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

Volume 9, Number 1March 2017

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2017

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Editors and Sections

Genetics and Breeding

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

Nutrition and Physiology

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Production Systems

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

Agriculture and Environment

Martin Banov (Bulgaria)Peter Cornish (Australia)Vladislav Popov (Bulgaria)Tarek Moussa (Egypt)

Product Quality and Safety

Stefan Denev (Bulgaria)Vasil Atanasov (Bulgaria)Roumiana Tsenkova (Japan)

English Editor

Yanka Ivanova (Bulgaria)

2017

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

Volume 9, Number 1March 2017

Product Quality and Safety

Accumulation of astaxanthin and canthaxanthin in muscle tissues of Rainbow trout (Oncorhynchus mykiss W.) fed with xanthophyll supplemented feed

M. Tzanova*

Deparment of Biochemistry, Microbiology and Physics, Faculty of Agriculture, Trakia University, 6000 Stara Zagora, Bulgaria

(Manuscript received 17 November 2016; accepted for publication 13 February 2017)

Abstract. In this study the rate of accumulation of both main pigments in Salmonidae - astaxanthin (AX) and canthaxanthin (CX) in different muscle tissues of Rainbow trout (Oncorhynchus mykiss W.) and their distribution in flesh and heart was researched. Experimental fish were reared 100 days in two tanks as two

-1 -1parallel experimental groups and fed with market feed, containing 40 mg.kg AX and 25 mg.kg CX. The diet was according to the manufacturer's recommendations, which are consistent with fish size and water temperature. The accumulated pigment quantities in the muscle tissues, were measured every twenty days by high performance liquid chromatography with photodiode array detection after selective xanthophyll extraction. The highest rates of AX

th th -1and CX deposition were recorded on the 60 and 80 day. The average contents of AX and CX were at the end of the trial 1.943 ± 0.167 mg.kg and 0.807 ± -1 -1 -10.021 mg.kg in skeletal muscle and 0.683 ±0.055 mg.kg and 0.280 ±0.017 mg.kg in cardio muscle, respectively.

Keywords: Salmonidae, Astaxanthin, Canthaxanthin, Flesh, Heart, HPLC-PDA

Abbreviations: AX – astaxanthin, CX – canthaxanthin, HPLC – high performance liquid chromatography, PDA – photodiode array detection, SD – standard deviation

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 9, No 1, pp , 2017DOI: 10.15547/ast.2017.01.013

77 - 82

Introduction demand for Salmon trout on the market is huge. However, salmonids are not able to synthesize carotenoids de novo, but these species are able to absorb carotenoids from their diet. The pigments Global fish production has grown steadily in the last five accumulated in largest quantities are astaxanthin and decades, with food fish supply increasing at an average annual rate canthaxanthin. A combination of both pigments in the diet gives a of 3.2 percent, outpacing world population growth at 1.6 percent higher total carotenoid deposition in the flesh than either one alone.(FAO, 2014). Of the 66.6 million tons of farmed food fish produced in

In 1994 Meyers (1994) reviewed the factors affecting 2012, two-thirds (44.2 million tons) were finfish species grown from carotenoid absorption in fish. Among them are pigment source, form inland aquaculture. Aquaculture has also been promoted in view of and concentration, diet composition, especially fat content, fish size, the fact that it provides consumers with safe, nutritious and high physiological state and stage of sexual maturation, and genetic quality food products (Staykov et al., 2013). High quality background. Astaxanthin absorption also depends on the aquaculture products must fulfill several requirements well concentration used and whether it is provided in its free form or as appreciated by consumers, including the adequate color, which is diester. The effects of abiotic factors, i.e. temperature and salinity among the most important fish quality parameters on the market have also been examined in studies involving immature and mature (García-Chavarría and Lara-Flores, 2013). Color is the first salmonids.characteristic perceived and is the determinant selection criterion,

The fish muscle tissues are responsible for the largest body directly related to the subsequent acceptance or rejection. pool of this carotenoid (Bjerkeng, 2000). The specific pink flesh color Pigments are responsible for the wide spectrum of colors in fish provided mostly by astaxanthin has always been associated with which is an essential prerequisite for quality as they fetch higher salmonids and has distinguished salmonids from other fish species.price on the commercial market.

