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Banat’s Journal of Biotechnology–(Biannual)ISO Abbreviation: Banats J BiotechnolPublication Start Year: 2010 Country of Publication: RomaniaPublisher: Timisoara, RomaniaLanguage: EnglishISSN–L: 2068–4673 (Print), ISSN–CD: 2068–4738 (Electronic), ISSN–E: 2344–4045 (Online)NLM ID: 101564899 [Serial]Contact: Banat’s Journal of Biotechnology, Calea Aradului no. 119, 300645 Timisoara, Romania, web: http://www.bjbabe.ro, e–mail: bjb@usab–tm.ro.Publication of works is FREE (no money).

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Banat s Journal of Biotechnology 2014, V(10)

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CONTENT, Volume 10, Jul.–Dec. 2014

THE FRESHWATER MUSSELS ARE A GOOD BIOINDICATOR FOR MONITORING THE WATER POLLUTION BY HEAVY METAL (THE SAINT–VICTOR–SUR–LOIRE LAKE, FRANCE), Essaid Bilal, Issam Guenolé–Bilal, Moussa Bounahkla, Luisa Elena Iatan, Fernando Machado De Mello, Marc Doumas, Mounia Tahri, Frédéric Gallice, Didier Graillot, Herve Piegay, Najla Lassoued ___5 BREEDING EVALUATION OF FORAGE PEA VARIETIES AND HYBRIDS BY THE TRAITS NUMBER SEEDS AND SEED WEIGHT PER PLANT, Valentin Ivanov Kosev _______________12 SERO–PREVALENCE, AND ASSOCIATED RISK FACTORS OF TOXOPLASMA GONDII INFECTION IN PREGNANT WOMEN AND HIV/AIDS PATIENTS IN SELECTED CITIES OF ETHIOPIA, Anteneh Hailu, Kassahun Negashe, Aweke Tasew, Medhint Getachew, Tesfaye Sisay, Tariku Jibat, Danel Fekadu ______________________________________________________17 HEPATOPROTECTIVE AND GASTRO PROTECTIVE STUDIES OF Terminalia arjuna LEAVES EXTRACT AND PHYTOCHEMICAL PROFILE. Ataa Said, Ahlam M. El-Fishawy, Siham El-Shenawy, Usama W. Hawas, Mohamed Aboelmagd __________________________________30 EVALUATION OF BREEDING POTENTIAL FIELD PEA (Pisum sativum L.) CULTIVARS AND THEIR PROGENIES, Valentin Ivanov Kosev ________________________________________37 OPTIMIZATION OF PHYSICO–CHEMICAL PARAMETERS FOR THE EXTRACTION OF CHEBULINIC ACID FROM MEDICINAL HERBS, Surya Prakash D.V., Meena V. ____________46 Molecular Characterization of Hyalomma dromedarii from Bikaner, India, Harish K. Changal, G. Nagarajan, R. K. Purohit, Shelesh Kumar Swami, S.C. Mehta, K.M.L. Pathak _______________52 STUDY OF THE INFLUENCE OF MICROBIOLOGICAL PREPARATION BIO–ONE IN SPRING FORAGE PEAS (Pisum Sativum L.) UNDER THE CONDITIONS OF ORGANIC FARMING, Ivan Pachev, Tatiana, Malyuk ________________________________________________________59 OVICIDAL EFFECT OF SOME INSECTICIDES ON BRUCHUS PISORUM L. (COLEOPTERA: CHRYSOMELIDAE) EGGS, Ivelina Nikolova ________________________________________65 EFFECTS OF THE MARL ON THE PERFORMANCE, CUTTING YIELD AND MEAT QUALITY OF BROILER CHICKENS, Ouachem, Derradji, Meredef Aissa, Kaboul Nourredine, Ahmed Gaid Zohra, Bakroune Faten, Bensalem Adel __________________________________________________71 BIODIESEL SOLID WASTE AND URBAN SLUDGE SEWAGE AS A SOIL AMENDMENT FOR THE SEEDLINGS PRODUCTION OF EUCALYPTUS CAMALDULENSIS and MORUS ALBA, Mariana Ferreira Rabelo Fernandes, Regynaldo Arruda Sampaio, Essaid Bilal, Jatnel Alonso Lazo, Luiz Arnaldo Fernandes _________________________________________________________77

November, 2014



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THE FRESHWATER MUSSELS ARE A GOOD BIOINDICATOR FOR

MONITORING THE WATER POLLUTION BY HEAVY METAL (THE SAINT–VICTOR–SUR–LOIRE LAKE, FRANCE)

DOI: 10.7904/2068–4738–V(9)–5

Essaid BILAL1, Issam GUENOLÉ–BILAL4, Moussa BOUNAHKLA3, Luisa Elena IATAN1,4, Fernando MACHADO de MELLO5, Marc DOUMAS1, Mounia TAHRI3,

Frédéric GALLICE1, Didier GRAILLOT1, Herve PIEGAY6, Najla LASSOUED7

1Ecole Nationale Supérieure des Mines de Saint Etienne, GSE, CNRS UMR 5600, F42023 Saint Etienne FRANCE; [email protected]

2Département de physiologie, Université de Lausanne–CHUV, Lausanne, SUISSE. 3Nuclear Centre of Energies, Sciences and Nuclear Techniques (CNESTEN), B.P. 1382, R.P.

10001, Rabat, MOROCCO 4Institute Geodynamic "Sabba S. Stefanescu" of Romanian Academy, Bucharest, ROMANIA. 5Institutos de Geociencias, Universidade de São Paulo–USP–Posdoc Research, BRASÍLIA

6UMR 5600 EVS / Site ENS de Lyon 69342 Lyon, FRANCE 7Faculty of Science of Tunis, Department Plant biology, on 1002 Tunis, TUNISIA

Abstract. The study has focused freshwater mussels to determine the risk of water

pollution. We have shown that freshwater mussels are a good bioindicator for monitoring over time this type of water pollution due to their ability to filter water from the river. The entire food chain (freshwater mussels, mollusc, shrimp, fish, birds and humans) is affected by the pollution.

The Saint Etienne (France) has a large industrial and mining history. The end of these activities has given way to industrial wasteland and mine dumps. Leaching by rain industrial and mining waste may generate a very large urban pollution. Rivers (Ondaine and Loire) and lakes (Saint–Victor–sur–Loire) downstream of this zone are highly polluted by heavy metals.

Key words: Heavy metals, freshwater mussels; Bioaccumulation; Bioindicator.

Introduction Due to their industrial and mining

history, several agglomerations in the Rhône–Alpes region have mining and industrial wastes containing several metals pollution sources of groundwater, public health and urban management problem of some sites.

The study of pollution of the Saint–Victor–sur–Loire Lake lacks.

Indeed, considering the difficulty of the collection of valid samples for trace metal analysis in water as well as the low spatial and temporal representativeness of these, use of quantitative bioindicator of the chemical contamination attracts more and more attention in ecotoxicology.

We have elaborated this work to estimate the degree of metal contamination at the level of the Saint–Victor–sur–Loire Lake and Ondaine river mouth (figure 1) this by dosage of seven

trace metals (Cu, Mn, Fe, Co, Cr, Pb and Cd) in fresh water Anodontes mussels.

In fact, freshwater mussels have been found to be suitable indicator species for trace metals GOLDBERG, 1975;

PHILLIPS, 1976; PHILLIPS, 1977; BROWN & LUOMA, 1995;

JUMSHAMN & GRAHL–NIELSEN, 1996 . It is capable to accumulate trace

metals such as cadmium, mercury and lead largely than for example fish and algae JUMSHAMN & GRAHL–NIELSEN, 1996; RIGET et al.,

1996; AIRAS et al., 2004, BUTNARIU et al., 2015 . Context Saint Victor Lake is bordered by two

castles; it is a reservoir of 66 million m3 of volume, 3.65 km2 surface, built in 1957.

Decennial drains in 1977, 1987 and 2000 were not performed.

They were conducted between 2011 and 2012.

Saint Victor Lake is located downstream of Aurec–sur–Loire (Haute–

Banat s Journal of Biotechnology Contact: web: http://www.bjbabe.ro, e-mail: bjb@usab–tm.ro

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Loire) and upstream of Saint–Just–Saint–Rambert (Loire).

The density of carp is low but includes catfish, crayfish and mussels.

In March 1995, the volume of sludge deposited at Ondaine mouth was estimated at 40 000 m3.

The cost of sludge removal was evaluated then at € 2 million.

The total volume of sludge deposited across the Grangent dam was between 1.5 and 2 million m3, which represents 1.3 Mt.

Two–thirds of the sediment would have been deposited by the Loire River and a third by tributaries (primarily Ondaine).

Among the sediments, there were found metal oxides in significant quantities.

The thickness of sediment was about 5 meters at the confluence of Ondaine–Loire.

Water quality is still a major problem for the Saint Victor Lake.

Indeed, during the heart of the great industrial period, Ondaine brought 20,000 tonnes of materials such as iron, manganese, zinc and nickel.

Ondaine is an affluent of the Loire River, crossing several small towns.

The Ondaine River itself has several tributaries:

The Cotatay has long time been renowned for the quality of its waters.

Twenty–two ancient factories located on its shores, ended their industrial activity.

The Egotay’s Ondaine tributary was also renowned for the quality of its waters.

Many laundresses and Unieux washerwomen were washing their line basins using its waters.

Figure 1. Localization of the Ondaine Valley

Since the late 1960, quality of

rivers has deteriorated. In November 2002, Egotay was still

receiving Charles’s area wastewater and ones of Beaulieu town, located in the town of Roche–la–Molière, and domestic

discharges of Bécizieux, small town of Saint–Victor–sur–Loire via a collector of sewage and other agricultural and industrial untreated waste.

The first traces of pollution of the Gampille tributary of the Ondaine were



7

identified in 1964. In 1989, the Agency of Loire–Brittany Basin is committed to Department of the Haute–Loire to stop pollution problems of the Gampille and its tributary Combobert, caused by the waste disposal of Saint–Just–Malmont and the malfunction of the treatment plant.

The causes of "historic" pollution of the Ondaine River have many origins:

– Industrialization of the valley from 1840, receives without any treatment wastewaters from many industrial plants.

– Mine tailing and industrial waste. – Direct discharges of wastewater into

the river. – The small size of the watershed that

provides the preponderance of urban discharges during low flow periods and the excessive and uncontrolled use of pesticides also contributes to the degradation of Ondaine and Loire water quality.

The mines are no longer operating in the Ondaine Valley since 1983.

The basement of valley contains 304 galleries.

Mine wastewater was pumped by

Charbonnages de France Company but rejected in part on bed of the Ondaine water course.

The slag wastes are still important. The management of these

historical slag wastes still constitutes to be a major problem for the region.

For 150 years, many companies operated production of special steels but dairy has accumulated in many slag wastes.

The sources of pollution of river were still potentially important in 2003.

Industrial equipment is not waterproof. Wastes were sometimes stored near watercourses.

A preliminary study by Geosciences & Environment department of Ecole Nationale Supérieur des Mines de Saint Etienne of a one hundred samples showed that the soils of the Ondaine valley are very rich in metal oxides, especially those growing on industrial waste.

Industrial waste and harvested soils are highly reactive (figure 2 and 3) and metal oxides are mobilized and easily pass into solution. Water filled metal oxides exceed safety standards.

Figure 2. Mo versus Cr content from slag, altered slag and soil of Ondaine valley.

Leaching tests conducted in laboratory have shown that water charged resided 3–4 days, depending on location,

before returning to streams and Ondaine River.


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investigation into the reactions of biochar in soil. Soil Research 48(7), 2009. 501–515.

12. Lopes, J.L.W; Guerrini, I.A.; Saad, J.C.C. Qualidade de mudas de eucalipto produzidas sob diferentes lâminas de irrigação e dois tipos de substrato. Revista Árvore, Viçosa, v. 31, n. 5, 2007, 845–853.

13. Maguire J.D. Speed of germination–aid in selection and evaluation for seedling emergence and vigour. Crop Sci. 2. 1962. 176–177.

14. Maia, C.M.B.F. (1999) Uso da casca de Pinus e o lodo biológico como substrato para produção de mudas de Pinus taeda. Boletim de Pesquisa Florestal, Colombo, n. 39, 81–82.

15. Martins, C.C.; Nakagawa, J.; Bovi, M.L. Efeito da posição da semente no substrato e no crescimento inicial das plântulas de palmito–vermelho (Euterpe espirito santensis Fernandes Palmae). Revista Brasileira de Sementes. v.21, n. 1, 1999, 164–173.

16. Meisiner, A.C. Diatomite, Mineral Commodity Summaries. USA. 1981. 46–47.

17. Mello, S.C.; Vitti, G.C. Influência de materiais orgânicos no desenvolvimento do tomateiro e nas propriedades químicas do solo em ambiente protegido. Horticultura Brasileira, 2002, 20: 452–458.

18. Miyazawa, M., Pavan, M.A., Franchini, J.C. Neutralização da acidez do perfil do solo por resíduos vegetais, Informações Agronômicas, Piracicaba: POTAFOS, 2000. 92: 1–8.

19. Oliveira Júnior, O.A. de; Cairo, P.A.R.; Novaes, A.B. de. Características morfofisiológicas associadas à qualidade de mudas de Eucalyptus urophylla produzidas em diferentes substratos. Revista Árvore, Viçosa, v. 35, n. 6, 2011, p. 1173–1180.

20. Simãon, S. Manual de fruticultura. São Paulo: Ed. Agronomia Ceres, 1971. 530pp.

21. Simonete, M.A.; Kiehl, J.C.; Andrade, C.A.; Teixeira, C.F.A. Efeito do lodo de esgoto em um argissolo e no crescimento e nutrição de milho. Pesquisa Agropecuária Brasileira, v. 38, n. 10, 2003. 1187–195.

22. Soares, E.R. Da Rui, T.L.R.; Braz, R.F.; Kanashiro Junior, W.K. Desenvolvimento de mudas de pepino em substrato produzidos com resíduos de algodão e de poda de árvores. In: Encontro Nacional sobre substratos para plantas materiais regionais como substratos, 6, Fortaleza, 2008. Anais. Fortaleza, Ed. Embrapa Agroindústria Tropical/ SEBRAE/CE/UFC.

23. Souza, G.P.; Filgueira, M.; Rosenthal, R.; Holanda, J.N.F. Caracterização de material compósito diatomáceo natural. Cerâmica, 2003. 49, 40–43.

24. Valeri S. V. and Corradini L. (2005) Fertilização em viveiros para a produção de mudas de Eucalyptus e Pinus. In: GONÇALVES J.L.M.; BENEDETTI V, Ed. Nutrição e fertilização florestal. Piracicaba: Instituto de Pesquisas e Estudos Florestais; 2005. 167–190.

25. Wendling, I.; Dutra, L.F. Produção de mudas de eucalipto por estaquia e miniestaquia. In: WENDLING, I.; DUTRA, L.F. Produção de mudas de eucalipto Colombo. Ed. Embrapa Florestas, 2010. Chap.2, 50–80.

26. Wendling, I.; Gatto, A. Substratos, adubação e irrigação na produção de mudas. Viçosa: Ed. Aprenda Fácil, 2002. 160pp.

Received: October 29, 2014 Accepted: November 11, 2014

Accepted: Last modified on November, 25, 2014



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The DQI values Morus Alba seedlings growing in test substrates with sewage sludge and diatomaceous earth, are all higher than the recommended minimum value of 0.2, with high value for T4 and T5 rich substrate diatomaceous earth. Treatments with manure does not meet the desired quality standard (fig. 3).

The DQI is obvious that the morphological parameters’ used to evaluate the quality of seedlings and morphological parameters should not be analyzed separately to determine the quality level.

The Eucalyptus and Morus Alba seedlings produced with test substrates (sewage sludge, diatomaceous earth and commercial substrate) showed similar results to those obtained with the use only commercial substrate.

The Eucalyptus and Morus Alba seedlings have good growth in substrates containing sewage sludge and diatomaceous earth. They show better results than using only the commercial substrate. They can be used up to 50% by volume of the substrate without compromising the seedlings quality with a reduction of cost of seedlings production.

Acknowledgements This work supported by FAPEMIG

(Fundação de Amparo a Pesquisa de Minas Gerais) and the Institute of Agricultural Sciences of the Federal University of Minas Gerais (UFMG), Regional Campus Montes Claros. We also thank the French Embassy and the Rectorate of UFMG for the grant of a visiting professor Essaid Bilal (ENSMSE, France) in the framework of the France–UFMG Academic Chair. The authors are grateful to Prof. Monica Butnariu for their assistance and for useful discussions.

References

1. Alvarez V., V.H; Novais, R.F.; Barros, N.F.; Cantarutti, R.B.; Lopes, A.S. Interpretação dos resultados das análises de solos. In: RIBEIRO, A. C.; GUIMARÃES, P.T.G.; ALVAREZ, V.H. (Org.). Recomendações para o uso de corretivos e fertilizantes em Minas Gerais: 5ª aproximação. Viçosa: Ed. CFSEMG, 1999, 25–32.

2. Caldeira Júnior, C.F.; Souza, R.A.; Santos, A.M.; Sampaio, R.S.; Martins, E.R.

Características químicas do solo e crescimento de Astronium Fraxinifolium Schott em área degradada adubada com lodo de esgoto e silicato de cálcio. Revista Ceres, v. 56, n. 1, 2009, pp 213–218.

3. Caldeira MVW, Peroni L, Gomes D.R., Delarmelina WM, Trazzi PA. Diferentes proporções de biossólido na composição de substratos para a produção de mudas de timbó (Ateleia glazioviana Bail). Scientia Forestalis 2012; 40(93) 15–022.

4. Caldeira, M.V.; Delarmelina, W.M.; Peroni, L.; Gonçalves, E.O.; Silva, A.G. Lodo de esgoto e vermiculita na produção de mudas de eucalipto. Pesq. Agropec. Trop., Goiânia, v. 43, n. 2, 155–163, abr./jun. 2013.

5. Carneiro, J.G.A. Influência dos fatores ambientais, das técnicas de produção sobre o desenvolvimento de mudas florestais e a importância dos parâmetros que definem sua qualidade. In: Florestas plantadas nos Neotrópicos como fonte de energia, Anais Viçosa. 1983. p. 10–24.

6. Daniel, O.; Vitorino, A.C.T., Aloisi, A.A.; Mazzochin, L.; Tokura, A.M.; Pinheiro, E.R.; Souza, E.F. Aplicação de fósforo em mudas de Acacia mangium. Revista Árvore, v.21, 1997, p.163–168.

7. Dickson, A.; Leaf, A.L.; Hosner, J.F. Quality appraisal of white spruce and white pine seedling stock in nurseries. Forest Chronicle, v. 36, 1960, p.10–13.

8. Eloi, C.M.A. Enquadramento das “zonas de vida” de HOLDRIDGE na classificação climática de Minas Gerais. Ms thèse Meteorlogy agrícola, Viçosa Univesity Federal, 2001, 48pp.

9. EMBRAPA. Manual de métodos de análise do solo. 2 eds. Rio de Janeiro: EMBRAPA–Centro Nacional de Pesquisa de Solos. 1997. 247pp.

10. Gomes, J.M. Souza, A.L. DE; Paula Neto, F. DE; Resende, G.C. DE. Couto, L.; Leite, H.G.; Xavier, A.; Garcia, S.L.R. Parâmetros morfológicos na avaliação da qualidade de mudas de Eucalyptusgrandis. Revista Árvore, Viçosa, v. 26, n. 6, 2002. 655–664.

11. Joseph S.D., Camps–Arbestain M., Lin Y., Munroe P., Chia C.H., Hook J., van Zwieten L., Kimber S., Cowie A., Singh B.P., Lehmann J., Foidl N., Smernik R.J., Amonette J.E. An


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Figure 3. Distribution of slag, altered slag and soil of Ondaine valley in CaO–SiO2–Al2O3 diagram. The SiO2/Al2O3 ratio is same in slag, altered slag and soil and CaO/SiO2 ratio

decrease substantially. The composition of soil from slag waste is near glass raw material.

The SiO2/Al2O3 ratio is same in slag, altered slag and soil and CaO/SiO2 ratio decrease substantially to slag at soil.

The composition of soil and altered slag is near glass raw material.

Material and methods The sampling of freshwater mussels

was elaborated at four stations belonging to the mouth of the Ondaine estuary and Saint Victor Lake.

The shells were carefully opened so as not to damage tissues. The samples were conserved in sterilized petri dishes, weighed and stored at a temperature of–18°C. The adopted procedure for preparation of mussels was as follows: lyophilization during 48 hours under vacuum and a temperature of –50°C, and under a pressure of about 4000 bar.

Grinding of each sample in a porcelain mortar until obtaining of a fine powder. Addition of 3 mL of concentrated

HNO3 (69 C) in each bomb to solubilize metals and 1 mL of H2O2 to oxidize the organic matter and digestion at high pressure and high temperature.

Analyses of final solutions were done for all trace elements detected by ICP–AES and Atomic Absorption Spectroscopy in graphite furnace.

The results are expressed in μg/g of dry weight (ppm). Scanning electron microscopy (SEM) coupled with energy dispersive spectroscopy (EDS).

Observations and elemental analyzes were performed on a scanning electron microscope Jeol JSM 6400 coupled to an Oxford EDS analyzer (ENSM–SE). Observations were carried out on secondary electrons mode in case of granular raw unpolished samples and backscattered electron mode. To obtain comparable results, all analyzes were performed under identical conditions with an accelerating voltage of 20 kV.



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Chemical analyzes are semi–quantitative. Mappings XR heavy metals helped to highlight their distributions in mussel shells. Previously, samples were metallized with carbon or gold depending on type of observations and analyzes made.

Results The organic portion of these

freshwater mussels is richer in metals than the shell (Figure 4). The freshwater mussels filter the water of the river and therefore concentrate metals in the organic part. Metal concentrations are above the safety and health standards BILAL, 2013 . Ondaine Valley drains the

major part of the heavy metals that we find in freshwater mussels. The contents of heavy metals in both cases, Loire River and at the mouth of Ondaine are

comparable. The same pattern is found in the freshwater mussels of the Ribeira de Iguape River in Brazil and the freshwater mussels from Sebu River in Morocco.

The processes of accumulation in tissues are extremely complex and depend on several factors, including properties of receiving tissue MAURY &

ENGRAND, 1986, FERENCZ et al., 2012 ; nature of the contaminant and intra and extracellular environment BOWEN, 1966 .

Contrary to fishes, the organs of the freshwater mussels are all exposed to chemical pollutants suspended in water; direct contamination could have the same significance as trophic contamination.

Heavy metals are in relationship with different pollution incidents and continuous leaching of industrial and mining and industrial waste.

Figure 4. SEM image and distribution of heavy metals in shells of freshwater mussels by

XR mapping BILAL, 2013 . Fe, V: yellow, Mn, Mo, Ni: white; Co, Cu: green and Zn: grey.


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of fertilizer must be applied in the production of seedlings.

The speed emergence index of Morus Alba shows (Table 7) no statistical difference between treatments.

Figure 3. Dickson Quality Index (DQI) versus total dry matter (g) of seedlings of Morus

Alba and versus Ca (mg/dm3) from different substrates tests and control substrate. At the stage of the germination and

emergence of seeds, nutrients are not necessary for the reactions leading to the formation stem and root. Only hydration and aeration of the substrate and good porosity of the substrate allows a movement of the air and water favoring the quickest germination SIMÃO, 1971 .

The GPDM/RDM ratios of Morus Alba seedling (Table 7) are not statistically different for all treatments. They indicate that the seedlings have the same probability of survival.

The GPDM/RDM ratio and TDM (fig. 1 and 3) is directly related to the composition of substrate tests strongly

influenced by the availability of water, which favors the flow of nutrients and seedling growth.

The HI ratio (stem height/stem base diameter) of seedlings Morus Alba (fig. 4) shows no statistically significant difference 2.35 to 3.12 (Table 7).

This ratio is less than that observed for the Eucalyptus (7.47 to 9.63) for the same experimental conditions.

The H ratio is likely to depend on the species of seedlings because in spite of the low values, seedlings showed good conditions for adaptation and resistance on the ground (fig. 4).

Figure 4. Dickson Quality Index (DQI) versus H Index (H/D) of Morus Alba seedlings and

pH versus RV root volume of Morus Alba seedlings from different substrates tests and control substrate.



83

The seedlings may be placed on the field after 120 days of growth, with an average diameter stem from 2.65 to 2.95 mm. The emergency speed index of T2 and T9 is statistically equivalent to treatment with substrate control (Table 5). This result differs from that observed by Oliveira OLIVEIRA JUNIOR et al. 2011 ; these authors studied the value of speed emergency index based on mixtures of the substrate with rice husk, cow manure, powdered coconut and fine vermiculite.

Different mixtures had no effect on the speed emergency index of Eucalyptus urophylla. It should be noted that the decline in the rate of seedling emergence is an undesirable feature, because most of the time in the early stages of growth, it makes plants more vulnerable to adverse environmental conditions MARTINS et al., 1999 . The faster root development is important, the better the vegetative development and stability of the plant SOARES et al., 2008 .

The GPDM/RDM ratio showed no statistical difference between the different substrates (Table 5).

However, DQI is more important in the substrate test mixture of sludge sewage and DE (0.17–0.23), whereas it is very low in substrates based on manure and DE. (0.05–0.09) and in the order of 0.12 in the control substrate (fig. 1); we have a good correlation with total dray matter TDM and DQI.

This links up the observations of Caldeira CALDEIRA JUNIOR et al., 2009 and CALDEIRA

et al., 2013 found that on the field the best results in the survival of eucalyptus seedling are those substrates containing sewage sludge. These authors also noted that 2/1 of GPDM/RDM is a good balance for the growth of plants.

