Online Version ISSN: 1314-412Xolume , Number

September 2012V 4 3

2012

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Editor-inChief

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Production Systems

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

English Editor

Yanka Ivanova (Bulgaria)

2012

Volume 4, Number 3September 2012

Online Version ISSN: 1314-412X

Research on the stimulating effect of Tribulus terrestris on the oviparous activity of the queen bees

I. Hristakov*

Department of Agricultural Machinery, Agrarian and Industrial Faculty, University of Ruse Angel Kanchev, 8 Studetska, 7017 Ruse, Bulgaria

Abstract. All major processes in the bee colony are closely related to nectariferous vegetation in the area. Oviparous activity of queen bees is directly dependent on the flow of nectar and pollen in the bee colony. When it rises it leads to increase of the oviparous activity of queens, and thus increases the power of the colony. Stimulating feeding with sugar syrup in the absence of pollen flow has little effect on the laying activity of the queens, as the syrup with biologically active substances which are necessary for bees and queens. This paper presents a research on the addition of extract of Tribulus terrestris L. as invert sugar syrup, by which we import some of the biologically active substances needed by bees and bee stimulating oviparous activity of the queen bees.

Keywords: honey bees, queens, oviparous activity, plant extracts, extract of Tribulus terrestris L.

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 4, No 3, pp 228 - 233, 2012

what extent the different herb stimulants, and particularly the extract Introductionfrom Tribulus terrestris L. affect the development of bee families and the oviparous activity of the queen bee.Providing the so-called macro nutritious substances:

The purpose of this study is to determine the effect of using an carbohydrates, proteins, vitamins and mineral substances as honey, extract from Tribulus terrestris L. on the oviparous activity of queen pollen or their substitutes is vitally significant for the bee family. bees.Besides those, however, a considerable number of nutrients has

been discovered to have an extremely beneficial effect on a number of life functions of the bee family either by complementing the nutritious value of the above-mentioned group, or by directly Material and methodsaffecting the bee organism. Most often they enhance the laying rate of the queen bee, give rise in the quantity of the brood and mobilize The empirical part of the study was conducted in bee hives bees for a constant activity in the family. Their chemical nature is located in the village of Brestovitsa, Borovo Municipality, Ruse quite diverse – they can be vitamins, microelements, various region. The village is located about 290 m above sea level. The area enzymes, phytohormones, phytoncides, etc. (Goloskov,1980; is rich in nectariferous vegetation. Close to the bee hives there are Zherebkin, 1965). Conducting stimulating feedings with sugar syrup linden and acacia woods, as well as sunflower and rapeseed fields. cannot replace the need of biologically active nutrients. That is why, it The climate in the village is favourable for bee keeping. The first part is exactly in the period when the physiologically active nutrients, of the empirical work started on 28.02.09 and continued until which stimulate laying and lead to a rapid growth of the families are 23.05.2009.insufficient in the food of the queen bees that the latter can have an In the summer of 2008 we equalized 21 bee families, intake of various phytohormones and hormone-like nutrients, introducing queen bees of similar age coming from pre-selected normally found in the pollen (Boicenyk, 2001; Cherevko et al., 2003). strong and productive families. Then we divided the bee families into

In Bulgaria there is a unique herb, which yields biologically three groups of seven. The three groups, which we equaled in active nutrients from its aerial parts, having a many-sided stimulating strength and honey stock, were placed in one apiary at equal effect on the reproductive functions of some livestock. The name of conditions. The bee families participating in the experiment were fed this herb is Tribulus terrestris L. The active substances extracted under the following scheme: from Tribulus terrestris L. are saponins, glucosides, scleroids, Group №1, the control group - 200 ml inverted sugar syrup flavonoids (Tomova et al., 1981; Saleh et al., 1982; Ren et al., 1994). every three days; The presence of the steroid furostenol and spyrostenol saponins – Group №2, experimental group - 200 ml inverted sugar syrup Protodioscin, Terrestrinins A and B, Gitogenin, Hecogenin, with 10 mg extract of Tribulus terrestris L. per one kilogram of bees; Diosgenin and others determine the pharmacological value of the the correction of the dose done every 12 days, depending on the bee herb as their quantity depends on the region of their growth and the mass in each family;part of the plants used (Bedir and Khan, 2000; De Combarieu et al., Group №3, experimental group - 200 ml inverted sugar syrup 2003; Huang et al., 2003). with a dose of 1 g per 2 litres of syrup of “Startovit”.

