Effect of Аrtemisia annua L. extract on growth performance, biochemical blood parameters and meat quality of rainbow trout (Oncorhynchus mykiss W.), cultivated in recirculating system

R. Koshinski*

Department of Biology and Aquaculture, Faculty of Agriculture, Trakia University, 6000 Stara Zagora, Bulgaria

(Manuscript received 30 June 2018; accepted for publication 20 August 2018)

Abstract. Medicinal herbs can be used as feed additives to improve animal growth performance and to optimize physiological processes. The purpose of this study was to trace growth performance, meat quality and biochemical blood parameters (glucose, urea, creatinine, total protein, albumin, ASAT, ALAT, Ca, P, Mg, triglycerides, cholesterol) of rainbow trout (Oncorhynchus mykiss W.), fed with additive sweet wormwood (Аrtemisia annua L.) extract. In a recirculation

-1system at the Aquaculture Base of the Faculty of Agriculture at Trakia University, a control group (no added) and an experimental (with added 204 mg.kg of sweet wormwood extract) option were set, each of them with two replicates and mean initial weight of fish 13.32±3.07g and 13.37±2.76g, respectively. Forty specimens of rainbow trouts in good health condition were placed in each tank and cultivated for 60 days. At the end of the experiment average final weight, specific growth rates, feed conversion ratio, meat quality and blood biochemical parameters were calculated. Trouts from the experimental group, fed with supplement had 5.5% higher average final weight compared to this one of individuals from the control (P<0.05). The blood biochemical parameters glucose, ASAT and ALAT in control variant were higher compared to the values of that parameters of the fish from the experimental group (P>0.05). Electrolytes of blood such as phosphorus had higher level in the experimental group (P>0.05) compared to the control group. Better growth performance and blood parameters were measured in trout fed sweet wormwood supplement.

Keywords: rainbow trout, sweet wormwood, biochemical blood parameters, growth, meat quality

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 10, No 3, pp 266 - 270, 2018DOI: 10.15547/ast.2018.03.051

Introduction

Many studies show that medicinal herbs can be used as feed additives to improve farm animals' growth performance as well as to optimize physiological processes. They are also used to increase the growth performance of hydrobionts, their survival rate and to reduce feed conversion ratio. Plant extracts are natural products and they are safe for fish and the environment (Gabor et al., 2011). These diet supplements are active substances, natural ingredients and they are not expensive. Since ancient times different parts of plants and their extracts are used as spices and for the treatment of various diseases (Farahi et al., 2012). Studies of their effects on the health status and growth of different species of fish are insignificant. Interestingly, many researches are being conducted in Asia and Africa, not so much in Europe. Studies are needed for perennial herbal supplements that can be used as an alternative to antibiotics in aquaculture (Sharif et al., 2017). Herbal therapy and proper dosing does not stress the fish. Sweet wormwood is the best natural remedy to remove threadworms because of its strong anti-parasitic properties. This herb also acts as a mild stimulant of bile secretion.

Iron-deficiency anemia of blood is among the diseases of human conditions in which treatment with sweet wormwood is practised. No data are available on the use of Аrtemisia annua L. extract in the cultivation of hydrobionts in the literary review. The purpose of this study was to trace growth performance, meat quality and biochemical blood parameters (glucose, urea, creatinine, total

protein, albumin, ASAT, ALAT, Ca, P, Mg, triglycerides, cholesterol) of rainbow trout (Oncorhynchus mykiss W.), fed with additive sweet wormwood (Аrtemisia annua L.) extract.