The aim of this paper was to study the rate of accumulation and Carotenoids are the most widespread and important pigment distribution of the two main pigments - astaxanthin and classes in living organisms.canthaxanthin in different muscle tissues of the most common As for animals, freshwater reared fish in Europe, including Bulgaria, Rainbow trout including fish, carotenoids(Oncorhynchus mykiss W.) fed with xanthophyll supplemented feed.Maoka, 2011). Xanthophyll-

supplemented diet of salmonids is recommended for prevention of reproductive syndrome M74 (Atanasov et al., 2015). In the

Material and methodsaquaculture industry, carotenoids have been included in diets of salmonids and other farmed fish mainly as pigments to provide a desirable coloration to these cultured organisms. Consumers found Study area better organoleptic qualities of the flesh from these fish and the This study is part of a greater experiment of research of

Besides the coloration properties, carotenoids have major biological functions.

are not essential in nutritional sense but they are useful for their health (

77

* e-mail: milenatzanova@hotmail.com

78

deposition of xanthophills in different tissues of Rainbow trout Supply water quality(Oncorhynchus mykiss W.) males and females in different The supply water for the farm facilities was from the upper-physiological states. The experimental fish was reared in a middle stream of Tundzha River. During the experimental period Bulgarian fish farm, situated near Tundzha River and Sredna gora water quality met the requirements of the Bulgarian standard for fish

3 farm (Regulation 44/ 20.04.2006). The data from the self-monitoring Mountain, south central part of the country, in 20 m concrete pools, in the fish farm for the two key quality indices (temperature and under ambient conditions suitable for the normal growth of these dissolved oxygen) are presented in Table 1.species.

Table 1. Water quality by the self-monitoring of the fish farm (Mean ± SD)

*According to Regulation 44/ 2006 (for cold water)

Temperature, °С-1Dissolved oxygen, mg.l

not more than 20

not less than 9.0

14.6 ± 3.1

10.4 ± 0.4

16.0 ± 2.1

9.9 ± 0.3

18.5 ± 1.4

9.2 ± 0.2

17.6 ± 1.8

9.5 ± 0.3

Monitoring periodOptimal*

Fish and feeding of methanol HPLC grade. The methanol extracts were stored The experimental fish were 36 months old males and females overnight at -12°C prior to the HPLC analysis. A small quantity of

with body weight of 829 ± 52 g at the beginning of the experiment. each extract was transferred into a cupped vial and placed in the The fish species were reared 100 days from August to November HPLC system autosampler.2015 in two tanks as two parallel experimental groups, each of them The content of AX and CX in skeletal and cardio muscle tissues comprising 20 specimens. The fish diet contained guaranteed of rainbow trout Oncorhynchus mykiss reared in a Bulgarian fish

-1 -quantities of both xanthophills: 40 mg.kg astaxanthin and 25 mg.kg farm was measured by HPLC-PDA method, developed by Tzanova 1 et al (2016). Analytical HPLC was performed with a C-18 column canthaxanthin delivered by “Aqua Garant”, Austria, a certificated

Hypersil Gold (5µm; 150 mm x 4.6 mm) on a Thermo system European producer. It was chosen because of the following composed of a Surveyor LC Pump Plus, Surveyor Plus, and advantages: extruded and slowly sinking; high content of fish meal, Surveyor photodiode array detector PDA Plus. The mobile phase animal proteins and fish oil of great quality; high digestibility; low methanol:water (97:3 v/v) was filtered through a 0.45 μm membrane water pollution and no content of GMOs, according to Regulation and degased before use. Under isocratic conditions, the analysis (EC) 1829/2003. The used raw materials for its production are: fish

-1meal, animal proteins, fish oil, sunflower concentrate, colza oil, was carried out at a flow rate of 1.0 ml.min at room temperature for 6 wheat, wheat meal, hemoglobin powder and rapeseed expeller. The min run time. Chromatograms were recorded at 474 nm with a diet was according to the manufacturer's recommendations, which photodiode-array detection system.are consistent with the size of the fish and the water temperature. The reference materials astaxanthin (min 97%, HPLC) from

®The fish were fed three times daily, and the average feed ratio was Haematococcus pluvailis and canthaxanthin OEKANAL (min 97%, 1.0 kg per 100 kg fish. HPLC), were purchased from Sigma-Aldrich (St. Louis, MO and

Seelze, Germany, respectively). All solvents – methanol and ®Sampling chloroform CHROMASOLV HPLC grade, ethanol p.a., n-hexane