In our case, the values are slightly higher but still reasonable for the different substrates tests. We find that the ratio between the height and diameter of the plant did not differ significantly between the substrates tests studied (H in Table 5). Greater the value of this ratio is, the better the ability to survival and establishment of seedlings

in the field CARNEIRO, 1983 . However, this ratio is influenced by the volume pots experience Gomes GOMES et al. 2003 .

The best treatments are those with sewage sludge and diatomaceous earth. The DQI is above the recommended minimum by Gomes GOMES et al. 2002 . These authors argue that DQI should be greater than 0.2. DQI than, the higher the quality standards seedlings. The study of seedlings of Eucalyptus urophylla by Oliveira OLIVEIRA

JÚNIOR et al. 2011 showed higher values for the DQI treatments with manure than what we got.

Morus Alba Yu vc–62 Tests substrates with sewage

sludge and diatoms favor the growth of white mulberry (Morus Alba) seedlings (fig. 3). They are usually better than the control substrate (Table 6).

For all treatments the chemical and physical properties were similar except for pH (Tables 2 and 3), we can consider that sewage sludge played an important role in nutrient level and diatoms have improved physical properties including porosity, water capacity and density of retention substrates tested (fig. 4).

These observations are consistent with the Maia MAIA, 1999 found that sewage sludge should not be used pure, despite its relative fertility CALDEIRA et al.,

2012 , sewage sludge cause compression of the substrate. Diatomaceous earth is an important addition to the growth of seedlings.

All treatments showed levels of potassium (Table 2) very good according to the classification of Alvarez (1999), which explains the high values of stem diameter of seedlings. Valeri and Corradini VALERI and CORRADINI 2005 showed that the potassium regulates the opening of the stomata and promotes the thickening of the stem of the seedling.

According to Daniel DANIEL et al. 1997 , the diameter of the stem is the most appropriate to evaluate the ability of seedling survival. It is also the most widely used to help determine the doses


10

Mo, V and Co gets to control the composition of mussel’s shells. Fe, Mn, Ni appear sporadically (Figure 5) and indicate an occasional pollution.

The striations of these shells show growth and it represents a very good bioindicator to monitor the pollution of water.

Figure 5. Distribution of metals in freshwater Andonata mussels at the Saint Victor Lake, Loire river (Shell 1, organs 1), at the mouth of Ondaine, Shell 2 and organs 2 BILAL, 2013

and freshwater mussels at the Ribeira de Iguape River, organs RIR, Brazil RODRIGUES et al.,

2013 and green: Organs from Sebou River Morocco BOUNAKHLA et al., 2011 .

Conclusions Understanding the phenomenon of

bioaccumulation of heavy metals in living substance is of extremely complex.

This is due to the interpenetration of several parameters, which influence the bioaccumulation namely in general:

– the physico–chemical characteristics of the environment,

– the chemical properties of contaminant and

– the biologic factors of the organism. The freshwater mussels are reliable

as bioindicator and easy to use to track the evolution of water pollution.

This study showed that all measured heavy metals are higher than normally permitted levels and reveal a health hazard for the population.

Notably, Saint–Victor–sur–Loire Lake is a station where take place a lot of nautical activities and swimming from spring to late summer.

In conclusion, the results of this study show the persistent degradation of the Ondaine Valley and Loire River as a result of industrial, mining, agricultural

and domestic discharges, which it would increase if no strategy of protection of this environment by concerned authorities, is developed and implemented.

Acknowledgements Funding for this project was

provided by a grant from the Conseil Régional de Rhone–Alpes and Post–doc grant from CAPES/PNPD.

The authors thank to Ecole Nationale Supérieure des Mines, the Unit of Pollution and Geochemistry of CNESTEN for their technical support.

References

1. Airas, S.; Duinker, A.; Julshamn, K. Copper, zinc, arsenic, cadmium, mercury, and lead in blue mussels (Mytilus edulis) in the Bergen harbor area, Western Norway. Bulletin of environmental contamination and toxicology, 2004, 73(2):276–84.

2. Bilal, E. Environment Hazards: The recharge areas of the Guarani Aquifer in the state of São Paulo and comparison with the Loire and the Rhône basins in the Rhône–Alpes region. 1st International Research



11

Workshop, 13–15 November 2013, Lyon, France.

3. Bounakhla, M.; Bounakhla, Z.; Tahri, M.; Zahry, F.; Zghaid, M.; Doukkali, A.; Benkirane, O.; Saidi, N.; Bilal, E. Evaluation of the Metallic Contamination Impact on 'Mytilus edulis' Mussel at the Level of the Mouth of Sebou's Estuary, Marocco. Carpathian Journal of Earth and Environmental Sciences, 2011, 6(2): 219–214.

4. Bowen, H.J.M. Trace elements in biochemistry. Academic Press, London. 1966, pp. 241.

5. Brown, C.L.; Luoma, S.N. Use of the Euryhaline Bivalve Potamorcorbula Amurensis as a Biosentinel Species to assess Trace Metal Contamination in San Francisco Bay. Marine Ecology Progress Series, 1995, 124:129–142.

6. Butnariu, M.; Goian, M.; Ianculov, I.; Gergen, I.; Negrea P.; Study regarding the influence of the Co (+2) on the development of soy plant and accumulation of some chemical elements (Fe, Mg, Ca, K and P). Rev Chim (Bucharest). 2005, 56(8), p. 837–841.

7. Ferencz, Á.; Juhász, R.; Butnariu, M.; Deér, A.K; Varga, S.; Nemcsók J. Expression analysis of heat shock genes in the skin, spleen and blood of common carp (Cyprinus carpio) after cadmium exposure and hypothermia. Acta Biologica Hungarica, 2012, 63(1), p. 15–25.

8. Goldberg, E.D. The Mussel Watch–A first Step in Global Marine Monitoring. Marine Pollution Bulletin, 1975, 6(7), 111.

9. Julshamn K.; Grahl–Nielsen, O. Distribution of Trace Elements from Industrial Discharges in the Hardangerfjord, Norway: A Multivariate Data Analysis of Saithe, Flounder, and Blue Mussel as

Sentinel Organisms. Marine Pollution Bulletin, 1996; 32:564–571.

10. Maury, R.; Engrand, P. Recherches en écotoxicologie expérimentale sur la bioaccumulation et les transferts de mercure dans un système "sédiment–eau–macrophytes. M.S. Thesis, University of Bordeaux 1, 1986, pp. 320.

11. Phillips, D.J.H. The Common mussel Mytilus edulis as an Indicator of Pollution by Zinc, Cadmium, Lead and Copper. II. Relationship of Metals in the Mussel to those Discharged by Industry. Journal of Marine Biology, 1976, 38, 71–80.

12. Phillips, D.J.H. The Use of Biological Indicator Organisms to Monitor Trace Metal Pollution in Marine and Estuarine Environments–a Review. Environmental Pollution Journal, 1977, 13, 281–311.

13. Riget, F.; Johansen, P.; Asmund, G. Influence of Length on Element Concentrations in Blue Mussels (Mytilus edulis). Marine Pollution Bulletin, 1996, 32, 745–751.

14. Rodrigues, V.; Sigolo, J.; Guimarães, E.; Availability of metals in bottom sediments and freshwater bivalve (Corbicula flumínea and Anondontites tenebricosus)–Ribeira de Iguape River Brazil. In Society for Environment geochemistry and health Symposium, Paris 8–12, 2013. p. 78.

Received: May 28, 2014

Article in Press: September 18, 2014 Accepted: Last modified on November, 17, 2014


82

diatomaceous earth were statistically lower, compared with the control

substrate (fig. 1).

Figure 1. Dickson Quality Index (DQI) versus total dry matter (g) of seedlings of

Eucalyptus Camaldulensis and Ca from different substrates tests and control substrate. Whereas the physical and chemical

properties of the substrates were similar in all treatments (Table 2 and 3), with the exception of pH (fig. 2), the effects observed may be attributed to the higher pH (higher than 6.5) of these substrates.

In more alkaline pH conditions, there may be nutrients and micronutrients are not available which will cause a physiological disequilibrium plants. The pH should be in the range of pH 5.5 to 6.5 to obtain the recommended quality of seedlings. According to Mello and Vitti MELLO and VITTI 2002 and Miyazawa MIYAZAWA et al. 2000 , the precipitation of the solution of aluminum ground causes, among other effects, the increase of pH.

Moreover, the application of manure may cause complexing Al by organic acids released by these materials. The high acidity of the soil caused by the sludge can be associated with the nitrification of ammonium nitrogen and the oxidation of sulphites and feasible production of organic acids during the degradation by microorganisms of the residue SIMONETE et

al, 2003 . If we consider that seedlings good quality must have a minimum stem diameter of 2.0 to 2.5 mm LOPES et al., 2007,

WENDLING and DUTRA, 2010 , the treatment with the substrate containing sludge sewage and diatomaceous earth corresponds to the desired quality (fig. 2).

Figure 2. Dickson Quality Index (DQI) versus H Index (H/D) of seedlings of Eucalyptus

Camaldulensis and pH versus RV root volume of seedlings from different substrates tests and control substrate.



81

Table 6. Means of the variables total height (H), collar diameter (D), number of branches (NB),

number of leaves (NL), root volume (RV), fresh biomass of the above ground part, (GPFM), root fresh matter (RFM), dry biomass of the above ground part (GPDM) and root

dry matter (RDM) seedlings of Morus Alba from different substrates tests and control substrate.

Substrates tests

H D NL RV GPFM RFM GPDM RDM cm mm -- no /seedling Cm3 g

T1 7.49 C 3.04 C 2.75 A 1.00 B 1.00 C 1.45 B 0.35 B 0.43 C T2 7.96 A 3.83 B 2.47 B 3.28 A 1.61 B 2.01 A 0.57 B 0.63 B T3 10.10 A 3.78 B 2.67 B 1.50 B 1.47 B 1.75 B 0.48 B 0.56 B T4 12.38 A 4.46 A 2.48 B 2.92 A 2.27 A 2.51 A 0.83 A 0.78 A T5 11.11 A 4.76 A 2.54 B 3.11 A 2.19 A 2.90 A 0.82 A 0.94 A T6 9.06 B 3.53 B 2.84 A 1.67 B 1.28 B 1.55 B 0.37 B 0.43 C T7 7.96 C 2.90 C 2.86 A 1.25 B 1.03 C 0.99 C 0.29 B 0.23 D T8 8.98 B 3.49 B 2.91 A 1.67 B 1.31 B 1.35 B 0.38 B 0.30 D T9 9.24 B 2.99 C 3.06 A 0.64 B 1.12 C 0.92 C 0.28 B 0.20 D

Means without letters or followed by the same letter are not statistically different according to the Scott-Knott test at 5% probability

Morus Alba Yu vc–62 The heights of rods, the diameter,

number of leaves and the root volume are higher in all the substrates tests than in the control substrate (Table 6).

However, all the morphological characteristics and weight biomass of the seedling in substrates with manure are less than those seedlings growing in substrates tests with sewage sludge and diatomaceous earth.

Seedlings treatments T4 and T5 have the largest diameter 4.46 mm and 4.76 mm respectively (Table 6). All other values are higher than the minimum required (2.0 to 2.5 mm). DQI seedlings Morus Alba is high (Table 7) in the treatment with sewage sludge and diatomaceous earth T4 (0.42) and T5 (0.55) and very low in treatment with manure T7 (0.13), T8 (0.18) and T9 (0.11). Results of the yield of cut up pieces and the meat quality in Table 3.

Table 7. Means Speed Emergency Index (SEI), GPDM/RDM ratio, H index (H/D) and Dickson

Quality Index (DQI) seedlings of Morus Alba from different substrates tests and control substrate.

Substrates tests SEI GPDM/RDM H index DQI T1 2.97 0.80 2.53 0.24 B T2 2.21 0.91 2.74 0.33 B T3 2.62 0.85 2.68 0.29 B T4 2.73 1.09 2.83 0.42 A T5 2.38 0.89 2.35 0.55 A T6 2.73 0.85 2.77 0.23 B T7 1.84 1.40 2.81 0.13 C T8 2.19 1.32 2.65 0.18 C T9 1.95 1.35 3.12 0.11 C

The broilers growth performance of the experimental group are characterized by a significant increase of body weight at slaughter age (+8.5%, P=0.02). Marl improves the feed conversion ratio by a decrease of about 5%.

Discussion and conclusions Eucalyptus Camaladulensis The observed values of the plant

height, diameter of the stem and the fresh and dry weight of roots and shoots in the treatment with a manure and


12

BREEDING EVALUATION OF FORAGE PEA VARIETIES AND HYBRIDS BY

THE TRAITS NUMBER SEEDS AND SEED WEIGHT PER PLANT

DOI: 10.7904/2068–4738–V(10)–12

Valentin Ivanov KOSEV

Institute of Forage Crops–Pleven, Gen. Vladimir Vazov 89 Street, 5800 Pleven, BULGARIA; e–mail: [email protected]

Abstract. Proved is the influence of the factors “genotypes” and “individual measurements”

on the variability of the components of productiveness weight of seeds and number seeds per plant in investigated varieties and hybrids forage pea. Vastly is the influence of the factor “genotypes” in the common variation of the traits–weight of seeds and number seeds per plant as in parents so and in F1 and F2 hybrids. The highest productiveness of weight and number of seeds per plant is obtained in the hybrids Кеrро х Мir from F1 and Мir х E.F.B.33 from F2 that surpass parents.

Key words: breeding, pea, seed productivity

Introduction The productiveness is main task in

the selection programs as biological potentials, but also as capacity for realizing at various combinations the factors of the environment.

The is main parameters are: number fertile nodes and pods per plant, number of seeds per pod and seed weight per plant КУМАНОВ и др., 1988, SAMFIRA et al., 2013 .

As every quantitative trait, which is determined by high number of gennes, they are influenced in high degree by environment conditions.

The success of the selection about these indicators is depended no insomuch on their main exhibition but from theirs optimum that will ensure high yields and qualitative production BOURION et al., 1998 .

The knowledge for the genotype of the parental forms, the conditions of the environment and the interaction between them determines the good choice and direction of selection.

The objective of this study was to determine importance of the genotype and the individual specimen in the general variation of the main productive components and the character of the interrelations between them.

Material and methods The experimental study was

conducted during the period 2009–2011

in second expеrimental field of the Institute of Forage Crops, Pleven.

As parental component are using the following varieties forage pea: spring form Kerpo (P. sativum subsp. Sativum L.) and winter types pea–Pleven 10, Mir and EFB33 (Pisum sativum ssp. sativum var. arvense L.).

These forms were crossed by hand in 2009.

The parental forms (P1 and P2) and first and second generation (F1 and F2) are sown at scheme P1, F1, F2, P2 on a row spacing 20 cm and distance in row 5 cm. Hand planting was applied with depth of sowing 5 cm.

The forage pea is grown by approved technology of the Institute of forage Crops–Pleven.

In the hybridization were included all direct and reciprocals crosses.

The follow quantitative traits was analyzed–number seeds per plant and seed weight per plant (g) of parental components (Р1 and Р2) and crosses of first and second hybrid generation (F1 and F2).

The individual number measurement of the each trait on one plant is respectively: from F1, F2, Р1 and Р2 40 plants.

Data were processed statistically by classical two factors analysis of variance (ANOVA) with using MS Excel for Windows XP.



13

Results and discussion The results of carry out analysis of

variance (Таble 1) show that watched variation of the traits is determined from high level of significance (р 0.001)

inherited talents on included varieties and hybrids forage pea.

The exception made pods per plant (F=1.25254) in F1 hybrids at that differences are not significant for factor “individual measurement”.

Table 1. Two–way analysis of variance on the investigated traits

Parental forms (P1 and P2) Seeds weight per plant Source of Variation SS df MS F P–value F crit Genotypes 1 118,20 3 372,73 147,72 1,265E–39 2,68 Individual measures 898,57 39 23,04 9,13 5,383E–21 1,50 Error 295,23 117 2,52 Total 2 312,00 159 seeds per plant Source of Variation SS df MS F P–value F crit Genotypes 211 073 3 70 357.7 1 016.7 1.350E–83 2.68 Individual measures 40 794.2 39 1 046.01 15.12 2.24E–30 1.50 Error 8 096.53 117 69.20 Total 259 963.73 159 F1 hybrids Seeds weight per plant Source of Variation SS df MS F P–value F crit Genotypes 7 302.21 11 663.84 57.07 6E–77 1.811 Individual measures 679.09 39 17.41 1.50 0.031 1.427 Error 4 989.69 429 11.63 Total 12 970.99 479 seeds per plant Source of Variation SS df MS F P–value F crit Genotypes 271436.3 11 24676 48.4885 2E–68 1.811 Individual measures 24859.53 39 637.424 1.25254 0.1478 1.427 Error 218320.1 429 508.905 Total 514615.93 479 F2 hybrids Seeds weight per plant Source of Variation SS df MS F P–value F crit Genotypes 7619.243 11 692.658 26.436 6.765E–42 1.810 Individual measures 3408.608 39 87.4002 3.33572 6.255E–10 1.427 Error 11240.37 429 26.2013 Total 22268.221 479 Seeds per plant Source of Variation SS df MS F P–value F crit Genotypes 585495.1 11 53 226.8 48.32 3.664E–68 1.81 Individual measures 129155.8 39 3 311.69 3.01 2.143E–08 1.43 Error 472576 429 1 101.58 Total 1187226.9 479

Legend: SS–Sum of squares; df–degree of freedom; MS–Mean square; F–criterion on Fisher; P–value–probability;

The cause for this is probably due of the smaller genetically diversity on included selection material in the study in

this trait. In all others traits for varieties and hybrids criterion F–experimental exceeds the corresponding values of F–


80

All data were subjected to analysis of variance and means were compared by the Scott–Knott test at 5% probability.

Results The pH in the control substrate

Commercial T1 chosen as substrates tests mixtures of T2 to T4 is the same 6.4; it passes to 6.7 in the mixture T5 (33% of DE).

The pH was higher in mixtures with manure between 6.9 and 7.6.

The electric conductivity (1.64 to 1.82 dS / m) in the mixtures T2 to T8 is higher, compared with control substrate (1.54 dS/m).

Except for the case T9, manure mixture, is very low 1.35 dS/m. K is high 880 mg/dm3 in the mixture T5. K remains high in the mixtures Manure (647–990 mg/dm3) compared with the control substrate (249 mg/dm3).

Table 4. Means of the variables total height (H), collar diameter (D), number of branches (NB),

number of leaves (NL), root volume (RV), fresh biomass of the above ground part, (GPFM), root fresh matter (RFM), dry biomass of the above ground part (GPDM) and root dry matter (RDM) seedlings of Eucalyptus Camaldulensis from different substrates tests

and control substrate. Substrates tests H D NB NL RV GPFM RFM GPDM RDM

Cm mm no /seedling Cm3 g T1 22.03 A 2.43 A 1.62 5.12 3.69 2.59 B 1.14 A 0.86 A 0.45 A T2 24.51 A 2.95 A 1.60 5.85 5.42 3.96 A 1.57 A 1.50 A 0.54 A T3 23.25 A 2.65 A 1.75 5.49 4.92 3.83 A 1.39 A 1.41 A 0.54 A T4 20.34 A 2.75 A 1.85 6.08 4.53 4.24 A 1.58 A 1.52 A 0.65 A T5 21.98 A 2.89 A 1.79 5.97 2.72 4.15 A 1.43 A 1.36 A 0.61 A T6 16.55 B 1.78 B 1.65 4.78 2.83 1.89 B 0.65 B 0.55 B 0.27 B T7 16.97 B 2.02 B 1.55 4.57 3.00 1.86 B 0.80 B 0.55 B 0.26 B T8 13.49 B 1.85 B 1.62 5.05 2.44 1.93 B 0.69 B 0.61 B 0.27 B T9 15.36 B 1.59 B 1.66 5.03 1.69 1.57 B 0.58 B 0.39 B 0.20 B

The organic matter (OM), total

porosity, water holding capacity (WHC) and density decrease significantly in mixtures T4, T5 and T9 (table 2 and 3).

Eucalyptus Camaladulensis The main observation is that the

mixture with manure (T6–T9) show

characteristics (Table 4–5) very different from the development of Eucalyptus relative to the mixture with D.E. and Sludge sewage (T2–T5).

The volume of the root is very low in the T5 (2.5 cm3) compared to the control substrate T1 (3.69 cm3) or T2, T3 e T4 (Table 4).

Table 5. Means Speed Emergency Index (SEI), GPDM/RDM ratio, H index (H/D) and Dickson

Quality Index (DQI) seedlings of Eucalyptus Camaldulensis from different substrates tests and control substrate.

Substrates tests SEI GPDM/RDM H INDEX DQI

T1 4.58 A 1.94 9.44 0.12 B T2 4.61 A 2.96 8.33 0.18 A T3 3.09 B 2.68 8.96 0.17 A T4 3.32 B 2.38 7.45 0.23 A T5 3.13 B 2.25 7.66 0.20 A T6 2.22 B 2.05 9.28 0.07 B T7 3.32 B 2.17 8.70 0.08 B T8 2.65 B 2.75 7.72 0.09 B T9 4.85A 2.26 9.63 0.05 B



79

after sowing took place clarified leaving only one plant per pot.

The experiment consisted of two tests, one for each species.

The experimental design was a randomized complete block design with three replications and four pots per experimental unit.

The compositions of the pots were made on April 30 and May 1, 2014.

The plants were sown on May 2. A shade sail was used with a rate

30% and 50% shade and was placed 1.5 m above the ground, oriented east–west to provide shade from the experimental unit. This shade sail was removed 30 days after the lifting of plant.

Both treatments were irrigated three times daily.

Physical and chemical analyzes of different substrates tests and control substrates (commercial substrate Bioplant) were performed at the Laboratory of Solid Waste UFMG following the methodology of Embrapa EMBRAPA, 1997 .

Were determined pH, electrical conductivity (EC), density (ρ), matter (OM), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and potential acidity (H + Al), the density of the particles and the water holding capacity (WHC).

The diameter and height of the

stem of the plants were measured from the 49th day to the 118th day. Starting at thirty days, we calculated the rate of emergence of two cultures.

Where the species reached a height greater than 10 cm and 42 days after planting, we add the ammonium sulphate fertilizer.

The Speed Emergence Index (SEI) was calculated based on the following formula of Maguire MAGUIRE, 1962 :

SEI=E1/N1+E2/N2+ +En/Nn,

Where E1, E2, En = are number of seeds germinated on first, second and nth day, respectively and N1, N2, Nn=are number of days from sowing to first, second and nth count, respectively.

Dickson DICKSON et al. 1960 , we determine total dry matter TDM (g), shoot height H (cm), collar diameter D (mm), dry biomass of the above ground part GPDM (g) and root dry matter RDM (g) following this formula:

DQI = TDM / ((H/D) + (GPDM/RDM)).

The number of leaves per plant was calculated. Fresh weight of shoots, leaves and roots were measured by weighing.

Table 3. Water retention capacity (WHC), density (ρ) and total porosity (TP) of substrate tests and

substrate control (T1). Substrate tests WHC ρ TP mL g-1 g cm3 % T1 0.56 0.35 64.73 T2 0.49 0.46 73.38 T3 0.42 0.47 62.16 T4 0.38 0.55 58.40 T5 0.43 0.55 56.30 T6 0.60 0.43 63.51 T7 0.53 0.43 64.28 T8 0.57 0.46 64.44 T9 0.44 0.50 62.60

After drying in an oven at 65°C for

72 hours to constant weight, the dry weight matter was weighed.

We evaluated the relationship between shoot dry weight and root dry weight.


14

crit. The accurate value of the (P) on sources of variation is lover than 0.05 that show considerable influence of the factors.

Established is that the source of variation “genotypes” is being on first from the factors influencing.

Into variation of the investigated traits. It exceed at sum of the squares the factor “individual measurement” for weight and number seeds per plant as parents so and F1 and F2 (Таble 2).

The factor “genotypes” in F1 proves larger influence in the traits toward F2.

Table 2. Influence of the factors of variation in the morphological traits seeds weight per plant (А)

and seeds per plant (B) of the varieties and hybrids forage pea Source of Variation А B А B А B

Parental forms F1 hybrids F2 hybrids Genotypes 55.45% 83.80% 91.49% 91.61% 69.09% 81.93% Individual measures 44.55% 16.20% 8.51% 8.39% 30.91% 18.07% Total 100% 100% 100% 100% 100% 100%

The productive possibilities of the varieties as regards of weight of the seeds and number seeds per plant are varied from 7 g. (Кеrpо) to 13 g. (Mir and

Pleven 10) and respectively from 29 (Кеrpо) to 116 (Mir) number seeds (Figure 1 A and B).

A B Figure 1. Average value on А–seeds weight per plant and B–seeds per plant in

parental forms The made relative assessment of

the hybrids of the two generations (Figure 2) shows that they are characterized with higher productiveness toward parental components.

This can due of genetic transgression or heterosis actions.

In cases in which the inbred depression in F2 is not significance the explanation must seek in behaviour of genetic transgression.

Established is that in hybrids F1 with the highest weight seeds pre plant are Кеrро х Мir and Кеrро х E.F.B.33, Mir x Kerpo and Pleven 10 x Mir are with the lowest.

The almost same tendency is watched in number seeds per plant. Мir х Кеrрo and Pleven 10 х Мir are with the smallest number of seeds, E.F.B.33 х Мir (188) and Kerpo x Mir (184) have the most seeds per plant. Кеrро х Мir E.F.B.33 х Мir (188) form the most seeds (Figure 2). In F2 is god the larger variation due disintegrating of the trait.

With the highest values as seed weights so and number seeds per plant is characterized Мir х E. F. B. 33.

The observed for hybrids good reproductive ability is import quality for all annual forage leguminous varieties, which except high quality and stability of yield



15

green mass must being have middle or high grain yield.