Material and methods

Experimental fish and feeding

Forty specimens from rainbow trout with an average weight of 13.32+3.07g (control group) and 13.37±2.76g (experimental group) in good health condition were placed in each tank and cultivated for 60 days. The concrete tanks have an effective water volume of 0.8

3m , which are part of a recirculation system. Fish from the control group, C (no added) and the experimental, A.a. (with added А. annua extract) option, each with two repetitions, were set in a recirculating system in the Aquaculture Base of the Faculty of Agriculture at Trakia University. Fish were fed with 6mm extruded pellets “Aqua UNI“, produced by “Aqua garant”, a product of Garant-Tiernahrung Gesellschaft m.b.H. – Austria. To the fish feed of trouts from the experimental group was added 204 mg/kg extract of А. annua, as well as oiling the granules with 5ml of sunflower oil for every 100g of pellets. Rainbow trout from the control group were fed with granules only greased with the same amount of sunflower oil. The nutrient content in the feed of the two groups is: 25% crude protein, 3.10% crude lipids, 6% crude fiber, 8% crude ash, 1.04% P, 11.10 MJ/kg

* e-mail:rkoshinski@abv.bg

266

ME, 4800 IU/kg Vitamin A, 900 IU/kg Vitamin D. The daily ration that the studied fish received was 1.8% of their live weight and they were fed three times per day. The tanks were daily cleaned and excreta were siphoned. Light was about 12:12 h light:dark cycle throughout the day.

Fish growth performance

The average individual weight (g) of the fish was calculated at the start, middle and end of experiment in order to study the extract of А. annua influence on the weight gain and feed conversion ratio in the rainbow trout, cultivated in recirculation system. At the end of the trial the weight gain (g), survival rate (%) and the feed conversion ratio in fish were determined.

The biometrical calculations were carried out according to the following formulas:

- Specific growth rate (SGR) (Zhou et al., 2006):

Where:SGR - specific growth rate, %;Wi – initial weight, g;Wf - final weight, g;n – number of days.

Feed conversion ratio (FCR):

Where:

FCR – feed conversion ratio;

Feed given – g;

Fish weight gain – g.

Hydrochemical parameters -1The oxygen content (mg.l ), pH, water temperature (°C) and

-1electrical conductivity (μS.cm ) were measured daily with a portable meter (HQ30D), accordingly with LDO, pH (liquid) and conductivity

-1electrodes. Other water quality parameters, ammonium (mg.l ) and -1phosphates (mg.l ) were monitored on a weekly in Ecolab

Agriculture Faculty.

Chemical analyses of meat samples

The musculature samples of rainbow trout were determined on atomic absorption spectrometer (AAS) “A Analyst 800” – Perkin Elmer. Crude protein content (%) was calculated by converting the nitrogen content, quantified by Kjeldahl's method, using an automatic Kjeldahl system (Kjeltec 8400, FOSS, Sweden). Lipid content (%) was determined by the method of Soxhlet, using an automatic system (Soxtec 2050, FOSS, Sweden). Ash content (%) was investigated by incineration in a muffle furnace (MLW, Germany) at 550°C for 8 h. Crucibles were brought about the room temperature and weighed (mg).

Biochemical blood analyses

Blood was taken from the examined fish directly from the heart

with disposable sterile plastic syringes (3ml) with a needle. As an anticoagulant Heparine sodium (1%) was used. The blood samples were instantly transmitted and analyzed in a hematological laboratory (NCPTC - Trakia University) and reported in Mindray BC – 120 hematology analyzer. The following biochemical blood

-1 -1parameters were determinated: glucose (mmol.l ), urea (mmol.l ), -1 -1 -1 -1creatinine (µmol.l ), total protein (g.l ), albumin (g.l ), ASAT (U.l ),

-1 -1 -1 -1ALAT (U.l ), Ca (mmol.l ), P (mmol.l ), Mg (mmol.l ), triglycerides -1 -1(mmol.l ) and cholesterol (mmol.l ).

Statistical analysis

The data received from the trial were statistically analyzed with ANOVA single factor (MS Office, 2010).

Results and discussion

Water temperature is an important indicator for the optimal development of the cultivated species. It was 16.8–17.9°C in control and experimental tanks which were within the optimum values for trout farming (Figure 1).

Figure 1. Water temperature in control (C) and experimental (A.a.) tanks

The dissolved oxygen during the experiment ranged between –1 –17.35 mg.l and 8.32 mg.l (Figure 2). The values of this parameter

during the trial period were higher by 3.85% in the experimental tanks in comparison to these of the controlled one.