Three fishes were sampled from each experimental groups at p.a., i-propanol p.a. were also delivered from Sigma-Aldrich. -1the beginning of the trial and every twentieth day of the experiment Deionized water (σ ≤ 0.4 µS.cm ) was used thoroughly.

period. From each fish skeletal and cardio muscle tissue was Stock solutions of AX and CX were prepared by dissolving 2.5 extracted and the prepared samples were immediately frozen and mg of each pigment (weighed to the nearest of ± 0.1 mg) in 25 ml stored for a maximum of 14 days at -12°C prior to the analyses. Xanthophyll extraction was prepared according to the method developed by Schweigert (2009).

Xanthophyll quantification The samples were thawed at room temperature, 2.0 – 2.5 g

were weighed to the nearest ± 0.0001 g, and homogenized with 3 3mechanical tissue homogenizer in 4 cm 1N urea and 10 cm of a

mixture of ethanol-i-propanol-n-hexane (1:2:6 v/v/v) for 3 min in ice obath. The homogenized samples were centrifuged for 2 min at 4 C

at 600 g. The suspended samples were vortexed for 30 s with the solvent mix and centrifuged at the same conditions. This procedure was repeated. The extraction step is crucial for the analysis of these compounds because they are light and oxygen sensitive. The extraction procedure was carried out as quickly as possible in a dark room to minimize xanthophyll exposure to air and sunlight and to avoid compromising the experimental results.

The solvents were removed from the collected supernatants by 3rotary evaporator at 40°C and the dry residue was dissolved in 2 cm

Figure 1. Typical chromatogram of standard solution -1 -1containing 1 mg.l AX and 1 mg.l CX The results in mg.kg

-1were calculated by the formula: Xanthophylls, mg.kg = -1 -1Xanthophylls, mg.l *V*M , where: V is the volume of the

3methanol solution in cm and M is the mass of the sample in g.

-1

AX

2.7283 min

160000

140000

120000

100000

80000

60000

40000

20000

0

-20000

Std_Lppm

PDA 474 nm

CX

4.2256 min

Minutes

79

chloroform (HPLC grade) and stored in dark at –12°C. The standard Windows. All analytical determinations were performed in triplicate solutions used for the calibration were obtained by diluting the stock and the mean values ± standard deviation (SD) were reported. All solutions in methanol yielding a final concentration of 0.10; 1.00; data were statistically compared by one-way variance analysis

-12.00; 4.0 and 10.00 mg.l ) of both, AX and CX. Each of the (ANOVA). The significant level was set as P ≤ 0.05.calibration standards was run triplicate. The correlation coefficients

2(r ) obtained for the regression lines were calculated: 0.9995 for AX and 0.9997 for CX demonstrated an excellent relationship between Results and discussionpeak area and concentration according to the International Conference on Harmonization (ICH) guidelines. Typical Typical chromatograms of sample solutions (row data of the chromatogram of standard solution is illustrated in Figure 1 (the quantity measurement by HPLC) are illustrated in Figure 2. Each of retention times were approximately 2.7 min for AX and 4.2 min for the chromatograms clearly shows the presence of two well CX). separated peaks at 2.7 and 4.2 min. This sufficiently proves the

Statistical analysis existence of the two pigments in the tested samples.The statistical analyses were performed using Statistica 6 for

Figure 2. Typical chromatograms of sample solutions of (A) and (B) - skeletal and cardio muscle at the beginning of the experiment, respectively; (C) and (D) - skeletal and cardio muscle at the end of the experiment, respectively

Fillet_RT_5PDA 474 nm

Fillet_RT_32PDA 474 nm

Heart_RT_33PDA 474 nm

Heart_RT_2PDA 474 nm

← Ax, 2.7312 min

Ax, 2.7309 min ↓

← Cx, 4.2256 min

← Ax, 2.7267 min

← Cx, 4.2198 min

↓ Ax, 2.7343 min

← Cx, 4.2114 min

Minutes

Minutes Minutes

MinutesA

C D

B

600

400

200

0

-200

-400

-600

-800

500

400

300

200

100

0

-100

-200

-300

12000

10000

8000

6000

4000

2000

0

-2000

4000

3500

3000

2500

2000

1500

1000

500

0

-500

-1000

The content of astaxanthin and canthaxanthin in skeletal The average contents of AX and CX were at the beginning -1 -1muscle of Oncorhynchus mykiss is presented in Table 2. 0.202 ± 0.014 mg.kg and 0.037 ± 0.006 mg.kg in skeletal and

