Journal of Animal Research and Nutrition

Keywords

Phytogenic additives; Productive traits; Carcass characteristics; Microbial counts; Rabbits

Introduction

The acute shortage of meat supply compels livestock farmers to improve feed utilization, health status and meat production from their animals. This shortage can be bridged by the farming of highly prolific animals with short production cycles, such as rabbits. Rabbits have high fertility rates and fast growth rates, making them good sources of meat and protein of animal origin with a highly digestible, tasty, low fat and cholesterol contents, rich polyunsaturated fatty acids, high nutritional advantage and low-calorie meat compared to other meats [1].

To enhance feed utilization of rabbits, feed additives play important roles in their production. Feed additives such as herbal plants and seeds have been used in ruminants and recently in the diets of rabbits to improve feed conversion and health status. Essential oils, spices and herbs have been successfully used in animal feed to increase the oxidative stability of meat and reduced total mesophilic aerobes, Pseudomonas spp. and Enterobacteriaceae. Combining herbal plants, special extracts and essential oils (phytogenic additives mixture) has attracted an increased attention from rabbit producers due to its notable antioxidative, antibacterial and antifungal activities and ability to enhance feed flavour, palatability, voluntary feed intake and weight gain. Studies on single components of herbal plants, spices, special extracts or essential oils have reported several positive effects on growth performance, feed utilization and animal health of rabbits. Who reported that mortality was reduced in growing rabbits fed diet supplemented with 0.5 g fennel and thyme essential oil per kg diet. Furthermore, a diet supplemented with 0.5% fennel seed increased the digestibility of organic matter, crude fibre and ether extract, final weight and body weight gain [2].

Plant extracts, essential oils and herbal plants possess antimicrobial and antioxidant activities and have been examined in broiler chickens and rabbits with mixed results on performance and animal health. During the period around weaning, young rabbits are threatened by frequently-occur in genteric diseases, leading to an extensive use of antibiotics. Therefore, there is an increasing interest in exploring new and safe antimicrobial agents that prevent and/or overcome infections and improve welfare and health with increasing feed utilization. From this point of view, the objective of our study was to test the effect of different doses of a mixture of herbal plants with special extracts and essential oils in the drinking water of growing rabbits on feed consumption and conversion, blood measurements, carcass characteristics and caecal bacterial profile measured at marketing age (63 days of age) during the fatting period [3].

Materials and Methods

Experimental site

The experiment was conducted in the nucleus breeding rabbit unit of the poultry research centre, poultry production department, faculty of agriculture, Alexandria university, Egypt during spring season 2018. The area has a climate with winter rains and an annual average rainfall of 22 mm and the ranges of ambient temperature and relative humidity during the experimental period (5 weeks) were 18°C-24°C and 68%-71%, respectively. Procedures for this research were approved by the Institutional Animal Care and Use Committee in AU-IACUC, Alexandria university, Egypt. Authors declare that the procedures in accordance with the guidelines of animal welfare of the European parliament (2010/63/EU) of the council of 22 September 2010 on the protection of animals used for scientific purposes. Authors also adhere to the EU regulations on feed legislation, the Regulation (EC) No 767/2009 of the European parliament and of the council of 13 July 2009 on the placing on the market and use of feed [4].

Additives mixture

A newly developed mixture (Aromix^® 2336, Masa Egypt company, Alexandria, Egypt) of natural finely ground herbs with special extracts and essential oils was composed to contain (per litre): 2.5 g carvacol, 0.75 g thymol, 17.8 g menthol and 50 g propylene glycol dissolved in 1 L of distilled water.