Based on the review of literature, it can be concluded that the The last stimulating feeding was conducted on 17.04.2009, application of stimulating substances on bee families can affect the since the first honey storehouses were installed. During 12 days oviparous capacity of queen bees, and hence, the entire since the beginning of the experiment, we measured the amount of development of the bee families. It is still not clear whether and to sealed brood for the three groups of bee families, using a measuring

* e-mail: ihristakov@ru.acad.bg

228

229

2 per number of cells was measured on 29.04.2009 for the three frame with squares of 5 x 5 cm (25 cm area). Each square has an groups. The group fed with the extract of Tribulus terrestris showed area corresponding to the area taken by 100 bee cells (4 cells per

2 25,24% growth compared to the control group while the “Startovit” cm ). The average oviparous capacity of the queens per day for the stimulated bee colonies showed 19,56% more brood. It can be seen period to be reported is obtained by multiplying the number of on Figure 2 that after 17.04.2009 the difference in percentage squares by 100, and then dividing that number by 12.between the two experimental groups and the control group is gradually diminishing, which is probably due to the stimulating effect of the blooming fruit trees and the rapeseed on the control families.

Results and discussion Comparing the average daily oviparous activity of the queen bees from the three groups of families shows that it is growing

When the sealed brood was measured for a second time on gradually from the beginning of the experiment, to reach its peak on 12.03.2009, the groups stimulated by the herb extract and by 29.04.2009 (Table 2). The average values in all three groups are the “Startovit” displayed a significant brood growth - 53,08% in the group highest 60 days from the start of the experiment. The queen bees fed with the extract of Tribulus terrestris and 36,12% in the group fed stimulated by the extract of Tribulus terrestris display the highest with “Startovit” more than the control group (Table 1). daily value of 2020,21 eggs, those stimulated by “Strartovit” -

The tendency of increasing the sealed brood continues through 1919,01 eggs, and for the control group the number is 1613,04. the following periods (Figure 1). The largest quantity of sealed brood Following this period, there is a slight decrease in the oviparous

Table 1. Sealed brood in a number of cells during 2009

Sealed brood, number of cell by:

Gro

ups

Gro

ups

1G

roup

s 2

28.02.09.

0

328.57

357.14

0.00

1000.00

116190.48

340.87

104.63

609.66

8.70

0.00

900.00

119047.62

345.03

72.97

584.17

12.03.09.

12

1508095.24

6485.71

9928.57

8500.00

12500.00

2202380.95

1484.04

8829.20

11027.94

53.08

4500.00

7600.00

1228.05

5575.98

7395.45

24.03.09.

24

2832380.95

10228.57

16514.29

10500.00

20600.00

11301428.57

3361.76

14023.91

19004.67

61.45

20071.43

18000.00

23000.00

3785714.29

1945.69

18630.07

21512.79

51.8951.89

23385.71

19600.00

27100.00

6304761.90

2510.93

21525.63

25245.80

41.73

24242.86

21600.00

26500.00

2942857.14

1715.48

22972.04

25513.68

25.24

23585.71

20600.00

26000.00

3934761.90

1983.62

22116.25

25055.18

29.19

24157.14

21100.00

27000.00

4956190.48

2226.25

22507.94

25806.34

29.98

17780.36

14987.50

20462.50

-

-

-

-

38.16

8000.00

12500.00

1682.97

8981.83

11475.31

05.04.09.

36

13214.29

10500.00

16000.00

4238095.24

2058.66

11689.23

14739.34

17.04.09.

48

4333333.33

16500.00

13000.00

18500.00

2081.67

14957.91

18042.09

29.04.09.

60

19357.14

15500.00

23000.00

6976190.48

2641.25

17400.51

21313.77

11.05.09.

72

2679523.81

18257.14

16500.00

20300.00

1636.93

17044.51

19469.77

1184761.90

23.05.09.

