ISSN 1313 - 8820Volume 7, Number 2

June 2015

2015

Scope and policy of the journalAgricultural Science and Technology /AST/ – an International Scientific Journal of Agricultural and Technology Sciences is published in English in one volume of 4 issues per year, as a printed journal and in electronic form. The policy of the journal is to publish original papers, reviews and short communications covering the aspects of agriculture related with life sciences and modern technologies. It will offer opportunities to address the global needs relating to food and environment, health, exploit the technology to provide innovative products and sustainable development. Papers will be considered in aspects of both fundamental and applied science in the areas of Genetics and Breeding, Nutrition and Physiology, Production Systems, Agriculture and Environment and Product Quality and Safety. Other categories closely related to the above topics could be considered by the editors. The detailed information of the journal is available at the website. Proceedings of scientific meetings and conference reports will be considered for special issues.

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Editor-in-Chief

Tsanko YablanskiFaculty of AgricultureTrakia University, Stara ZagoraBulgaria

Co-Editor-in-Chief

Radoslav SlavovFaculty of AgricultureTrakia University, Stara ZagoraBulgaria

Editors and Sections

Genetics and Breeding

Atanas Atanasov (Bulgaria)Nikolay Tsenov (Bulgaria)Max Rothschild (USA)Ihsan Soysal (Turkey)Horia Grosu (Romania)Bojin Bojinov (Bulgaria)Stoicho Metodiev (Bulgaria)

Nutrition and Physiology

Nikolai Todorov (Bulgaria)Peter Surai (UK)Zervas Georgios (Greece)Ivan Varlyakov (Bulgaria)

Production Systems

Dimitar Pavlov (Bulgaria)Bogdan Szostak (Poland)Dimitar Panaiotov (Bulgaria)Banko Banev (Bulgaria)Georgy Zhelyazkov (Bulgaria)

Agriculture and Environment

Georgi Petkov (Bulgaria)Ramesh Kanwar (USA)Martin Banov (Bulgaria)

Product Quality and Safety

Marin Kabakchiev (Bulgaria)Stefan Denev (Bulgaria)Vasil Atanasov (Bulgaria)

English Editor

Yanka Ivanova (Bulgaria)

2015

ISSN 1313 - 8820 Volume 7, Number 2June 2015

Effect of amitraz on varroosis in bees (Apis mellifera L.)

1 2 1 3K. Gurgulova , I. Zhelyazkova *, S. Takova , K. Malinova

1National Diagnostic and Research Veterinary Medical Institute, 1000 Sofia, Bulgaria2Department of Animal Science, Faculty of Agriculture, Trakia University, 6000 Stara Zagora, Bulgaria3Department of Ecology and Landscape Architecture, Faculty of Agriculture, Forestry University, 10 Kliment Ohridsky, 1756 Sofia, Bulgaria

Abstract. In European (Western) honey bee Apis mellifera L. Varroa mites must be continuously monitored to allow colonies to develop normally and realize high productivity and quality of bee products. A clinical trial of veterinary-medical product, based on amitraz, in the form of strips (VMP Apivar ® – 3.33% amitraz) was carried out in the autumn of 2011. High efficiency of the active substance against Varroa destructor in apiaries on the territory of Bulgaria is determined. After exposure of 46 to 78 days high efficiency on varroosis (Varroa destructor) is achieved – average in four apiaries from 99.00% to 99.86%.

Keywords: Varroa destructor, amitraz, clinical trial efficiency

Abbreviations: VMP – veterinary-medical product, LR – Langstrot-Rut hives, DB – Dadan-Blat hives, EI – extensinvazion, IL – infestation level

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 7, No 2, pp , 2015260 - 263

Introduction the soil in the presence of oxygen (Kidd and James, 1994).The application of “Apivar®” has a variety of advantages –

requires minimal work – two visits per hive for six weeks. One or two Varroosis is a parasite disease of the European honey bee Apis visits may be combined with other activities of the bee keeper. The Mellifera L., and represents one of the essential problems in bee necessary time to place the strips is about 2 minutes per hive for pathology. In Europe veterinary-medical products (VMP) from placing of one strip, while the spraying application requires at least different groups are used to control the Varroa destructor: “Apivar” 21 minutes per hive (Fries et al., 1997).(amitraz), “Perizin” (coumaphos), “Apistan” (fluvalinate) and

