietary modulation of the gastrointestinal microbiota by natural antimicrobial substances could result in an enhancement of colonization resistance against potentially pathogenic bacteria.

#### 3.3. Effect on Eimeria sp. Oocysts

Coccidiosis is one of the most frequent and prevalent parasitic diseases in rabbit farms. The most markedly and typical symptoms are weight loss, diarrhea (from mild intermittent to severe) with the presence of blood and/or mucus in feces, which leads, through dehydration, to the mortality of animals. The high morbidity and mortality rates among all ages, especially in the young rabbits may be responsible for important economic losses [62]. The oocysts are always present in the intestines of rabbits and they cannot be completely eliminated even by the use of coccidiostat because of the caecotrophy and the symptomless, but potential source of infections of adults. These oocysts are able to cause not only pure eimeriosis after their multiplication and massive infection, but they may also be the cause of multifactorial diseases, when associated with other bacterial or viral infections. Eimeria infections can cause severe disease depending on Eimeria species, especially in young animals and the highest incidence of oocysts was usually found around the weaning period [63]. The EU has banned the use of antibiotics as feed additives for growth promotion in animals since 2005 [64]. Today, these antibiotics are replaced with alternative anticoccidials, including prebiotics, based on their bactericidal and/or bacteriostatic activities, with immunostimulation and improved growth performance and productivity of the host organism. The experimental application of the CCM7420 strain and its Ent7420 was associated with the reduction of fecal Eimeria sp. oocysts. The different treatment period had no impact on oocysts reduction. When the fresh culture of the CCM7420 strain at concentration x109 CFU/mL/g was applied only for 2 weeks to compare its effect with the commonly used probiotic strain Lactobacillus rhamnosus GG, the decrease in oocysts counts was observed after probiotic application (8.3 × 10+ OPG; oocysts per 1 g of feces) compared to control data (1.5 × 104 OPG), but also compared to the initial counts in experimental group (7.5 × 10° OPG). This reduction effect was maintained until the end of the experiment, also after cessation of the CCM7420 strain. Moreover, at the end of the experiment (at day 42), the difference one order of magnitude in Eimeria sp. oocysts was found comparing the control (1.5 x 104 OPG) and the experimental groups (7.2 × 103 OPG; [26]). On the other hand, when CCM7420 was applied with its bacteriocin Ent7420 in rabbits through 3 weeks, after 1 week of their addition, oocysts showed a trend towards a numerical reduction (not significant; [25]) in both experimental groups compared to the control group (Table 1). Surprisingly, at the end of CCM7420 strain addition, increased oocyst counts was observed in this group, while Ent7420 administration was more effective due to a significant reduction in oocysts (v < 0.05) at the end of probiotic application (day 21). This finding could be explained by the irregular excretion of oocysts. In the group with Ent7420 addition, a decreasing tendency of oocysts occurrence (not significant) was observed up to the end of enterocin substance application. This could lead to

consideration that longer application of CCM7420 strain did not influence more the Eimeria sp. oocysts counts in rabbits.

While probiotics are widely used in animals because they improve the growth performance, productivity, health status and stimulate the immunity, studies concerning the protective effect against Eimeria sp. are still limited and focused on mainly the avian coccidiosis [65,66]. In rabbits, natural alternatives—prebiotics and herbal extracts—to coccidiostats have been studied [52,67-69]. To the best of our knowledge, only several works demonstrated the anticoccidial effect of beneficial microbes and/or probiotics as well as their antimicrobial products bacteriocins in rabbits [24,25,28,52,53,55,70]. The in vitro effect of four probiotic/bacteriocin-producing strains towards poultry Eimeria sp. oocysts was also documented by Strompfová et al. [71] and no differences in the reductive effect of bacteriocin-producing and non-producing strains (p < 0.05) were found in this experiment. The in vivo administration of bacteriocin-producing and probiotic strains decreased Eimeria sp. oocysts in rabbits. Outgoing from these results, we supposed that anticoccidial effect could be done due to the lactic acid production or by the effect of bacteriocins produced by the mentioned strains. Moreover, the potential protective effect of the CCM7420 strain against zoonotic Trichinella spiralis infection was also investigated in the framework of a new therapeutic strategy aimed at using probiotics to control parasitic zoonoses [72], when the authors demonstrated the reduced intensity of T. spiralis infection and female fecundity ex vivo and in vitro (about 60%) throughout the CCM7420 administration.