Animals, diets and managerial conditions

Ninety weaned unsexed V-line rabbits at an average age of four weeks and body weight range between 668.7 g ± 7.7 g were randomly allocated into three groups of 30 animals each. Each group was further divided into 10 replicates of 3 rabbits each. Rabbits of groups 2 and 3 were drank water supplemented with levels of 0.5 ml (LA) and 1 ml (HA) of phytogenic additives mixture (Aromix^®) per litre drinking water for 5 weeks while group 1 (A0) served as a control drank water without any supplementation. Throughout the experimental period, pelleted feed was offered ad libitum according to cover the nutritional needs of growing rabbits. The experimental diet contained 17.70% crude protein, 14.40% crude fiber, 3.63% fat, 1.21% calcium and 2584 kcal/kg and was further supplied with 0.3% rabbit premix which included all required vitamins and minerals [5].

Housing and management

All rabbits were kept under the same managerial, hygienic and environmental conditions in well ventilated in galvanized wire cage batteries with typical dimensions of 35 cm × 40 cm × 50 cm for length, width and height, respectively with grid wire floor to keep rabbits completely separated from excreta. Ranges of ambient temperature, relative humidity and day-light length during the experimental period were from 20°C to 25°C, 60%-70% and 14 h-16 h, respectively. All cages were provided with manual feeders and clean fresh water was excessively available through an automatic nipple system.

Collected productive data

Initial Body Weight (IBW) and Final Body Weight (FBW) of individual rabbit was recorded weekly, while Daily Body Gain (DBG) and Feed Consumption (FC) were recorded daily. Mortality rate was calculated at experimental period from four weeks to nine weeks. Feed Conversion Ratio (FCR) was calculated as the ratio of the FC (g) to DBG (g).

Blood collection and analysis

At the end of the experiment, 10 rabbits were sampled and about 3 ml blood from each rabbit was taken at 08:00 h-09:00 h from the marginal ear vein into non-heparinized tube [6]. Blood samples were centrifuged for 15 minutes on 4000 rpm and stored in a deep freezer at -20°C until biochemical analysis using specific kits (Stanbio laboratory, Boerne, TX, USA) according to manufacturers’ recommendations. Serum samples were analysed for total proteins, albumin, total lipids, triglyceride, total cholesterol, High Density Lipoprotein (HDL), Low Density Lipoprotein (LDL), Aspartate Amino Transferase (AST), Alanine Amino Transferase (ALT), Alkaline Phosphate (ALP), uric acid, creatinine, calcium, inorganic phosphorus, Immunoglobulin (IgG and IgM), glucose, Triiodothyronine (T₃), Thyroxin (T₄), Total Antioxidant Capacity (TAC) and activity of Malondialdehyde (MDA).

Carcass characteristics

At the end of the experimental period (9 weeks of age), ten rabbits from each group (one from each pen) were randomly taken, fasted for 12 hours, individually weighed, slaughtered and exsanguinated. After complete exsanguinations, the carcass was weighed again and the difference between the pre-slaughter weight and weight after exsanguinations was considered as the blood weight [7]. The slaughtered rabbits were skinned, the skin weight recorded and the carcass eviscerated. The liver, heart, lung, spleen, pancreas, kidney, thymus, thyroid and adrenal glands, abdominal fat, shoulder fat, full stomach, full intestine and full caecum were separated and weighed. The following carcass traits were determined: Dressing percentage (weight of hot eviscerated carcass including liver, heart, abdominal fat and head divided by the live body weight); organs weight as a percentage of live body weight liver, heart, lung, spleen, pancreas, kidney, thymus, thyroid and adrenal glands, abdominal fat, shoulder fat, full stomach, full intestine and full caecum. Slaughtering and dissection were carried out according to World Rabbit Science Association (WRSA) recommendations. Hot carcasses were chilled at 4°C for 24 h. Intestine, caecum and carcass lengths were also measured.

Caecal microbial count

Total number of caecum bacteria (total gut Bacilli and Lactobacillus populations) was counted according to. The isolation of coliforms and Escherichia coli from the caecum was completed using the method described by the food and drug administration. Dilution factor is a reciprocal value of dilution exponent. Such value is expressed as CFU/g (Colony Forming Units), i.e. units that form colonies. Technique of colony CFU was adopted and incubation took place at 30°C for 2-7 days [8]. Microbiological contents of the caeca from 6 rabbits/treatment were collected. The numbers of anaerobic bacteria, lactose entero-bacteria as coliform bacteria, were counted on appropriate selective and non-selective agar plates. The colony counts were expressed in log₁₀ CFU related to 1 g of sample. The pH of caecal content was determined with a glass electrode pH meter.