Figure 2. Dissolved oxygen in control (C) and experimental (A.a.) tanks

SGR=n

x100(LnWf-LnWi)

FCR=Fish weight gain

Feed given

18

17,8

17,6

17,4

17,2

17

16,8

16,6

16,4

16,2

14 7 10 13 16 19 22 25 28 3134 37 40 43 46 49 52 55 58

days

0CC

A.a.

8,6

8,4

8,2

8

7,8

7,6

7,4

7,2

7

6,8

14 7 10 13 16 19 22 25 28 3134 37 40 43 46 49 52 55 58

days

mg.l

1

C

A.a.

267

Young fish are extremely sensitive to pH levels and it is very important not to be under 5 and over 9. Water pH values in the recirculation system varied between 7.3 and 8.02, which were slightly alkaline (Figure 3).

Figure 3. pH in control (C) and experimental (A.a.) tanks

–1Electric conductivity of water varied from 263 μS.cm to 269 –1μS.cm (Figure 4). The conductivity values of the experimental

variant were by 0.8% higher compared to these of the control. The analysis of hydrochemical data showed that during the experiment they were optimum for the farmed species.

Figure 4. Electric conductivity in control (C) and experimental (A.a.) tanks

Tanks were cleaned three times per day, with addition of fresh water in amount of 10% from the total recirculation system volume. To maintain the optimum water chemical parameters during the experiment, the mechanical filter and the biofilter in particular was of major significance. This led to good results with respect to survival, weight gain and feed conversion ration in the experimental fish.

–1 –1Ammonium concentration was from 0.18 mg.l to 1.92 mg.l in the water of two variants for a 60 days' period. Phosphorus ranged

–1 –1between 0.504 mg.l – 1.64 mg.l for both groups during the trial (Table 1).

Growth performance and feed utilization efficiencyThe results of the growth performance of cultivated trout in both variants are presented in Table 2.

The average initial live weight of rainbow trout from control and experimental variants was 13.32±3.07g and 13.37±2.08g, respectively, and the differences were not statistically significant (P>0.05) (Таble 2). By the end of the experiment average live weight obtained of fish fed with sweet wormwood extract supplemented 43.34±0.20g, which was by 5.8% higher compared to the values of the same parameter of the control trouts (P<0.05). Survival rate during the experiment showed 100% in fish in experimental and control group.

The average individual weight gain of rainbow trout from the group fed А. annua extract supplemented was 29.97±1.05g, which was by 7.74% higher compared to this of the control fish and the differences were statistically significant (P<0.05). At the end of the trial, the analysis of the consumed feed amount showed that feed conversion ratio of trout in the experimental group was 1.67±0.001 and in control fish - 1.830±0.260 (P>0.05). The growth parameters of rainbow trout were higher in the group fed А. annua extract supplemented.

Biochemical blood parametersBiochemical blood parameters of rainbow trout in control (C)

and experimental group (A.a.) are presented in Table 3. In the present study, the value of glucose level was 2.17% lower in rainbow trout from the control group, but the differences were not statistically proven (P>0.05). The urea level was 28.09% higher for the control fish compared to these from the experimental variant, but with not statistically significant differences (P>0.05). The creatinine level was 41.16% higher in the experimental group versus of the control group (P>0.05). The level of the total protein was less by 0.61% in the rainbow trout fed the supplement compared to the control, although this difference did not reach statistical significance (P>0.05). The decreased concentration of plasma protein may be due to structural liver alternations that reduce aminotransferase activity, with a concurrent decrease in deamination capacity (Kavadias et al., 2004).

7,9

7,8

7,7

7,6

7,5

7,4

7,3

7,2

7.1

7

14 7 10 13 16 19 22 25 28 3134 37 40 43 46 49 52 55 58

8,0

8,1

days

pH C

A.a.

263

262

261

260

14 7 10 13 16 19 22 25 28 3134 37 40 43 46 49 52 55 58

264

265

266

267

268

269

270

days

1mS.sm

C

A.a.