Table 2. Content of astaxanthin and canthaxanthin in skeletal muscle of Oncorhynchus mykiss (Significance level, P ≤ 0.05,

n = 3)

DayGroup 1

Mean ± SDGroup 1

Mean ± SDGroup 2

Mean ± SDGroup 2

Mean ± SDAverage 1 & 2

Mean ± SDAverage 1 & 2

Mean ± SD

1

20

40

60

80

100

0.206 ± 0.016

0.263 ± 0.014

0.318 ± 0.022

0.750 ± 0.123

1.585 ± 0.173

1.906 ± 0.170

0.039 ± 0.005

0.074 ± 0.011

0.111 ± 0.012

0.240 ± 0.015

0.742 ± 0.061

0.815 ± 0.018

0.035 ± 0.007

0.075 ± 0.009

0.120 ± 0.017

0.232 ± 0.012

0.766 ± 0.097

0.799 ± 0.025

0.037 ± 0.006

0.075 ± 0.009

0.115 ± 0.014

0.236 ± 0.013

0.754 ± 0.074

0.807 ± 0.021

0.197 ± 0.013

0.259 ± 0.016

0.319 ± 0.018

0.747 ± 0.150

1.716 ± 0.090

1.981 ± 0.190

0.202 ± 0.014

0.261 ± 0.013

0.319 ± 0.018

0.748 ± 0.124

1.650 ± 0.143

1.943 ± 0.167

-1 Astaxanthin, mg.kg -1 Canthaxanthin, mg.kg

80

-1 -1 in the flesh. Presently, on the market feed supplemented with 0.254 ± 0.031 mg.kg and 0.047 ± 0.009 mg.kg in cardio muscle, -1 astaxanthin and canthaxanthin is available, because a combination respectively and at the end of the trial 1.943 ± 0.167 mg.kg and

-1 -1 of the two pigments in the diet gives a higher total carotenoid 0.807 ± 0.021 mg.kg in skeletal muscle and 0.683 ± 0.055 mg.kg -1 deposition in the flesh than either one alone. The quantities of these and 0.280 ±0.017 mg.kg in cardio muscle, respectively (Tables 2

pigments are guaranteed and they are in a form protected from and 3).sunlight and oxidation. To achieve the desired effect of reddish flesh, Data from similar xanthophyll-supplemented diet tests with the producer recommends a diet period of 4 - 6 weeks. Economically other fish species were reported: Garner et al. (2010) carried out a this period is reasonable.carotenoids-supplemented diet trial with female Chinook salmon

The obtained results correspond to those reported by Torrisson Oncorhynchus tshawytscha and the established carotenoids were et al. (1995) and Garner et al. (2010). Comparative results of stored primarily in the muscle early in the breeding season, with a

−1 accumulation of AX and CX in the skeletal and cardio muscle tissues concentration of 9.9 μg g in fish fed a high carotenoid diet compared −1 of rainbow trout Oncorhynchus mykiss) are presented in Figure 3.with 1.9 μg g in fish fed a low carotenoid diet. Torrisson et al. (1995)

Astaxanthin accumulations in salmonid in different muscle have studied the deposition of astaxanthin in flesh of Atlantic salmon tissues at every stage of the experimental period are larger than (Salmo salar L) in relation to dietary carotenoid levels in the 0 – 200

−1 those of canthaxanthin. They conform to the statement by Foss et al. mg kg range and feeding times of 3 – 21 months. The determined −1 (1987) and Torrissen (1989) that astaxanthin is deposited in the flesh amount of astaxanthin in the flesh ranged from 0.7 to 8.9 mg kg at

of rainbow trout Oncorhynchus mykiss more effectively than the termination of the experiment. The recommendation made is, canthaxanthin. Foss et al. (1987) explained this by the better ability Atlantic salmon should be fed astaxanthin-supplemented diets of the muscle actomyosin to bind astaxanthin, and the higher during the whole seawater stage in order to obtain maximal AX level

Table 3. Content of astaxanthin and canthaxanthin in cardio muscle of Oncorhynchus mykiss (Significance level, P ≤ 0.05,

n = 3)

