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Volume 8, Number 4December 2016

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2016

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2016

ISSN 1313 - 8820 (print)ISSN 1314 - 412X (online)


Heterosis manifestations by survival and larval duration of F Bombyx mori L. Hybrids reared 1

with artificial diet

1 1 2 2R. Guncheva *, M. Panayotov , P. Tsenov , Y. Dimitrova

1Department of Animal Science - Non-ruminants and other Animals, Faculty of Agriculture, Trakiа University, 6000 Stara Zagora, Bulgaria2Sericulture and Agriculture Experiment Station, 3000 Vratsa, Bulgaria

(Manuscript received 2 July 2016; accepted for publication 22 November 2016)

Abstract. The aim of this paper was to study the influence of the degree of heterosis (compared to higher parent value- HP and mean parental values- MP) on the signs, survival and duration of development in F hybrids of Bombyx mori L. from different geographical origin, fed by artificial diet. The study was conducted 1

at the Training Experimental Station of the Sericulture section of the Faculty of Agriculture at Trakia University. A total of 10 hybrids were tested featuring 6 breeds from the genetic bank of Sericulture and Agriculture Experiment Station (SAES)-Vratsa. Silkworms were reared with artificial diet containing 15% powder of dried mulberry leaf produced at SAES-Vratsa and prepared by methods, developed by the manufacturer, whereby 250g of dry substance and 675ml of distilled water are homogenized using a mixer. The mixture is cured thermally in a microwave for 10min. at ~800W. The receptivity of specimens was

-stdetermined based on the results from the survival and the duration in the I age of the larval stage. In heterogeneous crossbreds higher survival rate in the first age is exhibited by hybrids in which a Japanese type breed was used as dam. Homogeneous crossbreds created with the participation of Japanese breeds excel in both analyzed traits those involving a Chinese type. For the survival trait heterosis manifestations to higher parent value (HP) and mean parental values (MP), were detected in the majority of hybrids (90.91%). Regarding the duration of development, heterosis effect to MP was observed in all F hybrids, 1

while to HP heterosis is exhibited by 60% of the tested F hybrids.1

Keywords: Bombyx mori L. hybrids, artificial food, heterosis, receptivity, survival

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 8, No 4, pp , 2016DOI: 10.15547/ast.2016.04.056

292 - 296

Introduction

The introduction of technologies for the use of artificial diet in Impactsericulture practice, the increase of efficiency of their

implementation, testing breeds and creating highly productive hybrids suitable for rearing with artificial diets expands the range of

and Kumardistribution and the opportunities for rearing and experimenting with Bombyx mori L. in laboratory conditions (Ito and Kobayashi, 1978; Horie, 1981).

One of the limiting factors for growing artificial foods is the strong dependence of sericulture on the narrow food specialization of the mulberry silkworm. In order to adapt silkworms to switch from their natural food source (mulberry leaf) to the artificial diet and stabilizing yields it is necessary to create specialized hybrids with high degree of receptivity and productivity comparable to rearing et al. with mulberry leaf. According to Horie (1981) and Nair et al. (2004), not all breeds and hybrids are suitable for growing on artificial diet, which necessitates the creation of tailor-made mixes for them, on the forms in Bombyx mori L. have one hand, and testing and creating appropriate breeds for rearing on been intensified to such an extent that the entire production of artificial diet, on the other hand. Hybridization is one of the main industrial cocoons in the developed sericulture countries is based on methods for improving the productivity of silk butterflies. In this hybrids (Petkov, 1995). Industrial crossing between two or more regard, the use of the F hybrids is a major component in the 1 lines with different geographical and genetic origin is one of the most technological complex of factors for intensification of sericulture. effective methods for creating and using in the production of high

heterosis F hybrids in Bombyx mori L. (Kremky, 1970). In this 1

regard, many scientists (Kremky and Szuba, 1977; Hirata, 1985; Brasla and Matei, 1992; Osawa and Harada, 1994, etc.) have found that while in interbreed crossing the increase in cocoon and raw silk yield in F hybrids is within the range 5-10% compared to baseline 1

forms, in the interline F hybrids it reaches 15-30%. 1

combinations of crossing is an essential prerequisite for identifying the variants that have a high degree of heterosis manifestation and is important in making a decision on the guidelines of future breeding programs (Kumar et al., 2011). on the degree of heterosis manifestation has the creation system (di-, tri- and tetra-hybrid forms), and the results are different for the different traits (Tzenov, 2008; Tzenov et al., 2010; Lakshmanan , 2012; Singh et al., 2012). According to Brasla et al. (1992) and Ozama et al. (1994), hybrid forms obtained by two-breed crossing exhibit higher heterosis effect in F by three-breed and four-breed crosses. F hybrids of the 1 1

silkworm have the strongest heterosis manifestation, after which the thhybrid power disappears till about the 14 generation (F ). 14