Table 1. Ammonium and phosphorus concentration in control (C) and experimental (A.a.) tanks

Group N + mg l4

-1H . P-PO - mg l4

-1.

C

A.a.

0 55±0 12. .

0.67±0.59

0 8 ±0 54 . 0 .

0.93±0.37

Table 2. Growth performance of rainbow trout in control (C) and experimental (A.a.) tanks

Group

Initial body weight, g

Final body weight, g

Survival rate, %

SGR % per day

Average individual weight gain, g

FCR

40

40

40

nC A.a.

± SDx ± SDx

13.32±3.07

40.97±0.40*

100

1.86±0.16

27.65±1.03*

1.830±0.260

13.37±2.08

43.34±0.20*

100

1.96±0.01

29.97±1.05*

1.670±0.001

*P<0.05

268

According to Hrubec et al. (2001) the striped bass protein level increased with age. Albumin transports hormones, vitamins, and substances such as calcium throughout the body. It also keeps fluid from leaking out of blood vessels and nourishes the tissues. The amount of albumin in the experimental fish blood is higher by 18.42% compared to the control group (P>0.05). ASAT, ALAT and ALP values are higher by 48.08%, 46.51% and 6.25%, respectively, in the blood of trouts from the control group compared to the experimental variant (P>0.05). These results show that the А. annua extract has a positive effect on liver function. Cholesterol and triglyceride levels of fish may be affected by pollution agents (Yang and Chen, 2003). The triglyceride concentrations in serum of rainbow trout fed supplement were higher by 9.78% than in the control fish (P>0.05). The measured cholesterol was lower by 5.08% in the control variant compared to the А. annua extract supplemented experimental group, but with no statistically significant differences (P>0.05). The change in blood electrolytes may bring disturbances in the normal vital physiological functions of the fish and its growth rate (Prasad et al., 2011). Calcium is a very important mineral element and is present in larger amounts compared to the other electrolytes. It is contained in the bones and combines with phosphorus under the form of calcium phosphate. The ionized calcium is very important for the normal excitability of muscles and in blood coagulation (Kulkarni, 2015). In this study the blood calcium of rainbow trouts in the control variant is 10.31% higher than the experimental variant, but it is not statistically proven (P>0.05). Magnesium levels are similar in both

-1studied groups: 0.76±0.13 mmol.l in control fish and 0.75±0.14 -1mmol.l in trouts from the experimental group.

Chemical analysis of meat samples

The sweet wormwood supplementation in the diet led to 2.57% higher moisture content in the fillet of the rainbow trout compared to that of the control (P<0.001) (Table 4). The protein content in the fish of the experimental group was with higher value of the control group, but not statistically proven (P>0.05). The А. annua extract supplementation in the diet led to lower lipid content in the fillet of the

rainbow trout by 31.85%, but it is not statistically proven (P>0.05). This result show that fish fed with the supplement are more useful and dietetic in their quality as human food. The dry matter content in the meat of the experimental fish group was 23.85±0.39% and it was significantly lower than the control group by 7.41% (P<0.001). The ash content of the fillet of the control was higher by 7.43% compared to the experimental group (P>0.05).

Conclusion

For the first time the effect of A. annua extract as a supplement to the feed of rainbow trout was tested. Fish fed sweet wormwood extract supplement have better growth performance and blood parameters. Trouts fed the sweet wormwood extract are more useful and dietetic in their quality as human food.