Statistical analyses

Except for bacterial counts and mortality data, data collected were analysed using the GLM procedure (SAS Inst. Inc. Cary, NC, USA) for a complete randomized design. The treatment (Control, LA or HA) was the only source of variation (fixed effect). For the consumption and performance studies, pen within each treatment was the experimental unit, where as for the carcass characteristics and caecal bacterial count study, rabbit within each treatment was the experimental unit. Measurements (body weight changes, feed consumption and conversion and mortality) recorded daily or weekly were analysed as a repeated measure [9]. Bacterial count values were log transformed and analysed using the PROCGLIMMIX of SAS with log of bacterial counts as there sponse variables and diet (Control, LA or HA)as the factor explanatory variable, assuming that the random residual variance follows a normal distribution. When treatment effects were significant, the means were compared using Duncan’s multiple range test. Polynomial (linear and quadratic) contrasts (adjusted for the equal spacing of treatments) were used to examine dose responses to increasing doses of additives mixture and for comparison between control vs. the average of additives mixture treatments. Significance was declared when P<0.05.

Results

Principal chemical constituents in the additive mixture

A total of 39 peaks from the additives mixture were detected in the GC-MS chromatograms, with the retention time ranging from 5.167 to 42.495 min (Table 1).

Table 1: Examination of healthy and control rabbits.

Productive traits

Treatment LA showed greater (P=0.001) Final Body Weight (FBW) and Daily Body Gain (DBG) (P<0.001) compared with HA and control treatment (Table 2). The percentages of increase by LA and HA relative to A0 were (7.88% and 3.70%) and (12.87% and 6.29%) respectively. Data indicated that Aromix^® had no effect (P˃0.05) on rabbit feed consumption. Slight decrease (P=0.603) in Feed Consumption (FC) by increasing HA concentration was observed relative to A₀ at the end of experimental period [10]. Improved (P<0.001) Feed Conversion Ratio (FCR) was observed by LA treatment compared to the other treatments. The percentages of improve compared to the control were 12.28% and 1.17% higher for LA and HA in drinking water, respectively. The treatments LA and HA decreased mortality rate compared with the control treatment.

Table 2: Comparitive study of rabbits.

Blood biochemical measurements

Blood biochemical parameters of phytogenic additives mixture (Aromix^®) treated rabbits are shown in Table 3. LA and HA treatments decreased total lipid (P=0.013), cholesterol (P=0.002), triglycerides (P=0.023), LDL cholesterol (P=0.032) and malondialdehyde (P=0.041). The percentages of decrease compared to control were 19.65 and 20.52%; 17.00 and 12.43%; 18.98 and 14.60%; 13.84% and 16.38%; 5.84% and 4.38% for LA and HA, respectively. Further, LA treatment gave the highest mean of serum HDL (P=0.047) and TAC (P=0.049) concentrations followed by HA treatment. The percentages of increase compared to control were 26.59% and 10.60%; 26.85% and 18.12%, respectively [11].

Serum Immunoglobulin G (IgG) and Immunoglobulin M (IgM) concentrations were signi icantly (P=0.032 and P=0.044) higher in V-line growing rabbits in Table 3 drank water administrated with 0.5% and 1.0 ml Aromix^® per liter tab water than control group, by percentage of 8.93% and 4.37%; 8.29% and 7.32%, respectively compared with the control group. Data indicated that phytogenic additives mixture (Aromix^®) treated growing rabbits had no effect (P>0.05) on rabbit plasma total protein, albumin, globulin, glucose, AST, ALT, ALP, uric acid, calcium, phosphorus, T₃ and T₄ concentrations and T₄/T₃ ratio in Table 3.

