HUMIC-FATTY ACID PREPARATION IN GROWING RABBITS NUTRITION

Anna Rząsa¹, Dorota Miśta², Edyta Wincewicz², Wojciech Zawadzki², Zbigniew Dobrzański³, Agata Gelles^2
1 Department of Immunology, Pathophysiology and Veterinary Prevention, Wrocław University of Environmental and Life Sciences, Poland
² Department of Animal Physiology and Biostructure, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Poland
³ Department of Environment Hygiene and Animal Welfare, The Faculty of Biology and Animal Science, Wrocław University of Environmental and Life Sciences, Poland

ABSTRACT

The investigations were carried out on 24 rabbits divided into 3 groups: C (control), fed a basal diet; H5, fed a diet with a 5% humic-fatty acid preparation; H10, fed a diet with a 10% humic-fatty acid preparation. A lower total protein content and γ-globulin fraction were observed in the H5 and H10 groups. A higher albumin-globulin ratio was obtained in the serum of the experimental H5 and H10 animals, which may suggest an immunological activation. The obtained results indicate that using the humic-fatty acid additive in the rabbit feed can positively influence on health status of these species.

Key words: rabbit, red blood cell indices, white blood cell, albumin-globulin ratio.

INTRODUCTION

Trends towards so-called ecological or organic farming and the consequences of the withdrawal of antibiotic growth stimulators from animal feeding have prompted the search for novel solutions in production of additives used in animal nutrition. One area where research is developing is in the return to the use of natural feed additives that have a beneficial influence on the health and condition of the animals and that manage to meet the expectations of both the breeder and the consumer.

 There are obviously known some antibacterial and antiviral properties of humic compounds [1, 4, 13, 14]. A decrease in the accumulation of heavy metals in tissues after animals were given a humic acid supplement was also observed [10, 24]. These substances create a protective layer on the surface of the mucous membrane of the alimentary tract, protecting it against infections and toxins. The humic and fulvic acids in humic substances have a profitable influence on nutrient absorption and the homeostasis of the alimentary tract via the inhibition of bacteria and viruses growth and the reduction of mycotoxin level. Their influence on microflora stabilization and the maintenance of optimal pH in the intestines leads to better feed utilisation [11]. The humic substances administered as feed additives often contribute to improve production efficiency in animals [1, 16, 23]. Moreover, fulvic acids exhibit the capacity to chelate chemical elements, which increases the ability of mineral compounds to infiltrate from the blood to the bones and other tissues [20].

No study on the use of humic compounds in rabbit nutrition has been undertaken so far. The high nutritional value and the outstanding taste of rabbit meat has given rise to an increase in its consumption in a number of countries. This means that the search for the new ways to improve the production yield of this animal species are of considerable interest. The humic-fatty acid preparation used in this study is a mixture of plant oils on a humic-mineral carrier. Among other types, the raw materials like peat, humodetrinite, dolomite or bentonite were used to prepare this additive. The mineral composition of the preparation contains about 40 minerals, including the most important macro- and microelements for the rabbits’ diet.

The objective of the study was to investigate the influence of humic-fatty acid preparation on selected blood parameters and health status of rabbits. Presented paper is the supplement to the data showed by Miśta et al. [18]. Some data in the mentioned paper are described selectively and there are no to many such results in scientific papers, so summing up and showing all of estimated parameters in a presented paper with wider discussion seems worth doing.

MATERIALS AND METHODS

The study was carried out on 24 New Zealand White rabbits, fed ad libitum with a standard commercial feed for fast-growing rabbits (Granum), according to the recommendations given in the international feeding standards [6]. The animals were kept under vivarium conditions in underslung grate cages, with 4 individuals in each. They had free access to drinking water. After a 3-week adaptation period, the rabbits were divided according to the analogue rule (based on sex and body mass) into 3 groups, with 8 individuals in each: group C (control) consisted of animals to be given the basal diet without any additives, group H5 consisted of animals to be given the basal diet with a 5% humic-fatty acid additive (w/w) and group H10 consisted of animals to be given the basal diet with a 10% humic-fatty acid additive (w/w). The basal diet contained 13% dried grass, 15% dried alfalfa, 17% wheat bran, 15%  corn, 10% wheat grain, 10% barley grain, 10% extraction soybean meal, 5% rape meal, 1% rapeseed oil and 4% vitamin-mineral premix (containing, per kg: 15000 IU vitamin A, 1504.0 IU vitamin D3, 8.7 mg vitamin B1, 13.1 mg vitamin B2, 7.1mg vitamin B6, 0.01 mg vitamin B12, 57.2 mg vitamin E, 73 mg vitamin C, 39.9 mg vitamin K, 1.7 mg folic acid, 63.10 mg niacin, 30.1 mg pantothenic acid, 884 mg choline, 1334 mg inositol, 0.272 mg biotin, 80 mg Mn, 15 mg Cu, 197 mg Fe, 62 mg Zn, 0.523 mg Co,  0.157 mg Se). The diet contained 10.95 MJ/kg metabolizable energy, 17% crude protein, 11.8% crude fibre and 3.6% crude fat. The humic-fatty acid preparation (Humobentofet, Tronina PHW, Poland) was composed of 80% humic-mineral carrier and 20% plant oils (48% oleic, 20% linoleic, 5% linolenic and 15% palmitic acid). The main mineral components of this preparation were (g/kg of preparation dry matter): silica (80), Al (27.5), Fe (2.25), Ca (3.03), Na (2.2), Mg (3.3), P (6.6), Mn (0.08), Zn (0.04), Cu (0.006), K (0.003), Co (0.002), Se (0.0001); the energy value per 1 kg was 8 MJ/kg.