DayGroup 1

Mean ± SDGroup 1

Mean ± SDGroup 2

Mean ± SDGroup 2

Mean ± SDAverage 1 & 2

Mean ± SDAverage 1 & 2

Mean ± SD

1

20

40

60

80

100

0.262 ± 0.021

0.353 ± 0.019

0.398 ± 0.010

0.501 ± 0.008

0.515 ± 0.047

0.694 ± 0.072

0.044 ± 0.009

0.063 ± 0.005

0.088 ± 0.003

0.138 ± 0.017

0.190± 0.017

0.273± 0.013

0.050 ± 0.009

0.068 ± 0.008

0.089 ± 0.014

0.145 ± 0.022

0.188± 0.009

0.286± 0.018

0.047 ±0.009

0.066 ± 0.007

0.088 ± 0.009

0.141 ± 0.018

0.189 ± 0.012

0.280 ± 0.017

0.246 ± 0.042

0.338 ± 0.027

0.409 ± 0.021

0.509 ± 0.019

0.508 ± 0.018

0.672 ± 0.046

0.254 ± 0.031

0.346 ± 0.022

0.404 ± 0.016

0.505 ± 0.014

0.512 ± 0.033

0.683 ± 0.055

-1 Astaxanthin, mg.kg -1 Canthaxanthin, mg.kg

2.0

1.5

1.0

0.5

1 20 40 60 80 100

Ax Flesh

Cx Flesh

Ax Heart

Cx Heart

Accumulation of Ax and Cx in muscle tissues of rainbow troutWilks lambda = 00025, F(20, 90.499) = 50.871, p = 0.0000

Effective hypothesis decompositionVertical bars denote 0.95 confidence intervals

0.0

Day

-1m

g.kg

Figure 3. Accumulation of astaxanthin and canthaxanthin in the skeletal and cardio muscle tissues of rainbow trout Oncorhynchus mykiss

81

metabolic turnover of canthaxanthin. concentration levels have been almost reached (Table 4). The - thThe total xanthophyll content in the cardio muscle (0.301 mg.kg experiment was terminated on the 100 day, because the sexual

1 maturation/spawning migration period started shortly thereafter. ) of salmonid was greater than that in the skeletal muscle (0.239 -1 Garner et al. (2010) and Rajasingh et al. (2006) explain the mg.kg ) at the beginning of the trial (Figure 4). The obtained results

dynamics of asthaxanthin in the different organs of fish including correspond to the higher cell concentration per volume of cardiac th salmonids by thei r metabol ism: dur ing the sexual muscle tissue. This trend continues until the 40 day from the

maturation/spawning migration the astaxanthin is redistributed from beginning of the trial. After that the accumulation of the determined the muscle to the skin and gonads. pigments in the skeletal muscle was ahead of this in the cardio

muscle towards the end of the experimental period: in the cardio muscle the measured total xanthophyll concentration was 0.792

-1 -1 thmg.kg against 2.750 mg.kg in the skeletal muscle. After the 40 Conclusionday, the xanthophyll concentration level continued to increase in the two muscle tissues, but the accumulation rate in the skeletal muscle In this study for the first time in Bulgaria the rate of accumulation was greater than that in the cardio muscle. The measured deposited of the two main pigments in Salmonidae - astaxanthin and quantities of astaxanthin and canthaxanthin in the two muscle canthaxanthin in muscle tissues of rainbow trout Oncorhynchus tissues showed high significance, P<0.001. mykiss (Walbaum) and their distribution in flesh and heart was

Compared to accumulation of AX and CX in the cardio muscle researched. The highest rates of xanthophyll deposition were th th thtissues, this deposition in the skeletal muscle tissues after the 40 recorded on 60 and 80 day. The xanthophyll accumulation after the

-1 thday increased abruptly, from 0.319 ± 0.018 mg.kg to 0.748 ± 0.124 80 day in the skeletal muscle kept going, but not so intensively, -1 -1 -1mg.kg for AX and 0.236 ± 0.013 mg.kg to 0.236 ± 0.013 mg.kg for probably the maximum concentration levels have been almost

CX. reached. A period of 4 - 6 weeks, recommended by the producer, Astaxanthin and canthaxanthin concentration levels in flesh was not sufficient to accumulate the maximum xanthophyll amount

th thwere doubled once again after the 60 day and reached at the 80 in the muscle tissues and the accumulation continues after this -1 -1day 1.650 ± 0.143 mg.kg and 0.754 ± 0.074 mg.kg , respectively. period.