#### 3.4. Effect on Serum Biochemistry

The measurement of biochemical parameters is data mostly used for diagnostic investigations and presents a useful way for controlling the health of animals. However, in some cases, several "components" of the host biochemistry are less specific because of the reparation ability of healthy tissues/organs and/or metabolic processes.

The tested serum parameters were in the range of normal values defined for these parameters in previous studies with rabbits [73-75], although there are differences in physiological or reference ranges in rabbit serum. During the E. faecium CCM7420 application, increased (even though not significantly) concentration of the total protein (TP) was noted in most experiments, and remained stable or higher also three weeks after the strain ceasing (cessation). The highest increase in TP was measured through the fresh CCM7420 culture administration in rabbits (Table 3) [24-26], while the lyophilized (freeze dried form resolved in water and mixed in pellets did not affect the TP level [60]. Application of enterocin Ent7420 as well as the combinative application of CCM7420 with the E. senticosus extract also improved the TP concentration [25,26]; similarly as was reported by Fathi et al. [33] and Kalma et al. [51] who observed a slight increase in serum TP after probiotic supplementation in rabbits. The increased level of TP could be explained by better resorption and utilization from the gut; this finding could be also confirmed by higher ADWG. On the other hand, application of non-autochthonous probiotic strains E. faccium CCM4231 and AL41 as well as their enterocins did not affect the TP in blood serum [21,29,38]. Blood glucose is an important source of energy for many cells and this is a parameter of the balance between glucose source/availability and utilization. Similarly to TP, higher glucose content was observed during fresh (both 2 and 3 weeks) and lyophilized CCM7420 culture mixed in feed as well as during Ent7420 application. The increased glucose level can be explained by conversion of lactic acid to pyruvate through the gluconeogenesis in the liver. Oppositely, reduced glucose level was observed in the case of the rehydrated-lyophilized CCM7420 strain and its combination with E. senticosus (p < 0.01). It could be that the glucose concentration was reduced by increased H+ concentration due to higher organic acid values in the cecum content, which inhibited gluconeogenesis [76]. The increased H\* (lactate accumulation) in the organism first stimulates physicochemical mineral dissolution by increasing the osteoclast and osteoblast activity (bone resorption) and mostly the Ca2+ and Mg2+ reabsorption in renal tubules for pH neutralization; it is usually confirmed also with higher serum calcium levels. This hypothesis was confirmed also

by us, when the slight increase of calcium content was noted during three weeks CCM7420 fresh culture and its Ent7420 application. The rabbits' blood calcium levels fluctuate widely, dependent upon the level of calcium in their diet and the intestinal absorption as well [77]; this is a difference in the calcium metabolism from other mammals. While Lauková et al. [21] and Szabóová et al. [35] described no influence of probiotic strains on serum glucose, triglycerides and calcium levels, Fathi et al. [33] presented numerical increase of triglycerides associated with dietary probiotic treatment in rabbits, similarly to our achievements [25]. The hypocholesterolemic effect of probiotics in rabbits has been already presented [30,34,51]; surprisingly, our results did not confirm those findings. Moreover, increased/higher cholesterol levels (however, without significant changes) were measured during fresh culture CCM7420 and its combinative administration with E. senticosus [26,55] in rabbits; the detected levels were still within the physiological norm.

Table 3. The effect of Enterococcus faecium CCM7420 and its enterocin Ent7420 on the serum biochemistry of rabbits.


The fresh culture of CCM7420 strain was applied into water (at concentration of cells 1 x 10" CFU/mL; dose 500 µL/animal/day); 1yophilized (freeze-dried) from rehydrated in water (1 x 10° CFU/mL; dose 500 µL/animal/day) as well as mixed in feed and pelleted (15 g/100 kg feed). Statistical analysis was performed using one-way analysis of variance (ANOVA) with the post hoc Tukey test with the level of significance set at (p < 0.05), within experimental groups during each individual experiments.