The experiment lasted for 6 weeks. It was performed after approval from the Local Ethics Commission for Experiments on Animals in Wroclaw, Poland (license no. 78/2007). The blood samples were collected after 3 and 6 weeks of the experiment, into two testing tubes: one with an anticoagulant (EDTA) and the other with granules (in order to obtain serum). Apart of parameters described by Miśta et al. [18] additionally there were determined in whole blood and serum: the mean red cell volume (MCV), the mean corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin concentration (MCHC). The percentage of particular leukocyte forms in the blood smear, and the ratio of neutrophils to lymphocytes (N/L) were calculated. The above parameters were determined using a Pentra 400 analyser (HORIBA ABX). The concentration of total protein was determined in the serum via the burette method and the total protein fractions: albumins, alpha, beta and gamma globulins were determined using paper electrophoresis according to method described by Campbell [3].

The results of the study were analysed statistically using Statistica 7.1 software (Statistica for Windows, StatSoft, Tulsa, OK, USA). The one-factorial analysis of variance (ANOVA) was used with the level of significance set at P < 0.05. The significance of differences was confirmed by means of Tukey’s multiple comparison test. The data obtained is compiled in Tables 1–3 in the form of means and the standard error of the mean (SEM) for 8 animals.

RESULTS

Table 1 presents average values of red blood cell indices. The humic-fatty acid additive did not influence the red blood cell variables like MCH, MCHC or MCV.

Table 1. The average red blood cell indices of the rabbits blood

		Group C	Group H5	Group H10
MCV (fl)	After 3 weeks	65.17 ± 0.98	63.60 ± 0.68	65.60 ± 1.47
	After 6 weeks	64.50 ± 0.92	64.17 ± 0.48	64.83 ± 1.74
MCH (pg)	After 3 weeks	20.20 ± 0.21	19.86 ± 0.16	20.46 ± 0.44
	After 6 weeks	20.45 ± 0.29	20.33 ± 0.16	20.96 ± 0.34
MCHC (g/dL)	After 3 weeks	31.28 ± 0.14	31.18 ± 0.13	31.12 ± 0.30
	After 6 weeks	31.77 ± 0.12	31.73 ± 0.11	31.70 ± 0.16
C – animals fed the basal diet (control) H5 – animals fed the basal diet with a 5% humic-fatty acid additive H10 – animals fed the basal diet with a 10% humic-fatty acid additive The results are expressed as the mean ± SEM

The percent values of the particular forms of leukocytes are presented in Table 2. The calculated neutrophil-to-lymphocyte ratio increased between weeks 3 and 6 of the experiment in all of the groups. The highest value of this index was observed in the group H5, particularly after 6 weeks.