Table 3: Blood biochemical parameters of phytogenic additives mixture.

Carcass characteristics

Data of carcass characteristics presented in that rabbits drank water during experimental period LA and HA ml Aromix^® as phytogenic additives mixture was not significantly effect of different carcass characteristics among experimental groups.

Caecum microbial count

The LA treatment showed greatest (linear and quadratic effects, P<0.001) beneficial Lactobacillus spp. bacteria count followed by the HA treatment by 12.05% and 5.77% compared to control group which had the least count. Conversely, coliform bacteria and E. coli counts were lowest for the LA, followed by the HA and highest for the control (linear and quadratic effects, P<0.001).The percentages of decrease compared to control were 48.54% and 28.08% for LA treatment while were 33.42% and 23.40% for HA treatment, compared to control group, respectively.

Discussion

The present study aimed to explore effects of two levels in drinking water of some phytogenic additives mixture (Aromix^®) in rabbit ration on growth performance, feed consumption and conversion, carcass characteristics and some cecum microflora. Due to scarcity of available data on effect of phytogenic additives mixture on rabbits, comparison was done with studies that used different essential oils, spices and herbs either in rabbits or broilers.

Growth performance, feed consumption and feed efficiency

The insignificant differences among the treatments for the initial body weights reveal effective random distribution and homogeneity of the experimental rabbits at the beginning of the experiment. The low and high doses of the phytogenic additives mixture increased Daily Body Gain (DBG) compared with the control treatment. The increased DBG and FBW reveal enhanced feed utilization which may be partly explained by the increased nutrient absorption and improved digestive secretions of saliva, bile, mucus and enzyme activity. In addition, the observed decreased numbers of pathogenic bacteria in the cecum may improve the ability of epithelial cells to regenerate villus and thus enhance intestinal absorptive capacity, resulting in improved daily gains. Also, Aromix^® was enhanced the high level of immunity (IGA and IGM) in growing rabbits, which helps to increased vitality and less injury diseases. Similarly, the improved population of the beneficial lactic acid bacteria (Lactobacillus ssp) possibly enhanced the gut function and health status of the additives mixture administered rabbits which may have invariably improved feed utilisation and weight gains as a result of the substantial reduction in digestive disorders of the rabbits. Who reported that phenolic compounds in additives mixture give it antimicrobial activity against several microorganisms due to their hydrophobic characteristics by altering the permeability of the cytoplasmic membrane to hydrogen ions and potassium and depletion of the intracellular ATP pool leading to the disruption of essential cellular processes. These results are corroborated by the caecal microbial profile result, as it was discussed later. Who showed that the hydrophobic constituents of essential oils disintegrate the outer membrane of pathogenic bacteria (e.g. E. coli).

Phytogenic additives mixture did not affect feed consumption; however, the low dose of the additives mixture increased feed conversion, revealing unaffected palatability and enhanced feed efficiency of the diets with the additives. The unchanged feed consumption was not expected since essential oils, herbs and spices are often used to improve flavour and indirectly, palatability of feeds. Supplementation of phytogenic additives (essential oils) and herb extracts improve feed conversion ratio. In consonance with the current findings, Celia, et al. Observed unaffected feed consumption and enhanced feed conversion ratio with the feeding of herbal formulation containing a mixture of essential oils, herbs, spices and extracts. In addition, reported that the addition of essential oil improved feed efficiency in broilers. In the present experiment, the low dose of the phytogenic additives mixture was more effective than the high dose, suggesting the 0.5 ml additives mixture as the optimum level for the young rabbits. It appears the high additives mixture dose contained relatively high amount of phenolic compounds which depressed performance relative to the low dose but the phenolic compounds concentration was not as high as to induce toxicity or negatively affect the rabbits. This conjecture was confirmed by the superior performance of the high dose rabbits relative to the control rabbits. Earlier attributed relatively low performance of animals on high dose of phytogenic additives to the concentration of phenolic compounds in the additives.