The wide use of amitraz and its high effectiveness in other “Apiguard” (thymol) or “Apilife Var” (thymol and other etherical oils). countries define the aim to do a clinical test for determining the In Bulgaria to control the mite Varroa are registered: “Byvarol” and effectiveness and eventual side effects on the bee family of VMP “Varostop” (flumethrin), “Varotom” (fluvalinate), “Check mite+” and Apivar® – strips produced by “Veto – Pharma” (Arysta Life Science “Perizin” (coumaphos), “Apiguard” (thymol), “Еcostop” (thymol and Group).mint oil), “Formitom” (formic acid). In the country at the moment there

are no registered products based on amitraz. This substance is known to bee keepers, who in the past applied it sporadically through fumigation with “Varostat-R” (“Tactic”) with weaker effect. Material and methods

“Apivar” is a product containing amitraz – chemical class Amadine, which is included in a carrier-plastic strip and can stay a The investigation was conducted according to the long time in the bee-hive (6 – 10 weeks), where it destroys up to 99% requirements of Directive 81/852/EEC (Demonstration of Efficacy of of the mites. Amitraz is a lardaceously dissolvable substance, but ectoparasiticides. 7AE17a), Regulation of the requirements for the unlike other similar substances used as varroacides, it is volatile and data contained in the documentation for license use issuing of VMP unstable in honey. The low values of the residues, which are 60.09.05.2006 and Guidelines for Evaluation of Treatment for determined in the honey even when amitraz is applied directly in the Control of Varroa Mites in Honey Bee Colonies” – Ca 3686 Working hive, are probably due to its quick degradation of 2 – 4 weeks Group, 2006.(Jimenez et al., 1997). In more recent experiments residues higher

–1than 0.01 mg.kg have not been established (Floris еt al., 2001). ApiariesResearching honey samples in Slovenia in 2000 – 2001 Maver et al. The trials were conducted in August – October 2011 in 4

–1(2003) determined residues of amitraz up to 0.02 mg.kg , which are apiaries from regions in the North part of the country as follows:–1quite below the established level of 0.2 mg.kg , according to · Apiary in the village of Erden, region Montana – 30 bee

Directive 81/852/ЕЕС (Demonstrat ion of Eff icacy of colonies in Langstrot-Rut (LR) hives, 1 corpus, 10 frames. spring ectoparasiticides, 7АЕ17а). The official maximum levels of the treatment was conducted with “Perizin®”, Bayer.

–1 remainders (MRL's) in honey vary from 0.01 mg.kg in Italy, · Apiary in the village of Slivovik, region Montana – 20 bee –1 Germany and Switzerland, to 1 mg.kg in the USA. The levels of colonies in LR hives, 10 frames, spring treatment was conducted

–1 MRL's in the EU are established at 0.2 mg.kg (Wallner, 1999). MRL with “Varostop®”, Primavet-Sofia.for amitraz in the beewax are not established, because the · Apiary in Gulyantsi town, region Pleven – 20 bee colonies in substance has never been found as a remainder in the wax. Amitraz different types of hives. The experimental group treated with isn't dangerous for the environment because it degrades quickly in “Apivar®” included 10 bee colonies in LR hives, 1 corpus, 10 frames.

“

260

* e-mail: izhel@uni-sz.bg

261

Control bee colonies were 10 in Dadan-Blat (DB) hives – 12 frames. bee hives) at intervals of 7-10-12 days in different apiaries and on the Spring treatment was conducted with formic acid. day before the controlled treatment. The acaricidal effectiveness of

VMP “Apivar®” in trial and control groups we established through · Apiary in the village of Ralevo, region Pleven – 33 bee thcontrol treatment with “Perizin®” respectively on the 67 day in the colonies in LR hives, 2 corpuses. Spring treatment was conducted

thwith “Perizin®”, Bayer. trial groups in the village of Erden, and on the 45 day in the apiary in

ththe village of Slivovak, Montana region; on the 53 day in the apiary thVeterinary medical products in the town of Gulyantsi, and on the 77 day in the apiary in the village

“Apivar®” is a product containing 0.5 g (3.33%) amitraz, which of Ralevo, Pleven region, respectively.is included in a carrier – plastic strips, production of Veto-Pharma To determine precisely the effectiveness of tested VMP the (Arysta Life Science Group). “Perizin®” was used for control control product must show effect >95% (Directive 81/852/EEC). In treatment. This VMP is a fluid in phials of 10 ml (1 ml contains 32 mg the case of establishing the effectiveness of “Apivar®” – strips, coumaphos), production of Bayer, Germany. Both products were “Perizin®” from Bayer was selected like a control product with >95% used according to the instructions of the producer. The dose is effectiveness, declared by the manufacturing company. The consistent with the strength of the bee colonies. effectiveness was determined in percent as the fallen from the