Table 2. The percentages of different forms of leukocytes in blood smear

		Group C	Group H5	Group H10
Neutrophils	After 3 weeks	27 ± 1.8	30 ± 3.8	26 ± 1.7
	After 6 weeks	39 ± 4.2	49 ± 2.7	41 ± 4.7
Lymphocytes	After 3 weeks	70 ± 1.6	68 ± 4.1	71 ± 2.2
	After 6 weeks	60 ± 3.5	48 ± 2.2	57 ± 4.5
N/L*	After 3 weeks	0.38 ± 0.03	0.47 ± 0.10	0.38 ± 0.04
	After 6 weeks	0.69 ± 0.11	1.04 ± 0.10	0.77 ± 0.14
Neutrophilic band forms	After 3 weeks	0	0.67 ± 0.42	0.33 ± 0.33
	After 6 weeks	0	0	0.17 ± 0.17
Eosinophiles	After 3 weeks	3.33 ± 1.26	1.17 ± 1.17	3.17 ± 0.83
	After 6 weeks	1.14 ± 0.86	2.5 ± 1.45	0.83 ± 0.40
Basophiles	After 3 weeks	0	1.17 ± 1.17	0.50 ± 0.34
	After 6 weeks	0	0.67 ± 0.42	1.00 ± 0.68
Monocytes	After 3 weeks	0.29 ± 0.29	0.33 ± 0.33	0
	After 6 weeks	0.29 ± 0.29	0.17 ± 0.17	0
C – animals fed the basal diet (control) H5 – animals fed the basal diet with a 5% humic-fatty acid additive H10 – animals fed the basal diet with a 10% humic-fatty acid additive The results are expressed as the mean ± SEM * the neutrophil-to-lymphocyte percentage ratio

Table 3 presents the mean contents of total protein and its fractions in the blood serum. After 3 weeks, a significantly lower concentration of total protein was noted in the experimental groups compared to the control group. The mean values of this parameter at the end of the experiment were similar in all of the groups. The mean concentration of the albumin fraction decreased during the course of the experiment by 10 g/l. The lowest contribution of that fraction in the first sample collection was observed in the group H10, while in the last collection, the highest value was found in the same group. During the course of the observations, the globulin fractions increased in all of the animals. The highest concentrations of the gamma globulin fraction were observed in the control group in both collections of the blood samples.

Table 3. The total protein and its fraction in the rabbit serum (g/l)

		Group C	Group H5	Group H10	Total
Total protein	After 3 weeks	88.42 ± 2.54a	72.90 ± 3.65b	68.55 ± 1.72b	76.62 ± 2.55
	After 6 weeks	73.52 ± 3.05	68.03 ± 2.02	75.62 ± 3.09	72.32 ± 1.69
Albumin	After 3 weeks	54.95 ± 2.15a	51.21 ± 2.41ab	45.54 ± 2.91b	50.53 ± 1.65
	After 6 weeks	39.15 ± 1.78	39.65 ± 1.64	42.41 ± 1.47	40.29 ± 0.96
Alpha globulin	After 3 weeks	9.63 ± 1.86	6.29 ± 1.01	7.46 ± 1.17	7.79 ± 0.83
	After 6 weeks	10.81 ± 0.85	10.01 ± 0.65	11.64 ± 1.11	10.77 ± 0.49
Beta globulin	After 3 weeks	8.85 ± 0.64	7.20 ± 0.55	7.50 ± 0.30	7.85 ± 0.33
	After 6 weeks	10.64 ± 0.82	9.10 ± 0.25	12.73 ± 0.72	10.71 ± 0.50
Gamma globulin	After 3 weeks	13.31 ± 1.65	8.20 ± 1.11	8.06 ± 1.51	9.86 ± 0.98
	After 6 weeks	12.03 ± 0.66	10.36 ± 0.83	10.49 ± 0.73	10.99 ± 0.45
C – animals fed the basal diet (control) H5 – animals fed the basal diet with a 5% humic-fatty acid additive, H10 – animals fed the basal diet with a 10% humic-fatty acid additive The results are expressed as the mean ± SEM a, b – values in rows with different letters differ significantly at P < 0.05

DISCUSSION

All of the haematological indices determined in the study were within the range of reference values [15, 17]. Most of obtained haematological results are presented in paper Miśta et al. [18]. In those study the authors found that the humic-fatty acid feed additive increased red blood cell counts (RBC), the haemoglobin concentrations and the hematocrit level in rabbits. The authors of numerous studies on the application of humic preparations in animal feed did not often observe changes in the erythrocytic indices under the influence of the given substances. Wang et al. [22] did not observe any changes in RBC after the addition of humic substances to the feed for fatteners (5 and 10%). Similarly, in the study by Dobrzański et al. [8], the addition of a humic-fatty acid preparation did not significantly influence the hematocrit or the haemoglobin level in the blood of finishing pigs. The study by Rath et al. [19] also did not demonstrate any differences in RBC, or in HCT index between control animals and broilers that were given humic acids. A similar increase like in our own research in erythrocytic indices was noted by Jasek et al. [12] in a study on piglets. These differences may be explained by the age of the animals in the studies. Younger animals, characterised by a higher rate of metabolic changes, react with an increase in erythropoiesis. No significant changes concerning WBC influenced by humic additives were observed in broiler chickens [19] or fatteners [22]. The higher number of leukocytes after 3 weeks in the control group of the present study may suggest the presence of inflammatory processes in the organisms of these animals, which may also be interpreted for the advantage of the researched additives.