The LA and HA treatments decreased mortality rate by 59.88% and 40.12%, respectively. This effect was expected since the health status of the rabbits supplemented with the additives mixture was not affected negatively. As discussed later, the additives increased the count of the beneficial Lactobacillus spp. and decreased the pathogenic bacteria numbers. The presence of pathogenic bacteria causes digestive disturbances that are often responsible for high morbidity and mortality of young rabbits after weaning and economic losses in rabbit farms. The higher mortality of rabbits on high dose of additives mixture relative to the low dose further confirms the efficacy of the low dose. It appears the high dose increased the concentration of active substances which mildly affected the rabbits but the effect was not pronounced as to raise mortality rate above that of the control rabbits. As previously stated, the low additive dose was more effective than the high dose in enhancing rabbit performance and reducing mortality. However, the high dose was superior to the control (no additive supplementation). The better gain and feed utilisation efficiency and lower mortality of the high dose additives than the control indicate that the concentrations of the phenolic compounds and other active substances in this treatment were below intoxicating levels.

Blood measurements

Blood metabolic profiles have been used for diagnosis and prognosis of diseases in animals and are also useful to assess the welfare condition of animals or to understand if some changes in a diet can affect animal physiology. The concentrations of total protein, albumin, globulin, creatinine and uric acid were within the ranges for healthy rabbits. Who noted unchanged serum total protein, albumin and globulin with feeding rabbits on a diet supplemented with a mixture of natural finely ground herbs and spices enriched with special extracts and essential oils at 300 g/ton and 400 g/ton feed. In addition, the insignificantly affected concentrations of immune globulins of the LA and HA rabbits are an indicator of the unaffected immune defences or health status and performance with the additives mixture. The additives mixture did not affect glucose, T₃ and T₄ hormones concentrations; however, observed that phytogenic additives mixture increased blood glucose and T₃ hormone concentrations. The observed glucose concentrations were within the range indicated for healthy rabbits. Liver enzymes (AST, ALT and ALP) are indicators conventionally used for diagnosing domestic animal hepatic damage. The unchanged values suggest that no damage occurred in the liver. The reported concentrations fell within reference ranges for healthy rabbits.

Both of LA and HA treatments decreased cholesterol, triglycerides and LDL cholesterol, suggesting beneficial modulatory influence on cholesterol metabolism and turnover, protection of tissues from lipid peroxidation and significant lipid lowering activity in rabbits on these treatments. These effects support the cardiovascular protective influence with the additives supplementation. The active components and phenolic in the additives have the ability to affect lipid metabolism in animal tissues by increasing the antioxidative enzymes activity and preventing the production of reactive oxygen species and off-flavours derived from the peroxidation of polyunsaturated fatty acids. Moreover, lowering of the cholesterol and LDL cholesterol concentrations could possibly be associated with cellular cholesterol biosynthesis in rabbits and decrease in intestinal absorption of cholesterol resulting in an increase in faecal excretion of neutral lipids in broiler. Phytogenic (polyphenolicsand flavonoids) have the ability to inhibit the hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, a key regulatory enzyme in cholesterol synthesis. Who reported that reduction of cellular cholesterol biosynthesis is associated with increased activity of the LDL receptor, leading to enhanced removal of LDL from blood and thus causing a decreased serum or plasma cholesterol concentration [12].

The LA treatment increased HDL cholesterol and TAC and decreased MDA because the additives mixture contains phenolic compounds that elicit antioxidant action by scavenging reactive oxygen species, enhancing the cellular antioxidant enzyme (e.g., superoxide dismutase, catalase and glutathione peroxidase) and increasing glutathione in the cells. The MDA is an indicator of cell membrane injury and its concentration depends on lipid peroxidation. Increasing HDL cholesterol reveals reduced the rogenic risk by virtue of increased reverse cholesterol transport from peripheral organs to liver. The antioxidative activity of some phenol components in feed additives mixture has been attributed to scavenging superoxide anion and hydroxyl radicals of the antioxidant compounds, phenolic ketone derivatives, catechins and volatile oils present in the additives mixture. Although the phytogenic additives mixture altered some blood metabolic profiles, the occurrence of all the blood parameters within the normal physiological ranges ruled out the possibility of any intoxication, especially with the high dose. Therefore, the results indicate the safety of the additives mixture doses used in the current study.