84

18585.71

17500.00

20600.00

1088.47

17779.38

19392.05

-

Average for the period

12869.64

10687.50

14925.00

-

-

-

-

Parameter

Duration, days

Average values

Average values

Minimum values

Minimum values

Max. values

Max. values

Evaluationof displ. σ

Evaluationof displ. σ

Average deviation Sx

Average deviation Sx

Confidence

Confidence

Interval

Interval

% to the control group

230

activity in all three groups, with the most insignificant value the three groups. It can be seen clearly that by day 25 from the start measured in the experimental group fed with the plant extract of the experiment the highest dispersion is demonstrated in the bee (Figure 3). This slight decrease is probably due to blocking a large colonies stimulated by the extract of Tribulus terrestris and by number of cells with nectar and pollen from the rapeseed and the first “Startovit”. This period coincides with the mass blooming of fruit acacia. trees, which are the first source rich in pollen. Another such peak in

On Figure 4 it can be seen that the queens from the group the dispersion values for the two experimental groups can be noted stimulated by the extract of Tribulus terrestris display the highest around day 50, which coincides with the blooming of rapeseed. oviparous activity percentage compared to that of the control group. Probably the differences in egg laying of the queen bees in the This percentage is the highest in the beginning of the period up to experimental groups are influenced more strongly by the outside 24.03.2009, when there is still no mass blooming of honey-yielding stimulus of the blooming honey-yielding vegetation and the higher vegetation. After that this percentage gradually decreases. From the egg laying of queen bees in the group, the greater the dispersion. Table 2 it can be seen that the queens from the families that were It can be assumed that it is also enhanced by the existing hereditary stimulated by the extract of Tribulus terrestris show 38,61% higher differences between the queens although they are sisters. With the oviparous activity on average during the whole experiment queen bees from the control group, such a peak of dispersion is compared to the control group, while those stimulated by “Startovit” - noticed not earlier than day 60 from the beginning of the experiment, 23,45% more. when they have reached their highest oviparous capacity.

On Figure 5 the graph shows the dispersion in the families from As can be seen from the results, feeding the families with

Gro

ups

3

314.29

0.00

1000.00

144761.90

380.48

32.43

596.14

-4.35

8828.57

6600.00

10500.00

1749047.62

1322.52

7848.86

9808.29

36.12

13500.00

10700.00

16300.00

4530000.00

2128.38

11923.30

15076.70

31.98

17200.00

14500.00

19600.00

3603333.33

1898.24

15793.79

18606.21

30.16

22028.57

18000.00

25200.00

6872380.95

2621.52

20086.56

23970.59

33.51

23142.86

20000.00

26000.00

5392857.14

2322.25

21422.54

24863.17

19.56

21500.00

18500.00

24500.00

4416666.67

2101.59

19943.15

23056.85

17.76

20785.71

20000.00

23000.00

1238095.24

1112.70

19961.43

21610.00

11.84

15912.50

13537.50

18262.50

-

-

-

-

23.64

Average values

Minimum values

Max. values

Evaluationof displ. σ

Average deviation Sx

Confidence

Interval

% to the control group

Dis

trib

utio

n of

bro

od

Period, days

Control Extract Sta tovitr

Figure1. Distribution of brood for 2009. The dotted lines illustrate the confidence intervals

30000.00

25000.00

20000.00

15000.00

10000.00

5000.00

0.000 10 20 30 40 50 60 70 80 90

Table 2. Oviparous capacity of queen bees, average per day 2009

Oviparous capacity of queen bees, average per day by:

Gro

ups

Gro

ups

1

28.02.09

0

27.45

0.00

75.60

836.24

28.92

6.02

48.87

12.03.09

12

544.01

375.00

658.30

11336.16

106.47

465.14

622.89

24.03.09

24

852.21

666.60

1041.00

19630.95

140.11

748.42

956.01

05.04.09

36

1101.16

875.00

1333.30

29428.90

171.55

974.07

1228.24

17.04.09

48

1374.96

1083.30

1541.60

30089.35

173.46

1246.46

1503.46

29.04.09

60

1613.04

1291.60

1916.60

48448.48

220.11

1449.99

1776.10

11.05.09

72

1521.40

1375.00

1691.60

18602.20

136.39

1420.36

1622.44

23.05.09

84

1548.79

1458.30

1716.60

8224.95

90.69

1481.60

1615.97

Average for the period

1072.88

890.60

1246.83

-

-

-

-

Parameter

Duration, days

Average values

Minimum values

Max. values

Evaluationof displ. σ

Average deviation Sx

Confidence

Interval

231

80.0070.0060.0050.00

40.0030.00

20.0010.00

0.00-10.00

0 20 40 60 80 100

Icre

ase o

f egg layin

g

Extract

Period, days

Stratovit

Figure 4. Increase of egg laying compared to control group for 2009

Figure 2. Distribution of brood increase in comparison to the control group - 2009

Period, days

70.0060.0050.0040.0030.0020.0010.000.00

-10.00

Bro

od incre

ase

0 10 20 30 40 50 60 70 80 90

Extract Startovit

60000.00

50000.00

40000.00

30000.00

20000.00

10000.00

0.000 20 40 60 80 100

Period, days

Dis

pers

ion

Control StratovitExtract

Figure 5. Distribution of egg laying dispersion values forthe three groups of families – 2009

Figure 3. Distribution of oviparous capacity for 2009. The dotted lines illustrate the confidence intervals

Period, days

Control Extract Startovit2500.00

Ovip

aro

us c

apacity 2000.00

1500.00

1000.00

500.00

0.000 20 40 60 80 100

232

inverted syrup and extract from Tribulus terrestris leads to increasing with a supplement of “Startovit” and extract of Tribulus terrestris L. the oviparous capacity of the queen bees, and from there the amount are affected more by the external stimulus of the blooming of sealed brood is increased too, compared to the families that vegetation, and the higher it is, the greater the dispersion. receive only inverted sugar syrup (the control group) and those that get “Startovit” as a supplement to the syrup.

As a consequence of the increased oviparous capacity of the Referencequeen bees in the families stimulated with extract of Tribulus

terrestris, the potency of the families themselves should be increased. Bedir E and Khan IA, 2000. New steroidal glycosides from de fruits

of Tribulos terrestris. Journal of Natural Products, 63, 12, 1699 -1701.Boicenyk LI, 2001. Epibrassinolide: results and perspectives // Conclusionbeekeeping, 3, 35-36.Cherevko YA, Antimirov S, Timasheva O and Boicenuk L, 2003.

With the supplement of “Startovit” and extract from Tribulus About the use of synthetic analogs of phytohormones for regulation terrestris in the inverted sugar syrup, the oviparous activity of queen development of colonies. Status and development of beekeeping in bees is increased, and the increase is higher in the families fed with North-West Russia. Proceedings of the second Russian scientific the plant extract (38,61% more). Toward the end of the period of and practical conference. Moscow, 104-107 (Ru).active development, the oviparous activity of the queen bees De Combarieu E, Fuzzati N, Lovati M and Mercalli E, 2003. diminishes and becomes almost the same for the three groups. The Furostanol saponins from Tribulus terrestris. Phitoterapia, 74, 6, differences in the oviparous capacity of the queens in the groups fed 583-591.