Exogenous factors such as manipulation, nutritional, weather and temperature changes (mainly hot environmental conditions) often induce physiological oxidative stress, which is avoided by the host's defense system. The host's reaction to stress can be marked mostly by the glutathione-peroxidase (GPx) enzyme activity in blood. In addition, there were no significant differences in GPx activity in blood among experimental groups whereas they were differently affected by CCM7420 (Table 3). Application of CCM7420 strain did not disturb the GPx level; while Ent7420 has a reducing effect on GPx during its application (day 21; p < 0.05; [25]). Comparing our previous experimental applications of probiotic strains and their enterocins in rabbits, we suppose that enterocins were more active to protect the host organism; our assumptions were confirmed by reduced GPx levels during Ent7420 and EntM application [25,36]. Outgoing from results we suppose that application of CCM7420 and its bacteriocin Ent7420 did not evoke oxidative stress in the rabbits, similarly to other probiotic strains or enterocins administration [21,29,38].

#### 3.5. Effect on Organic Acids

In rabbits, approximately 40% of digested organic matter of the feed is digested in the caeco-colic segment; so the caecum and the proximal colon are the primary fermenters [78]. The digestion process of nutrients continues in the small intestine by the digestive enzymes of the host, but some components, e.g., plant cell walls and fibers (mainly lignins, cellulose, pectins) are hydrolyzed by bacterial enzymes into soluble smaller compounds and fermented into the end products: volatile fatty acids (VFA: acetic, propionic and butyric acid), ammonia, intermediary metabolites (lactic, succinic and formic acid) and gas (CO2, CH4 and H2; [79]). The stable microbial fermentation is essential for rabbit health, and only small dietary and environmental changes can lead to increased morbidity and/or mortality via microbial dysbiosis and digestive disturbances. Natural substances applications could prevent those disturbances [12-14]. The concentrations of VFA are usually measured in the cecal content of rabbits [21,28-30,38,78], in our first experiment we decided to follow the VFA and organic acids concentrations in feces (it was a model experiment with a low number of rabbits in the experimental groups). Application of E. facium CCM7420 to rabbits led to an increase of fecal levels of acetic acid (v < 0.001) compared to control animals [24], while other tested organic acids (butyric, succinic and lactic acids) were unaffected with the CCM7420 treatment. Similarly to our results, application of other probiotics (E. facium CCM4231; [28] or bacteriocins (EntM and nisin; [21,38]) in rabbits did not influence the molar proportion of VFA in caecum, while the total VFA production and cecal fermentative activity was increased after L. plantarum spray application [29]. Concluded from these results-enhanced enzymatic activity and organic/fatty acid production, better feed conversion ratio and improved jejunal morphology (data shown below) during the CCM7420 strain application, we hypothesized a positive correlation between weight gain and cecal fermentation, improved gut functionality (jejunal morphology) and nutrient absorption. Despite many reports presenting the beneficial results during natural substance application in rabbits, there is a need to existing knowledge and find new possibilities to improve the cecal fermentation and rabbit gut health.