These pigment quantities in the heart remained almost equal, ca -1 -10.50- 0.68 mg.kg for AX and 0.15- 0.28 mg.kg for CX, as if the

saturation of the cardio muscle tissue was achieved. Information Acknowledgmentsabout deposition of astaxanthin and canthaxanthin in the heart of Salmonidae has not been reported until now. Aoi et al. (2003) found This work is part of the Project No 11.G.15 “Study of new tools to out that astaxanthin shows accumulation in the gastrocnemius and overcome reproductive M74 syndrome in salmonids under the heart of mice from the 3 week supplementation, but exact results contamination conditions with fungicide, mancozeb, containing of concentration levels have not been published. Fassett and heavy metals manganese and zinc”, financially supported by the Coombes reviewed (2012) experimental investigations in a range of Faculty of Agriculture, Trakia University, Stara Zagora, Bulgariaspecies using cardiac ischaemia-reperfusion model demonstrated cardiac muscle preservation when astaxanthin is administered either orally or intravenously prior to the induction of ischaemia. ReferencesObviously, the heart needs to accumulate a lot of astaxanthin. In this

Aoi W, Naito Y, Sakuma K, Kuchide M, Tokuda H, Maoka T, Toyokuni S, Oka S, Yasuhara M and Yoshikawa T, 2003. Astaxanthin limits exercise-induced skeletal and cardiac muscle damage in mice. Antioxidants and Redox Signaling, 5, 139-144. doi: 10.1089/ 152308603321223630Atanasov V, Tzanova M, Valkova E, Krastev B and Dimitrov Zh, 2015. Ecology-biochemical aspects of the reproductive syndrome M74 in salmonids. Agricultural Science, LII, 63-77 (Bg).Bjerkeng B, 2000. Carotenoid pigmentation of salmonid fishes - recent progress. (Cruz -Suárez, L.E., Ricque-Marie, D., Tapia-Salazar, M., Olvera-Novoa, M.A. y Civera-Cerecedo, R., Eds.). Avances en Nutrición Acuícola V. Memorias del V Simposium Internacional de Nutrición Acuícola, 19-22 Noviembre, 2000. Mérida, Yucatán.FAO, 2014. The state of the world fisheries and aquaculture. FAO, Rome.Fassett RG and Coombes JS, 2012. Astaxanthin in Cardiovascular Health and Disease. Molecules, 17, 2030-2048; doi:10.3390/ molecules17022030Foss P, Storebakken T, Austreng E and Liaaen-Jensen S, 1987. Carotenoid in diets for salmonids V. Pigmentation of rainbow trout study it is established that canthaxanthin is also deposed in the and sea trout with astaxanthin and astaxanthin palmitate in cardio muscle during a supplementation diet.comparison with canthaxanthin. Aquaculture, 65, 293-305.Xanthophyll accumulation in the skeletal muscle kept going

th García-Chavarría M and Lara-Flores M, 2013. The use of after the 80 day, but was not so intensive, probably the maximum

3

2.5

-1To

tal c

onte

nt A

x an

d C

x, m

g.kg

2

1.5

1

0.5

01 20 40

Day60 80 100

Flesh Heart

Figure 4. Chart of distribution of astaxanthin and canthaxanthin (summary) in the skeletal and cardio muscle tissues of rainbow trout Oncorhynchus mykiss