29.42

4.38

47.97

-4.65

123.70

735.66

918.94

52.07

189.38

1289.35

1569.93

67.76

154.02

1552.53

1780.73

51.35

209.24

1793.77

2103.77

41.73

142.94

1914.32

2126.10

25.24

165.31

1842.97

2087.89

29.19

185.52

1875.63

2150.49

29.98

-

-

-

38.61

Average deviation Sx

Confidence

Interval

% to the control group

Gro

ups

3

26.17

0.00

83.30

1004.79

31.70

2.69

49.65

-4.65

735.70

550.00

875.00

12148.00

110.22

654.05

817.35

35.24

1124.96

891.60

1358.30

31461.39

177.37

993.56

1256.35

32.00

1433.30

1208.30

1633.30

25021.30

158.18

1316.12

1550.48

30.16

1833.30

1500.00

2083.30

46288.43

215.15

1673.92

1992.68

33.34

1919.01

1666.60

2100.00

32798.66

181.10

1784.85

2053.18

18.97

1791.63

1541.60

2041.60

30673.15

175.14

1661.89

1921.37

17.76

1732.00

1666.60

1916.00

8560.50

92.52

1663.46

1800.54

11.83

1324.51

11966.40

1511.35

-

-

-

-

23.45

Average values

Minimum values

Max. values

Evaluationof displ. σ

Average deviation Sx

Confidence

Interval

% to the control group

Gro

ups

2

26.17

0.00

83.30

865.62

827.30

708.00

1041.60

15302.92

1429.64

1166.60

1716.00

35863.35

1666.63

1500.00

1916.60

23723.15

1948.77

1633.30

2258.30

43780.19

2020.21

1800.00

2208.30

20431.98

1965.43

1716.60

2166.60

27328.12

2013.06

1758.30

2250.00

34417.21

1487.15

1285.35

1705.09

-

Average values

Minimum values

Max. values

Evaluationof displ. σ

233

Goloskov V, 1980. Мicroelements influence on the vit. C content in Saleh NA, Ahmed AA and Abdalla MF, 1982. Flavanoid Glucosides bee organism. Prevention and eradication of diseases of livestock of Tribulos terrestris. Phytochemistry, 21, 1995-2000.and poultry. Ulyanovsk, 85-89 (Ru). Tomova M, 1981. Steroidal saponins from Tribulos terrestris L. with Huang JW, Tan CH, Jiang SH and Zhu DY, 2003. Terrestrinins A a stimulating action on the sexual functions. First In. Conference and B, two new steroidsaponins from Tribulos terrestris. Journal of Chem. Biotehnology Biologic Active Natural products, Proceedings, Asian Natural Products Research, 5, 4, 285-290. Varna, September, 3, 299-303.Ren YJ, Chen HS, Yang GJ and Zhu H, 1994. Isolation and Zherebkin MV, 1965. Age and seasonal changes in some of the identification of a new derivative of cinnamic amide from Tribulos processes of digestion in honeybee // notes of scientists. Journal II. terrestris. Acta Pharmaceutica Sinica, 29, 204-206. Moscow, 72. (Ru).

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Research on the stimulating effect of Tribulus terrestris on the oviparous activity of the queen beesI. Hristakov

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CONTENTS 1 / 3

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 4, No 3, 2012

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196

201

203

208

211

215

220

223

228

234

241

246

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Change of some chemical properties of alluvial-meadow soil (Mollic fluvisol) after long term fertilizationS. Todorova, N. Simeonova, K. Trendafilov, V. Valcheva

Investigation on the effect of the environment on some new common winter wheat varieties E. Penchev, K. Kostov, I. Stoeva, V. Dochev

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Physico-chemical quality characteristics of royal jelly from three regions of BulgariaR. Balkanska, I. Zhelyazkova, M. Ignatova

Microscopic method for qualification of the cut surface of white brined cheeseP. Boyanova, P. Panayotov, V. Ganchovska, A. Bosakova – Ardenska

Characterization of enzyme with carboxymethyl cellulase activity produced by Trichoderma reesei NRRL 3652B. Zhekova, G. Dobrev, V. Dobreva, M. Hadjikinova

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Investigations on production traits of mulard ducks with experimentally induced aflatoxicosis I. Valchev, N. Grozeva, L. Lazarov, D. Kanakov, Ts. Hristov, R. Binev, Y. Nikolov

Study on levels of some heavy metals in water and liver of carp (Cyprinus carpio L.) from waterbodies in Stara Zagora region, BulgariaV. Atanasov, E. Valkova, G. Kostadinova, G. Petkov, N. Georgieva, Ts. Yablanski, G.Nikolov

Comparative electronmicroscopical study of the enterocytes of the duodenum of the Japanese quail (Coturnix japonica) and the wild type (Coturnix coturnix) R. Mihaylov, R. Dimitrov, V. Yordanova

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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, Sept.11 – 14, Berlin, Germany, p. 302 (Abstr.).Thesis:Penkov D, 2008. Estimation of metabolic energy and true digestibility of amino acids of some feeds in experiments with muscus duck (Carina moshata, L). Thesis for DSc. Agrarian University, Plovdiv, 314 pp.

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

Volume 4, Number 3September 2012