A

B

C

D 1–Mir х Pleven 10; 2–Mir х E.F.B.33; 3–Mir х Kerpo; 4–Pleven 10 х Mir; 5–Pleven 10 х E.F.B.33; 6–Pleven

10 х Kerpo; 7 E.F.B.33 х Mir; 8–E.F.B.33 х Pleven 10; 9–E.F.B.33 х Kerpo; 10–Kerpo х Mir; 11–Kerpo х Pleven 10; 12–Kerpo х E.F.B.33

Figure 2. Average value on А–seeds weight per plant and B–seeds per lant in F1; C–

seeds weight per plant and D–seeds per plant in F2


78

study aims to evaluate substrate test (DE and sludge sewage) as an alternative for improving the composition of soils for plant production.

Material and methods The study was conducted in the

period May–August 2014 at the Institute of Agricultural Sciences–ICA, of the Federal University of Minas Gerais–UFMG, Montes Claros, located in northern Minas Gerais state, latitude 16°51'38'' S and longitude 44°55'00' W and 647 m of altitude. The climate of the region is semi–arid, hot and dry tropical.

The rainy season is concentrated between Octobers to March. The average annual rainfall is 1060 mm and uneven ELOI, 2001 . The experimental substrate was prepared from different proportions of commercial substrate control (Bioplant), DE, sludge or manure (table 1).

Manure from breeding ICA–UFMG. The sewage sludge from the

treatment plant wastewater Montes Claros–MG.

Diatomaceous earth from the disposition of Petrobras biodiesel plant in Montes Claros, after use as a filtering agent of vegetable oils (soybean).

Table 1. The materials used and their proportions by volume for each substrate tests

Substrate tests (T) T1 = 100% substrate control (Bioplant) T2 = 75% substrate control + 12.5% sludge + 12.5% Diatomaceous earth T3 = 75% substrate control + 8.3% sludge + 16.7% Diatomaceous earth T4 = 50% substrate control + 25% sludge + 25% Diatomaceous earth T5 = 50% substrate control + 16.7% sludge + 33.3% Diatomaceous earth T6 = 75% substrate control + 12.5% manure + 12.5% Diatomaceous earth T7 = 75% substrate control + 8.3% manure + 16.7% Diatomaceous earth T8 = 50% substrate control + 25% manure + 25% Diatomaceous earth T9 = 50% substrate control + 16.7% manure + 33.3% Diatomaceous earth

Diatomaceous earth, before use, was submitted to the combustion process for the removal of residual oil.

Chemical and physical characterization of the substrates is shown in Tables 2 and 3.

Table 2. The chemical characteristics of substrate tests and substrate control (T1)

The pots had a volume of 55 cm3

and the species studied were Camaladulensis Eucalyptus and white mulberry (Morus Alba Yu vc–62).

The seeds of Eucalyptus

Camaladulensis forum obtained from the ICA / UFMG and seeds of white mulberry Morus Alba vcYu–62 were CIA / Cuba.

At the time of sowing three seeds were placed in each pot and 21 days

Substrate tests pH CE P K Ca Mg H + Al SB t T V MO C

dS m-1 - mg dm-3 -- ------------------------ cmolc dm-3 ------------- % --- dag kg-1 --

T1 6.4 1.54 900 249 7.3 5.4 0.91 13.34 13.34 14.25 94 18.57 10.79

T2 6.4 1.72 1.020 497 8.2 3.4 2.32 12.88 12.88 15.2 85 38.44 22.34

T3 6.4 1.68 980 298 9.3 3.0 2.32 13.07 13.07 15.38 85 18.19 10.57

T4 6.4 1.64 960 497 9.6 3.5 1.38 14.38 14.38 15.75 91 13.6 7.9

T5 6.7 1.71 880 895 8.0 3.0 1.39 13.30 13.3 14.69 91 14.6 8.48

T6 6.9 1.82 510 970 7.0 3.6 1.55 13.09 13.09 14.64 89 16.34 9.5

T7 6.8 1.78 960 990 6.0 4.0 1.55 12.54 12.54 14.09 89 16.7 9.7

T8 7.6 1.65 960 846 4.0 4.8 0.85 10.97 10.97 11.82 93 16.34 9.5

T9 7.5 1.35 700 647 4.6 2.8 0.85 9.06 9.06 9.91 91 13.6 7.9



77

BIODIESEL SOLID WASTE AND URBAN SLUDGE SEWAGE AS A SOIL

AMENDMENT FOR THE SEEDLINGS PRODUCTION OF Eucalyptus Camaldulensis AND Morus Alba

DOI: 10.7904/2068–4738–V(10)–77

Mariana Ferreira Rabelo Fernandes1, Regynaldo Arruda Sampaio1, Essaid Bilal1,2,

Jatnel Alonso Lazo3 Luiz Arnaldo Fernandes1

1Instituto de CiênciasAgrárias, Universidade Federal de Minas Gerais, Caixa Postal 135, CEP

39404–006, Montes Claros, MG, BRASIL. 2Ecole Nationale Supérieure des Mines de Saint Etienne, GSE, CNRS UMR 5600, F42023 Saint

Etienne, FRANCE (e–mail: [email protected]) 3Instituto de Ciência Animal, Apartado Postal 24, San José de las Lajas, Mayabeque, CUBA.

Abstract. We examine in this study a possibility of use the biodiesel industry waste as a

source of nutrients for the production and development of seedlings seems like a very good option. We added the diatomaceous earth residues of Biodiesel with sludge from urban wastewater treatment or manure to stimulate the activity of soil bacterial. The Dickson Quality Index (DQI) values Eucalyptus Camaladulensis and Morus Alba seedlings growing in test substrates with sewage sludge and diatomaceous earth are all higher than the recommended minimum value of 0.2. The treatments with manure and diatomaceous earth do not meet the desired quality standard. The Eucalyptus Camaladulensis and Morus Alba seedlings have good growth in substrates containing sewage sludge and diatomaceous earth than only the commercial substrate. They can use up to 50% by volume of the substrate without compromising the seedlings quality with a reduction of cost of seedlings production.

Keyword: diatomaceous earth, Dickson Quality Index, Eucalyptus Camaladulensis and Morus Alba.

Introduction Recently, with the growing demand

for biodiesel in the world, several plants for biofuel production, mainly from vegetable oils, are being installed in Brazil. For the elimination of various impurities, both the first and itself biodiesel material, these industries use filters of various types, particularly those using diatomaceous earth (D.E.) as a filter element. The organic compounds formed from industrial waste can be used as sources of organic matter and nutrients in a substrate Drill WENDLING and GATTO, 2002 .

They increase the water retention capacity by improving aeration of the roots of plants and they increase the availability of nutrients from the growth of beneficial microorganisms. Can interfere with the increase of pH and levels of exchangeable cations of substrates.

To avoid environmental problems associated with deposits of residues biodiesel industry, we can use as a

source of nutrients for the production and development of plants seems like a very good option. The residue D.E. has specific physical properties that can improve the soil properties. The D.E. is a naturally occurring, soft, siliceous sedimentary rock, consisting of shells or frustules seaweed, which has many properties. It is a lightweight material with low–density porous structure with low thermal conductivity and non–toxic MEISINER, 1981; SOUZA et al., 2003 .

At the end of the production, process of biodiesel, it is as a residue impregnated of organic material. We added the DE residues of Biodiesel with sludge from urban wastewater treatment or manure to stimulate the activity of soil bacterial JOSEPH et al, 2009 .

The advantage of this operation is to eliminate both wastes, residues Biodiesel industry and sludge treatment plants. Given the economic and environmental importance of reusing these resources considered as waste, this


16

This reduce the costs for sowing material on making of mix green forage ALEKSIEVA, NAIDENOVA, 2012, GHICA et al., 2012 .

The obtained results show that differences in the components of productivity are due on genetic diversity of varieties and genetic combinations and recombination’s in hybrids. The results are similar with these from other researchers RANALLI et al., 1997, ABDOU et al., 1999,

SHARMA et al., 2000, BUTU et al., 2014 . According to Жученко Жученко 1980 the character of interaction genotype–year, independently from of phenotypic plasticity of organisms is under genetic control. This force bear in mind influence and this factor in variability of investigated traits in creation on more adaptive selection forms. From obtained results can be done the next conclusions:

Proved is influence of factors “genotypes” and “individual measurements” on variability of components of productiveness weight of seeds and number seeds per plant in investigated varieties and hybrids forage pea. Vastly is influence of factor “genotypes” in common variation of traits–weight of seeds and number seeds per plant as in parents so and in F1 and F2 hybrids. The highest productiveness of weight and number of seeds per plant is obtained in the hybrids Кеrро х Мir from F1 and Мir х E.F.B.33 from F2 that surpass parents.

Conclusions From the obtained results can be

done the next conclusions: Proved is the influence of factors

“genotypes” and “individual measurements” on variability of components of productive ness weight of seeds and number seeds per plant in investigated varieties and hybrids forage pea. Vastly is influence of factor “genotypes” in common variation of traits–weight of seeds and number seeds per plant as in parents so and in F1 and F2 hybrids. The highest productiveness of weight and number of seeds per plant is obtained in the hybrids Кеrро х Мir from F1 and Мir х E.F.B.33 from F2 that surpass parents.

References 1. Abdou, A., M. Mohamed, N. Kandeel,

Breeding implications on cultivar–selection in garden pea (Pisum sativum L.) towards enhancing earliness and pod–yield. Assiut Jour. of Agri. Sci. 1999. 30(3): 117–132.

2. Aleksieva A.; Naidenova, G. New lines of spring forage pea (Pisum sativum L.) suitable for green forage, Journal of Mountain Agriculture on the Balkans, 2012. 15(5):1057–1066.

3. Bourion, V.; Duparque, M.; Munier–Jolain, N.; Lejeune–Henaut, I. Genetic variability of development rates in pea (Pisum sativum L.). 3rd European conference on grain legumes, 1998. 192–193, 3 ref.

4. Butu, M.; Rodino, S.; Butu, A.; Butnariu M. Screening of bioflavonoidand antioxidant activity of Lens culinaris Medikus. Digest Journal of Nanomaterials and Biostructures. 2014. 9 (2), 519–529

5. Ghica, A.; Samfira, I.; Butnariu, M.; Corneanu M. The morphological features influence on Phalaris arundinacea green mass and dry matter yield. Journal of Biotechnology. 2012, 161, 35.

6. Ranalli, P.; Di Candilo, M.; Fdeti, V. Perfomance in northern Italy of pea breeding lines for dry seed yield. Adv Hort Sci, 1997. 11, 85–90.

7. Samfira, I.; Gaitin, D.; Butnariu M. The microelements dynamics in a collection of varieties of Alfalfa in the Romanian Banat plain conditions. Current Opinion in Biotechnology. 2013, 24.

8. Sharma, T.; Sharma, J. Stability for green pod yield in garden pea (Pisum sativum). Indian Jour of Agri Sci, 2000. 70, 40–41.

9. Ward, J.H. Hierarchical grouping to optimize an objective function. Journal of American Statistical Association, 1963. 48, 236–244.

10. Жученко, А. Екологическая генетика культурных растений. Кишинев, 1980. 587 in Bulgarian .

Received: June 17, 2014 Article in Press: September 19, 2014




17

Sero–Prevalence, and Associated Risk Factors of Toxoplasma gondii

Infection in Pregnant Women and HIV/AIDS Patients in Selected Cities of Ethiopia

DOI: 10.7904/2068–4738–V(10)–17

Anteneh HAILU1*, Kassahun NEGASHE2, Aweke TASEW2, Medhint GETACHEW2,

Tesfaye SISAY1, Tariku JIBAT3, Danel FEKADU4

1Department of Medical Biotechnology, Institute Biotechnology, Addis Ababa University, P.O.Box

1176, Addis Ababa, ETHIOPIA; e–mail: [email protected], 2African Medical and Research Foundation [AMREF–Ethiopia], P.O.Box 20855/1000, Addis Ababa,

ETHIOPIA 3Addis Ababa University, College of Veterinary Medicine and Agriculture, P. O. Box 34, Debre zeit,

ETHIOPIA 4Addis Ababa University, College of Health Sciences, School of Medicine, P.O.Box1170, Addis

Ababa, ETHIOPIA

Abstract: Toxoplasma gondii infection is main cause of abortion, congenital defects and fatality in pregnant women and HIV/AIDS infected individuals respectively. The seroprevalence and risk factor assessment of toxoplasmosis in pregnant women and HIV/AIDS infected individuals in Addis Ababa, Jinka, Mojo and Awash towns of Ethiopia was conducted in this study. The study also assessed knowledge and perception of health professionals including physician, nurses and gynecologist, working in antenatal care in selected health institution of different parts of Ethiopia. A cross–sectional study was conducted from October 2011 to March 2012. Sera of 293 pregnant women and 190 in HIV/AIDS infected individuals were analyzed by serological method called Indirect Enzyme Linked ImmunoSorbent Assay [ELISA]. The total seropostivity of IgG and IgM T. gondii were 89.9% and 28.3% respectively. From 293 pregnant women of study 9.5% were IgG negative and IgM positive and 25.5% were both IgG and IgM positive, which means 28.6% of pregnant women had detectable IgM antibodies during pregnancy. Univariate logistic regression analysis showed that study areas, age, residential places, educational status, HIV/AIDS status, ART utilization, pregnancy status, number of pregnancy, stage of pregnancy, history of abortion and number of abortion, cat at home, contact with cat, separate cat house, raw milk and vegetables consumption were significantly associated with seropositivity of T. gondii. From health professionals, 63% know health risk of domestic animals like cat with regards to toxoplasmosis. Consumption of raw or undercooked meat and vegetables (71.2%) were recognized as common source of T. gondii infection by health professionals consumption of raw milk (18.4%), contact with cat faces (14.3%), and drinking unboiled water (6.1%) were also mentioned as important modes of transmission. In this study, 52.7% of health professionals thought toxoplasmosis as important pathogen in HIV infected patients and pregnant women. Seropositivity of T. gondii infection in HIV/AIDS infected individuals and pregnant women in Addis Ababa, Jinka, Mojo and Awash towns of Ethiopia is high. Abortion, exposure to cat faces and HIV/AIDS status are main determining risk factors to acquire T. gondii infection in study population. There is also urgent need of incorporating zoonotic diseases concept in medical education and training. These findings are helpful for optimal design of strategies in contribution of health professionals in relation to toxoplasmosis, pregnant women and immunocompromised individuals. Furthermore, health Education, screening pregnant women during their antenatal care and in depth epidemiological studies are recommended.

Key words: Cats; HIV/AIDS; Pregnancy; Health professionals; T. gondii, Zoonosis

Introduction Toxoplasmosis is one of major

global zoonotic diseases TORGERSON and

MACPHERSON, 2011; PETERSEN et al, 2010 . Toxoplasma gondii can be transmitted to human after consuming raw or

undercooked meat, by ingesting cat shed oocysts via contaminated soil, food or water, or congenitally by transplacental transmission of tachyzoites TORGERSON and

MACPHERSON, 2011; DUBEY, 2010 . Congenital toxoplasmosis is a major


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absorbents in diets for broiler chickens.Anim. Feed. Sci. Technol., 2007. 132:103–110.

22. Parasini, P.; Sardi, L.; Panceroli, A.; Coppa,C.;. Effetti della sustituzione con sepiolite di parte de la mangime nell’alimentazione del suino pesante. Atti 10 Congresso Nazional ASPA., 1993. 459–464.

23. Quentin, M.; Bouvarel, I.; Bastianelli,D.; Picard,M.; Quels besoins du poulet de chair en acides aminés essentiels. INRA. Prod. Anim. 2004. 17:19–34.

24. Salari, S.; Kermanshahi, H.; Moghaddam, H.N.; Effect of sodium bentonite and comparison of pellet vs mash on performance of broiler chicken. Int. Jour. Poul. Sci. 2006. 5(1):31–34.

25. Tauqir, N.sA.; Sultan, J.I.; Nawaz, H.; Effect of Different Levels of Bentonite with Varying Energy Levels on the Performance of Broilers. Int. Jour. Agri. Biol., 2001. 3(1):85–88.

26. Trckova, M.; Matlova, L.; Dvorska, L.; Pavlik, I.; Kaolin, bentonite, and zeolite as feed supplements for animals: health advantages and risks. Vet. Med. Czech. 2004. 49 (10):389–399.

27. Xia, M.S.; Hu, C.H.; Xu, Z.R.; Effects of Copper–Bearing Montmorillonite on Growth Performance, Digestive Enzyme Activities, and Intestinal Microflora and Morphology of Male Broilers. Poult. Sci. 2004. 83:1868–1875.

Received: September 30, 2014 Accepted: October 29, 2014




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filets du poulet de chair. 6 eme

Journées de la Recherche Avicole. St. Malo, 30&31 mars 2005. 234–238.

3. Castaing, J.; Larroudé, P.; Peyhorgue, A.; Hamelin, C.; Maaroufi, C.; Incidence de deux niveaux d’apports en vitamines sur les performances du poulet de chair. 5 eme Journées de la Recherche Avicole. Tours, 26&27 mars 2003. 130–133.

4. De Vries, M.; Kwakkel, R.P.; Kijlstra, A.; Dioxins in organic eggs: a review. Netherlands Journal of Agricultural Sciences., 2006. 54(2):207–221.

5. Doussat, Y.; Bouvarel, I.; Relandeau, C.; Magdelaine, P.; Catteau, H.; Raisonner les teneurs en lysine des aliments poulet en vue d’optimiser le résultat économique de la filière.2005. 6 eme Journées de la Recherche Avicole. St. Malo, 30&31 mars 2005. 189–193.

6. Gigaud, V.; Le Bihan–Duval, E.; Berri, C.; Facteurs de variation de l’aptitude à la transformation de la viande de volaille. 8 eme Journées de la Recherche Avicole. St. Malo, 25&26 mars 2009. 124–131.

7. Guerder, F.; Parafita, E.; Debut, M.; Vialter, S.; Première approche de la caractérisation technologique de la viande de poule pour une valorisation innovante. 8 eme Journées de la Recherche Avicole. St. Malo, 25&26 Mars 2009. 507–511.

8. Haddad, S., L’argile dans un aliment émietté: Conséquences sur les performances de croissance du poulet de chair. Mémoire Ingénieur Agronome. Dépt Agro Univ de Batna, Algérie., 2009.65 p.

9. Hesham, M.T.; Ali, A.H.; Yehia, A.H.; Efficiency of Kaolin and Activated Charcoal to reduce the toxicity of low Level of Aflatoxin in Broilers. Sci. Jour. King Fayçal Univ, 2004. 5:145–160.

10. Jlali, M.; Sibut, V.; Sellier, N.; Tesseraud, S.; Metayer, C.S.; Gigaud, V.; Duclos, M.; Le Bihan–Duval, E.; Berri, C.; Moduler les réserves en glycogène musculaire via l’alimentation pour améliorer la qualité de la viande de poulet. 8 eme Journées de la Recherche Avicole. St. Malo, 25&26 mars 2009. 512–516.

11. Kidd, M.T.; Fancher, B.I.; Lysin needs of starting chicks and subsequent effects during the growing period. Jour. Appl. Poult. Res., 2001.10:385–393.

12. Katsoulos, P.D.; Roubies, N.; Panousis, N.; Christaki, E.; Karatzanos, P.; Karatzias, H.; Effects of long–term feeding dairy cows on a diet supplemented with clinoptilolite on certain hematological parameters. Vet. Med. Czech. 2005. 50(10):427–431.

13. Luca, S.; Giovanna, M.; Fernando, E.; Paola, P.; Paolo, P.; The effects of sepiolite–SPLF on piglet and heavy pig production. Ital. Jour. Anim. Sci., 2004. 3:225–234.

14. Mallet, S.; Delord, P.; Juin, H.; Lessire, M.; Effet d’une supplémentation de l’aliment en Talc (STEALIM®) sur les performances du poulet de chair. 6 eme Journées de la Recherche Avicole. St. Malo, 30&31 mars 2005. 254–258.

15. Mekaoussi, S., L’argile source biologique dans le régime du poulet de chair. Mémoire Ingénieur Agronome. Dépt. Agro. Univ de Batna, Algérie, 2007. 52 p.

16. Nowar, M.S.; Ouadia, L.; Harb, M.; Chakib, A.; El Fatafta, A.; Al Shawabekh, K.; Khoury, H.N.; Sayed, K.; Effect on feeding Jordanian clays on digestibility and daily feed intake to lambs. Final report of clay. Univ of Oman. 1989. 18–19.

17. NRC. Nutrients Requirements of Poultry, National. Acad. Sci, National Research Council. 1994. 177 pp.

18. Ouachem, D.; Soltane, M.; Kalkil, T.; Mekaoussi, S.; Abdessemed, F.; Soualah, Z.; Berghouti, F.; Yakhlef, I.; La marne un produit naturel dans le régime du poulet de chair: conséquences sur les performances et l’état des fientes. 8 eme Journées de la Recherche Avicole. St. Malo, 25&26 mars 2009. 507–511.

19. Ouachem, D.; Soltane, M.; Hadjar, A.; Bakroune, F.; Kalkil, T.; Bensalem, A.; Smaili, A.; Haddad, S.; Abdessemed, F.; Effects of the marl on the performances of chicken feeding starting diet containing acid oil. Banat’s Journal of biotechnology, 2011. II(3):3–6.

20. Ouhida, I.; Perez, J.F.; Gasa, J.; Sepiolite (Exal) decreases microbial colonization in the gastrointestinal tract of young broilers fed barley–wheat based diets. Arch. Zootech., 2000c. 49:501–504.

21. Pasha, T.N.; Farooq, M.U.; Khattak, F.M.; Effectiveness of sodium bentonite and two commercial products as aflatoxin


18

problem in most communities with a high prevalence of T. gondii infection.

T. gondii infection in pregnant women can result in still birth, congenital defects, such as microcephalus, congenital defects and chorioretinitis.

Trans–placental transmission mostly occurs during acute maternal infection. In newborns, T. gondii infection is asymptomatic REMINGTON et al., 2006 .

In immunosuppresed patients deaths due to Toxoplasmosis usually results from rupture of cysts that lead to continued multiplication of tachyzoites.

Hence, encephalitis is reported to be predominant sign and symptom of toxoplasmosis in HIV/AIDS patients and is known to be due to reoccurrence of latent infection MONTOYA and LIESENFELD, 2004; DUBEY,

2010 . Detection of T gondii antibodies in sera of pregnant women and HIV/AIDS patients allows its magnitude and those at risk of getting infection. A study shows magnitude of toxoplasmosis in Ethiopia population ranges from 20.2% to 97.7% ESHETE et al., 1994 . But still no adequate study has reported its prevalence in pregnant women of study areas in Ethiopia.

Because of asymptomatic nature of primary toxoplasmosis infection, counseling of pregnant women is of paramount importance to reduce risk of fetal infection. Knowledge of most likely sources of infection in a given population is a prerequisite for development of effective strategies to decrease, and perhaps eliminate, infection risks.

Effective counseling for prevention requires knowledge of risk factors associated with transmission of parasite.

Knowledge of life cycle of T. gondii is necessary to understand how to advice women to decrease their risk of primary toxoplasmosis KRAVETZ and DANIEL, 2005; JONES et

al., 2003 . The knowledge and perception of

health professionals towards toxoplasmosis is best tool to reduce risk of infection in human. In few surveys carried out elsewhere among obstetricians, a deficit in knowledge about diagnostic, clinical, and epidemiological aspects of toxoplasmosis was

demonstrated, with inherent risk of inadequate management LAURA BERRIEL da

SILVA et al., 2011 . Alvarado–Esquivel ALVARADO–

ESQUIVEL et al., 2011 indicated that physicians surveyed showed an incomplete knowledge about diagnosis and treatment of toxoplasmosis. A survey showed that vast majority of obstetricians counseled pregnant women on avoiding cat litter and undercooked or raw foods, but fewer provided counseling on safe gardening and over 50% responded that keeping a cat outdoors would lower risk of toxoplasmosis JONES et al., 2001 .

Education of obstetricians, nurses and physician on risk factors for toxoplasmosis transmission is needed and may lower rate of congenital toxoplasmosis as well as decrease frequency of cat abandonment during pregnancy. There are few studies addressing degree of knowledge on toxoplasmosis of health professionals.

In these regards, assessing knowledge and perception of health professionals towards to toxoplasmosis in Ethiopia is very crucial but no similar study conducted in country before.

Lack of adequate studies on sero–epidemological pictures, potential risk factors and knowledge and perception of health professionals towards toxoplasmosis in country justified importance of this study.

In Ethiopia, causes of most abortions, stillbirths and neonatal mortalities in human are unexplored and relationship with seroprevalence of toxoplasmosis not well studied.

In addition, segmented or not well known on epidemiology and public health importance of T. gondii infection in Ethiopia. Particularly importance of this disease in pregnant women and HIV/AIDS infected individuals is not studied well in Ethiopia.

Besides, there is tradition of eating raw meat, unavoidable contact between humans and small ruminant’s animals and very high prevalence of HIV/AIDS.

Therefore, objectives of this study were to determine prevalence and identify potent risk factors associated with T.



19

gondii infection in pregnant women and HIV/AIDS infected individuals in Addis Ababa, Jinka, Awash and Mojo towns of Ethiopia. This study will also explore knowledge and perception of health professionals working in antenatal care in selected health institution in mentioned towns.

Material and methods 2.1 Study Areas and Population The study was done at selected

health facilities of Addis Ababa, Jinka, Mojo and Awash towns of Ethiopia from October 2011 to March 2012. Addis Ababa, capital city of Ethiopia, is described by a biannual rainfall. Jinka is capital of South Omo Zone of South nation, nationality people. Mojo and Awash are located in East Shoa Zone 74 and 202 kms from Addis Ababa respectively.

2.2 Study Population The current study done on pregnant

women and HIV/AIDS patients who were visiting health institution for seroprevalence and risk factor assessment were all patients who were visiting health institution for follow up of antenatal care services.

Blood samples were taken from pregnant women and HIV/AIDS patients who were visiting selected health institution of respective study areas for antenatal follow up or medication.

For assessment of knowledge and perception of health professionals, we considered health professionals who are gynecologist, physician and nurses working in prenatal and Anti–Retroviral Treatment services of selected health institution of different parts of Ethiopia.