82

carotenoid in aquaculture. Research Journal of Fisheries and Regulation 44/ 20.04.2006 on the veterinary requirements housing Hydrobiology, 8, 38-49. of farm animal in Bulgaria, http://lex.bg/laws/ldoc/2135525777 Garner SR, Neff BD and Bernards MA, 2010. Dietary carotenoid (accessed July 18, 2016).levels affect carotenoid and retinoid allocation in female Chinook Schweigert F, 2009. Patent Reg. No WO 2009115352 A1.salmon Oncorhynchus tshawytscha. Journal of Fish Biology, 76, Staykov Y, Atanasov V, Zaikov A and Hubenova T, 2013. 1474-1490. doi:10.1111/j.1095-8649.2010.02579.x, Innovative aquaculture technologies, Collection of scientific works. ICH, Q2B, FDA, Federal Register, 1997. Validation of Analytical Scientific conference with international participation "Innovation and Procedures: Methodology. http://www.fda.gov/downloads/drugs/ development of agriculture in Bulgaria” Stara Zagora, 16-guidancecomplianceregulatoryinformation/ guidances/ucm 17.05.2013 (Bg).073384.pdf (accessed May 20, 2016). Torrissen OJ, 1989. Pigmentation of salmonids: interactions of Maoka T, 2011. Carotenoids in Marine Animals. Marine Drugs, 9, astaxanthin and canthaxanthin on pigment deposition in rainbow 278-293. trout. Aquaculture, 79, 363-374.Meyers SP, 1994. Developments in world aquaculture, feed Torrissen OJ, Christiansen R, Struksnæs G and Estermann R, formulations, and role of carotenoids. Pure and Applied Chemistry, 1995. Astaxanthin deposition in the flesh of Atlantic Salmon, Salmo 66, 1069-1076. salar L., in relation to dietary astaxanthin concentration and feeding Rajasingh H, Øyehaug L, Vage DL and Omholt SW, 2006. period. Aquaculture Nutrition, 1, 77-84. doi: 10.1111/j.1365-Carotenoid dynamics in Atlantic salmon, BMC Biology, 4, 10-23, 2095.1995.tb00022.xdoi:10.1186/1741-7007-4-10. Tzanova M, Argirova M and Atanasov V, 2016. HPLC Regulation (EC) 1829/2003 on genetically modified food and feed. Quantification of Astaxanthin and Canthaxanthin in Salmonidae http://eurlex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2003: Eggs. Biomedical Chromatography, doi: 10.1002/bmc.3852.268:0001:0023:EN:PDF (accessed July 18, 2016).

Review

Genetics and Breeding

Nutrition and Physiology

Production Systems

Agriculture and Environment

Antimicrobial activity of Lactobacillus acidophilus against pathogenic and food spoilage microorganisms: A reviewT. Dinev, G. Beev, S. Denev, D. Dermendzhieva, M. Tzanova, E. Valkova

Heterosis and degrees of dominance of grain yield and grain yield elements in maize hybrids in different groups of ripenessM. Ilchovska

Use of recurrent selection of early flowering in late maize synthetic population. Results of second cycle of breeding.N. Petrovska, V. Valkova

Productivity and adaptability of new genotypes field pea (Pisum sativum L.) cultivated under environmental condition of Southern RomaniaR. Sturzu, A. M. Ene, Cr. Melucă, J. M. Cojocaru

Nitrogen uptake and expense in durum wheat depending on genotype and nitrogen fertilizationG. Panayotova, M. Almaliev, S. Kostadinova

Haematological investigations upon acute intoxication with carbofuran in dogsR. Binev, I. Valchev, R. Russenov, Y. Nikolov

Phytosanitary status and yield of kamut (Triticum turgidum polonicum L.) grown in organic and biodynamic farmingV. Maneva, D. Atanasova, T. Nedelcheva

Hot-water treatment of gladiolus cormels for control of corm-borne fungal diseasesS. Bistrichanov, T. Vatchev, Z. Avramov

Productivity of common wheat (Triticum aestivum L.) grown after various predecessors and nitrogen fertilization ratesM. Gerdzhikova

Agro-ecological assessment of manure from different farm animals by content of biogenic elementsD. Dermendzhieva, G. Kostadinova, G. Petkov, D. Dimov, T. Dinev, T. Penev, Ch. Miteva, J. Mitev

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Screening of cucurbitaceous rootstocks against root-knot nematodes (Meloidogyne spp.) and soilborne pathogens (Fusarium spp. and Pythium spp.) V. Yankova , D. Markova, N. Velkov, S. Masheva

Animal hygiene assessment of microclimate in semi open free-stall barns for dairy cowsD. Dimov, Ch. Miteva, I. Marinov, Zh. Gergovska, Т. Penev, A. Enchev

Accumulation of astaxanthin and canthaxanthin in muscle tissues of Rainbow trout (Oncorhynchus mykiss W.) fed with xanthophyll supplemented feedM. Tzanova

Chemical composition and technological characteristics of wines from red grape varieties, selected in BulgariaV. Haygarov, T. Yoncheva, Z. Nakov, M. Ivanov, D. Dimitrov

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”.

Volume 9, Number 1March 2017

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