In an initial selection of parental forms for hybridization to achieve heterosis effect the level of difference between the parents is of crucial importance (Maqbool et al., 2015). Arunachalam (1984), reported that for heterosis manifestation in F optimum level 1

of genetic divergence between the parental forms is needed. The efforts to create and use F hybrid 1

The achievement of a high level of heterosis manifestation has been recognized as the most appropriate methodology for breeding silkworms to increase yields and improve the quality parameters. In this regard, the selection of suitable parents and assessment of the degree of heterosis is an important step (Ridey et al., 2003). Information on the extent of the hybrid power in the various

* e-mail: [email protected]

292

293

The aim of this paper was to investigate the influence of the where, V is survival of hibryds (%), N is number of viable silkworms in degree of heterosis (compared to higher parent value - HP and mean the relevant instar, n is total number of silkworms laid.parental values - MP) on the signs, survival and duration of In silkworm rearing with artificial diet it is of great economic development in F hybrids of Bombyx mori L. from different importance to trace the duration of larval development. Data 1

analysis has been conducted on the basis of the generally geographical origin, fed by artificialestablished standards for the duration of the larvae development in hours, which is 96 h (incl. 24h sleep) first age. In order to establish the duration of larval development date and time for the beginning Material and methodsand end of the respective physiological states (instar and sleep) have been registered individually for each repetition.

The study was carried out in 2016 at the Training Experimental The heterosis effect has been calculated by the formula of

Station of the Sericulture section of the Faculty of Agriculture, Trakia Kremky (1970), both by the mean parent value (MP) of the trait and

University, Stara Zagora, Bulgaria. It was tested a total of 10 hybrids to the higher parent value (HP):

featuring 7 breeds from the genetic bank of Sericulture and · by MP:

Agriculture Experiment Station (SAES), Vratsa. Silkworms were reared with artificial diet containing 15% powder of dried mulberry leaf produced at SAES, Vratsa. In addition mulberry meal, consisting of artificial food included soybean meal, corn starch, citric acid,

where, MP is mean parent value of the trait, F is trait value in the 1 askorbic acid, agar agar, trace elements and vitamins, sitosterol, hybridantibiotics and preservatives. The preparation of the diet to feed the

· by HP:silkworms was done by methods developed by the manufacturer, whereby 250g dry substance and 675ml of distilled water are homogenized using a mixer. Placed in a box with a lid, the mixture is treated thermally in a microwave for 10min. at ~800W. The ready diet

where, HP is the higher parent value, F is trait value in the hybrid.1 was stored in a refrigerator at temperature 2-5°C until using it for Data obtained were processed by using relevant modules in feeding.

STATISTICA software of StatSoft and Microsoft Excel 2010.Of all breeds and hybrids object of this study, it was formed groups in 3 reps of 50 normal silkworm eggs previously disinfected with a 2% formalin solution. Incubation of the silkworm eggs, rearing and feeding the silkworms was done in sterile petri dishes, in Results and discussionthermostat with air temperature 29-30°C and air humidity 85%, as

stthe requirements for I instar. During the growing, food was served The classical form of hybridization in sericulture is crossing immediately after the mass hatching of larvae and on the third day of breeds of the Japanese type with breed of the Chinese type. For the the first age. purposes of selection other schemes of crossing can also be used.