Therefore, in current study 100 health professionals were included for knowledge and perception study towards toxoplasmosis.

Those health professionals who are physician, nurses and gynecologist and had more than or equal to two years’ work experience were included in study but others health professionals except aforementioned health professionals who had less than two years work experience were excluded.

2.3 Study Design The cross–sectional study was

conducted from October 2011 to March 2012 by using design. The formula on Thrusfield (2007) formula was used to calculate required sample size According to Yimer [YIMER et al., 2005] prevalence of T. gondii infection in different parts of Ethiopia was 74.4% and according to prevalence of T. gondii infection in HIV infected was 93.3%. Accordingly, 293 sera of pregnant women were included in this study. The calculated sample size was distributed to four towns using a proportional allocation based on annual pregnant women flow to respective selected health institution. In addition, calculation resulted 156 samples from HIV/AIDS infected individuals. However, increase representativeness, sample size was inflated by 20% to enhance precision of study. Therefore, a projected sample size of 190 HIV infected patients was used to collect sera samples.

2.4 Blood Specimen Collection and Transportation Method

Five ml of Blood Specimen were collected from study population by using sterile plain Vacutainer tubes [BD Vacutainer

systems, Plymouth, UK]. Specimen were collected from eight

health facilities namely Addis Ababa Black line specialized and referral hospital, Kebena health center, Jinka hospital, Jinka health center, Awash Health Center, Mojo health center, werrer health center]. At room temperature Blood samples were put overnight to allow clotting and centrifuged for 10 minutes at 3000 rpm. Finally sera were collected in Eppendorf tubes [Eppendorf–AG, Hamburg, Germany] and stored at 4°C for 48–78 hours until transported in an ice box to Microbiology laboratory of college of veterinary medicine and agriculture, Addis Ababa University, where they were kept at –20°C until tested.

2.5 Indirect Igg and Igm ELISA Serologically serum was tested for

presence of anti– T. gondii IgG and IgM antibodies by indirect enzyme linked immunosorbent assay [ELISA] kit [Demeditec

Diagnostics GmbH, Germany]. According to manufacturer instruction kit has reported


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The Enhancement in growth performance was explained by the positive effects of antimicrobial [XIA et al., 2004] and detoxifying [PASHA et al., 2007] clay properties on the gut pathogenic flora and through improving digestive efficiency and nutrient absorption in the gastrointestinal tract [OUACHEM et al., 2009].

The response on growth rehabilitates muscle growth observed across the meat yield and in particular, the chicken ready to cook (+9.13%, P<0.001) and the breast (11%, P=0.01).

Results are also in conformity with the positive correlation (+0.73) described by [GUERDER et al., 2009] between the yield of chicken ready to cook and breast meat.

Furthermore, this performance can be assigned to the improved mineral metabolism balance as explained by [CASTAING et al., 2003].

According to these authors, the supply of minerals as premix induces a significant increase of the chicken ready to cook and the breast meat yield.

Thus, it is not excluded that this effect be observed in our conditions.

In fact, clays are considered as a real sieve able to regulate mineral exchanges [OUACHEM et al., in press World Poultry

Science Journal]. Moreover, these yields may be

partially explained by the positive effect of the clay on the digestibility of proteins previously described by [XIA et al., 2004,

OUACHEM et al., 2009 and 2010]. This effect does probably not

exclude improvement digestibility of some amino acids such as lysine.

In fact, according to many authors, the lysine constitutes a limiting factor for yields of breast meat and chicken ready to cook [KIDD and FANCHER, 2001; QUENTIN et al., 2004;

BERRY et al., 2005; DOUSSAT et al., 2005]. Regarding the quality of meat, the

use of marl significantly reduce the rate of abdominal fat (–11.8%, P=0.005).

This confirms the previous results achieved in chicken with marl [OUACHEM et al.,

2009 and 2010] or in pigs fed on a diet containing sepiolite [LUCA et al., 2004].

The reduce of abdominal fat may be explained by a better availability of protein [JLALI et al., 2009].

Moreover, although not significant, the marl effect seems to be better on reducing water losses of breast meat (2.04 vs 2.40) than on the value of the ultimate pH (5.84 vs 5.81).

Anyway, this pH value coincides with the optimum value (5.8) and is situated in the interval (5.7–6.2) required to produce a light–colored meat suitable for processing [GIGAUD et al, 2009].

Indeed, according to these authors, the meat acid with an ultimate pH less than 5.7 have a very pale colour, low water–holding capacity and therefore are poorly suited for processing.

According to the sepiolite effect on producing of light–colored meat reported by [PARASINI et al., 1993], we can retain the possible effect of marl on the ultimate pH and the lightness of meat.

In terms of quality, the combined effects of marl on the breast pH and the decreased drip loss provide an interesting contribution to the meat ability for processing which depends mainly on its water holding capacity.

Conclusions The results of this trial confirm the

marl effects on broilers performance described in previous experiments and highlighted its importance on yield of chicken ready to cook and the breast meat.

Moreover, marl helps to improve the technological meat quality and seems to provide solutions to the current consumer preferences looking for leaner meats and health requirements.

However, to validate these first observations, it would be interesting to pursue additional studies testing amino acid digestibility. References 1. Andrews, P.L.R.; Horn, C.C.; Signals for

nausea and emesis: implications for models of upper gastrointestinal diseases. Qutonomic Neuroscience–Basis & Clinical, 2006. 125:100–115.

2. Berri, C.; Relandeau, C.; Le Bellego, L.; Picard, M.; Effet de la teneur en lysine de l’aliment sur les performances, le pH ultime et les pertes en eau des



73

ultimate pH and the yields of chicken ready to cook (CRC), the breast and the thigh–drumstick.

To assess the chicken breast meat quality, three indicators were used: ultimate pH, the chilling drip loss and the abdominal fat. In order to recover the full fat, abdominal fat were withdrawn after cooling carcasses for 12 hours at 2ºC.

The breast pH was measured according to the method described by [BERRI et al., 2005] by insertion of the pH meter electrode into the thickest portion of the pectoral muscle.

The breast muscle was subsequently weighed, packaged in a polyethylene–zipped bag and suspended by a hook for 4 days at 2ºC.

At the end of chilling, the muscle was dried and reweighed in order to assess the chilling drip loss.

The analytical methods used were those described by [AFNOR, 1985 and CARRE et al.,

2002]. Statistical analysis was carried out

using T–Student test. Values represented in the tables

are the means ±standard deviation. Statistical significance was set to

P≤0.05. Results and discussion The effects of dietary treatments on

broilers slaughter body weight, feed intake and feed consumption ratio data obtained from one to 56 days are presented in Table 2.

Table 2. Growth performances of broiler at 56 days of age (n = 6)

Performances Control Experimental P value

Live Body Weight (g) Feed Intake (g) Feed Consumption Ratio (g/g)

3222b ± 90 6489 ± 230 2.03 ± 0.07

3491a ± 122 6457 ± 213 1.93 ± 0.17

P = 0.02 NS NS

(a, b): The means affected of different letters in the same line are statistically different at the 5% significance level; (NS): Not Significant.

Results of the yield of cut up pieces and the meat quality in Table 3.

The broilers growth performance of the experimental group are characterized

by a significant increase of body weight at slaughter age (+8.5%, P=0.02).

Marl improves the feed conversion ratio by a decrease of about 5%.

Table 3. Yield at slaughter and meat quality of broiler at 56 days of age (n = 6)

Performances Control Experimental P value Slaughter yield (% of LBW) CRC 69b ± 3.09 75.3a ± 1.7 P < 0.001 Breast 18b ± 1.87 20a ± 2.54 P = 0.01 Thigh 14 ± 1.73 15 ± 1.94 NS Drumstick 10.04 ± 0.84 10.8 ± 1.84 NS Meat Quality Abdominal Fat (% CRC) 2.54a ± 0.45 2.24b ± 0.74 P = 0.05 Ultimate pH 5.81 ± 0.08 5.84 ± 0.05 NS Drip Loss (% of breast weight) 2.40 ± 0.13 2.04 ± 0.66 NS

(a, b): The means affected of different letters in the same line are statistically different at the 5% significance level; (LBW): Live Body Weight; (CRC): Chicken Ready to Cook; (NS): Not Significant.

This response confirms the results

observed in broiler chickens at the age of 56 days by [MEKAOUSSI, 2007; HADDAD, 2009] with the same clay type and the same level of incorporation.

Similar effects have also been reported by [NOWAR et al., 1989] with kaolinite, [TRCKOVA et al., 2004; SALARI et al., 2006] with bentonite and [TAUQIR et al., 2001; XIA et al., 2004] with montmorillonite.


20

sensitivity and specificity of 98% and 99%, respectively.

At a wavelength of 450 nm optical densities of wells were measured by a photometer. To be result is positive Values should be higher than cut–off [10IU/mL]. Where as values ±20% of cut–off were equivocal.

2.6 Questionnaire Survey A pretested and organized

questionnaire was used to identify risk factors, which were: sociodemographic variables, host related factors, feeding habit and hygienic condition, study participants awareness about toxoplasmosis and HIV status.

A separate questionnaire was used to collect data from health professionals to collect information on their age, sex, academic qualification, area of work, awareness of toxoplasmosis, exposure of toxoplasmosis case on patient, clinical sign and symptoms, prevalence of case and provision of health education to pregnant women. The other important part of study was questionnaire survey based investigations to assess various risk factors to acquire T. gondii infection in study participants and knowledge and perception of health professionals.

2.7 Data Management and Analysis

Once data were recorded in Microsoft Excel spreadsheet [Microsoft

Corporation], it is transferred and analyzed using STATA version 20.0 for Windows.

By dividing number of serologically positive samples by total number of samples tested seroprevalence was calculated. To identify predictive values of potential risk factors A logistic regression model was employed.

To determine associations between seropositivity and potential risk factors chi–square test was used. Odds ratios and 95% confidence intervals [CI] were also used. Results were considered statically significant at P ≤ 0.05.

2.8 Ethical Clearance The principal investigator were

made brief explanation for all study population about study protocol and after that written informed consents were obtained from all study population.

By using codes confidentiality was maintained. The study proposal ethically approved by Ethical clearance committee from School of Medical Laboratory Sciences Addis Ababa University.

Results and discussion 3.1 Seroprevalence of

Toxoplasmosis The mean age of study population

was 20.4 ± 5.04 years. A total of 483 sera; 293 pregnant women and 190 HIV/AIDS patients were included.

During study, total anti–T. gondii IgG and IgM seropositivity were 89.9% and 28.3% respectively. Tested sera, 137/483 [28.3%] were positive for IgM antibodies suggestive of recent infection.

Among IgM positive study population 28.6% accounts for pregnant women. T. gondii IgG positive and lgM negative results [7%] indicate chronic exposure to parasite infection, whereas IgM positive results were [28.3%].

From serologically tested pregnant women of study 9.5% were IgG negative and IgM positive and 25.5% were both IgG and IgM positive, which means 28.6% of women had detectable IgM antibodies during pregnancy. Most of, 89.8%, women were reactive for IgG and non–reactive for IgM; 25.2% were both IgG and IgM reactive. It can be observed that most of study population had pre–existing infection with parasite [89.8%]. Around 10.1% [seronegative] of study population is has chance of getting infection in future. The prevalence of latent toxoplasmosis [IgG] in Pregnant women and HIV/AIDS patients in study areas were 92.1% in Awash, 91% in Jinka, 90.4% in Mojo and 87.2 % in Addis Ababa.

3.2 T. gondii Seropositivity Risk Factors

Analysis of e risk factors to get T. gondii among study population was done using logistic regression analysis.

Univariate logistic regression analysis indicated that study areas, age (years), residential places, educational status, HIV/AIDS status, ART utilization, pregnancy status, number of pregnancy, stage of pregnancy, history of abortion and number of abortion, cat at home,



21

contact with cat, separate cat house, raw milk and vegetables consumption were

statically significant association with T. gondii seropositivity [P<0.05] (Table 1).

Table 1. Seroprevalence of IgG and IgM anti–T. gondii antibodies in pregnant women and HIV

infected patients in different parts of Ethiopia Sero reaction Total [N=483] Pregnant [N=293] HIV infected [N=190] P–value Positive % Positive % Positive % IgG positive only 434 89.8 256 87.3 178 93.6 0.283 IgG and IgM Positive 122 25.2 75 25.5 47 24.7 0.099 IgG pos and IgM Neg 34 7 28 9.5 6 3.1 <0.001 IgG Neg and IgM pos 15 3.1 9 1.8 6 3.1 0.005 Total seronegativity 49 10.1 37 12.6 12 6.3 0.008

Out of 293 tested pregnant women

87.3% [256] and 28.6% [84] were positive for IgG and IgM T–gondii antibodies respectively. Among IgG positive pregnant women, seropostivity of

IgG antibody increased with duration of pregnancy but that of IgM or recent T. gondii infection was relatively higher in second trimester [Figure 1].

Figure 1. Seroprevalence of T.gondii IgG and IgM in pregnant women and HIV infected

patients in Addis Ababa, Jinka, Mojo and Awash towns of Ethiopia Among IgM positives pregnant

women 24.5 % were in first trimester, 31.2 % second trimester and 24.6% third trimester [Figure 2].

According to current study, seropositivity of T. gondii infection had statistically significant association with stage of pregnancy [P=0.022].

Figure 2. T. gondii seropositivity in pregnant women verses with stage of pregnancy

Using multivariate analysis, study area [P = 0.005], Residential place [P = 0.002], HIV/AIDS status [P = 0.015], Pregnancy status [P = 0.001] consumption of raw vegetables [P = 0.000], Raw meat consumption [P = 0.029], Raw milk consumption

[P = 0.000] and Abortion [P = 0.048] were independent predictors of toxoplasmosis according to Multivariate logistic analysis [Table 2]

From current study, it is also observed that chance of getting T. gondii

infection is enhanced with number of children. The seropostivity of T. gondii


72

were not studied and the scientific information’s on this subject are very few.

This contribution intends to study on chicken broilers, over a rearing cycle of 56 days, the effects of 3% of marl addition on the growth performance, the carcass yield and the meat quality.

Material and methods Diets and Clay During a trial of 56–days, the effects

of two treatments (control without addition of marl and experimental with addition of 3% of marl in dilution) were compared. Diets (Table 1) were prepared according to the NRC (1994) recommendations.

Table 1. Percentual and chemical composition of experimental diets

Starter Grower Finisher Corn 58.0 61.0 63.5 Soybean meal 48 31.0 27.5 25.0 Wheat bran 4.0 5.0 5.0 Marl 3.0 3.0 3.0 Premix 1.0 1.0 1.0 Dicalcium–phosphate 1.90 1.50 1.60 CaCO3 0.90 0.80 0.70 Salt 0.2 0.2 0.2

Nutritional and chemical composition EM (Kcal/kg) 3000 3100 3150 Crude Proteins (%) 21 20 18 Lysine (%) 1.1 0.90 0.70 Methionine (%) 0.42 0.38 0.35 Methi+Cyste (%) 0.85 0.83 0.78 Ca 1.2 0.85 0.65 P 0.75 0.55 0.50

The nutritional characteristics of these foods were: 3000 kcal ME/kg and 21% CP for the starter diet (1–14 days); 3100 Kcal ME/kg and 20% CP for the grower diet (15–42 days) and 3150 Kcal ME/kg and 18% CP for the finisher diet (43–56 days).

The different diet ensures the needs for essential amino acids and minerals of chickens.

The clay used in this trial is a gray marl, used in the artisan making of pottery, abundant in the area of study (Aures–Algeria), it contains 65% of clay, low rate of organic matter (0.6%) and its physicochemical composition (in milli equivalents/100g of soil) is: (Ca2+=4.6); (Mg2+=2.87); (Na+=0.33); (K+=0.1); (Cation Exchange Capacity=20.5).

Animals, Methods and Analysis This experiment was carried out in

an open sided house, in the research poultry unit of Agronomic and Veterinary

Sciences Institute, Batna University–Algeria. A total of 240–day–oldchicks ISA15 commercial broilerwere individually weighed, identified and randomly allocated to two treatments groups (Control and Experimental) with six replicates of 20 birds each group.

From 1 to 56 days, chicks were raised ground on a wood shavings litter.

All chicks (2X120) were given ad–libitum access to feed and water, feed intake (FI), slaughter body weight (SBW) and feed consumption ratio (FCR) were recorded at the end of the trial (d56).

At the age of 56 days, the marl effects on the cut up pieces yield and the meat quality were also determined.

For this purpose, by each repetition, eight broilers having the mean body weight of the group (48 broilers per flock) were slaughtered, bled, feathered and eviscerated.

The carcass were weighed and dissected in order to measure the



71

EFFECTS OF THE MARL ON THE PERFORMANCE, CUTTING YIELD AND MEAT

QUALITY OF BROILER CHICKENS

DOI: 10.7904/2068–4738–V(10)–71

Ouachem, Derradji1,2, Meredef Aissa1;2, Kaboul Nourredine1,2, Ahmed Gaid Zohra1, Bakroune Faten3, Bensalem Adel1

1 Institute of Agronomic and Veterinary Sciences, Hadj Lakhdar University, Batna 05000 ALGERIA

2 Laboratory of Food Sciences, Hadj Lakhdar University, Batna 05000 ALGERIA 3Direction of Agricultural Service of Khenchela, 040000 ALGERIA, [email protected]

Abstract. Following the economic and social pressures that have affected the poultry market, and after prohibition of the antibiotic growth promoter’s use, various natural additives have been proposed as an alternative for the poultry feed industry. Among these products, some clay types or derivatives were used in order to improve the digestive health and to optimize the growth performances. However, studies on the effects of clays on carcass cut up pieces yield and meat quality are unavailable. The aim of this trial is to study in broiler chickens during a rearing period of 56 days, the effects of 3% of marl addition on growth performance (slaughter weight and feed consumption ratio), the carcass and cut up pieces yield and meat quality through three indicators (abdominal fat, ultimate pH and water loss).Results showed firstly, that marl increases significantly the slaughter body weight (+ 8.3%, P=0.02) and provides an enhancement to the feed consumption ratio (–5%). Parallel, marl incorporation significantly improves the ready cook chicken yield (+ 9.13%, P<0.001), the breast meat (+11%, P=0.01) and reduce the abdominal fat (–11.8%, P=0.005). Furthermore, the effect of marl on the ultimate pH was not marked statistically (5.84 vs. 5.81) and water loss of breast muscle was less important at chickens fed with marl diet (2.04 vs. 2.40). Results of this trial suggest that marl can be used as a natural supply to promote an efficient chicken’s portion and to produce a technological meat quality adapted to further processing. Keyword: Marl; Ultimate pH; Yield carcass; Growth performance; abdominal fat; Water loss; Broilers.

Introduction Following the removal of antibiotic

growth promoters and the increase in raw material prices of poultry feeds, as well as new trends in consumer looking for more natural poultry products, various alternatives based on the use of probiotics, prebiotics, plant extracts and enzymes were suggested for the poultry feed industry in order to promote digestive tract health and to optimize the poultry performance.

The clay is a natural product that can be economically used to achieve similar goals.

Indeed, it is naturally abundant, cheap and so widely voluntarily used by the free–range hens or through ingestion of earthworms and insects soil fauna.

As an indication, [DE VRIES et al., 2006] estimated the consumption of soil by lying hens raised in open access to almost 10% of the total dry matter intake.

The spontaneous consumption of clay has been shown in other situations, especially in cases of digestive disorders or for reducing a state of unrest [ANDREWS

and HORN, 2006]. Moreover, studies in recent years

have shown excellent nutritional and antibacterial properties, detoxifying effects and technological aspects of various clay types like bentonite, kaolinite, sepiolite and zeolite [OUHIDA et al. 2000c; HESHAM et al., 2004;

XIA et al., 2004; KATSOULOS et al., 2005; MALLET et al., 2005;

PASHA et al., 2007; OUACHEM et al., 2009 and 2010]. For these reasons, clays are

recommended for their pro digestive properties to increase feed efficiency and healthy digestive tract and for their antitoxic capacity to many undesirable substances in the gut (biogenic amines, mycotoxins, endotoxins).

However, clay effects on the yield of cut up pieces and the on the meat quality


22

infection is 76.6%, 77.7% and 100% in those female study populations who had one, two and three and above children respectively. This difference was found to be statistically significant [p<0.05].

Questionnaire survey showed that all study participants do not have any prior knowledge about toxoplasmosis and health risk of petting cats [Table 2].

Table 2. Potential risk factors T. gondii infection in pregnant women and HIV infected patients by

logistic analysis Variable Category IgG+/total [%] Univariate Multivariate

OR[95% CI] P–value OR[95% CI] P–value

Study Areas

Mojo Addis Ababa Awash Jinka

104/115[90.4] 137/157[87.2] 71/77[92.2] 122/134[91]

– 1.08[0.291,2.371] 5.2[0.209,3.531] 9.58[0.185,3.237]

– 0.760 0.017 0.890

– 0.59[0.251,1.4] 0.34[0.237,1.919] 1.99 0.59 ,1.85]

– 0.005 0.003 0.012

Sex Male Female

34/35[97] 400/448[89]

– 2.66[0.305,23.27]

– 0.375

– 6.5[1.4,11.6]

– 0.68

Age[years] ≤ 24 25–48 >48

131/153[85.6] 285/312[91.3] 18/18[100]

– 2.95[1.46, 5.97] 0.476[0.182,1.245]

– 0.003 0.130

– 4.5[1.4,14.6] 0.35[1.195,6.652]

– 0.112 0.78

Residential place Urban Rural

241/270[88.8] 193/213[90.6]

– 2.88[0.908,0.992]

– 0.05

– 2.62[0.419–4.828]

– 0.002

Educational status

Tertiary Secondary Primary Illiterate

8/14[57] 118/133[88.7] 138/151[91.3] 167/182[92]

– 4.8[1.42,16.48] 5.9[0.48,72.622] 4.9[0.198,12.87]

– 0.012 0.162 0.329

– 2.02[2.127–9.631] 1.05[ 4.59 – 8.85] 0.541[1.337–8.8]

– 0.97 0.66 0.45

HIV/AIDS status No Yes

256/293[87.3] 178/190[93.6]

– 0.466[0.237,1.919]

– 0.028

– 9.59[1.544,59.27

– 0. 015

ART utilization Yes No

42/54[78] 128/136[94]

– 2.6[0.085,0.987]

– 0.049

– 1.541[3.33–6.86]

– 0.66

Pregnancy status No Yes

126/135[93.3] 256/293[87.3]

– 2.3[0.901,0.991]

– 0.045

– 0.13[0.128,4.341

– 0.001

Stage of pregnancy First trimester Second trimester Third trimester

49/57[85.9] 134/154[87] 76/82[92]

– 1.08[0.618,1.92] 1.233[0.764,1.958]

– 0.009 0.003

– 0.071[3.28,11.86] 0.746[5.198,12.8]

– 0.225 0.78

History of abortion No Yes

274/309[88.6] 108/119[90.7]

– 0.356[0.195,0.652]

– 0.001

– 0.4[2.182,11.245]

– 0.45

Number of abortion

One Two Three and above

23/30[76.6] 7/9[77.7] 4/4[100]

– 0.225[0.065,0.776] 0.122[0.027,0.51]

– 0.031 0.006

– 15.8[1.024,24.6] 0.5[0.834–0.986]

– 0.048 0.033

Cats at home No Yes

275/309[88.9] 159/174[91.4]

– 1.205[0.481–0.90]

– 0.034

– 2.02[6.127–10.6]

– 0.88

Number of Cats One More than one

113/132[85.6] 53/57 [92.9]

– 0.5[0.834–0.986]

– 0.005

– 4.09[1.1–2.631]

– 0.77

Contact with cat No Yes

69/78[88.4] 92/96[95.8]

2.02[0.127–0.631]

– 0.045

– 0.5[1.37–8.89]

– 0.99

Precautions Precaution No Precaution

5/6[83.3] 160/168[95.2]

– 0.12[0.127–4.648]

– 0.108

– 4.9[0.19,12.8]

– 0.33

Raw meat consumption

No Yes

279/313[89.1] 155/170[91.1]

– 1.190.602,2.37]

– 0.012

– 0.47[0.248,0.928

– 0.029

Raw Vegetables consumption

No Yes

321/359[89] 113/124[91.1]

– 0.325 [0.046–0.84]

– 0.011

– 0.21[0.104,0.446

– 0.000

Wash Vegetables before consumption

yes No

290/326[88.9] 31/34[91.1]

– 0.245[0.046–0.675]

– 0.02

– 4.2[1.209,3.531]

– 0.88

Raw milk consumption

No Yes

194/219[88.5] 240/264[90.9]

– 0.541[0.337–0.869]

– 0.002

– 3.95[2.154,7.245

– 0.000

Source of water Tap Well + river

350/398[87.9] 76/85[98.8]

– 2.4[0.630,9.204]

– 0.002

– 3.05[ 4.5 – 8.8]

– 0.56

Exposure with soil No Yes

259/292[88.6] 175/191[91.6]

– 1.05[ 0.59 – 1.85]

– 0.001

– 0.7[5.18,12.7]

– 0.46

Lack of knowledge about disease was predictor of T. gondii seropositivity [P≤0.05]. Relation seropositivity to T. gondii was positively associated with abortion, exposure to cat faces and HIV /AIDS status and negatively associated with study areas, residential place, Pregnancy status, unwashed vegetable consumption, raw meat consumption, raw

milk consumption, abortion, and HIV infection.

3.3 Socio Demographic Characteristics of Health Professionals

A total of one hundred physicians, nurses and gynecologist attending pregnant women in selected health institution of different parts of Ethiopia were surveyed. Male to female ratio is



23

0.53. The health professionals consisted mostly of women (65%). The mean age of respondents was 27.2 years ranging from 21 to 47 years old and half of respondents were belongs to 25–40 years age group.