product “Apivar®” mites were calculated compared to the total Groups of bee colonies number of fallen mites using the following formula:The experimental groups included: 20 bee colonies for apiary in

the village of Erden; 10 bee colonies for each of the apiaries in Effectiveness (% reduction) =Slivovik and Gulyantsi; 23 bee colonies for apiary in Ralevo. All colonies in the experimental groups were treated with 2 strips where VMP is number of mites killed by VMP are all mites collected “Apivar®”. The control groups included 10 bee colonies each – at the time of exposition to the tested product “Apivar®”, P is number untreated. of mites killed by “Perizin®” are these, which fall after the treatment

with the control product “Perizin®”.Method for determination the acaricidal effectiveness of VMP During the whole period of the experiments the treated colonies

“Apivar®” were observed for normal development, manifestation of side To determine the acaricidal effectiveness of VMP “Apivar®” effects and harmful influence of the tested product “Apivar®” on the

against V. destructor mite we used field test, applied by Lodesani et queen, the bees and the brood. Monitoring of bee mortality was al. (1995), with the difference that in our tests in the colonies there conducted. The side factors which could have been influencing the was a presence of sealed brood, which was finished at the end of the effectiveness of the tested VMP “Apivar®” such as temperature, experiments. We used the instructions of guideline “Demonstration colony development, presence of brood, etc. were reported.of Efficacy of Ectoparasiticides (7AE17a) and Veterinary Medical The presented results were statistically processed with Excel Products Controlling Varroa and Acarapis Woodi Parasitosis in Bees computer software from Microsoft Office.(7AE16a), based on Directive 81/852/EEC, and the good clinical practices for conducting clinical tests on VMPs in the EU.

For more complete and complex evaluation of “Apivar®” – strips Results and discussioneffectiveness we determined EI (extensinvazion in %) of the bees (on 10 g bees = 100 bees) and of the brood (unsealed 100 cells of Infestation level (extensinvasion – IL) of bee coloniessealed brood) of the trial and control groups before and after the The results on infestation level (IL) of colonies from treated and treatment. The dynamics of the fallen mites in the trial and control control groups at the beginning and the end of study are presented in groups were reported (by placing sticky paper at the bottom of the Figures 1 and 2.

VMPVMP+P

.100

Figure. 1. Infestation level (IL) of bees and brood from the experimental groups in tested apiaries

%

16

14

12

10

8

6

4

2

0

v. Erden v. Slivovik t. Gulyantsi v. Ralevo

IL - beesbeginning

IL - bees end IL - broodbeginning

IL - broodend*(IL=0)

Figure 2. Infestation level (IL) of bees and brood from the control groups in tested apiaries

%

16

18

14

12

10

8

6

4

2

0IL - beesbeginning

IL - bees end IL - broodbeginning

IL - broodend*(IL=0)

v. Erden v. Slivovik t. Gulyantsi v. Ralevo

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The percentages of the infestation level (extensinvasion-IL) defined at the beginning of the experiment in apiaries in the village of Erden showed relatively low invasion of bees and brood in all colonies in the trial group, 0.930.34% and 1.60.48%, respectively. Low percentage of IL due to the fact that the colonies included in the experiment are not basic, but swarms made one month before the trial. When comparing IL of bees in the experimental group and the control one in the experiment revealed no reliable differences, indicating that invasion of bees is similar. After completion of the treatment at the end of the experiment, a high degree of confidence in the infestation of bees between treated and control groups was

observed (p≤0.001). After treatment with “Apivar” in the trial group of bees IL decreased with high confidence, from 0.93±0.34% to

0.11±0.08%, respectively (p≤0.001).In the experimental group in the apiary in the village of Slivovik

before treatment was established low invasion of bees – 2.460.24%. In the control group IL was 3.340.24%. The values of IL in the brood were higher in both the experimental and control groups, 10.401.60% and 10.002.00%, respectively. After treatment at the end of the experiment IL of bees and brood in the experimental group

decreased reliably (p≤ 0.001). At the same time in the control untreated group IL for bees and brood increased and differences

were reported with a high degree of confidence (p≤0.001). These colonies treated with “Apivar”. Data on the number of fallen mites for data indicated that “Apivar” had high effect on mite control of the apiary in the village of Erden showed that the total amount in the colonies and reduced IL of bees to 1.29±0.20%. The effect is even group treated with “Apivar” was small due to the low infestation of higher as brood rearing is discontinued and all mites in brood are colonies (an average of 116.85±4.85 units). Natural mortality of destroyed as a result of strips being left in the nest. Meanwhile mites mites in the control group during the study ranged from 3 to 25 units in the colonies in the untreated control group are reproduced and the (average for the period 23.10±2.71 units). After the control treatment degree of infestation of bees was increased approximately 3.5 times most mites fell in the control group – 247.70±11.81 units, and in the - from 3.34±0.24% to 11.13±0.71%. trial group at least – 1.05±0.20 mites. This could be explained by the