To establish the receptivity of specimens from the analyzed According to Singh et al. (2012) the length of the larval development breeds and hybrids to artificial food with reduced content of dried and the survival rate are among the traits characterized by a high mulberry leaf it was identified their survival and duration of degree of heterosis manifestation.

st-stdevelopment in the I instar of the larval stage. Data on the average survival rates of specimens in I instar at

Taking into account the receptivity of silkworms to artificial diet, crossbreds of the type: Japanese x Japanese; Chinese x Chinese; one of the main signs is considered to be the number of normally Japanese x Chinese, the reciprocal Chinese x Japanese and their developing specimens, calculated as percentage of survival. Data parental forms are presented in Table 2. The baseline breeds are analysis has been performed on the basis of the criteria set out in characterized by vitality of specimens above the accepted lower limit Table 1, defining the degree of receptivity of larvae to artificial diet. of survival when reared with artificial diet - 70%. The results of this

study confirmed the high survival rate (over 70%), established by Guncheva (2016) when rearing the breeds with artificial diet with reduced content (15%) of mulberry flour.

The predominant part of hybrids (90%) demonstrate very high receptivity (over 90%) and superiority over the baseline breeds. Superiority of the hybrid forms over the parental ones in terms of receptivity is reported by Nair et al. (2011) and Saviane et al. (2013). Rather high, but less than 90% and lower receptivity than the parents has hybrid "Nig 2 x Merefa 6" (88.37% ± 4.00).

The comparative analysis of the data in Table 2 shows that heterogeneous by geographical origin hybrid forms are characterized by higher average survival rate (95.76%) to artificial diets by the homogenous ones (93.13%). Homogeneous To determine the survival rate it was recorded the number of crossbreds created with the participation of Japanese type breeds hatched viable specimens and the number of silkworms after mass (94.02% mean survival rate) outperform those with the participation awakening from the first sleep calculated in percentages according of Chinese ones (92.25%). Results obtained are unidirectional with to the following formula:the higher receptivity established by Azizov and Gulamova (1982) and Kouno and Ohdachi (1985), of the breeds of Japanese origin compared to those of Chinese origin. In the heterogeneous variants

diet.

Table 1. Criteria for assessing the degree of receptivity of silkworms to artificial diet

Degree of receptivity

Very high

High

Normal

Low

Very low

Survival (%)

Over 90

85 - 90

70 - 85

60 - 70

Under 60

V(%) = x 100, Nn

MP(%) = x 100, F1 - MP

MP

HP(%) = x 100, F1 - HP

HP

294

higher survival rate is typical of hybrids in which the maternal breed results obtained with regard to the duration of the first age. In used is of Japanese type and the paternal one is of Chinese type heterogeneous hybrid forms better results are obtained when the (straight form). maternal breed used is of Chinese type and the paternal one is of

From the data presented in Table 3, it is evident that the duration Japanese type (reciprocal form). Higher values on some traits in the of the development in the first instar in all studied hybrids is shorter or reciprocal variants are reported by Kumar et al. (2011).equal to the generally established 96 hours. The shortest first age Results for heterosis manifestations in F hybrids by the traits 1

(79h) is typical of the hybrid "Vratsa 55 x Merefa 6" followed by survival rate and duration of larval development in the first instar are hybrids "Line 22 x Vratsa 63"; "Vratsa 63 x Line 22" and "VBAS x Nig given in Table 4. The highest heterosis effect in the survival rate by to 2", which have duration of the first age 81 hours. The most extended, the higher parent value (HP) and to the mean parental value (MP) but not more than the generally established is the development in was observed in the Japanese type hybrid "Line 22 x Vratsa 63", specimens of the hybrid forms "Nig 2 x Merefa 6" and "Line 22 x Nig exceeding by 8.02% the better parent (HP) and by 15.01% the mean 2" (96 h). The analysis of the data for development duration shows parental value (MP), followed regarding HP's heterogeneous that 70% of the hybrids have shorter larval development than the straight form "VBAS x Nig 2" (6.28 % for survival and -13.83% at baseline breeds. The superiority of the Japanese type crossbreds instar development). By the survival rate trait the majority of the over those of Chinese origin established by Azizov and Gulamova analyzed hybrids exhibit high heterosis, both as to the mean parental (1982) and Kouno and Ohdachi (1985) and confirmed in the present values (MP), and to the higher parent value (HP). An exception is the study concerning the survival rate of specimens is identical to the hybrid form "Nig 2 x Merefa 6", in which heterosis manifestations are

Table 2. Mean values of the survival (%) in F hybrids and the baseline breeds P and P1 21

No.