Among health professionals, 40% of them had diploma, 30% of them had

bachelor degree, 20% of them had medical degree and 6% of them specialize in their profession.

Most of health professionals (60%) had 4–6 years of work experiences in their professions (Table 3).

Table 3. Socio–demographic characteristics of health professionals in selected health institution of

different parts of Ethiopia Professions Nurses (N=74) Physicians (N=20) Gynaecologists (N=6) Total Sex M F

20(27) 54(73)

13(65) 7(35)

2(33.3) 4(66.7)

35(35) 65(65)

Age group (years) <25 25–40 >40

20 (27) 54 (73) 0 (0)

2(10) 14(70) 4(20)

0(0) 4(66.7) 2(33.3)

22(22) 72(72) 6(6)

Educational status Diploma BSc MD MSc

40(54) 34(46) 0(0) 0(0)

0(0) 0(0) 20(100) 0(0)

0(0) 0(0) 0(0) 6(100)

40(40) 34(34) 20(20) 6(6)

Service years(Years) 2–4 years 4–6 years >6 years

19(25.7) 34(46) 21(28.3)

4(20) 8(40) 8(40)

2(33.3) 3(50) 1(17.7)

25(25) 45(45) 30(30)

Number in parentheses is percentage of respondents *– Shows that significance difference occur Of them, 74 were Nurses, 20

general practitioners, and 6 gynecologists. They were asked about [i] general aspects about toxoplasmosis including clinical manifestations, diagnosis and, treatment, [ii] their practices and experiences on toxoplasmosis.

3.4 General Knowledge And Perception Of Health Professionals About Toxoplasmosis

From health professionals, 65 % and 63% of them had knowledge of health risk of cat and toxoplasmosis respectively.

Consumption of raw or undercooked meat and vegetables were recognized as common source of T. gondii infection by 71.2% health professionals, but consumption of raw milk (18.4%), contact with cat faces (14.3%), and drinking unboiled water (6.1%) were also mentioned as important modes of transmission. Of health professionals, 42.5% respondents considered as avoid contact with cat faces is most commonly used method for preventing spread and transmission of toxoplasmosis. More over 35% respondents mentioned that transmission of toxoplasmosis could be prevented by

keeping personnel hygiene, avoid undercooked food substances (meat, vegetables and milk) (15%) and avoid unboiled water for drinking purpose (7.5%). In current study, 56.7% and 47.3% of health professionals not knew importance of testing toxoplasmosis during pregnancy and symptoms of toxoplasmosis respectively.

The four commonly recognized symptoms of toxoplasmosis mentioned by health professionals were hydrocephalus [33.3%], mental/brain retardation (26.9%), microcephaly (25.4%) and hearing loss (14.3%). Majority of health professionals (93%) do not screen pregnant women but 78% of them had case or exposure of toxoplasmosis in HIV patient and pregnant women. All health professionals didn’t give health education for pregnant women regarding modes of transmission and prevention of toxoplasmosis.

Most commonly used method of diagnosis known by health professionals for toxoplasmosis were clinical examination (66.7%) but serology (17.5%), ultra sound (11.1%) and CT scan (4.7%) were also mentioned by respondents as method of diagnosis for toxoplasmosis. 70% (11/63) of health


70

11. Horowitz, A.R.; Mendelson, Z.; Weintraub, P.G.; Ishaaya, I. Comparative toxicity of foliar and systemic application of acetamiprid and imidacloprid against the cotton whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae). Bulletin of Entomological Research, 1998, 88: pp. 437–442.

12. Lee, A.M. Characterization of the insecticidal properties of acetamiprid under field and laboratory conditions. (Under the direction of Dr. J.R. Bradley, Jr. and Dr. J. W. Van Duyn.). A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Master of Science, Department of Entomology, Raleigh, 2003, 71 pp.

13. Parrish, M.D.; Ayad, H.; Holmes, K., Ovicidal activity of acetamiprid (Assail brand 70WP insecticide) on economic pests of cotton. In: Proc. Beltwide Cotton Conf., Anaheim, CA. 9–13 January 2001. Natl. Cotton Counc. Am., Memphis, TN. pp. 904–905.

14. Seidenglanz, M.; Rotrekl, J.; Cejtchaml, J., Complicated aspects of pea (Pisum sativum) protection to Bruchus pisorum L. (Coleoptera; Chrysomelidae). In: kolektiv autorů., Sborník příspěvků.

Proceedings from the XVII. Czech and Slovak Plant Protection Conference, 11–14 September 2006, ČZU Praha, Praha, Czech Republic, ISBN 80–213–1564–4. pp. 522–527.

15. Smith, A.M. Modeling the development and survival of eggs of pea weevil (Coleoptera: Bruchidae). Environmental Entomology, 1992, 21: pp. 314–321.

16. Smith, E.H.; Sakeld, E.H., The use and action of ovicides. Annual Review of Entomology, 1996, 11: pp. 331–368.

17. Sparks, T.C.; Leonard, R.B.; Schneider, F.; Graves, J.B. Ovicidal Activity and Alteration of Octopamine Titers: Effects of Selected Insecticides on Eggs of the Tobacco Budworm (Lepidoptera: Noctuidae). Journal of Economic Entomology, 1993, 86(2): pp. 294–300(7).

Received: September 8, 2014




69

There was significant interaction between the products and the conditions

of years–АхВ–5.0%.

Table 3. Influence of the factors of variance on the eggs viable level of Bruchus pisorum

Souгce of variation Degrees of freedom Sum of squares Iпfluеnсе of factor Меаn square df SS % MS

Тоtаl 55 16544.6 – – Vагiаnts 13 12698.1 76.8 976.8 * Factor А – Year 1 5470.0 33.1 5470.0 * Factor В –Insecticide 6 10409.1 62.9 1734.9 * А х В 6 650.0 5.0 108.3* Рооlеd еггог 39 3615.8 – 92.7

With regard to the fact that the ovipositing activity of B. pisorum females can take about two to three weeks and can shift onto the upper nodes, it is necessary to spray additionally when the first spray timing is derived from the first eggs occurring on pods.

Conclusions The females of Bruchus pisorum

laid more than 60% of the eggs in the form of two eggs clusters.

It was found that among the tested insecticides Mospilan 20 SP (300 g ha–1 dose) had the lowest rate of Bruchus pisorum eggs survival (37.7%) and the highest ovicidal efficacy (51.63%).

From the comparison of the mean pea weevil eggs survival and efficacy, expressed separately for the group of pyrethroid and neonicotinoid insecticides, the neonicotinoid group emerged as significantly more effective. References 1. Abbott, W.S., A method of computing the

effectiveness of an insecticide. Journal of Economic Entomology 1925, 18: pp. 265–267.

2. Alekhine, B.; Ivanova, I., Pea weevil in Russia. Protection and Plant Quarantine, 2007, 6: pp. 28–29 (Ru).

3. All, J.; Lance, K.; Lohmeyer, K., Ovicidal properties of Leverage for bollworm in cotton. In: Proc. Beltwide Cotton Conf., Anaheim, CA. 9–13 January, 2001. Natl. Cotton Counc. Am., Memphis, TN. pp. 802–803.

4. Clement, S.L.; Wightman, J.A.; Hardie, D.C.; Bailey, P.; Baker, G.; McDonald, G., Opportunities for

integrated management of insect pests of grain legume. In: R. Knight (ed.) Linking research and marketing opportunities for pulses in the 21st century. Kluwer, Dordrecht, The Netherlands, 2000, pp. 467–480.

5. Demkin, V.; Dobronravova, M., Improved protection against pea pests. Plant Protection and Quarantine, 2007, 12: pp. 25–26.

6. Dochkova, B.; Naneva, D., Study on losses caused by Bruchus pisi L. and the role of Sigalphus thoracicus West for decrease of these losses in different varieties and lines in Forage Pea. Jubilee International Conference–90 years institute "Obrazchov chiflik"–Ruse, Problems of selection, Seed Science, seed and agricultural practices, scientific papers, Ruse, 1995, II, pp. 186–190.

7. Flores, D.; Murúa, F.; Acosta, J.C., Susceptibility of Latrodectus geometricus Koch eggs (Araneae, Theridiidae) to two piretroids in a laboratory environment. Boletín Sociedad Entomológica Aragonesa, 2007, 40: pp. 477–479.

8. Elzen, G.W. Changes in resistance to insecticides in tobacco budworm populations in Mississippi, 1993–1995. Southwestern Entomologist, 1997, 22: pp. 61–72.

9. Ermakov, A. Tactics of pea crop protection from pests based on the forecast of their number. Agro XXI, 1998, 12: 16.

10. Horne, J.; Bailey, P. Bruchus pisorum L. (Coleoptera. Bruchidae) control by a knockdown pyrethroid in field peas. Crop Protection, 1991, 10 (1): pp. 53–56.


24

professionals only requested laboratory tests for detecting toxoplasmosis (Table

4).

Table 4. Health professional’s knowledge about general aspects of Toxoplasmosis in different

parts of Ethiopia Profession Nurse (N=74) Physician(N=20) Gynaecologist (N=6) Total P=value Know disease human could get from cats No Yes

35(47.3) 39(52.7)

0(0) 20(100)

0(0) 6(100)

35(35) 65(65) 0.000*

Know Toxoplasmosis No Yes

35(47.3) 39(52.7)

2(10) 18(90)

0(0) 6(100)

37(37) 63(63) 0.009*

Do you know that women could get toxoplasmosis from cats No Yes

44(59.4) 30(40.6)

2(10) 18(90)

0(0) 6(100)

46(46) 54(54) 0.009*

Methods of transmission from animals to human beings No Yes

12(30.7) 27(69.3)

1(5) 17(95)

0(0) 6(100)

13(20) 50(80) 0.003*

Methods of transmission Contact with cat faces Raw meat consumption Raw milk consumption Raw vegetables consumption Unboiled water

2(7.4) 8(29.6) 7(26) 9(33.3) 1(3.7)

5(29.4) 6(35.3) 1(5.8) 4(23.5) 1(5.8)

1(16.7) 1(16.7) 1(16.7) 2(33.2) 1(16.7)

7(14.3) 15(30.6) 9(18.4) 15(30.6) 3(6.1)

0.078* 0.006*

Know Prevention Method No Yes

10(25.6) 29(74.4)

12(66,7) 6(33.3)

0(0) 6(100)

22(35) 42(65) 0.003*

Method of Prevention Avoid cat contact Avoid consuming raw food Personal hygiene Avoid unboiled water for drinking

8(44.5) 1(5.5) 9(50) 0(0)

8(50) 3(18.7) 4(25) 1(6.3)

1(16.7) 2(33.3) 1(16.7) 2(33.3)

17(42.5) 6(15) 14(35) 3(7.5)

0.000*

Importance of testing toxoplasmosis during pregnancy No Yes

42(56.7) 32(43.3)

0(0) 18(100)

0(0) 6(100)

42(42) 58(58) 0.000*

Know symptoms No Yes

35(47.3) 39(52.7)

2(10) 18(90)

0(0) 6(100)

40(40) 60(60) 0.009*

Symptoms of Brain /mental retards

Hearing loss Hydrocephalus Microcephaly

7(18) 6(15.4) 17(43.6) 9(23)

8(44.4) 2(11.1) 3(16.6) 5(27.7)

2(33.3) 1(16.7) 1(16.7) 2(33.3)

17(26.9) 9(14.3) 21(33.3) 16(25.4)

0.007*

Case of toxoplasmosis No Yes

15(20.3) 59(79.7)

5(25) 15(75)

2(33.3) 4(66.7)

22(22) 78(78) 0.711

Providing health education for pregnant for toxoplasmosis No Yes

74(74) 0(0) 20(20) 0(0) 6(6)

0(0) 100(0) 0(0)

Screen pregnant women for toxoplasmosis No Yes

73(90) 1(10)

18(90) 2(10)

2(33.3) 4(66.7)

93(93) 7(7) 0.000*

Diagnosis of toxoplasmosis Clinical CT scan Serology Ultrasound

24(61.5) 0(0) 9(23) 6(15.5)

14(77.7) 3(16.7) 1(15.6) 0(0)

4(66.7) 0(0) 1(16.7) 1(16.6)

2(66.7) 3(4.7) 11(17.5) 7(11.1)

0.000*

Importance of Toxoplasma in pregnant women Important Not Important Don’t know

10(25.6) 5(12.8) 24(61.6)

14(77.7) 3(16.7) 1(15.6)

6(100) 0(0) 0(0)

30(47.6) 8(12.6) 25(39.8)

0.000*

Importance in HIV infected patient Important Not Important Don’t know

13(33.3) 6(15.3) 20(51.4)

14(77.7) 4(32.3) 0(0)

6(100) 0(0) 0(0)

33(52.4) 10(15.9) 20(31.7)

0.000*

Response for Medication Refer to specialist Don’t know

10(25.6) 15(38.4) 14(36)

12(66.6) 6(33.4) 0(0)

6(100) 0(0) 0(0)

28(44.4) 21(33.3) 14(22.3)

0.094

Number in parentheses is percentage of respondents*– Shows that significance difference occur



25

The greatest number of health professionals concentrated on clinical issues. In studying altitudes and practices of respondents, 52.7% and 47.6 % of health professionals thought toxoplasmosis as important pathogen in HIV infected patients and pregnant women respectively but Less than half of professionals, 44.4%, knew medication of toxoplasmosis in pregnant and neonates but 22.3 of respondents they don’t knew what they do if pregnant women and neonats became toxoplasmosis positive.

Comparing number of answers according to type of profession, professionals who are gynecologist and physician showed total number of correct answers on issues of clinical manifestation, prevention and diagnosis, higher than those who are nurses however nurses had a better experience (23%) of using laboratory test relative to other professionals. In comparison according to professions, gynecologist had highest number of correct answers in diagnostic and clinical issues.

Regarding prevention, no significant differences were found between two professional categories. Mostly nurses had better knowledge than Physician but in medication physician had more awareness relative to nurses.

Results Overall, a very high seroprevalence

of toxoplasmosis of 89.9% was found in pregnant women and HIV infected patients in different parts of Ethiopia.

This high findings is agree with other seroprevalence figures from general or selected populations in different parts of country that ranged from 60.0–97.7% [XIAO et al., 2010; YIMER et al., 2005].

The high seroprevalence due to cat contact, feeding habits and inadequate hygienenic condition, and the suitable climatic factors for the sporulation and survival of oocysts in environment.

When we compare results of our study with other similar studies, T. gondii infection in study population is still on increase in Ethiopia perhaps due to lack of awareness about disease. The anti–T. gondii IgG positive and lgM negative

results indicate past exposure to chronic infection, while positive results for IgM are suggestive of acute (recent) exposures.

Though positive IgM results are unique indicators of recent infections. Of course confirmation to exclude reaction of natural IgM antibody with Toxoplasma antigen is mandatory. Besides, Pregnant women and HIV infected patients negative for IgG and IgM antibodies are at chance of getting primary infection, so monitoring for seroconversion is needed [DIZA et al., 2005]. From pregnant women of study were IgM positive during their pregnancy with potentially high chance of getting congenital toxoplasmosis suggest need to design preventive measures.

From 21 Potent risk factors examined for T. gondii seropositivity, study area, residential place, pregnancy status and consumption of raw vegetables, raw meat, raw milk, abortion and HIV/AIDS status were found to be main predictors of T. gondii seropositivity.

The study areas association of seropostivity T. gondii infection can be a suggestive for way of life of people.

In jinka and Awash high seroprevalence of T. gondii infection in pregnant women and HIV infected patients relative to Addis Ababa may be using of T. gondii contaminated water for drinking and high interest of feeding raw vegetables. In Awash there is high seroprevalence of toxoplasmosis than mojo may be due to poor sanitation and exposure with cats [83.5%] instead raw vegetables consumption [15.3%].

Furthermore, free movement of people of Awash leading to acquiring of infection from other areas might additionally support this condition.

In Gubre–Xiaber report [GUBRE–XIABER

et al. 1994], during pregnancy there is a low risk of T. gondii infection, but in our findings recent infections [28.6%] IgM positives is common in pregnancy women.

The high prevalence of T. gondii infection in pregnant women is indicates need for launching prevention and control strategies in antenatal program for toxoplasmosis in Ethiopia.

The high prevalence of T. gondii


68

respectively and the ovicidal efficacy reached 40.2 and 30.0%.

The efficacy of Duet was close to that of the Nurelle D and Calypso and had comparatively low value (17.6%).

The obtained results in 2012 were identical to those in 2011, but ovicidal efficacy was significantly lower.

It was found that only the application of Mospilan, Fury and Proteus led to significantly lower rate of pea weevil egg survival (53.7, 57.1 and 60.5%) and the better ovicidal efficacy–34.1, 30.0 and 25.8%.

The differences between Mospilan, Fury and Proteus and the untreated control were statistically significant.

One of the reasons for the lower effect of the insecticides was related to different weather conditions.

The amount of rainfall during the growing season in 2012 was 21.3% higher compared to 2011.

During the second and third decade of June, when the insecticides were applied the amount of rainfall reached 27.2 mm which was 106.6% higher compared to 13.2 mm in 2011.

That explained to some degree the lower efficacy of the insecticides during the second year of the study.

The ovicidal effect of tested insecticides could be influenced by other factors: crop density, position of eggs on

the pod in relation to the direction of spraying, the form of the eggs, pea variety and also the prevailing morphological stage of eggs at the time of spraying SMITH 1992 .

In the trials (2011 and 2012) the eggs on bottom pods were either at stage 1 or mostly at stage 2 (according to the scale described in Smith SMITH 1992

during the spraying. Black–spotted eggs (stage 3) were

not observed at those times. The mean data for the period

2011–2012 showed that the treatment with the insecticides decreased the level of pea weevil eggs survival (%).

There were statistically significant differences between the untreated control and Mospilan, Fury, Proteus and Calypso.

Mospilan had the highest ovicidal efficacy (51.63%) followed by Fury (35.08%) and Proteus (27.93%).

As ineffective insecticides were differed Nurele D, Duet.

The separation of the insecticides into two groups: neonicotinoids and pyrethroids showed that a lower level of surviving eggs (51.76%) and higher efficacy (32.24%) average for the period had neonicotinoids (Table 2).

The neonicotinoid group emerged as significantly more effective.

Table 2.

Ovicidal efficacy of some Neonicotinoids and Pyrethroids against Bruchus pisorum

Group of insecticides

Level of B. pisorum eggs survival (%)

Efficacy %


Efficacy, %


Efficacy, %

2011 2012 Mean Neonicotinoid 43.56 a* 37.97 59.95 a 26.51 51.76 a 32.24 Pyrethroids 54.01 a 23.09 70.57 a 13.50 62.29 b 18.30 LSD0.05% 35.867 21.227 19.234

*Means in each column followed by the same letters are not significantly different (P > 0.05)

By analysis of variance regarding to the numbers of weevil egg was established that the factors year and used products had different effects (Table 3).

In terms of eggs products had the strongest effect on the average number–62.9% of the total variance of the variants.

The power of influence of years was 33.1% and it is also statistically significant.



67

highest level of pea weevil eggs survival (%) was recorded in the control –70.23%.

There were differences between the untreated control and other treatments.

The treatment with Nurelle D and Calypso was associated with lower percentage of surviving eggs from 62.2 and 59.9% respectively but significant differences compared to control were not

found. The efficacy of insecticides was low and unsatisfactory –11.5 and 14.8%. However, significant insecticide effect on the pea weevil eggs was observed in the other treatments.

Mospilan had the lowest rate of Bruchus pisorum egg survival–21.7% and respectively the highest efficacy–69.1%.

Table 1. Numbers of Bruchus pisorum eggs before treatment, proportions of eggs laid one on

another (couple), ovicidal efficacy of some insecticides Insecticides–Dose (g, ml ha–1)

Mean number of eggs (individuals) per assessed pod

Proportion of eggs (individuals) laid in form of two eggs clusters (%)


Efficacy, %

2011 Мospilan 20 SP–300g 3.31 a* 65.84 21.68 a 69.13 Calypso 480 SC–300 ml 3.88 а 69.04 59.86 cde 14.76 Proteus 110 ОD–700 ml 3.44 а 63.11 49.15 bc 30.02 Duet 530 EC–500 ml 3.48 а 59.60 57.90 cd 17.56 Nurelle D–400 ml 3.44 а 60.91 62.15 de 11.51 Fury 10 ЕC–100 ml 3.99 а 60.43 42.00 b 40.19 Control 3.90 а 60.71 70.23 e – Mean 3.63 62.81 51.85 30.53 LSD0.05% 0.876 11.652 2012 Мospilan 20 SP–300g 3.28 а 59.00 53.74 a 34.13 Calypso 480 SC–300 ml 3.18 а 60.60 65.61 abc 19.58 Proteus 110 ОD–700 ml 3.00 а 53.93 60.51 ab 25.83 Duet 530 EC–500 ml 2.72 а 59.86 75.21 bc 7.82 Nurelle D–400 ml 3.11 а 56.94 79.38 c 2.70 Fury 10 ЕC–100 ml 2.84 а 55.45 57.13 a 29.97 Control 3.36 а 62.47 81.58 c – Mean 3.07 58.32 67.59 20.0 LSD0.05% 0.745 16.134 Mean 2011–2012 Мospilan 20 SP–300g 3.30 а 62.42 37.71 a 51.63 Calypso 480 SC–300 ml 3.53 а 64.82 62.74 cd 17.17 Proteus 110 ОD–700 ml 3.22 а 58.52 54.83 bc 27.93 Duet 530 EC–500 ml 3.10 а 59.73 66.56 de 12.69 Nurelle D–400 ml 3.28 а 58.92 70.77 de 7.11 Fury 10 ЕC–100 ml 3.41 а 57.94 49.57 b 35.08 Control 3.35 а 61.59 75.91 e – Mean 3.31 60.57 59.72 25.27 LSD0.05% 0.810 11.036

* Means in each column followed by the same letters are not significantly different (P > 0.05)

The differences between Mospilan and the other treatments were statistically significant.

Significant toxic effect showed Proteus and Fury where the level of eggs survival was 42.0 and 49.2%,


26

infection in study population shows pathogen is highly circulating in Environment. Toxoplasma gondii infection was more common in individuals who are using vegetables than those who didn’t. The using of inadequate hygienic method of transport and using of less hygienic water to wash vegetables might have contribution for contamination by T. gondii oocysts.

Similar result, Liu [LIU et al., 2009] from China and Kapperud [KAPPERUD et al., 1996]

from Norway reported raw vegetable consumption as an important risk factor for contracting toxoplasmosis.

Seroprevalence of anti–T. gondii antibodies was higher in study population who used un–boiled river and well water for drinking purpose [98.8%] than those who used un–boiled tap water [87.9%], indicating contamination of river and well waters by oocysts from felids’ feces and inadequate water management as reported by Petersen [PETERSEN et al., 2010]. HIV infected patients [93.6%] are mostly acquires T. gondii relative to HIV negative women [87.3%]. This may be due to high chance of reactivation of latent infection and development of toxoplasmic encephalitis [MONTOYA and LIESENFELD, 2004; JONES

et al., 2009]. The high seroprevalence of

toxoplasmosis in HIV infected patients might partly be due to early child hood and teenage infection. In contrast, Biedermann [BIEDERMANN et al., 1995] and Woldemichael [WOLDEMICHAEL et al., 1998] reported similar seroprevalence rates between HIV infected individuals and normal controls.

As age increased, it was observed that there was a relative increase in seroprevalence due to exposure to infective stages of parasite. Around 91.3% of women seroconvert by time they reach 25 – 48 of age may be due to acquiring of oocyst from environment and consumption of raw meat for this age group is more common.

According to Gubre–Xiaber [GUBRE–

XIABER et al., 1993] report, 75% of children in Ethiopia were seroconvert before puberty. Due to deep rooted tradition of raw and undercooked meat consumption in

Ethiopia and high seroprevalence of toxoplasmosis in sheep and goats there was association between seroprevalence and raw/undercooked meat consumption of pregnant women and 35.5% of all studied pregnant and HIV infected patients consume raw / undercooked meat.

Similarly, earlier studies elsewhere [DIAZ–SUAREZ and ESTEVEZ 2009; JONES et al., 2009] demonstrated significant association between seropositivity and behavior of raw meat consumption.

The seroprevalence of toxoplasmosis was significantly associated with presence of domestic cats in household than in their absence.

The high seroprevalence observed in households where cats are absent suggest a high environmental contamination. In our finding like that of Negash [NEGASH et al., 2008] who reported strong association of seroprevalence and presence of cats. However, Sroka [SROKA et

al., 2010] and Guebre–xabier [GUEBRE–XABIER et

al., 1993] reported absence of association between seropositivity and presence of cats at home.

Failure to include species of food animals used for meat to correlate seropositivity with food producing animals and failure to do IgG avidity test to exclude reaction of natural IgM antibodies and to know exactly current status of patient are recognized as limitations of study.

In present study we detected 89.9% anti–T. gondii IgG and 28.3% anti–T. gondii IgM seroprevalence rates, indicators of latent and recent T. gondii infections, respectively.

Our study illustrated a reasonably high IgM positive pregnant women indicating vulnerability to congenital transmission. A questionnaire based survey was conducted among health professionals for assessment of knowledge and perception towards toxoplasmosis on selected health facilities of different parts of Ethiopia.

This study demonstrated that majority of health professionals [63%] recognized human get disease from cat and knew toxoplasmosis respectively.



27

This finding was in agreement with others studies LAURA BERRIEL da SILVA et al., 2011; JONES et

al., 2001 . Since knowledge and awareness of health professionals is not adequate, there is a need for improvement among health professionals in both patient education and self–education.

As numerous studies have shown, eating undercooked meat during pregnancy is most important risk factor for toxoplasmosis sero–conversion KRAVETZ and

DANIEL, 2009; ALVARADO–ESQUIVEL et al., 2011; LAURA

BERRIEL da SILVA et al., 2011 . Similarly, in current study majority of health professionals recognize consumption of raw or undercooked meat and vegetables as common source of T. gondii infection.