Carcass characteristics

The weak effects of additives on carcass weight, organs relative weights, cecum length, carcass length are in agreement with previous studies in which feeding of phytogenic additives did not affect carcass characteristics and relative organ weights or meat pH value. Observed that supplementation of rabbits with thyme essential oil did not affect the percentage of head, liver, heart, lungs and kidneys of the chilled carcass as well as the percentage of fore, mid and hind parts.

Caecum microbial count

The LA and HA treatments increased the beneficial Lactobacillus spp. bacteria counts (by about 12% and 6%, respectively). It is well documented that essential oil, herbs and phytogenic extracts suppress harmful microorganisms and stimulate beneficial microbes such as Lactobacillus spp. Lactobacillus spp. is a normal member of the rabbit intestinal microflora, with a positive effect on regular gut function. Therefore, increasing the number of Lactobacillus spp. could play a role in preventing the mortality of the rabbits on the treatment diets. Lactobacilli bacteria activate the intestinal immune system and increase the resistance to diseases through the releasing of low-molecular weight peptides which induce immune activation. Moreover, increasing the count of Lactobacilli bacteria contributes to the colonization resistance against pathogenic microbes by modifying the receptors used by them.

The LA and HA treatments decreased coliform bacteria and Escherichia coli counts by about 49% and 33% and 28% and 23%, respectively, compared to the control. Who noted a decreased number of coliforms in the caecum of rabbits fed a diet supplemented with 0.5 g/kg DM of thyme essential oil. Who observed a reduced bacterial diversity in the caecum of rabbits fed a diet supplemented with feeding herbal formulation containing a mixture of essential oils, herbs, spices and extracts. A reduction in the number of pathogenic bacteria changes the microbial ecology in favour of beneficial species in the intestine resulting in improved ability of epithelial cells to regenerate villus and thus enhances intestinal absorptive capacity. Such effects reveal the antimicrobial activity of additives mixture to control pathogenic bacteria. Phenolic compounds in essential oils, herbs and extracts display antimicrobial action against Escherichiacoli and many other pathogens and prevent their adhesion, colonization and proliferation in the gut of broilers. Essential oils, phytogenic extracts and aromatic plants are well known to exert antibacterial, antifungal and antiviral activity. The pathogenic bacteria are normal inhabitants of the intestinal tract of rabbits and can cause digestive disturbances that are often responsible for high morbidity and mortality of young rabbits after weaning and consequent economic losses in rabbit farms.

Decreasing pathogens such as E. coli contributes to healthy microbial metabolites, improves intestinal integrity and ensures protection against enteric disease. Phenolic compounds of essential oils, extracts and herbal plants cause structural and functional damage to cytoplasm membranes of harmful bacteria. Who showed the in vitro antibacterial activity of carvacol and thymol (the main components of oregano essential oil) on multi resistant strains of E. coli. The mechanisms of reduction of such pathogenic bacteria in the intestinal tract after administration of phytogenic additives have not been rigorously studied up till date. However, the hypothesis that plant extracts or essential oils inhibit target cells in the membrane and deplete the trans membrane potential and or the pH gradient which result in the leakage of cellular materials and destruction of bacterial cells has been confirmed.

Conclusion

Under the conditions of the present study, feeding additives mixture of growing rabbits enhanced feed conversion, daily gain and positively altered caecal bacteria profile. Better effects of the low dose of the phytogenic additives mixture (0.5 ml/L drinking water) compared with the high dose (1 ml/L drinking water). Additional studies, involving in vitro and in vivo evaluations, are recommended to investigate different levels of the additives mixture on the performance of growing rabbits.

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