Data for infestation of bee colonies in the town of Gulyantsi high acaricidal activity of “Apivar”, which caused the death of 99 – showed that IL for bees in the experimental group was 1.03±0.18% 100% of mites in bee colonies. The monitoring results of the fallen and 1.16±0.40% for the control, and IL for brood, 8.80±1.77% for the mites in the test group in the village of Slivovik showed that under the experimental and 9.60±1.90% for the control one, respectively. action of “Apivar” an average number of 321.80±5.61 mites fell. These differences in the initial values of IL in both groups have low Natural mortality of mites in the control group during the experiment

averaged 40.90±3.15 units. After the control treatment an average level of confidence (p ≤ 0.05). At the end of the experiment IL for bees of 2.80±0.39 mites fell in the experimental group, while in the control compared between trial and control groups decreased with high one they were 222.10±7.03 mites. The results obtained confirmed confidence (P0.001) from 1.030.18% to 0.190.13%. When the effectiveness of “Apivar”. The monitoring of fallen mites in the comparing the IL at the end of the experiment highly reliable apiary in the town of Gulyantsi showed that in the group treated with differences were established between the experimental group and “Apivar” an average number of 661.40±55.23 mites fell despite the the control one (0.001R). Therefore, the invasion in the group lower infestation of colonies. After the control treatment a relatively treated with “Apivar” greatly reduced. In brood reliable differences large number of mites fell in the control group – 34.10±6.22 and on change in IL of the trial and control groups could not be accounted minimum number in the group treated with “Apivar” – 0.80±0.20 on due to the discontinuity of the brood at the end of the experiment.average. In the apiary in the village of Ralevo under the action of The infestation data for groups in the apiary in the village of “Apivar” fell an average number of 348.83±6.33 mites in the Ralevo showed that both groups have similar values of IL for bees at colonies. The natural mortality of mites in the control group was an the beginning of the experiment, 1.360.47% in the trial and average of 53.83±1.96 units. After the control treatment in the 1.710.68% in the control group, respectively. The values assigned to experimental group with “Perizin” the fallen mites were 1.96±0.17 on the brood are 13.743.53% in the trial group and 15.604.28% in the average, and in the control group – 237.90±7.06.control one, respectively. The invasion of the brood in the control

group was higher, but the difference in both groups is less than 2% Efficiency of applying “Apivar”and does not show reliability. The degree of infestation for brood is Figure 4 presents the data for the effectiveness of VMP “Apivar” the highest compared to other apiaries included in the experiment,

in the tested apiaries. The graph shows that in all apiaries for which can be explained by the fact that the major colonies are 2 “Apivar” efficiency higher than 99% is established, which for the corpuses with 20 frames and raised a large amount of brood during different apiaries is in the range of 99.00±0.18% to 99.86±0.03%, the season. At the end of the experiment IL for bees in the and in some colonies in the treated groups reached 100%. The experimental group reduced reliably by the action of “Apivar” (p≤results show that the test acaricidal VMP “Apivar”, applied against 0.001) from 1.360.47% to 0.77 ± 0.18%.Varroa mites in the apiary in the village of Erden has high efficiency of over 95% – an average of 99.00 ± 0.18% (min / max 97.20 – Number of mites fallen after the treatment with “Apivar”100.00%). It can be seen that the effectiveness of “Apivar” and in the Figure 3 shows the number of mites fallen in groups of bee

Figure 3. Number of mites fallen in groups of bee ®colonies treated with “Apivar ”

Number

700650600550500450400350300250200150100500

v. Erden

Number of mites killed by “Apivar”

Number of mites killed by “Perizin” (control treatment)

v. Slivovik t. Gulyantsi v. Ralevo

116.85

1.05

321.8

2.8

661.4

0.8

348.83

1.96

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Conclusion

The conducted clinical test of VMP “Apivar” in the fall of 2011 showed high effectiveness of the product against varoosis in bee arapiaries on the territory of our country. After exposition with VMP “Apivar” containing 3.33% amitraz for 46 to 78 days, average effectiveness is achieved in 4 bee farms, from 99.000.18% to 99,860,03%, respectively and in some bee colonies it reaches even 100%.