With regard to direct advice given to pregnant women, all health professionals didn’t give health education for pregnant women regarding modes of transmission and prevention of toxoplasmosis however studies in USA KRAVETZ and DANIEL, 2009 showed almost all health professionals (98%) advised them to avoid raw meat and vegetables.

Since consumption of raw vegetables in current study was primary risk factor for toxoplasmosis transmission, all pregnant women should be counseled to thoroughly cook all vegetables during pregnancy. About prevention, although 42.5% of professionals recognize avoid contact with cat faces is most commonly used method for preventing spread and transmission of toxoplasmosis, which is similar to other studies AKYAR, 2011; KRAVETZ

and FEDERMAN, 2005 . Regarding to diagnosis, more than 83% of professionals never request lab test for diagnosis of toxoplasmosis which is very high (25%) relative to studies in Mexico ALVARADO–

ESQUIVEL et al., 2011 . The fact that overall median number of correct answers and diagnosis from professionals working in hospitals were higher than those of health center may in part be explained by fact that professionals working in hospitals have more training in high–risk prenatal care. However, a higher number of correct questions of prevention among professionals health center were expected, since health center has as one

of its main missions educational activities aimed at preventing health problems DIAZ–

SUAREZ and ESTEVEZ, 2009 . Likewise, median number of correct answers among physicians higher than that of nurses is expected in diagnosis and clinical issues, as these issues are object of focus in medical schools than in schools of nursing. However, basic knowledge of nurses in issues related to prevention is inconsistent with role of nurses as health educators ALVARADO–ESQUIVEL et al., 2011 .

The greatest number of correct answers among professionals with less training time is consistent with literature that indicates an inverse correlation between knowledge and years of professional practice, justifying need for recertification exams in some countries KRAVETZ and DANIEL, 2005; LAURA BERRIEL da SILVA et al.,

2011 . Continued education seems especially useful when targeted to specific groups and disciplines ALVARADO–ESQUIVEL et

al., 2011 like our target population. However, in this study only 100 data of health professionals are analyzed to assess their knowledge and perception but it is preferable to analyze all data of health professionals for better findings.

Conclusions In conclusion, high sero–prevalence

of toxoplasmosis in present seroepidemological study indicate need of preventive measures, mainly education about identified risk factors, in order to reduce associated morbidities and mortalities. The results of present study help to alert public health delivery system to undertake large scale studies and uncover economic and health impacts and formulate guidelines and policies leading to mitigation of potentially devastating outcomes of this zoonosis.

Health education should be given with special consideration to pregnant women and immunosuppresed individuals regarding minimizing contact with cats, avoidance of consumption of raw meat, vegetables and milk and un–boiled river water, proper hygienic practices should be exercised with aim of reducing contamination of drinking waters and food


66

The trail was conducted by split plot method (plot size 6.5m2 in four replications).

The following insecticides were used: Neonicotinoids:

– Mospilan 20 SP–3000 g ha–1 (200 g a.i. kg–1 acetamiprid);

– Calypso 480 SC–300 ml ha–1 (480 g a.i.l–1 thiacloprid);

– Proteus 110 OD–700 ml ha–1 (100 g a.i. l–1 thiacloprid +10 g a.i. l–1 deltamethrin);

– Pyrethroids: Duet 530 EC –500 ml ha–1 (50 g a.i. l–1 cypermethrin + 480 g a.i.l–1 chlorpyrifos–ethyl);

– Nurelle D–400 ml ha–1 (50 g a.i. l–1 cypermethrin + 500 g a.i. l–1 chlorpyrifosethyl);

– Fury 10 EC–100 ml ha–1 (100g a.i. l–1 zeta–cypermethrin).

Spraying was carried out after the appearance of the first eggs on pods on two bottom nodes according to method of Horne and Bailey [HORNE and BAILEY, 1991].

The average day temperature was 20.8 and 24. 40C and the relative air humidity–71 and 55 % % respectively in 2011 and 2012 in the day of the treatment.

Immediately after spraying a minimum of 20 pods (1 pod per plant) from two bottom nodes carrying 2–4 eggs (individuals) were chosen on each plot.

These chosen pods were marked (tagged with an identification number) and covered with a pocket from transparent net (avoiding any damage to plants and eggs) to stop further eggs being laid on them.

The number of eggs on the chosen pods and the portion of eggs (individuals) laid in the form of two–egg clusters were recorded.

At the time of ripening the marked pods were picked and then analyzed in the laboratory.

The sum of all larval entries to all seeds per pod was determined individually for each pod.

The entry openings are clearly visible on the round green seeds.

Thus the survival of eggs (+ first instar larvae) was determined on the

basis of comparing the recorded number of eggs per pod (immediately after treatment) with the number of larval entry openings per pod (after harvest in the laboratory) for each chosen pod individually.

The pods with only two seeds (and less) were excluded from the assessment.

The obtained data were statistically analysed using the Statgraphics Plus for Windows Ver. 2.1. Software program. Ovicidal efficacy of the insecticides was calculated by the formula of Abbott [ABBOTT,

1925]. Results and discussion It is obvious from Table 1 (2nd

columns) that the choice of pods resulted in a small variability among the mean numbers of eggs per affected pod.

It is possible to say that the starting situation was always well–balanced in the trials.

The mean number of eggs (individuals) per assessed pod varied from 3.31 to 3.99 (2011) and from 2.72 to 3.36 (2012).

There were no statistically differences among the variants.

The proportion of eggs (individuals) laid in the form of two–egg clusters varied slightly for the individual treatments in each of the years (from 59.6 to 69.0% in 2011 and from 53.9 to 62.5% in 2012).

The mentioned proportion of eggs compared to single laid eggs was considerably higher.

This means that most of the newly hatched larvae belong to of the couple laid eggs.

The results confirm the preference of females of Bruchus pisorum to lay the eggs in the form of two–egg clusters.

This is probably related to the protection of the lower eggs from the stress factors.

According to Seidenglanz SEIDENGLANZ et al., 2006 above eggs serve as a shield and contribute to lower mortality of pea weevil eggs and larvae located directly on the surface of the pods.

In 2011 the results from analyzed samples after harvesting showed that the



65

OVICIDAL EFFECT OF SOME INSECTICIDES ON BRUCHUS PISORUM L.

(COLEOPTERA: CHRYSOMELIDAE) EGGS

DOI: 10.7904/2068–4738–V(10)–65

Ivelina NIKOLOVA

Institute of Forage Crops, 5800–Pleven, V l. Vazov street, 89, BULGARIA, e–mail: [email protected]

Abstract. It was studied the effect of insecticides Neonicotinoids: Mospilan 20 SP–300 g ha–

1, Calypso 480 SC–300 ml ha–1, Proteus 110 OD–700 ml ha–1; Pyrethroids: Duet 530 EC–500 ml ha–1, Nurelle D–40 ml ha–1 and Fury 10 EC–100 ml ha–1 on Bruchus pisorum eggs under field conditions during 2011 and 2012. The insecticides were applied at the time of the first egg occurrence on lower pods. It was found that the females of B. pisorum laid more than 60% of the eggs in the form of two eggs clusters. The treatment with Mospilan, Fury, Proteus and Calypso significantly reduced the rate of pea weevil eggs survival compared to control. Mospilan 20 SP (300 g ha–1 dose) had the lowest level of B. pisorum eggs survival (37.7 %) and the highest ovicidal efficacy (51.63 %). From the comparison of the mean pea weevil eggs survival and efficacy, expressed separately for the group of pyrethroid and neonicotinoid insecticides, the neonicotinoid group emerged as significantly more effective. Keyword: Bruchus pisorum, egg, neonicotinoids, pyrethroids, ovicidal effect

Introduction Pea weevil Bruchus pisorum L. is

being one of the most serious insect of the spring pea CLEMENT et al., 2000 .

Many authors ascertained amount of damage that can cause weevil, witch reducing grain yield more by 40% DOCKOVA

and NANEVA, 1995; ERMAKOV, 1998; ALEKHINE and

IVANOVA, 2007; DEMKIN and DOBRONRAVOVA, 2007 . Damage caused by larvae, are

expressed in destroyed much of the grain and part of the embryo. As a result, damaged seeds have low germination and are not suitable for sowing.

Still the main method to control of B. pisorum is the chemical method.

When using a chemical method it is necessary to use effective, selective insecticides with low toxicity to humans and useful fauna.

Although the egg is most vulnerable stage on development of insects, little is known about pea weevil egg susceptibility to insecticides SMITH and SALKELD, 1966 .

Known information on this issue is found in the work of Sparks et al. (1993) where the pyrethroid lambda–cyhalothrin; and the carbamate thiodicarb were evaluated for ovicidal activity and effects on octopamine titers in eggs of the tobacco budworm, Heliothis virescens.

Horowitz HOROWITZ et al. 1998 reported ovicidal activity of neonicotinoids against the cotton whitefly, Bemisia tabaci, while Elzen ELZEN, 1997 and All ALL et al. 2001 reported that imidacloprid possessed ovicidal activity on tobacco budworm, Heliothis virescens.

Parrish and Lee PARRISH et al. 2001; LEE,

2003 reported ovicidal activity of neonicotinoids including acetamiprid against a variety of crop pests including the tobacco budworm, bollworm, cabbage looper, green stinkbug and Colorado potato beetle in greenhouse tests.

Flores et al. (2007) FLORES et al. 2007 reported a low susceptibility of Latrodectus geometricus eggs to deltamethrin and cypermethrin.

The aim of this study was to investigate the ovicidal effect of certain piretroids and neonicotinoids on Bruchus pisorum eggs.

Material and methods The trials were conducted in 2011–

2012 in the experimental field of the Institute of Forage Crops to control of weevil pea Bruchus pisorum in spring pea (Pisum sativum L.), variety “Pleven 4” with sowing rate 120 seeds m–2.


28

by cat feaces, team work between veterinarian and human health physician should be encouraged to increase public awareness about toxoplasmosis and its associated risk factors and transmission pathways via health extension service in rural and urban communities and it is recommended that if screening test for prenatal follow considers toxoplasmosis.

As to knowledge of health professionals especially nurses is not adequate as their main role is providing health education for their pregnant women to maintain safe delivery.

Only half of respondents were aware of treatment and more than 83% never request laboratory test for detecting toxoplasmosis.

The health professionals surveyed showed an incomplete knowledge about diagnosis and treatment of toxoplasmosis.

All health professionals didn’t give health education for pregnant women regarding disease. Such findings prove toxoplasmosis is consider as neglected disease even though it had various complication during pregnancy and on newborn health. Therefore, urgent providing of medical education and self learning is needed. Because such findings are useful for optimal design of strategies in medical education about toxoplasmosis. At last authors would like to recommend, Public health awareness through public media, assessment of health professional’s knowledge, attitude and practice in large scale towards toxoplasmosis is also recom

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15. da Silva, L.B.l de Vasconcelos, R. de Oliveira, C. da Silva, M.P. Bueno, W.F. Reis Amendoeira, M.R. de Souza Neves, E. Knowledge of Toxoplasmosis among doctors and nurses who provide prenatal care in an Endemic region, Infect Dis Obstet and Gynecol. 2011, 19:12–15.

16. Liu, Q. Wei, F. Gao, S. Jiang, L. Lian, H. Yuan, B. Yuan, Z. Xia, Z. Liu, B. Xu, X. Zhu, X.Q. Toxoplasma gondii infection in pregnant women in China. Trans R Soc Trop Med Hyg, 2009, 103:162–166.

17. Montoya, J.G. Liesenfeld, O: Toxoplasmosis. Lancet. 2004, 363: 1965–1976.

18. Njunda, A.L. Assob, J.C.N. Nsagha, D.S. Kamga, H.L. Nde, P.F. Yugah V.C. Seroprevalence of Toxoplasma gondii infection among pregnant women in Cameroon. J Public Health Afr. 2011.

19. Petersen, E. Vesco, G. Villari, S. Buffolano W. What do we know about risk factors for infection in humans with Toxoplasma gondii and how can we prevent infections?. Zoonoses Public Health, 2010, 57: 8–17.

20. Remington, J.S. McLeod, R. Thulliez, P. Desmonts, G. Toxoplasmosis In Infectious Diseases of the Fetus and Newborn Infant, edited by Remington JS, Klein JO, Wilson CB, Baker CJ. Philadelphia: Elsevier Saunders; 2006, 947–1081.

21. Sroka, S. Bartelheimer, N. Winter, A. Heukelbach, J. Ariza, L. Ribeiro, H. Oliveira, F.A. Queiroz, A.J.N. Alencar, JrC. Liesenfeld, O. Prevalence and Risk Factors of Toxoplasmosis among Pregnant Women in Fortaleza, Northeastern Brazil. Am J Trop Med Hyg, 2010, 83(3):528–533.

22. Thrusfield, M. Veterinary Epidemiology, 3 rd edition, Blackwell Science Ltd, Oxford, UK, 2007.

23. Torgerson, P.R. Macpherson, C.N.L. The socioeconomic burden of parasitic zoonoses: Global trends. Vet Parasitol, 2011, 182:79–95.

24. Woldemichael, T. Fontanet, A.L. Sahlu, T. Gills, H. Messele, T. Rinke de Wit, T.F. Yeneneh, H. Coutinho, R.A. Gool T.V. Evaluation of Eiken latex agglutination test for Anti–Toxoplasmosis antibodies and seroprevalence of Toxoplasmosis infection among factory workers in Addis Ababa, Ethiopia. Trans R Soc Trop Med Hyg: 1998, 92:401–403.

25. Xiao, Y. Yin, J. Jiang, N. Xiang, M. Hao, L. Lu, H. Sang, H. Liu, X. Xu, H. Ankarklev, J. Lindh, J. Chen, Q. Seroepidemiology of human Toxoplasma gondii infection in China. BMC, Infect. Dis. 2010, 10:4.

26. Yimer, E. Abebe, P. Kassahun, J. Woldemical, T. Bekele, A. Zewudie, B. Beyene, M. Seroprevalence of human toxoplasmosis in Addis Ababa, Ethiopia. Ethiop Vet. J, 2005, 9:109–122.

27. Zewdu, E. Agonafir, A. Tessema, T.S. Tilahun, G. Medhin, G. Vitale, M. Dorny, P. Seroepidemiological study of caprine toxoplasmosis in East and West Shewa Zones, Oromia Regional State, Central Ethiopia. Res Vet Sci, 2013. 94(1):43–48.

Received: July 17, 2014 Article in Press: August 30, 2014



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matter yield, 2012, Journal of Biotechnology, 161, 35.

3. Bashan Y., Holguin G. Proposal for the division of plant growth–promoting rhizobacteria into two classifications: biocontrol–PGPB (plant growth–promoting bacteria) and PGPB. Soil Biology and Biochemistry, 1998. 30, 1225–1228.

4. Bostan, C.; Butnariu, M;. Butu, M.; Ortan, A.; Butu, A. Allelopathic effect of Festuca rubra on perennial grasses. Romanian Biotechnological Letters. 2013, 18 (2), 8190–8196.

5. Brown, M.E., Seed and root bacterization. Annu. Rev. Phytophatol. 1974. 12, 181–197.

6. Butnariu, M. Armon, E. Tonea R. Quantity determination of molybdenum from Pisum sativum plants and the influence of heavy metal to chemical elements accumulation. Jurnal Lucrări Ştiinţifice–Zootehnie şi Biotehnologii, Universitatea de Ştiinţe Agricole şi Medicină Veterinară a Banatului Timişoara, 2008, 41(1), p. 735–743.

7. Butnariu, M.; Bostan C. Antimicrobial and anti-inflammatory activities of the volatile oil compounds from Tropaeolum majus L. (Nasturtium).

African Journal of Biotechnology, 2011, 10 (31), 5900–5909.

8. Pacovsky R.S. Development and growth effects in the Sorghum–Azospirillum association. Journal of Applied Bacteriology 1990. 68, 555–5.

9. Postgate J.R. Biological nitrogen fixation: fundamentals. Philosophical Transactions of the Royal Society of London, 1982. Series B 296, 375–385.

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Received: July 31, 2014 Article in Press: September 2, 2014




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microbiological preparation BIO–ONE showed that the highest yield was obtained with the dose of treatment 0.9 and 0.12 g/100 m2–203.0 kg/da, followed

by the dose of treatment 0.6 g/100 m2–202.0 kg/da, while for the rest variants the results were almost similar to those of the control.

Table 4. Yield of wheat after harvesting of peas treated with the microbiological preparation BIO–

ONE, field experiment Yield Variants

Hopper kg/da

Siftings kg/da

Yield kg/da

Yield exceeding the control kg/da

1. Control 471 129 343 – 2. Fertilized with N4P8 500 100 400 + 57 3. 0.3 g/100 m2 443 86 357 + 14 4. 0.6 g/100 m2 471 86 386 + 43 5. 0.9 g/100 m2 486 100 386 + 43 6. 0.12 g/100 m22 486 100 386 + 43 7. 0.15 g/100 m2 486 129 357 + 14

For tracing the differences in the effects of the various doses of treatment with the microbiological preparation BIO–ONE after harvest of peas, the soil was prepared for sowing of wheat.

The soil was not fertilized before and after wheat sowing which was on 27 September, 2012.

Wheat germination occurred simultaneously as the phase of tillering occurred in late October and early November 2012.

In the spring of 2013 the vegetation proceeded normally.

The harvest was on 20 June 2013, with grain moisture 11.1%. The results of the harvest are shown in Table 5.

It is seen that the treatment of peas with the preparation BIO–ONE at a concentration of 0.6 to 0.12 g/100 m2 increased the yield of wheat by 43kg/da compared to the untreated control.

It was also observed that at lower doses of treatment there was a smaller amount of siftings while the ratio of the siftings increased with at the higher doses of the treatment.

Conclusions It could be concluded that the

microbiological preparation BIO–ONE had a positive effect on:

– The yield of pea seeds after treatment of the crop with the microbiological preparation BIO–

ONE showed that the highest yield was obtained at a dose of treatment of 0.9 g/100 m2–185.0 kg/da, followed by the variants treated at a dose of 0.12 g/100 m2–167.0 kg/da and 0.6 g/100 m2–165.0 kg/da.

– At the lowest dose of treatment of 0.3 g/100 m2 and the fertilized control the yield was equal–158.0 kg/da.

– The lowest yield was obtained from the control variant. It increased the wheat yield as

after–effect after treatment of peas at concentration of 0.6 to 0.12 g/100 m2 as the wheat yield was raised by 43 kg/da compared to the untreated control.

The highest yield was obtained for the variant of peas fertilized with N4P8–as the yield exceeded by 57 kg/da the control that was not fertilized and treated.

It was noticed also that at lower doses of treatment the quantity of siftings is lower as it goes up at higher doses of treatment. References 1. Abbass, Z., Okon, Y. Plant growth–

promotion by Azotobacter paspali in the rhizosphere. Soil Biol. Biochem. 1993. 25, 1075–1083.

2. Andreea, G. Samfira, I. Butnariu, M. Corneanu M. The morphological features influence on Phalaris arundinacea green mass and dry


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HEPATOPROTECTIVE AND GASTRO PROTECTIVE STUDIES OF Terminalia

arjuna LEAVES EXTRACT AND PHYTOCHEMICAL PROFILE

DOI: 10.7904/2068–4738–V(10)–30

Ataa SAID1, Ahlam M. El-FISHAWY2, Siham El-SHENAWY3, Usama W. HAWAS4, Mohamed ABOELMAGD1

1Pharmacognosy Department, National Research Center, Dokki, Giza, EGYPT

2 Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Giza, EGYPT 3 Pharmacology Department, National Research Center, Dokki, Giza, EGYPT

4Phytochemistry and Plant Systematic Department, National Research Center, Dokki, Giza, EGYPT; e–mail: [email protected]

Abstract. The methanol extract of T. arjuna leaves was evaluated as hepatoprotective and

gastroprotective against Swiss albino rats. In hepatoprotective study the liver damage induced by paracetamol while the gastric lesion induced by absolute ethanol in the gastro protective study. The extract was given orally (250,500 and 1000 mg/kg) in different experimental models in both studies. The extract at dose (500mg/kg) showed significant reduction in ALT serum level by 19.9% while at dose (1000mg/kg) it reduced significantly serum ALT, AST and ALP levels by –26.15,–25.46 and –23.69 % respectively as compared with paracetamol treated group. In the gastro protective study the extract produced significant reduction in the number and severity of mucosal lesion by (–52.1 &–67.3 %, –66.7 &–71.2 % and –68.8 &–77.6%) respectively.

Key words: T. arjuna, hepatoprotective, gastroprotective, mucosal lesion.

Introduction Liver diseases considered one of

worldwide problem with major threats to public health up till now there is no effective drug available that stimulates liver function, offer protection to the liver from damage or help to regenerate hepatic cells.

The medicinal plants and their derivatives are still used all over the world in one form or another for the treatment of liver diseases.

The main constituents in liver protective plants are mainly glycosides, flavonoids, triterpenes and phenolic compounds ADEWUSI and AFOLAYAN 2010 .

Gastric ulcer is an illness that affects a considerable number of people worldwide.

Peptic ulcers are caused when natural balances between aggressive factors of acid and pepsin and defensive mechanisms of mucus, bicarbonate, mucosal turnover and blood supply (mucosal barrier) are disturbed MAHMOOD et

al., 2010 .

Genus Terminalia belongs to family Combretaceae which is mostly found in tropical and subtropical regions.

In Egypt several Terminalia species have been introduced since 1890 and grown as shade and ornamental trees in public gardens and streets YOUSIF, 1996 .

In Indian system of medicine the bark is used as astringent, cooling, aphrodisiac, cardiotonic, tonic, anti ulcer, spermatorrhoea, leucorrhoea, diabetes, cough, tumour, excessive perspiration, asthma, inflammation and skin disorders PAARAKH, 2010 .

Recently, it has been reported that T. arjuna exhibit hypocholesterolemic RAM

et al., 1997 , hypolipidemic SHAILA et al., 1998 , antioxidant, antimutagenic KAUR, 2001 , antibacterial SAMY et al., 1998 , antiviral CHENG

et al., 2002 , and anticancer activities NAGPAL et

al., 2000 . The bark is responsible for nearly all medicinal uses of T. arjuna GUPTA et al.,

2001, CHENG et al., 2002 and there is a little bit of work done on leaves so, it will be of great interest to investigate gastro and hepatoprotective activity of T. arjuna leaves.



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Material and methods Plant material The leaves of T. arjuna

(Combretaceae) were collected from El–Zohria Botanical Garden, Giza, Egypt.

In March 2007, and were kindly authenticated by, Dr. Salwa El–Kawatchy, Department of Plant Systematic, National Research Centre (NRC) Giza, Egypt and by Mrs. Tereez Labib Consultant of Plant Taxonomy at Ministry of Agriculture and Director of Orman Botanical garden.

A voucher specimen was deposited in the herbarium of El–Zohria Garden, Giza, Egypt.

Preparation of the extract: The air–dried powdered leaves of T.

arjuna (1.6 kg.) were extracted by maceration with 70 % MeOH (3 × 3 L) at room temperature, and the extract was filtered and evaporated to give residue (140 g). The crude extract (100 g) was suspended in H2O (250 mL) and partitioned with hexanes (6 × 500 mL), DCM (6 × 500 mL), EtOAc (6 × 500 mL), and n–BuOH (6 × 500 mL). The extracts were evaporated to yield 19, 16, 8.6, and 14 g, respectively.

Each extract was tested for the presence of the phytoconstituents according to following standard tests, Molisch's test for carbohydrates, Shinoda test for flavonoids, forth test for Saponins, Salkowski's for terpenes and sterols, FeCl3, and Mayer's reagents for detecting of tannins and alkaloids, respectively SOFOWRA, 1993; TREASE, and EVANS, 1989; HARBORNE,

1973, ROSS et al., 2013 . Reagents Drugs and Chemicals The 1–Paracetamol (Paramol) (Misr

Co. for pharmaceutical industries, Cairo, Egypt). 2–Silymarin (SEDICO, for pharmaceutical industries, Cairo, Egypt).

They were used in the experiments. The doses employed were based

upon the human dose after conversion to that of rat according to Paget and Barnes. PAGET and BARNES 1964 . 3–absolute ethanol (AL–Gomhoryia Co. chemical industry)2–2 Biochemical kits: ALT (Alanine aminotransferase) (Biodiagnostic, Egypt) AST (Aspartate aminotransferase).

(Biodiagnostic, Egypt) ALP (alkaline phosphatase). (Biodiagnostic, Egypt).

Animals Adult rats of both sexes weighing

150–200gm were used in the experiments.

Animals were housed under standardized conditions of light and temperature and received standard rat chow and tap water ad libitum.

Animals were randomly assigned to different experimental groups each of six, kept in separate cage.

All animal procedures were performed after approval from the Ethics Committee of the National Research Centre and in accordance with the recommendations for the proper care and use of laboratory animals NIH publication No. 85–

23, revise 1985 . Hepatoprotective study Thirty six rats were divided into six

groups of six animals each as following: Group 1: Normal control group

received a daily oral dose of 1ml saline. Group 2, 3, 4: Received a daily oral

dose of methanol extracts of T. arjuna leaves (250, 500 and 1000 mg/kg) alone for successive 7 days before paracetamol injection (1000 mg/kg)

Group 5: Received single oral dose of paracetamol (1000 mg/kg) according to SILVA et al., 2005 .

Group 6: Received a daily oral dose of Silymarin (25 mg/kg) alone also for successive 7 days before paracetamol injection (1000mg/kg) used as a reference drug.

At the end of the experimental period (24h after paracetamol injection), the blood was obtained from all groups of rats after being lightly anaesthetized with ether by puncturing rato–orbital plexus SORG and BUCKNER 1964 , the blood was allowed to flow into a clean dry centrifuge tube and left to stand 30 minutes before centrifugation to avoid hemolysis.