1

2

3

4

5

6

7

8

9

10

Line 22 х Vratsa 63

Vratsa 63 х Line 22

Merefa 6 х Nig 2

Nig 2 х Merefa 6

Vratsa 63 х Merefa 6

Merefa 6 х Vratsa 63

Line 22 х Nig 2

Nig 2 х Line 22

VBAS х Nig 2


89.75 ± 3.52

78.84 ± 3.27

94.44 ± 3.40

90.82 ± 3.13

78.84 ± 3.27

94.44 ± 3.40

89.75 ± 3.52

90.82 ± 3.13

91.89 ± 3.69

83.08 ± 3.94

78.84 ± 3.27

89.75 ± 3.52

90.82 ± 3.13

94.44 ± 3.40

94.44 ± 3.40

78.84 ± 3.27

90.82 ± 3.13

89.75 ± 3.52

90.82 ± 3.13

94.44 ± 3.40

96.94 ± 2.49

91.09 ± 2.28

96.12 ± 3.47

88.37 ± 4.00

97.36 ± 1.74

95.80 ± 1.15

95.47 ± 1.39

92.13 ± 3.34

97.66 ± 0.13

96.12 ± 1.35

HybridsP1

Mean ± SE Mean ± SE Mean ± SE

P2 F1

Homogenous crossbreds type "Japanese x Japanese"

Homogenous crossbreds type "Chinese x Chinese"

Heterogeneous crossbreds type "Japanese х Chinese" and "Chinese x Japanese"

Table 3. Average values of the trait of development duration (h) in F hybrids and the baseline breeds P and P21 1

No.

1

2

3

4

5

6

7

8

9

10



Merefa 6 х Nig 2

Nig 2 х Merefa 6



Line 22 х Nig 2

Nig 2 х Line 22

VBAS х Nig 2


98 ± 0.50

93 ± 1.00

119 ± 10.20

94 ± 0.50

93 ± 1.00

119 ± 10.20

98 ± 0.50

94 ± 0.50

96 ± 0.71

92 ± 0.00

93 ± 1.00

98 ± 0.50

94 ± 0.50

119 ± 10.20

119 ± 10.20

93 ± 1.00

94 ± 0.50

98 ± 0.50

94 ± 0.50

119 ± 10.20

81 ± 6.17

81 ± 4.67

92 ± 7.09

96 ± 0.88

94 ± 2.08

82 ± 6.00

96 ± 12.20

94 ± 0.88

81 ± 4.00

79 ± 7.42

HybridsP1

Mean ± SE Mean ± SE Mean ± SE

P2 F1




295

negative (-4.60% compared to MP and -6.43% compared to HP). (MP), which gives reason to believe that higher heterosis effect for The data analysis shows that both by HP and MP, the majority of the duration of development occurs, when for the maternal form hybrids (90%) in F exhibit heterosis effect. In the heterogeneous used a Chinese type, and the paternal is of a Japanese type.1

Data about the heterosis manifestations by HP and MP in F crossbreds higher heterosis manifestations are typical for the 1

hybrids in which the maternal breed used is of Japanese type and hybrids by the survival rate of specimens, grouped by type of the paternal one is of Chinese type. The shorter larval development crossing are graphically presented in Figure 1. The figure shows that is economically more profitable. Therefore, the negative sign of the with the exception of hybrids of the type "Chinese x Chinese", in all data obtained in this case reflects a desired effect of hybridization other groups heterosis effect is manifested both as to the mean (Table 4). parental values (MP) and as to the higher parent value (HP). The

The data presented in Table 4 shows that by MP all F hybrids highest heterosis both compared to MP and to HP is observed in 1

crossbreds of the type "Japanese x Japanese".exhibit heterosis effect in terms of duration of development ranging Graphical information about the heterosis manifestations by from -0.35% to -24.80%. Heterosis effect by HP is observed in 60%

MP and HP in F hybrids, the groups by type of crossbred by the of the tested F hybrids. The straight heterogeneous hybrid forms 11

duration of larvae development is given in Figure 2."Vratsa 63 x Merefa 6" and "Line 22 x Nig 2" compared with their In general, from the data presented in the Figure 2, it is evident reciprocal crossbreds have weaker heterosis manifestations, both

that by larval development duratio, crossbreds type "Japanese x as to the higher parent value (HP), as and to the mean parent value

Table 4. Heterosis manifestations (%) compared to HP and MP on signs survival and larval development duration in F 1

hybrids

*HP - higher parent value; MP - mean parental values

No.