The higher ratio of the percentage content of neutrophils to lymphocytes observed in this study in the group of rabbits receiving the 5% addition of the humic-fatty acid preparation was not reflected in other studies on humic preparations. It correlated with the highest neutrophil number in this group. The noted increase in the N/L index in the whole population over the course of the study was probably connected to the further growth and maturation of animals. A decrease in that index under the influence of the humic acids was observed in the broiler chickens [19], while in the fatteners that were given a 10% addition of the humic compounds in their feed, a higher percentage of lymphocytes was noted with respect to the control [22].

The stimulation of lymphocytes influenced by humic compounds (oxihumate) in an in vitro study was demonstrated by Joone and van Rensburg [13]. The same authors also observed the anti-imflammatory activity of potassium  humate manifested in the inhibition of the adherence and degranulation of neutrophils [14]. The above results might suggest that the humic compounds activate lymphocytes to a higher degree than neutrophils. However, in a study by Chen et al. [4], the influence of the humic acid on adhesion to endothelium cells, and the formation of peroxide radicals by neutrophils were observed.

As stated in the CVMP guidelines, an intramuscular injection of HA influences the ratio of albumins to globulins in the blood serum, which is the result of an increase in the globulin fraction [5, 11]. This tendency was also confirmed in this study. The mean increase in the globulin fraction in the group H10 was 11.84, while in the groups H5 and C it was 7.69 and 1.69 g/l, respectively, which certainly proves that the protective properties of an organism are supported by the humic- and fatty-acid additive. Ahamefule et al. [2] reported in their study that the diet and its digestibility influenced the activation of the immunological system, which was evidenced by globulin synthesis, among other processes.

An important effect of the humic-fatty acid additive used in this paper was the reduction of the cholesterol level in the blood serum, especially its LDL fraction [18]. The influence of the humic- and fatty-acid additive on the lipid profile of the blood was also analysed in fatteners by Dobrzański et al. [8]. They only observed a significant decrease in the triglyceride level, and did not note its influence on cholesterol. A preparation containing plant and fish oils was used in the mentioned study.

The chelated bioelements contained in the humic compounds may be transferred to animal products, for example the iron level was elevated in hens’ egg yolks as the result of humic additives to the feed [7]. The high content of biologically important elements proves the good absorption of these elements from the alimentary tract. Fuchs et al. [9] demonstrated that the iron level in the blood serum of rats increased under the influence of humic acids, which was also confirmed in this study [18]. The influence of the humic- and fatty-acid additive on the content of the minerals in the blood serum was also analysed by Dobrzański et al. [8] in the nutrition of fatteners. A preparation containing plant and fish oil caused an increase in the iron level, as in this study. It also contributed to an increase in the calcium and phosphorus level, which was not observed in this study.

SUMMARY

Summing up, the humic-fatty acid additive used in this study positively influence on estimated parameters in rabbit’s blood. The higher ratio of albumins to globulins was observed in the serum of the experimental animals, which may confirm lower immunological activation. The results obtained allow the assumption to be made that the application of the humic-fatty acid additive in the nutrition of growing rabbits will profitably affect the health status of these animals.

REFERENCES

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Accepted for print: 5.07.2014

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Anna Rząsa
Department of Immunology, Pathophysiology and Veterinary Prevention, Wrocław University of Environmental and Life Sciences, Poland
C.K. Norwida 31
50-375 Wrocław
Poland
email: anna.rzasa@up.wroc.pl

Dorota Miśta
Department of Animal Physiology and Biostructure, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Poland
C.K. Norwida 31
50-375 Wrocław
Poland

Edyta Wincewicz
Department of Animal Physiology and Biostructure, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Poland
C.K. Norwida 31
50-375 Wrocław
Poland

Wojciech Zawadzki
Department of Animal Physiology and Biostructure, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Poland
C.K. Norwida 31
50-375 Wrocław
Poland
Phone: +48 71 320 5401
email: wojciech.zawadzki@up.wroc.pl

Zbigniew Dobrzański
Department of Environment Hygiene and Animal Welfare, The Faculty of Biology and Animal Science, Wrocław University of Environmental and Life Sciences, Poland
J. Chełmońskiego 38 C
51-630 Wrocław
Poland
Phone: +48 71 320 5865
email: zbigniew.dobrzanski@up.wroc.pl

Agata Gelles
Department of Animal Physiology and Biostructure, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Poland
C.K. Norwida 31
50-375 Wrocław
Poland

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