Then blood samples were centrifuged for 15 minutes at 2500, rpm the clear supernatant serum was separated and collected by Pasteur pipette into a dry clean tube for the following biochemical tests: Alanine


62

Reduction was observed in the organic carbon amount, respectively, the content of humus.

That phenomenon could be explained by the increased microbiological activity aiming to mineralize larger quantities of organic matter and residues to nourish the growing needs of the crop during the vegetation season SAMFIRA et al., 2013, BUTNARIU

et al., 2008; BOSTAN et al., 2013 .

The structural analysis (Table 4) revealed that the phase onset of flowering occurred the earliest in the control variant –55 days.

In the fertilized control the phase onset of flowering occurred on the 67th day while in treated variants that period continued 66 days–in variants treated with a dose of 0.3 and 0.6 g/100 m2.

At a dose of treatment of 0.3 g/100 m2 it was 59 days, as it gradually went down for highest doses of the treatment.

Table 4.

N, P, K, рН and organic carbon content during vegetation after treating of the experiment with the microbiological preparation BIO–ONE in spring forage peas

Variants Indicators Control Control treated

with N4P8 0.3 g/100m2

0.6 g/1002

0.9 g/100m2

0.12 g/100 m2

0.15 g/100m2

Height during phase onset of flowering (cm) 42.0 44.0 41.0 45.0 39.0 40.0 38.0

Plant height (cm) 96.3 88.8 101.8 96.4 113.1 120.0 123.5 Height of the 1st pod (cm) 43.1 34.2 53.4 49.1 47.4 66.2 55.4 Number of offshoots per 1 plant 0.6 0.6 0.2 0.1 0.3 0.3 0.4 Offshoot length (cm) 27.3 42.5 16.6 2.7 32.1 13.8 22.9 Number of pods from 1 plant 11.2 12.1 7.9 10.8 10.8 11.8 11.2 Number of seeds pr 1 plant 37.6 45.2 34.9 37.2 39.9 45.0 43.9 Weight of seeds from 1 plant (g) 5.36 5.41 6.00 6.89 6.80 6.63 6.72 Mass of 1000 seeds (g) 153.2 153.6 156.1 150.1 154.7 155.7 153.9 Yield kg/da 194.0 199.0 199.0 202.0 203.0 203.0 194.0

The microbiological preparation BIO–ONE had a positive effect on the plant height.

Plants treated with a dose of 0.12 and 0.15 g/100 m2 were the highest–120.3 and 123.5 cm, followed by the variant of 0.3 g/100 m2.

The fertilized control–88.8 cm had the lowest result.

The indicator height of the first pod, it was set the highest in the fertilized variant–104.0 cm, followed by the variants with a dose of treatment–0.12 and 0.15 g/100 m2–66.2 and 55.4 cm.

The preparation did not have an impact on the pea offshoots.

The highest number of offshoots on the average–0.6 pieces was found in the variant fertilized with N4P8, while the lowest number was in the variant with dose of treatment 0.6 g/100 m2.

The offshoot length however was affected by the concentration of the microbiological preparation.

The offshoot length decreased with increasing the concentration as the length was the smallest–0.2 cm with the dose of treatment of 0.6 g/100 m2.

Data on the number of seeds per plant were comparatively close, as the greatest number of 45.2 and 45.0 pieces was found in the variant, fertilized with N4P8, and the one with a dose of treatment 0.12 g/100 m2.

For the indicator seed weight per plant it was observed a raise in the weight after the treatment of the plants.

Data showed that the highest weight was obtained in the variants treated with a dose of 0.6 and 0.9 g/100 m2–respectively 6.89 and 6.80 g, followed by 0.12 g/100 m2–6.63g.

Concerning the mass of 1000 seeds those treated with a dose of 0.3 and 0.12 g/100 m2 were the heaviest–156.1 and 155.7g.

The yield of pea seeds after the treatment of the crop with the



61

The phase full germination occurred fifteen days later after sowing of peas.

Crop was treated with microbiological preparation BIO–ONE

with increasing doses from 0.3 to 0.15 g/100 m2 plus the organic component “VARNA” (as prescribed by the manufacturer crops were treated before or after precipitation) (Table 2).

Table 2. N, P, K, рН and organic carbon content before setting and treating of the experiment with

the microbiological preparation BIO–ONE in spring forage peas Sample No. pHН2O Total Nitrogen

mg/1000g soil Phosphorus mg/100 g soil

Potassium mg/100 g soil Dry Humus

% 1. Control 8.13 16.80 4.81 42.22 1.64 2.83 2. Fertilized with N4P8 8.29 18.90 4.47 43.47 1.30 2.24 3. 0.3 g/100 m2 8.30 16.10 4.84 45.34 1.63 2.81 4. 0.6 g/100 m2 8.19 18.20 5.44 50.96 1.59 2.74 5. 0.9 g/100 m2 7.67 19.95 4.50 42.72 1.85 3.19 6. 0.12 g/100 m2 7.34 20.30 4.26 50.34 1.60 2.76 7. 0.15 g/100 m2 7.00 20.65 3.70 34.63 1.58 2.72

The phase full germination occurred fifteen days later after sowing of peas.

Crop was treated with microbiological preparation BIO–ONE with increasing doses from 0.3 to 0.15 g/100 m2 plus the organic component “VARNA” (as prescribed by the manufacturer crops were treated before or after precipitation).

Ten days after crop treatment soil samples were taken for monitoring the changes in the soil under the effect of the microbiological preparation (Table 3).

The analysis showed that the soil acidity changed from slightly acidic to neutral. The nitrogen content in control variant increased to 19.9 5 mg/1000 g soil.

In the treated variants with the lowest dose the rate of increase was up to 26.60 mg/1000 g soil while in the rest variants with increasing the dose of treatment, the raise of nitrogen went down

as it reached its minimum at the highest dose of 0.9 g/100 m2 of 24.50 mg/1000 g soil.

Phosphorus content during pea vegetation season had decreased both in the control variant as well as in the variants treated with lower doses as it was within the range from 1.80 for the control to 2.76 mg/100 g soil at a dose of treatment of 0.9 g/100 m2.

In the variants with higher doses of treatment its quantity was preserved as in the last variant with a dose of treatment of 0.15 g/100 m2 its quantity increased from 2.53 to 2.73 mg/100 g soil SAMFIRA et al., 2013,

ANDREEA et al., 2012; BURNARIU and BOSTAN, 2011 . That suggested the mobilization of

the reserves in the soil under the influence of anaerobic microorganisms of the genus–Clostridium pasterianum during drought in the summer months.

Table 3. N, P, K, рН and organic carbon content during vegetation after treating of the experiment

with the microbiological preparation BIO–ONE in spring forage peas Sample No. pHН2O Total Nitrogen

mg/1000g soil Phosphorus mg/100 g soil

Potassium mg/100g soil Dry Humus %

1. Control 6.63 19.95 1.77 30.80 1.63 2.81 2. Fertilized with N4P8 6.50 24.15 1.80 32.40 1.62 2.79 3. 0.3 g/100 m2 6.55 26.60 1.86 33.50 1.58 2.72 4. 0.6 g/100 m2 6.60 25.55 1.80 40.00 1.62 2.79 5. 0.9 g/100 m2 6.60 24.50 2.76 35.40 1.62 2.79 6. 0.12 g/100 m2 6.57 24.85 2.53 33.50 0.78 1.34 7. 0.15 g/100 m2 6.60 25.20 2.73 33.50 0.76 1.31


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aminotransferase (ALT) accord to BERGMEYER et al., 1985 .

Aspartate aminotransferase, (AST) accord to KLAUKA et al., 1993 .

Serum Alkaline phosphatase (ALP) accord to TIETZ and SHUCY 1986 .

Gastroprotective study Gastric lesions was induced in rats

by ethanol (1 mL of 100 % orally). Rats were divided into four groups

each of six, one group received ethanol as control, and the remaining groups received 70 % methanol extract of T. arjuna leaves (250, 500 and 1000 mg/kg) one hour before ethanol was given.

Rats were killed 1/2 h after ethanol administration by cervical dislocation after being lightly anaesthetized with ether and stomach was excised, opened along the greater curvature, rinsed with saline, extended on a plastic board and examined for mucosal lesions.

The number and severity of mucosal lesions were noted and lesions were scaled as follows: –Petechial lesions = 1 –lesions less than 1 mm = 2 –lesion between 1 and 2 mm = 3 –lesions between 2 and 4 mm = 4 –lesions more than 4 mm = 5.

A total lesion score for each animal is calculated as the total number of lesions multiplied by respective severity scores. Results were expressed as the severity of lesions/ rat MÓZSIK et al., 1982 .

Results and discussion Hepatoprotective study Both concentrations 500 mg/kg and

1000 mg/kg of the70% methanol extracts of T. arjuna leaves showed significant reduction in elevated serum ALT levels by

–19.9, –26.15 % while The extract at concentration 250 mg/kg didn't achieve significant improvement in AST, ALT or ALP enzyme levels.

Only dose 1000 mg/kg showed significant reduction in serum AST and ALP levels by –25.46 and –23.69 % respectively as compared with paracetamol treated group which exhibited significant serum elevation of ALT, AST and ALP by 43.14, 32 and 38.48% respectively indicating enzymes leakage due to disturbed transport function of hepatocytes as a result of paracetamol hepatotoxicity leading to and increased serum levels.

Both doses 500 and 1000 mg/kg gave statistically significant improvements in AST, ALT and ALP when compared to paracetamol group.

The over dosage of paracetamol is known to be hepatotoxic in experimental animals, at normal doses, about 80% of it is eliminated mainly as sulphate and glucoronide conjugates before oxidation and only 5% is oxidized by hepatic cytochrom P450 (CYP2E1) to a highly reactive and toxic electrophile N–acetyl–p–benzoquineimine (NAPQI), glucoronidation and sulfation routes become at over dosage of paracetamol saturated and as a consequence, paracetamol is increasingly metabolized into NAPQI DAR et al., 2012 .

The elevated Serum ALP levels are related to function of hepatic cell and indicating presence of billary pressure.

So it could be concluded that T. arjuna extract at concentration 1000mg/kg improve secretory mechanism of hepatocytes.

y

In hepatic cells ALT, AST found in higher concentration in cytoplasm and ALT particularly in mitochondria.

So T. arjuna at concentration 500, 1000 mg / kg significantly reduce the elevated levels of AST, ALT and so

consequently stabilize plasma membrane as well as repair hepatic tissue damage caused by paracetamol.

This is probably because of free radical scavenging activity of flavonoids



33

and polyphenols present in Terminalia arjuna DOORIKA and ANANTHI 2012 .

Phytochemical screening of leaves of T. arjuna revealed the presence of

Flavonoids which have been previously reported to have hepatoprotective activity.

Table 1. Effect of oral administration 70 % methanol extracts of T. arjuna W. and A.(250 mg, 500

mg and 1000 mg/kg ) on ALT, AST and ALP serum level in paracetamol induced hepatotoxicity in rats, (n=6).

Group ALT(IU/L) AST(IU/L) ALP(IU/L)

X ± S.E % of change X ± S.E % of

change X ± S.E % of change

Saline 1mL 42.23±2.1 ––––– 63.74±1.5 ––––– 47.17 ± 2.4 –––––– Paracetamol 1000mg/kg 60.45±3.5● 43.14 84.14±2.8● 32.00 65.32±3.1● 38.48

70% MeOH extract (250mg /kg) 56.31±2.3 33.34 80.2±5.1 25.82 62.71±4.2 32.94

70% MeOH extract (500mg /kg) 50.64±2.2* 19.91 72.99±3.5 14.51 56.85±2.4 20.52

70% MeOH extract (1000mg /kg) 44.64±1.8* 5.71 62.72±1.9* –1.60 49.84±3.2* 5.66

Silymarin 25mg/kg 43.91±1.4* 3.98 62.89±3.2* –1.33 50.73±2.3* 7.55 Values represent the mean ± S.E. of six animals for each group.

● = P <0.05: Statistically significant from saline control group. * = P <0.05: Statistically significant from paracetamol group by using (Student´s t test)

Percent of change was calculated as regard saline control group.

We reported previously the isolation of flavonoids apigenin, luteolin, Vitexin Isovitexin luteolin 3`glucrornic from the T. arjuna leaves ROSS et al., 2013 .

So the hepatoprotective effect of T. arjuna leaves.

May be due to it’s flavonoids content. The results were recorded in table 1 and showed in figure 1.

Figure 1. Effect of oral administration 70 % methanol extracts of T. arjuna W. and A. (250 mg, 500 mg and 1000 mg/kg ) on ALT, AST and ALP serum level in paracetamol induced

hepatotoxicity in rats. Gastro protective study All tested concentrations (250,500

and 1000mg/kg) of the70% methanol extracts of T. arjuna leaves showed significant reduction by –52.1 & –67.3 %, –66.7 &– 71.2 % and –68.8 &– 77.6 % in number and severity of gastric lesions

induced by absolute ethanol in control group respectively.

Ethanol is commonly used for induction of ulcer in experimental rats; it leads to intense gastric mucosal damage resulting in increased vascular permeability, edema formation and produces necrotic lesions in gastric


60

It facilitates the absorption of the residual phosphorus and potassium and retains moisture. It does not contain genetically modified organisms.

It is recognized by all organizations controlling the organic farming, including OMRI Institute. It could be used both in organic and traditional agriculture.

Objective: Study of the influence of preparation Bio–One of getting healthy agricultural products, increasing the productivity of the arable land and reducing the production costs.

Material and methods For the achievement of the set

objective, a field experiment was carried out in the period 2012–2013 with peas in the Second Experimental Field of the Institute of Forage Crops (IFC) with yield plots of 4 m2, in four replicates and the following variants: 1) Control–not treated 2) Control–fertilized with N4P8, in

accordance with the assumed technology for peas growing.

3) Background fertilizing components + 1 dose +“VARNA”–biological activation product humic acid

4) Background fertilizing components + 2 doses + “VARNA”–biological activation product humic acid



7) Background fertilizing components + 5 doses + “VARNA”–biological activation product humic acid Solution for 100 sq. m.–1 dose of

0.3 g/100 m2, 2,3,4,5–fold increasing of

the dose. The areas were treated before or after precipitation.

Simultaneously with the microbiological preparation BIO–ONE it was obligatory to be added the organic component “VARNA”–biological activation product humic acid.

The biometrical indicators for seed productivity (yield) were determined after the methodology of Nikolov [Nikolov, et al. 1981 .

The monitored indicators were: 1) Seeds yield and structural analysis of

seed yield; 2) Observed diseases on peas 3) Soil indicators–humus; phosphorus

and potassium content; physical characteristics of the soil

Results and discussion The experiment was located on the

second terrace of the Vit River. The soil is leached chernozem

(Haplic Chernozems, loamy) with medium strong humus horizon.

The mechanical structure was medium to heavy sandy–clay as the content of physical clay in the top layer was 47–49% and of clay about 29% as large powdery fraction and fine sand prevailed.

Data in Table 1 provide information about the agrochemical soil condition before setting the trial. The soil had very poor nitrogen content, poor phosphorus content and good potassium content.

Soil pH was neutral to slightly alkaline. In content of organic carbon it ranged from 1.30 (2.24%) to 1.85 (3.19%).

The agrochemical soil characteristic is presented in:

Table 1. Agrochemical soil characteristic

Sample pHН2O pHKCl Nitrogen mg/1000g soil Phosphorus mg/100 g soil

Potassium mg/100 g soil Dry Humus

% ammonia nitrate 1. 6.26 5.03 9.80 7.28 2.64 42.22 1.00 1.72 2. 6.25 5.04 8.68 16.24 2.40 43.47 1.34 2.31 3. 6.44 5.24 7.84 16.52 2.50 45.34 1.33 2.29 4. 6.66 5.44 7.84 4.76 2.56 50.96 1.07 1.85 5. 6.61 5.33 11.06 8.68 2.59 42.72 1.08 1.86 6. 6.48 5.34 5.46 1.68 2.81 50.34 1.34 2.31



59

STUDY OF THE INFLUENCE OF MICROBIOLOGICAL PREPARATION BIO–ONE IN

SPRING FORAGE PEAS (Pisum SativumL.) UNDER THE CONDITIONS OF ORGANIC FARMING

DOI: 10.7904/2068–4738–V(10)–59

Ivan PACHEV1, Tatiana, MALYUK2*

1Institute of Forage Crops–Pleven, BULGARIA; e–mail: [email protected] 2*Melitopol Research Fruit Growing Station named after M.F. Sidorenko of the Institute of

Horticulture of National Academy of Agrarian Sciences of Ukraine, UKRAINE

Abstract. The microbiological preparation BIO–ONE is a 100% natural liquid concentrated microbiological product providing 20 kg of active nitrogen/decar. It facilitates the absorption of the residual phosphorus and potassium and retains moisture. BIO–ONE does not contain genetically modified organisms. It is recognized by all organizations controlling the organic farming, including OMRI Institute. It could be used both in organic and traditional agriculture. With its gradual application in soil the microbiological preparation BIO–ONE contributes to increasing soil organic matter and also protects it from wind erosion. The application of the microbiological preparation BIO–ONE helps for obtaining the maximum efficiency of the used mineral fertilizers. The microbiological preparation BIO–ONE has a positive impact on the yield of peas seeds. After treatment of the crop with the microbiological preparation BIO–ONE the highest yield was obtained at a dose of treatment of 0.9 g/100 m2–185.0 kg/da, followed by the variants treated at a dose of 0.12 g/100 m2–167.0 kg/da and 0.6 g/100 m2–165.0 kg/da. At the lowest dose of treatment of 0.3 g/100 m2 and the fertilized control the yield was equal–158.0 kg/da. The lowest yield was obtained from the control variant. It increases the wheat yield as after–effect after treatment of peas at concentration of 0.6 to 0.12 g/100 m2 as the wheat yield is raised by 43 kg/da compared to the untreated control. The highest yield was obtained for the variant of peas fertilized with N4P8–as the yield exceeded by 57 kg/da the control that was not fertilized and treated. It is noticed also that at lower doses of treatment the quantity of siftings is lower as it goes up at higher doses of treatment.

Key words: fertilizing, bacterial fertilizers, peas.

Introduction Preparation Bio–One was created

and produced in the United States as a result of eight years of research and development and it is a microbiological product. It consists of live organisms.

It is harmless as microorganisms live freely in nature. They are of two kinds:

Aerobic–Azotobactervinlandii Anaerobic–Clostridiumpasterianum

The microorganisms capture nitrogen from the air and process it to an extent to be absorbed and thus they nourish the plants. Conditions for anions and cations exchange in the soil are obtained [POSTGATE 1982, ABBASS at al., 1993 .

It is effective in pastures, gardens, flowers, etc. Animals prefer pastures treated with microbiological preparation BIO–ONE [PACOVSKY 1990 .

The oil extraction from unit production is increased for the oleaginous plants.

In fruits and vegetables it facilitates sugar accumulation resulting in better taste and aroma of the fruit [BROWN, 1974,

BASHAN, 1998 . Gradually with its application in soil

the microbiological preparation BIO–ONE contributes to increasing soil organic matter and also protects it from wind erosion.

Application of microbiological preparation BIO–ONE helps also for the maximum impact of the used mineral fertilizers.

The microbiological preparation BIO–ONE is a 100% natural liquid concentrated microbiological product providing 20 kg of active nitrogen/decar.


34

mucosa by its direct toxic effect, reducing secretion of bicarbonates and production of mucus.

It is well known that many flavonoids display anti–secretory and cytoprotective properties in different experimental models of gastric ulcer.

Flavonoids possess anti–oxidant properties in addition to strengthening mucosal defense system through stimulation of gastric mucus secretion it also can scavenge for reactive oxygen species (super–oxide anions) and free radicals produced by ethanol which are

potentially implicated in ulcerogenicity MAHMOOD, 2010 .

Tannins could also prevent ulcer development via their abilities to complex proteins where it precipitate micro proteins at ulcer sites, protecting under laying mucosa from irritation they also showed strong radical scavenging activities HASLAM, 1996 .

Phytochemical screening of leaves of T. arjuna revealed presence tannins which we reported previously its isolation (Gallic acid, Ellagic acid and methyl gallate) from T. arjuna leaves ROSS et al.,

2013 . Table 2.

The effect of oral administration of 70% methanol extracts T. arjuna W. and A. (250, 500 and 1000 mg/kg) on gastric mucosal injury induced by 100 % ethanol (1 ml) in rats (n=6). Group Number of lesions/

Rat X±S.E % Change Severity of lesions/ Rat X ±S.E %Change

Ethanol 100% 9.6 ±0.8 –––––– 19.6 ±1.5 ––––––– 70% MeOH extract (250mg /kg) 4.6 ± 0.3* –52.1 % 6.4 ±0.5* –67.3 % 70% MeOH extract (500mg /kg) 3.2 ±0.2* –66.7% 5.6 ±0.4* –71.2 % 70% MeOH extract (1000mg/kg) 3 ± 0.1* –68.8 % 4.4 ±0.3* –77.6 % Data represent the mean value ± SE of six rat per group. Statistical comparison of difference between ethanol control group

and treated groups. Values represent the mean ± S.E. of six animals for each group. P< 0.05: Statistically significant from Control. (Dunnett's test)

Oxidative stress plays an important

role in the pathogenesis of various diseases including gastric ulcer.

The antioxidants play a significant role in protection of gastric mucosa against various necrotic agents it could be concluded that the gastroprotective effect exerted by T. arjuna could be attributed to

its antioxidant property which counteract oxidative damage caused by absolute ethanol toxicity.

And it’s the ability to strength mucosal barrier, which is first line of defense against endogenous and exogenous ulcerogenic agents.

Figure 2. The effect of oral administration of 70 % methanol extracts T. arjuna W. and A.

(250, 500 and 1000 mg/kg) on No. of lesions in gastric mucosal induced by 100 % ethanol The results were recorded in table 2

and showed in figure 2. The phytochemical analysis revealed presence of flavonoids, tannins, sterols



35

/or triterpenes, carbohydrate/or glycosides and absence of alkaloids table 3. Table 3.

Results of phytochemical screening of the extracts of T. arjuna W. and A. leaves. Constituents Dichloromethane Ethyl acetate 70% MeOH Carbohydrate /or glycosides – – + Coumarins – – – Flavonoids – + + Alkaloids – – – Sterols/or triterpenes + – + Saponins ± – – Anthraquinone – – – Tannins – – +

(+): present, (–): absent, (± ): traces

Conclusions T. arjuna powdered leaves was

extracted by maceration with 70% MeOH, the phytochemical screening revealed the presence flavonoids and tannins as major constituents.

The 70% MeOH extract showed very promising gastro protective activity in all tested concentrations also T. arjuna 70 % methanolic extract showed a dose dependant hepatoprotective effect and at dose 1000 mg/kg managed to render the liver enzyme levels back to normal and its effect at that dose is similar to Silymarin.

The Hepato and Gastro protective activities of 70% MeOH extract of T. arjuna leaves t is due to their flavonoids and tannins contents.

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Received: September 16, 2014 Article in Press: September 22, 2014




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C.; Wang, Q.; Mei L.; Lin, X.; Assessment of four DNA fragments (COI, 16S rDNA, ITS2, 12S rDNA) for species identification of the Ixodida (Acari: Ixodida). Parasit Vectors. 2014. Mar 3; 7:93. doi: 10.1186/1756–3305–7–93.

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12. Mahmood, A.A. Mariod A.A. Al–Bayaty F. Abdel–Wahab S.I. Anti–ulcerogenic activity of Gynura procumbens leaf extract against experimentally–induced gastric lesions in rats, Journal of Medicinal Plants Research 2010, 4(8):685–691.

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Received: July 1, 2014




37

EVALUATION OF BREEDING POTENTIAL FIELD PEA (Pisum sativum L.)

CULTIVARS AND THEIR PROGENIES

DOI: 10.7904/2068–4738–V(10)–37

Valentin Ivanov KOSEV

Institute of Forage Crops–Pleven, Gen. Vladimir Vazov 89 Street, 5800 Pleven, BULGARIA;

e–mail: [email protected]

Abstract: The inheritance and gene effects structure were investigated for ten quantitative traits in field cultivars by a full diallel model of four parents–the cultivars Kerpo, Pleven 10, Mir and EFB33. Over dominance was prevalent for most of the traits in both hybrid generations (F1 and F2) except for 1000–seed weights. A non–additive gene action appeared to be more predominant for the inheritance of most traits studied. The fraction of h2/H2 indicated that at least one group of genes controlled dominance for all the traits, namely for seed weight per plant (6), seeds per pod (3). The Н2/4Н1 ratio was close to the expected value of 0.25 for seeds per plant and seed weight per plant, indicating a symmetrical distribution of dominant and recessive genes. Dominant alleles were more frequent in parental forms for the inheritance of most traits except seeds per pod in F1. In the case of seeds per pod (F1) and 1000–seed weight (F1 and F2) a comparatively low difference was established between the coefficient of heritability in narrow and broad sense, indicating that the selection in early generations may be effective. The genotypes with exhibited dominant alleles were EFB33 for plant height, first pod height, internode distance and fertile nodes per plant, Mir for pods per plant, nodes per plant and 1000–seed weight and Pleven 10 for seeds per plant and seed weight per plant. Kerpo had recessive alleles for all the tested traits except seeds per pod.

Key words: breeding, field pea, gene effects, genetic analysis, inheritance

Introduction Pea (Pisum sativum L.) is an

important forage and vegetable crop grown in various regions worldwide.

However, its productivity has become static over the years despite the fact that its total harvested area has increased tremendously.

This can be attributed to the lack of suitable improved cultivars for different agroecological conditions.

In order to increase the yield potential in pea to a greater extent, it is desirable to develop new genotypes through a recombination breeding for which it is necessary to know the mode of inheritance and the genetic background of the desirable traits.

The structure of the gene effects is an indicator for assessing the selection value of the parental components and is used widely in breeding and applied genetic research.