References

Farahi A, Kasiri M, Sudagar M, Soleimani M and Zorriehzahra SM, 2012. Effect of dietary supplementation of Melissa officinalis and Aloe vera on hematological traits, lipid oxidation of carcass and performance in rainbow trout (Oncorhynchus mykiss). Online Journal of Animal and Feed Research, 1, 1-5.Gabor E, Şara A, Molnar F and Benţea M, 2011. The influence of some phytoadditives on growth performances and meat quality in rainbow trout (Oncorhynchus mykiss). Animal Science and Biotechnologies, 44, 12-18.Hrubec T, Smith S and Robertson J, 2001. Age related in haematology and biochemistry of hybrid striped bass Chrysops morone axatilis. Veterinary Clinical Pathology, 30, 8-15.Kavadias S, Castritsi-Catharios J and Dessypris A, 2004. Annual cycles of growth rate, feeding rate, food conversion, plasma glucose and plasma lipids in the population of European sea bass (Dicentrarchus labrax) farmed in floating marine cages. Journal of Applied Ichthyology, 19, 29-34.Kulkarni R, 2015. Comparative studies on blood electrolytes of the fresh water fish, Notopterus notopterus from three aquatic bodies. International Letters of Natural Sciences, International Letters of Natural Sciences, 40, 1-5.Prasad M, Kumar A, Mishra D, Srivastav S and Srivastav A, 2011.Blood electrolytes of the freshwater catfish Heteropneustes fossilis in response to treatment with a botanical pesticide (latex of Euphorbia royleana). Integrative Zoology, 6, 150-156.

Table 4. Chemical composition of the fillet of rainbow trout (O. mykiss) in control (C) and experimental (A.a.) groups (%)

Group

CA.a.

Parameter

Moisture Dry matter Grude protein Fat Ash

74.24±0.6376.15±0.39***

25.76±0.6323.85±0.39***

18.66±0.318.58±0.17

5.36±0.223.65±0.46

1.75±0.161.62±0.09

***P<0.001

Table 3. Biochemical blood parameters of rainbow trout in control (C) and experimental group (A.a.)

Blood

parameters

-1GLU mmol.l-1UREA mmol.l-1CREA µmol.l

-1Total protein, g.l-1ALB g.l

-1ASAT U.l-1ALAT U.l

-1ALP U.l-1CA mmol.l

-1P mmol.l-1Mg mmol.l-1TG mmol.l

-1CHOL mmol.l

5

5

5

5

5

5

5

5

5

5

5

5

5

nC A.a.

± SDx ± SDx

5.06 ± 0.67

0.64 ± 0.31

6.80 ± 4.21

38.42 ± 8.36

17.10 ± 1.91

71.67 ± 107.04

23.20 ± 43.89

416.00 ± 206.35

2.23 ± 0.49

4.93 ± 2.02

0.76 ± 0.13

1.77 ± 0.16

5.83 ± 2.25

4.95 ± 1.08

0.82 ± 0.16

9.60 ± 5.41

38.20 ± 4.80

20.25 ± 1.91

37.00 ± 67.70

12.4 ± 12.52

390.00 ± 293.50

2.00 ± 0.72

6.02 ± 2.11

0.75 ± 0.14

1.86 ± 0.18

6.40 ± 0.42

269

Sharif M, Haghighi M and Bazari M, 2017. Study on nanoparticles of Aloe vera extract on growth performance, survival rate and body composition in Siberian sturgeon (Acipenser baerii). Iranian Journal of Fisheries Sciences, 16, 457-468.Yang JL and Chen HC, 2003. Effects of gallium on common carp

(Cyprinus carpio): acute test, serum biochemistry, and erythrocytemorphology. Chemosphere, 53, 877-882.Zhou CQ, Wu HZ, Tan PB, Chi YS and Yang HQ, 2006. Optimal dietary methionine requirement for juvenile cobia (Rachycentron canadum). Aquaculture, 258, 551-557.

270

Genetics and Breeding

Usability of metadata analysis of goat genetic resources among five countries from Africa, Asia and Europe: Metadata analysis of goat geneticM.M. Musthafa, T. Hussain, M.E. Babar, R.S. Aljumaah, M.A. Alshaikh, I. Muritala, V. Landi, A. Martinez, M. Amills, O. Dadi, J.V. Delgado, A.B.J. Aina, A.A. Onasoga, O.A. Adebambo, C. Visser, E. Van Marle-Köster, A.O. Adebambo, F.M.M.T. Marikar

Knezha 560 – a new mid-late maize hybridV. Valkova, N. Petrovska

Sources of resistance in chickpea (Cicer arietinum L.) to ascochyta blight (Ascochyta rabiei)M. Koleva, Y. Stanoeva, I. Kiryakov, A. Ivanova