#### 3.6. Effect on Immunity and Jejunal Morphometry

Knowledge of the immune response and homeostasis in farm animals represents important information to protect animals from especially bacteria-derived diseases, to improve their health and productivity. The overall organization of the rabbit's digestive immune-lymphoid system is mostly similar to other species, but at the same time it is very special. There are two additional structures identified only in this species, the sacculus rotundus and the vermiform appendix (a place of lymphoid cells differentiation and maturation); they generally act synergistically. The gut microbiota contribute to intestinal homeostasis via inducing the intestinal immune cells and also influence the systemic host immunity. The probiotic consumption shows a beneficial effect in several ways, including intestinal microbiota balance and ability to modulate host innate and specific immune response. Their effect on non-specific immunity was reported as enhanced phagocytosis of pathogenic bacteria and modified cytokine production [80], while the specific way is usually followed through immunoglobulins testing. Nevertheless, the effect of probiotic administration on the immune system of rabbits has been reported on a limited scale [61,81]. Our studies with E. faccium CCM7420 alone and in combination with Eleutherococcus senticosus demonstrated significant increases in total phagocytic activity (PA) of leukocytes and PA of neutrophils at the end of the treatment period (21 days) and also after three weeks of the ost-treatment period (42 days; p < 0.0001; Table 3; [26]). The freeze-dried CCM7420 strain has not influenced the PA in rabbits during its application, either resolved in water or composed in pellets. However, the prolonged effect of CCM7420 strain rehydrated in water was observed at the end of the experiment (42 days; p < 0.0001; [55]). Another rabbit studies with non-autochthonous strains E. facium CCM4231 (ruminal isolate) and AL41 (CCM8558; isolate from animal waste) also showed stimulation of non-specific immune reaction in rabbits; the significant increase of PA (p < 0.001) was noted in both experiments during the treatment and increased several weeks after the strain's cessation. Fathi et al. [33] also represented improved cell-mediated immunity adding 400 g probiotic(t feed in rabbits´ diet. Contrary to results reported above, Wang et al. [57] noted no probiotic influence on the number of mast cells in duodenum and jejunum, but increased the number of mast cells in caecum and also, increased IgG and IgM in serum. During enterocin Ent7420 administration in rabbits, the prolonged immuno-stimulative effect was observed, which was demonstrated by a significant increase of PA in the experimental group (p < 0.05) compared also to the control data [82]. The same immunomoderate influence was noted during experimental applications of enterocins produced by

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rabbit meat quality. On the other hand, Fathi et al. [33] presented a significant effect of probiotic supplementation on moisture, dry matter, organic matter and ash content. Only limited information about rabbit minerals are available, in response to natural feed additives. Our result showed increased iron content (p = 0.0011) in treated groups with freeze-dried CCM7420 strain (both, rehydrated in water and enriched in feed pellets), in contrary to other findings with reduced iron content during microbial fermented feed utilization with Lactobacillus plantarum and Pediococcus acidilactici [90] and after enterocin administration [42]. Although the pH of the luminal content was not measured, we hypothesized a more acidic environment in the gut due to the previous results of higher lactic acid production and lower pH in the caecum during CCM7420 strain administration in rabbits [48]. This acidic environment can enhance the ionization of minerals, which in turn results in passive diffusion [91] and could be one alternative explanation of the higher iron absorption from the gut. Another hypothesis could be the larger absorption surface due to enterocyte proliferation, which is confirmed by improved morphometry parameters-villus height, crypt depth and villus height:crypt depth ratio-also recorded during our previous experiments with CCM7420 administration [82] and lantibiotic=nisin application to rabbits [38]. The enlargement of the luminal surface could ensure better mineral absorption and their inclusion to rabbit meat. On the other hand, significantly decreased concentrations of copper (v = 0.0004) and calcium (v < 0.0001) were noted. Similarly to these results, the copper concentrations in rabbit meat also reduced during the enterocin M addition to rabbits, however, not significant but only numerically changes were recorded. Similarly to us, lower concentrations of copper, zinc and manganese was found by Shah et al. [90] after probiotic supplementation. Copper is an essential trace element, performing important biochemical functions; its level in rabbit meat varies widely [92,93]. Regarding the higher iron concentration, we hypothesized an iron competitive influence on the copper intestinal absorption and its lower deposition to meat. The calcium metabolism in rabbits is very unique, widely fluctuates and its intestinal absorption is very vitamin D independent, in contrast to most mammals [77]. Despite the generally known fact that probiotics can increase the organic and short chain fatty acids in caecum, which also stimulate the minerals ionization and diffusion through the intestine, we noted decreased calcium concentration, although, still in the range presented in the literature [86,92]. Inferring from achieved results, we assumed no adverse effect of the CCM7420 strain on the meat characteristics; in addition, it could enhance the mineral quality of meat and also increased its value to the functional food level.