1

2

3

4

5

6

7

8

9

10



Merefa 6 х Nig 2

Nig 2 х Merefa 6



Line 22 х Nig 2

Nig 2 х Line 22

VBAS х Nig 2


8.02

1.50

1.78

-6.43

3.09

1.43

5.12

1.44

6.28

1.77

15.01

8.06

3.77

-4.60

12.38

10.57

5.74

2.04

6.90

8.29

-13.26

-13.26

-2.13

2.48

1.08

-11.83

1.77

0.35

-13.83

-13.77

-15.53

-15.53

-13.62

-9.55

-11.32

-22.64

-0.35

-1.74

-14.74

-24.80

HybridsSurvival

HP, % HP, %MP, % MP, %

Duration




12

10

8

6

4

4.76

11.54

8.33

6.31

4.07

1.44

-0.42

-2.33

HP MP

“Japanese x Japanese" Japanese х Chinese"

“Chinese x Chinese" “Chinese x Japanese"

2

0

-2

-4

Figure 1. Heterosis manifestations by survival rate of F 1

hybrids of different type

2%

0%

-2%

-4%

-6%

-8%

-10%

-12%

-14%

-16%

-13.26

-15.53

0.18

-12.80 -12.19

-6.19-5.74

-11.58

HP MP

“Japanese x Japanese" Japanese х Chinese"

“Chinese x Chinese" “Chinese x Japanese"

Figure 2. Heterosis manifestations by development duration in F hybrids of different type1

296

Japanese" have the highest heterosis manifestations, both Bombyx mori L. to Artificial Diet low in Mulberry Powder. compered to MP and HP. Analysis of the results of the studies show International Journal of Current Microbiology and Applied Sciences, that compered to MP, F crossbreds in all groups by type exhibit high 5, 10-18. doi: http://dx.doi.org/10.20546/ijcmas.2016.505.0021

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Production properties of flax (Linum usitatissimum L.) cultivated in Strumica region, Republic of MacedoniaP. Vuckov, M. Ilievski, D. Spasova, L. Mihajlov, N. Markova-Ruzdić

Quality of grain and flour of foreign bread wheat cultivars (Triticum aestivum L.) under the conditions of south Dobrudzha regionP. Chamurliyski, N. Tsenov, I. Stoeva, S. Doneva, E. Penchev

Breeding programme for developing new sweet cherry cultivars in the Fruit Growing Institute, Plovdiv, BulgariaS. Malchev, A. Zhivondov

Heterosis manifestations by survival and larval duration of F1 Bombyx mori L. hybridsR. Guncheva, M. Panayotov, P. Tsenov, Y. Dimitrova

Reproductive performance of breeding rabbits fed by graded levels of cassava (Manihot esculenta) leaf mealT. Ahemen, I.I. Bitto, O.I.A. Oluremi

Clinical toxicological investigations on acute carbofuran intoxication in quails (Coturnix coturnix)R. Binev, I. Valchev, R. Mihaylov, Y. NIkolov

Efficacy and timing of some new products against pear psylla (Cacopsylla pyri L.) (Hemiptera: Psyllidae): II. SpirodiclofenV. Arnaudov

Biochemical and chemical investigations of pikeperch fingerlings (Sander Lucioperca L.) after wintering A. Ivanova, R. Atanasova

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Effect of fluorescence on the technological characteristics of cocoons at different cooking temperaturesM. Panayotov

Comparative analysis of plane geometric parameters of various types of cow milking parlorsD. Georgiev

Panthaleus major /Duges/ of cereals in BulgariaV. Maneva, D. Atanasova

Selectivity and stability of herbicides and their tank mixtures for the seed yield of sunflower (Helianthus Annuus L.)G. Delchev, T. Barakova

Effect of green manure cover crops on tomato greenhouse production I. Tringovska, V. Yankova, D. Markova

Reclamation of lands disturbed by mining activities in BulgariaI. Kirilov, M. Banov

Fish production and meat quality traits in rainbow trout (Oncorhynchus mykiss) farmed in different production systems St. Stoyanova, Y. Staykov, G. Zelqzkov, I. Sirakov, G. Nikolov

Agriculture and Environment

Product Quality and Safety

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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”.


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