Based on the obtained results from the conducted experiments we may conclude that a resistance of Varroa destructor towards “Apivar” was not established in the studied regions of the country. When applying “Apivar” harmful influence is not observed on the development forms and morphological groups of the treated bee colonies. It can be assumed that because of the high effectiveness (99% – 99.86%), its harmlessness to the bee colonies, its easy application, VMP “Apivar” is a very suitable product for control of varoosis on bees and may respond to the needs of the conventional beekeeping practice in the conditions of our country.

apiary in the village of Slivovik meets the requirements for highly Referencesadvanced miticidal means (greater than 95%). This indicates that

the test product performed much better control of Varroa destructor in this area. The effectiveness which “Apivar” showed in the apiary in Baxter JR, Ibarra J, Wilson WT, Arther RG, Kellerby JD and the town of Gulyantsi averaged 99.86±0.03%, and the minimum Stewart J, 1999. Amitraz or Coumaphos Efficacy Tests in efficiency in individual colonies in the group is higher than 99%. The Guatemala for Control of Varroa jacobsoni in HoneyBees. graph (Figure 4) shows that “Apivar” indicated efficiency higher than Southwestern Entomological Society(Dec.): 309-313. 95% in the apiary in the village of Ralevo as well, meeting the Directive 81/852/ЕЕС, Demonstration of Eff icacy of requirements of EU regulations for acaricidal VMP (Directive 81/852 Ectoparasiticides. 7АЕ17а./ EEC, Demonstration of Efficacy of ectoparasiticides. 7AE17a.). Floris I, Satta A, Garau V, Melis M, Cabras P and Aloul N, 2001. Compared to other apiaries here high efficiency of the preparation Effectiveness, persistence, and residue of amitraz plastic strips in was recorded – an average of 99.42±0.05%, close to the efficiency the apiary control of Varroa destructor. Apidologie, 32, 577-585.set out in the apiary in the town Gulyantsi, which is the highest of all Fries I, Munn P and Jones R, 1997. Organic Control of Varroa. surveyed apiaries (99.86±0.03%). The minimum value in the apiary Varroa! Fight the Mite. London, IBRA, 16-21. in the village of Ralevo was more than 98.88% and the maximum Guidelines for “Evaluation of Treatment for Control of Varroa was 99.72%, which indicated that all colonies in the group showed Mites in Honey Bee Colonies”, 2006. Ca 3686 Working Group.similar effectiveness. Jiménez JJ, Bernal JL, Nozal MJ and Toribido L, 1997.

The results obtained for high efficiency (99% to 99.86%) from Characterization and monitoring of Amitraz degradation products in field experiments carried out in four apiaries from areas of the honey. Journal of High Resolution Chromatography, 20, 81-84.Northwest and Central North Bulgaria give us a reason to say that Kidd H and James DR, 1994. The Agrochemical Handbook, Third Varroa destructor showed no resistance against VMP “Apivar” in the Edition, update 5, January 1994, Cambridge CB4 4WF. apiaries on the territory of our country, so than seven years no Maver L and Poklukar J, 2003. Coumaphos and amitraz residues registered product based on this active substance. These data in Slovenian honey. Apiacta, 38, 54-57.indicate that VMP “Apivar” is a product of very high acaricidal activity Merrington O, 1990. Bibliography on the use of Amitraz for Varroa against Varroa destructor and is suitable to combat Varroa mites in Control in Bees (Apisspp.)(1979-1989). Cambridge, UK, Cambridge Bulgaria. Rates the effectiveness of this VMP confirm results Animal and Public Health Ltd. 36 pp.obtained by other researchers for varoocidal activity of amitraz and Regulation of the Requirements for the Data Contained in the “Apivar” (Merrington et al., 1990; Baxter et al., 1990). Side effects Documentation for License use Issuing of VMP – and harmful influence of the tested VMP “Apivar” and “Pericin” on №60/09.05.2006 г.bee colonies were not established during the period of treatment, as Wallner K, 1999. Varroacides and their residues in bee products. well as at a later stage. The treated bee colonies developed Apidologie, 30, 235-248. normally and prepared for winter in good condition.

Figure 4. Effectiveness of “Apivar®” in the tested apiaries in 2011

Effectiveness, %

101

100

99 99.14

99.8699.42

99

98

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96

95v. Erden

Apiaries

v. Slivovik t. Gulyantsi v. Ralevo

Review

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Effect of amitraz on varroosis in bees (Apis mellifera L.)K. Gurgulova, I. Zhelyazkova, S. Takova, K. Malinova

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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 for Animal Science and Heal th, 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 7, Number 2June 2015

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