Variability and grain yield potential of maize (Zea mays L.) genotypes under irrigated condition in central SudanM.B. Alhussein, S.H. Suliman, A.A. Mohammed

Nutrition and Physiology

Effect of monosodium glutamate dietary supplementation on some productive traits of common carp (Cyprinus carpio L.), cultivated in net cagesG. Zhelyazkov

Effect of experimentally induced aflatoxicosis on haematological parameters and bone marrow morphology in mulard ducksI. Valchev, N. Groseva, D. Kanakov, Ts. Hristov, L. Lazarov, R. Biinev

Effect of dietary phytoextracts supplementation on the chemical composition and fatty acid profile of rainbow trout (Oncorhynchus mykiss W.), cultivated in recirculation systemK. Georgieva, G. Zhelyazkov, Y. Staykov, D. Georgiev

Production Systems

Yield and seed quality of some soybean (Glycine max. L) varieties, cultivated in Osmaniye region, TurkeyF.F. Aşik, R. Yildiz

Productivity and yield stability at late treatment of durum wheat (Triticum durum Desf.) with antibroadleaved herbicides.

-stI. Influence at treatment during 1 stem node stageGr. Delchev, D. Delchev

The effects of inoculation and N fertilization on soybean [Glycine max (L.) Merrill] seed yield and protein concentration under drought stressO. Basal, A. Szabó

Soil structure after treatment with different operation modes of spading machineY. Stoyanov, K. Trendafilov, N. Delchev, G. Tihanov

CONTENTS 1 / 2

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 10, No 3, 2018

183

191

195

208

215

222

232

227

204

199

236

Application of herbicides on common winter wheat (Triticum aestivum L.) at different doses and their reflection on the structural elements of spikeZ. Petrova, M. Nankova

Agriculture and Environment

Differences in carbon forms under two land use types in Abia State, South-east NigeriaB.N. Ndukwu, D.N. Osujieke, C.M. Ahukaemere, P.E. Imadojemu

Theoretical analysis of the heat energy savings in wood pellets productionR. Georgiev, K. Peychev, V. Dimova, D. Georgiev

Agricultural characteristics of sugar factory waste productsB.B. Aşık, S. Dorak

Product Quality and Safety

Ontogenetic and diurnal variations of essential oil content of Hypericum montbretii Spach, cultivated in Kazdağı (Edremit/Balıkesir), TurkeyC. Paşa, E. Esendal, T. Kiliç

Effect of Аrtemisia annua L. extract on growth performance, biochemical blood parameters and meat quality of rainbow trout (Oncorhynchus mykiss W.), cultivated in recirculating systemR. Koshinski

CONTENTS 2 / 2

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 10, No 3, 2018

241

246

253

257

262

266

Instruction for authors

Preparation of papersPapers shall be submitted at the editorial office typed on standard typing pages (A4, 30 lines per page, 62 characters per line). The editors recommend up to 15 pages for full research paper ( including abstract references, tables, figures and other appendices)The manuscript should be structured as follows: Title, Names of authors and affiliation address, Abstract, List of keywords, Introduction, Material and methods,Results, Discussion, Conclusion, Acknowledgements (if any), References, Tables, Figures.The title needs to be as concise and informative about the nature of research. It should be written with small letter /bold, 14/ without any abbreviations. Names and affiliation of authorsThe names of the authors should be presented from the initials of first names followed by the family names. The complete address and name of the institution should be stated next. The affiliation of authors are designated by different signs. For the author who is going to be corresponding by the editorial board and readers, an E-mail address and telephone number should be presented as footnote on the first page. Corresponding author is indicated with *.Abstract should be not more than 350 words. It should be clearly stated what new findings have been made in the course of research. Abbreviations and references to authors are inadmissible in the summary. It should be understandable without having read the paper and should be in one paragraph. Keywords: Up to maximum of 5 keywords should be selected not repeating the title but giving the essence of study. The introduction must answer the following questions: What is known and what is new on the studied issue? What necessitated the research problem, described in the paper? What is your hypothesis and goal ?Material and methods: The objects of research, organization of experiments, chemical analyses, statistical and other methods and conditions applied for the experiments should be described in detail. A criterion of sufficient information is to be possible for others to repeat the experi-ment in order to verify results.Results are presented in understandable