#### 4. Conclusions

The strain E. faecium CCM7420 in different application forms (fresh culture at concentration 1 × 10° CFU/mL of cells in a dose 500 µL/animal /day applied into drinking water) was lyophilized (freeze-dried) from rehydrated water (1 x 10° CFU/mL; dose 500 µL/animal/day) as well as mixed in feed and pelleted (15 g/100 kg feed), either alone and in combination with Eleutherococcus senticosus and its enterocin Ent7420 (50 µL/animal/day applied into drinking water) were tested in rabbits. During these experiments, the following effects of the strain and its enterocin were observed: improved average daily weight gain and feed conversion ratio, good colonization ability of the tested strain with maximum counts in the first 2-3 weeks of application, increased lactic acid bacteria and reduced coagulase-positive staphylococci including S. aureus, coliforms and clostridia population as well as the Eimeria sp. oocysts counts in the rabbit's gut. Improved biochemical blood parameters (total proteins, glucose and triglycerides) have been noted during the CCM7420 strain application; however, the glutathione-peroxidase level was not disturbed and oxidative stress was not evoked through the additives application. Another interesting finding was the significant stimulation of blood phagocytic activity and also the improved morphometry parameters (enlargement of the absorption surface in jejunum and higher villi height: crypt depth (VH:CD) ratio). The physicochemical properties of rabbit meat were not negatively affected by the CCM7420 strain, while the meat iron content significantly increased during its application, which improved the rabbit meat quality. It could be also emphasized that knowing the probiotic properties and the ability of Enterococcus faccium CCM7420 to

produce enterocin Ent7420 with an antimicrobial effect is of great interest mainly in the case of several disease/pathologies, such as epizootic rabbit enteropathy, which are difficult to prevent and combat because their etiology is not known and there is no vaccine. This strain is the main component of the Prorabbit probiotic preparation, which is often used in Slovak rabbit farms (at dosage 1-2 g/animal/day for 21 days as prevention and 3 g/animal/day with a therapeutic effect; resolved in water or mixed into feed). Moreover, to the best of our current knowledge, our team is the first that deals with experiments regarding the morphological changes in the jejunum of rabbits during enterocins administration; the first reports regarding the effect of beneficial/probiotic strains and bacteriocins on the mineral and amino acid concentrations as well as the effect of bacteriocins on fatty acid content of rabbit meat were also published by our team.

Author Contributions: Conceptualization, M.P.S. and A.L.; data curation, M.P.S. and L.C.; funding acquisition, A.L.; investigation, M.P.S., A.L. and L.C.; project administration, A.L.; validation, A.L.; visualization, M.P.S.; writing-original draft preparation, M.P.S.; writing-review and editing, A.L., M.P.S. All authors have read and agreed to the published version of the manuscript.

Funding: This work was financially supported by the national Slovak Grant Agency VEGA project 2/0006/17.

Acknowledgments: This work was supported by the project VEGA 2/0006/17. All care and experimental procedures involving animals followed the guidelines stated in the Care and Use of Laboratory Animals approved by the State Slovak Veterinary and Food Administration and the Ethics Committees of both institutions (permission code: SK CH 17016 and SK U 18016). None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. We would like to thank Mrs. M. Bodnárová and Mr. P. Jerga for their skillful technical assistance with sample collection and processing. We are grateful to A. Kopčáková for the E. fiecium CCM7420 strain sequencing, L. Ondruška, V. Párkányi and R. Jurčík, from the National Agricultural and Food Centre (NAFC) in Nitra for blood sampling and Mr. J. Pecho for animals slaughtering, to Ž. Formelová and M. Chrenková (NAFC) for meat samples processing and meat quality properties analyses. We also thank to V. Strompfová for blood biochemistry analyses, K. Cobanová and S. Faix for glutathione-peroxidase activity measuring, I. Plachá for phagocytic activity determination, S. Gancarčíková (University of Veterinary Medicine and Pharmacy, Laboratory of Gnotobiology, Košice) for organic acids measurement, Z. Vasilková (Parasitological Institute of SAS) for Eimeria sp. oocysts detection, R. Zitňan for intestinal morphometry testing.

Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### References


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