tables and figures, accompanied by the statistical parameters needed for the evaluation. Data from tables and figures should not be repeated in the text.Tables should be as simple and as few as possible. Each table should have its own explanatory title and to be typed on a separate page. They should be outside the main body of the text and an indication should be given where it should be inserted.Figures should be sharp with good contrast and rendition. Graphic materials should be preferred. Photographs to be appropriate for printing. Illustrations are supplied in colour as an exception after special agreement with the editorial board and possible payment of extra costs. The figures are to be each in a single file and their location should be given within the text. Discussion: The objective of this section is to indicate the scientific significance of the study. By comparing the results and conclusions of other scientists the contribution of the study for expanding or modifying existing knowledge is pointed out clearly and convincingly to the reader.Conclusion: The most important conse- quences for the science and practice resulting from the conducted research should be summarized in a few sentences. The conclusions shouldn't be numbered and no new paragraphs be used. Contributions are the core of conclusions. References:In the text, references should be cited as follows: single author: Sandberg (2002); two authors: Andersson and Georges (2004); more than two authors: Andersson et al.(2003). When several references are cited simultaneously, they should be ranked by chronological order e.g.: (Sandberg, 2002; Andersson et al., 2003; Andersson and Georges, 2004).References are arranged alphabetically by the name of the first author. If an author is cited more than once, first his individual publications are given ranked by year, then come publications with one co-author, two co-authors, etc. The names of authors, article and journal titles in the Cyrillic or alphabet different from Latin, should be transliterated into Latin and article titles should be translated into English. The original language of articles and books translated into English is indicated in parenthesis after the bibliographic reference (Bulgarian = Bg, Russian = Ru, Serbian = Sr, if in the Cyrillic, Mongolian =

Мо, Greek = Gr, Georgian = Geor., Japanese = Jа, Chinese = Ch, Arabic = Аr, etc.)The following order in the reference list is recommended:Journal articles: Author(s) surname and initials, year. Title. Full title of the journal, volume, pages. Example:Simm G, Lewis RM, Grundy B and Dingwall WS, 2002. Responses to selection for lean growth in sheep. Animal Science, 74, 39-50Books: Author(s) surname and initials, year. Title. Edition, name of publisher, place of publication. Example: Oldenbroek JK, 1999. Genebanks and the conservation of farm animal genetic resources, Second edition. DLO Institute f o r A n i m a l S c i e n c e a n d H e a l t h , Netherlands.Book chapter or conference proceedings: Author(s) surname and initials, year. Title. In: Title of the book or of the proceedings followed by the editor(s), volume, pages. Name of publisher, place of publication. Example: Mauff G, Pulverer G, Operkuch W, Hummel K and Hidden C, 1995. C3-variants and diverse phenotypes of unconverted and converted C3. In: Provides of the Biological Fluids (ed. H. Peters), vol. 22, 143-165, Pergamon Press. Oxford, UK.Todorov N and Mitev J, 1995. Effect of level of feeding during dry period, and body condition score on reproductive perfor-

thmance in dairy cows,IX International Conference on Production Diseases in Farm Animals, September 11–14, Berlin, Germany.Thesis:Hristova D, 2013. Investigation on genetic diversity in local sheep breeds using DNA markers. Thesis for PhD, Trakia University, Stara Zagora, Bulgaria, (Bg).

The Editorial Board of the Journal is not responsible for incorrect quotes of reference sources and the relevant violations of copyrights.

Animal welfareStudies performed on experimental animals should be carried out according to internationally recognized guidelines for animal welfare. That should be clearly described in the respective section “Material and methods”.

Volume 10, Number 3September 2018

www.agriscitech.eu