A diallel scheme of crossing based on the data of the F1 hybrids is an accurate method for its determination

CSIZMADIA, 1994; SHARMA et al., 1999; SRIVASTAVA et al.,

2000; BOURION et al., 2002 . It is determined as the additive

effects and the quantity of the effective factors at inheritance of the specific traits.

Of a special importance is the choice of the parents used in developing novel, stable and heterotic lines.

The many authors reported on the inheritance of yield–related traits in field pea. Number of days to flowering is said to be additive PARMAR and GODAWAT, 1990 and non–additive SINGH et al., 1986 for days to flowering, number of pods per plant additive RATHORE et al., 1995 and epistatic NARSINGHANI et al., 1982 , number of seeds per pod additive and non–additive SINGH and

SINGH, 1991 , plant height additive KUMAR et al.,

1996 , both additive and non–additive DIXIT,

1999 and PANDA et al., 1996 . All these findings were probably the

major bottleneck towards the formulation of efficient breeding strategies SOOD and

KALIA, 2006 .


56

We compared the sequences of COXI gene of H. dromedarii from Bikaner with the corresponding sequences of H. dromedarii from various parts of the world and other ixodid tick species available in the database.

Sequence analysis revealed that COXI gene of H. dromedarii from Bikaner shared 99.1 & 99.2% sequence identity at the nucleotide level with H. dromedarii isolates from Kenya and Ethiopia, respectively.

With other species of Hyalomma, H. dromedarii from Bikaner exhibited 86.2–90.2 nucleotide identity. With other genera of the family Ixodidae, H. dromedarii from Bikaner showed 82.9–84.78% nucleotide identity (Table 1).

Although a low level of intraspecific variation in COX1 and ITS2 was observed among individuals of Ixodes holocyclus in Clade II, Song and his team [SONG et al., 2011] noted that the ticks from different geographic ranges could be genetically distinguished.

As the standard DNA barcode, COI is the first choice for species identification of ticks, while 16S rDNA, ITS2 and 12S rDNA could be used as complementary to COI, thereby circumventing situations where COI fails to produce reliable results.

Moreover, either NN (Nearest Neighbour) or BLASTn could be used for tick species identification because both methods outperformed tree–based methods [LV et al., 2014].

The length of the P–18 gene sequenced was 461 bp. BLAST search analysis in NCBI database showed that it has closely matched with P–18 gene of H. asiaticum (Accession No. EU000252), which is the only one available sequence in NCBI database.

Pair–wise comparison of these two sequences showed that 461 bp of H. dromedarii from India was matching with the nucleotide base, 26 to 486 of P–18 gene of H. asiaticum from China (data not shown).

These partial 461 bp gene sequences compared with the corresponding nucleotide sequences of P–18 gene of H. asiaticum (GenBank

accession No. EU000252) and it was found that they had 90.4 % sequence identity with H. asiaticum.

The nucleotide sequences of the salivary gland protein (P–18) gene of H. dromedarii were submitted to GenBank, NCBI database and assigned accession number HM051110.

The results of the present study indicate that the tick species infesting the one humped camels, maintained at NRCC herd, India is Hyalomma dromedarii.

Further the baseline information about the salivary gland protein P–18 gene of the present study affords the avenues for the exploration of sialomics of H. dromedarii and other ixodid ticks from different geographical areas of India and thereby the development of a new generation vaccine for the control of ticks would be feasible in India.

Therefore, it is recommended that extensive research work on the analysis of the genes involved in the phylogenetic analysis as well as various salivary gland protein genes of different ixodid tick species infesting the camels of different geographical areas of India needs to be carried out for the elucidation of evolution of hard ticks and the development of a common vaccine candidate gene for their control.

Further, it is proposed that the vector potentiality of H. dromedarii in the transmission of the viral diseases among the Dromedary camels of the NRCC herd.

Conclusions From the major findings of the

present study, it is concluded that the tick species infesting the one humped camels, maintained at National Research Centre on Camel, India is Hyalomma dromedarii.

Acknowledgements The authors are thankful to Dr. P.N.

Sivalingam, Scientist, CIAH, Bikaner, India, for critical reading and preparation of the manuscript.

The help rendered by Jeetendar Kumar, Technician, M.L. Kiradoo, Lab Attendant, NRC on Camel, Bikaner, Shahid Hussain and Shokat Ali in the



55

which the assigned Accession No. is GQ483461.

The percent nucleotide identity of COXI gene of H. dromedarii Bikaner

isolate with different ixodid tick species from various parts of the world are shown in Table 1.

Table 1. Percent nucleotide identity of cytochrome oxidase gene of Hyalomma dromedarii from

Bikaner with different species of Hyalomma and other ixodid ticks Sl.No. Tick species NCBI Accession No Percent nucleotide identity 1 Hyalomma dromedarii–Bikaner GQ483461 – 2 H.dromedarii–HY01–Ethiopia AJ437061 99.2 3 H.dromedarii–HY02–Ethiopia AJ437062 99.2 4 H.dromedarii–HY13–Kenya AJ437071 99.1 5 H.detritum EU827694 90.2 6 H.marginatum EU827692 88.3 7 H.aegyptium AF132821 87.8 8 H.lusitanicum EU827732 87.1 9 H.truncatum–HY86 AJ437087 86.2 10 Rhipicephalus appendiculatus AF132833 84.7 11 Rhipicephalus sanguineus AF132839 84.3 12 Dermacentor variabilis AF132831 84.4 13 Boophilus annulatus AF132825 82.9

The nucleotide sequences of COXI

gene of H. dromedarii from Bikaner showed a higher homology to H. dromedarii isolates from Ethiopia.

A phylogenetic tree constructed using the partial nucleotide sequences of COXI gene of different ixodid tick species

revealed that the H. dromedarii from Bikaner clustered with H. dromedarii isolates from Kenya and Ethiopia.

Boophilus annulatus was considered as the out group in the phylogenetic tree (Figure 1).

Figure 1. Phylogenetic tree based on nucleotide sequences of cytochrome oxidase gene

from different Hyalomma species and ixodids, constructed by the neighbor–joining method using MEGA 4 (Molecular Evolutionary genetics Analysis software with bootstrap values

calculated for 1,000 replicates. Horizontal distances are proportional to the genetic distances. Vertical distances are arbitrary. The numbers at each branch represent

bootstrap values (1000 replicates).


38

By this reason, four field pea cultivars were hybridised in a full diallel model in order to study the genetics components of the traits related to seed yield.

Material and methods An experimental study was

conducted from 2009 to 2011 at the Second Experimental Field of the Institute of Forage Crops, Pleven.

The following field pea (Pisum sativum L.) cultivars were used as the parental components: the spring–sown Kerpo and three autumn–sown ones, namely Pleven 10, Mir and EFB33. The hybridisation was done by hand in 2009.

The experiment was set up as a complete block design with two replications during the winter of 2010/2011.

The parental forms (P1 and P2) and the first and the second hybrid generations (12 F1 and 12 F2) were sown according to a scheme P1 F1 F2 P2, with a plot size of 2 m × 1 m, at a row spacing of 20 cm, a distance within row of 5 cm and a plot to plot distance of 0.70 m.

The seeds were sown by hand at a depth of 5 cm.

All the agronomy practices during the trial were ordinary and officially approved by the Institute of Forage Crops.

The hybridisation included all direct and reciprocals crosses, that is, a full diallel scheme. From each genotype in Р1, Р2, F1 and F2, a sample of 40 plants was used for analysing the following quantitative traits: plant height (cm), first pod height (cm), number of pods per plant, number of seeds per plant, number of seeds per pod, number of nodes per plant, internode distance (cm), seed weight per plant (g), 1000–seed weight (g), and number of fertile nodes per plant.

The obtained data were analysed according to Hayman HAYMAN, 1954a , while the genetically properties of the parental components were determined by the graphical method by Hayman HAYMAN, 1954a;

HAYMAN, 1954b , using the software DIALL UKAI, 1989 .

The diallel analysis of variance (ANOVA) graphical analysis (Wr/Vr; Wr^/Vr) by Hayman is based on the variance–covariance matrix (Vr–Wr), where: a is primarily additive effects; b is primarily dominance effects; b1 is mean deviation of F1

’s from their mid–parental value; b2 is variation of deviation of F1

’s from their mid–parent value over arrays; b3 is that part of dominance variation unique to each F1; c is average maternal or cytoplasmic effect of each parental line; d is reciprocal differences not ascribable to c.

The mean square of casual deviations of initial data on ANOVA analysis of diallel crosses was used for estimating σe

2. Estimating includes the values of

the following genetically parameters: – D are additive gene effects (additive

variance); Н1 and Н2 are dominance gene effects (dominance variance 1 and dominance variance 2);

– F is covariance of additive and non–additive effects in all the arrays that has a positive or negative sign depending on whether dominant genes or recessive genes were more. When F > 0 predominate dominant alleles, when F < 0 the recessive, when F = 0 alleles are equal representative.

In addition, h2 is dominance effect as the algebraic sum over all loci in heterozygous phases in all cases; Hbs is heritability for diallel in a broad sense; Hnsis heritability for diallel in a narrow sense; Н1/D is average degree of dominance in experimental material; Н2/4Н1 is mean value (piqi) on all polymorphic locuses show dominating (di ≠ 0); h2 /H2 is number of groups of genes which controlled character and exhibited dominance; and [(4DH1)½+F/(4DH1)½–F] is proportion of dominant and recessive genes in the parents.

The conclusions from the Wr, Vr graph are:

1) Wr is related to Vr by a straight regression line of unit slope in the absence of non–allelic interaction and with independent distribution of genes among the parents;



39

2) The distance between the origin and the point where the regression line cuts the Wr–axis provides a measure of the average degree of dominance;

3) D > H1 (partial dominance) when the intercept is positive; D = H1 (complete dominance) when line passes through the origin; D < H1 (over dominance) when intercept is negative; and no dominance when the regression line touches the parabola limit.

Results and discussion One dealing with applied genetics

and plant breeding needs to know the genetically structure of a specific trait, that is, what genetically effects prevail, number of the polymorphic locuses, how the alleles are shared, and so on.

Such information gives opportunity of including novel cultivars in hybridisation designs and breeding programmes.

The theoretical base on diallel analysis of the additive–dominant model according to Hayman is founded upon the following presumptions:

– the investigated genotypes are diploid and homozyguous,

– every locus can be in two allelic positions,

– no exhibition of epistatic effects, no difference in inheritance in reciprocal crosses and independently distribution of the genes between the parents HAYMAN,

1954a . Diallel ANOVA The components of the genetically

variation were established with the dispersion analysis of the traits in the tested field pea genotypes (Table 1 and Table 2).

The analysis of variance revealed highly significant differences among the progenies indicating that the parents were diverse for the studied traits.

In the diallel ANOVA, all the genetic effects were significant without b3 and d in F1 for 1000 seed weight, c for height first pod, b2 for seeds per plant and a, b, b2 for seeds weight per plant in F2.

Dominant gene effects prevailed in a majority of the traits in both F1 and F2.

The additive gene effects prevailed solely for 1000–seed weight in F1 and seeds per pod and 1000 seed weight.

Wr/Vr analysis The estimate of the components of

genetically variation for the different quantitative traits in forage pea is given in Table 3.

Plant height The line of regression intercepted

the negative part of axis Wr, showing the importance of over dominance (H1/D > 1; F > 0) in F1 and F2 (Figure 1 and Figure 2).

The regression coefficient was different from zero in both generations, showing the absence of epistatic gene effects and that the genes are independently distributed between the parents.

One is the number of the group of genes controlling this trait and exhibiting dominance (h2/H2) in both generations.

The true value of H2 in principle can not be higher than H1. The ratio Н2/4Н1 can be done by concluding that plus and minus alleles are distributed in a non–uniform way between parental forms.

The ratio of (4DH1)½+F/(4DH1)½–F showed a prevalent dominant gene effect.

First pod height A regression dotted line intercepted

the positive part of vertical axis solely in F1.

This was an indication that mean degree of dominance is partial and that one of the parents was rare genotype.

A solid line in F1 and F2 intercepted the negative part indicating the importance of over dominance.

The genetically parameter D was lower than H1 (H1/D > 1) in F1 (1.18) and F2 (1.27).

The ratio h2/H2 was similar to the one in plant height (0.62 in F1 and 0.84 in F2) indicating that one group of genes had control over the trait as Н2/4Н1 (0.20) for both generations.

The ratio of dominant and recessive genes was close or more than one, assuming the action of dominant genes.

The heritability (Hns and Hbs) could be estimated as medium to high.


54

described by El–Kammah and his team [EL–KAMMAH et al., 2005].

Genomic DNA and total cellular RNA were extracted from the tick salivary glands using respective Genei UltrapureTM Mammalian Genomic DNA Purification Kit–Tissues and Total RNA isolation kit–Cells and Tissues (Bangalore GeNei Pvt. Ltd, India) according to the manufacturer’s instructions.

Polymerase chain reaction The Exon 1 region of the

mitochondrial cytochrome oxidase gene of H. dromedarii was amplified from the total genomic DNA isolated from the salivary glands of female ticks using the forward primer of COI 5’ TTA CCG CGA TGA TTA TAT TCA ACA AAT CAT 3’ and reverse primer of COI 5’ GTT CTT TTT TTC CAG TAT TAA AAC AAA TAA 3’.

The sequences of the primers were deduced from the GenBank accession No. AJ437083.

Using total cellular RNA extracted from the tick salivary glands as a template, cDNA was synthesized by Easyscript First Strand cDNA Synthesis Kit according to the manufacturer’s instructions From the resultant cDNA synthesized, the P–18 protein gene of H. dromedarii was amplified using the forward primer of

5’ GAG CGG ATC CAT GAT TTT ATG GGC GCT TTG 3’

and reverse primer of 5’ CGC GCT CGA GTT ACC ACT CAA

TCT TGA CTG 3’. The sequences of the primers

were deduced from the GenBank accession No. EU000252.

PCR amplification was performed with the following thermal profiles: initial denaturation of 94 C for 3 min. followed by 35 cycles of denaturation at 94 C for 1 min., annealing at 55 C for 1 min for COXI gene; 57 C for 1 min for P–18 gene and extension at 72oC for 1 min. and final extension at 72 C for 10 min.

The PCR amplified products were checked on 1% agarose gel.

Cloning and sequencing of PCR amplified DNA fragments

The PCR amplified products were checked in 1.2% agarose gel.

The purified amplicons corresponding to genes encoding COXI and P–18 of H. dromedarii were cloned into pGEM–T Easy vector (Promega Corp., Medison, USA).

The ligated mixtures for both genes were individually transformed into Escherichia coli DH 5α [SAMBROOk et al., 1989].

The positive clones were confirmed by colony PCR using gene–specific primers and restriction analysis with EcoRI.

The positive clones were sequenced at the sequencing facility, Delhi University (South campus), Delhi.

Since pGEM–T easy vector was used for the cloning purpose, universal T7 and SP6 primers were used for the sequencing of recombinant clones.

The primer sequences used for the sequencing were based on respective promoter sequences.

The determined nucleotide sequences and the deduced amino acid sequences of the GIF and UDG were analyzed with the BLAST program (NCBI) search of GenBank.

Nucleotide identity and comparison of the sequences with published sequences of members of Ixodidae available in the GenBank database were carried out using the computer software BioEdit version 7.0.9.

These sequences were compared in Clustal X [THOMPSON et al., 1997] and a phylogenetic tree was constructed based on the amino acid sequences by the neighbor–joining method using Mega 4 (Molecular Evolutionary genetics Analysis software with bootstrap values calculated for 1,000 replicates [TAMURA et al., 2007].

Results and discussion The size of COXI gene of

H.dromedarii from Bikaner is 793 bp in length, which is only partial gene sequence.

The resultant gene sequences were submitted to GenBank, NCBI database for



53

This approach allows for the description of secretory products involved in blood–feeding of hematophagous organisms.

The analysis of sialomes in terms of their protein domain compositions allows for a comparative analysis of sialome complexity and diversity that gives us insights into evolution of blood–feeding behavior in arthropods [MANSA et al., 2008].

Research works have been carried out for the characterization of the salivary gland proteins of H. dromedarii at protein level [KANDIL and HABEEB, 2009].

Said and his team recently undertook the molecular characterization of Bm86 orthologue from H. dromedarii [SAID et al., 2012].

National Research Centre on Camel (NRCC), Bikaner, Rajasthan state, India, is a premiere institute carrying out the excellent research activities exclusively on camels. At NRCC herd, inspite of the regular application of the acaricides, tick infestation is an unavoidable menace in the Dromedary camels.

In Indian Dromedary camels, the clinical cases of only species specific viral infections such as camelpox [BALAMURUGAN et

al., 2009] and contagious ecthyma [NAGARAJAN

et al., 2010] have been reported both in organized farms and field conditions.

Various studies have demonstrated that during the rainy seasons, the incidence of both camelpox [WERNERY et al.,

1997] and contagious ecthyma [NAGARAJAN et

al., 2010] outbreaks increases during rainy seasons with the appearance of more severe forms of the disease.

This may be due to the fact that besides the moisture affords the conducive environment for the virus stability and subsequent transmission to susceptible animal, the involvement of arthropods abundant during rainy seasons may serve as a mechanical vector of the virus.

This idea was evidenced by the isolation of camelpoxvirus (CMLV) from H. dromedarii [WERNERY et al., 1997].

An outbreak of camelpox in September 2008 and contagious ecthyma in September 2010 occurred among the Dromedary camels maintained at NRCC

herd, Bikaner, Rajasthan state, India (unpublished data).

In Rajasthan state of India, September month is the monsoon period.

I t was speculated that ticks infested the Dromedary camels of the NRCC herd could have served as the mechanical vectors in the above mentioned both camelpox and contagious ecthyma outbreaks.

Prior to the studies on the vector potentiality of the ticks in the transmission of the diseases, it is imperative to identify the species of the ticks infesting the camels of NRCC herd.

Till date, there is no published report on work related to the identification of tick species infesting the one humped camels (Camelus dromedarius) maintained at NRCC herd, India.

Further, no information is available about the salivary gland protein (P–18) gene of H. dromedarii.

Keeping these in view, in the present study, the cytochrome oxidase (EC 1.9.3.1) subunit I gene and P–18 protein gene of the ticks infesting the Dromedary camels (Camelus dromedarius) maintained at NRCC herd, India, were cloned and sequenced and its phylogenetic relationship with other tick species is described.

Material and methods A field experiment was carried out

in the period 2009–2011 with Ticks Six adult ticks were collected from

the ground of camel pens of NRCC herd, India.

Morphological features were examined under stereo microscope and identified using the guide to identification of species [KANDIL and HABEEB, 2009] and morphological features of H. dromedarii was identified as per the keys described by Apanaskevich and co–workers [APANASKEVICH et al., 2008].

Isolation of salivary glands,

genomic DNA and cellular RNA Salivary glands of six H. dromedarii

were isolated as per the protocol


40

Table 1. Analysis of variance for combining ability of the field pea hybrids traits in F1 generation (by Hayman)

Source of variation df

Mean square Plant height (cm)

First pod height (cm)

Pod number per plant

Seed number per plant

Seed number per pod

Node number per plant

Internode distance (cm)

Seed weight per plant (g)

1000–seed Weight (g)

Fertile node number / plant

Replication 3 31.25 4.27 0.89** 28.79 0.18* 1.18 0.13 3.06 57.58 1.18 a 3 8315.25** 1760.96** 186.61** 6110.33** 0.61** 116.55** 1.44** 15.47** 11933.33** 57.89** b 6 9279.99** 2132.31** 371.96** 10593.63** 1.61** 234.26** 9.32** 333.68** 3074.56** 97.10** b1 1 2346.40** 2662.63** 1535.67** 46376.33** 1.88** 923.13** 27** 1698.13** 4116.26** 376.88** b2 3 15374.74** 2352.30** 186.86** 4134.69** 1.70** 114.80** 8.77** 91.55** 4676.09** 58.80** b3 2 3604.66** 1537.17** 67.75** 2390.68** 1.33** 69.02** 1.31** 14.65** 151.42 14.65** c 3 1319.19** 283.06** 156.88** 2889.23** 1.21** 180.15** 1.52** 33.48** 2017.15** 43.52** d 3 2932.41** 1203.06** 294.25** 2846.52** 2.54** 322.98** 1.90** 130.15** 651.56 74.02** Error 45 43.88 25.62 3.84 113.14 0.16 3.67 0.14 2.68 392.25 0.87

* significant at 5% level, ** significant at 1% level Table 2.

Analysis of variance for combining ability of the field pea hybrids traits in F2 generation (by Hayman) Source of variation df

Mean square Plant height (cm)




Seed number per pod





Fertile node number /plant

Replication 3 3 367.92* 163.60 32.68 1939.71 0.18 34.89* 0.23 67.10** 4009.04** a 3 3 8225.25** 1471.90** 83.09** 8806.50** 2.36** 62.39** 1.85** 6.28 17037.58** b 6 6 9060.83** 2551.62** 524.35** 12229.00** 1.50** 339.85** 9.73** 402.54 2625.60** b1 1 1 5676.75** 4265.76** 1782.42** 49987.2** 1.51* 1097.30** 38.52** 1969.92** 1819.17** b2 3 3 13821.42** 2607.30** 281.93** 1855.85 2.28** 213.89** 6.19** 29.95 3922.78** b3 2 2 3612.00** 1632.01** 258.94** 8909.46** 0.33 150.06** 0.65** 177.75** 1083.06** c 3 3 650.08** 66.15 135.40** 4906.10** 3.25** 88.77** 1.06** 51.25* 693.54** d 3 3 3835.58** 276.98* 171.56** 8402.40** 1.08* 135.69** 1.10** 85.21** 691.88** Error 45 45 95.56 95.91 13.99 824.63 0.26 8.69 0.12 12.42 64.41

* significant at 5% level, ** significant at 1% level Table 3.

Estimates of components of variation for different quantitative traits in field pea cultivars Genetic parameters

Plant height (cm)




Seed number per pod





Fertile node number/ plant

F1 F2 F1 F2 F1 F2 F1 F2 F1 F2 F1 F2 F1 F2 F1 F2 F1 F2 F1 F2 D 5213.7 5203.38 965.81 951.62 88.39 78.18 2112.03 1555.80 0.10 0.17 51.76 50.51 1.63 1.63 6.62 4.18 3049. 766.45 28.18 22.92 HI 6534.3 6198.37 1344.17 1545.27 206.94 288.66 5738.10 5831.07 0.91 1.09 129.18 191.22 5.67 5.56 176.60 197.25 1876. 1762.8 55.27 68.64 H2 4619.3 4485.62 1054.14 1234.35 184.18 255.61 5240.15 5727.94 0.72 0.82 115.41 165.84 4.59 4.80 165.58 195.44 1353. 1282.6 48.08 58.56 F 6096.1 5902.90 1039.73 1093.55 88.42 103.04 1865.07 686.99 0.23 0.06 51.55 69.45 2.54 2.17 16.13 7.14 2141. 2727.1 28.30 30.23 D–H1 –1320.6 –994.99 –378.36 –593.65 –118.55 –210.48 –3626.0 –4275.2 –0.81 –0.92 –77.42 –140.71 –4.04 –3.93 –169.98 –193.07 1172. –996.43 –27.09 –45.72 h2/H2 0.12 0.31 0.62 0.84 2.07 1.73 2.20 2.15 0.59 0.84 1.99 1.64 1.46 1.99 2.56 2.50 0.69 0.34 1.95 1.73 Hl/D 1.12 1.09 1.18 1.27 1.64 1.92 1.64 1.93 3.01 2.52 1.58 1.94 1.86 1.84 5.16 6.86 0.78 0.63 1.40 1.17 Н2/4Н1 0.18 0.18 0.20 0.20 0.22 0.22 0.23 0.25 0.20 0.19 0.22 0.22 0.20 0.22 0.23 0.25 0.18 0.18 0.22 0.21 [(4DH1)½+F/ (4DH1)½ –F] 1.0002 1.0002 1.0008 1.0007 1.0048 1.0046 1.0002 1.0001 –8.583 1.3863 1.0077 1.0072 1.3186 1.2720 1.0139 1.0087 1.000 1.0020 1.0183 1.0194

Hns 0.30 0.30 0.28 0.20 0.19 0.05 0.21 0.22 0.10 0.41 0.19 0.06 0.06 0.08 0.01 0.00 0.62 0.76 0.22 0.08 Hbs 0.99 0.98 0.98 0.94 0.98 0.95 0.98 0.90 0.83 0.87 0.97 0.95 0.97 0.97 0.98 0.94 0.91 0.98 0.98 0.98



41

Number of pods per plant Intercepting the regression line with

the vertical axis (Wr) showed overdominance in both the generations (Figure 1 and Figure 2).

The regression coefficient was near one and very different from zero in both generations, indicating the absence of non–allelic interactions.

In the case of the component analysis, the dominance (b) and the additive (a) components were significant in both the generations.

The magnitude of H1 was higher than the D, (H1/D > 1; F > 0) showing overdominance.

One–two groups of genes controlled this trait and exhibited dominance.

Figure 1. Diagram of climate in the vegetation period by years

The positive and negative alleles were distributed almost uniformly between parental forms Н2/4Н1 (0.22) in both hybrid generations.

The ratio between dominant and recessive genes favoured the dominant genes.

The estimate of narrow sense heritability was very low and broad sense high.

Number of seeds per plant The regression line on Wr in the F1

and F2 is given in Figure 1 and Figure 2. The coefficient a of the regression

equation was negative and H1/D > 1 indicating over dominance.

The coefficient b of the regression equation was close to unity indicating the absence of non–allelic interaction.

The estimates of components of variation revealed high values for dominance (b) as compared to additive genetically components (a) in the F1 and F2.

The ratio h2/H2 (2.20 in F1 and 2.15 in F2) indicated that two or three groups of genes controlled number of seeds per plant.

The ratio Н2/4Н1 in F1 (0.23) and especially in F2 (0.25) showed that the alleles were distributed uniformly between the parents and that the successful selection for increasing the value of this trait was 50%.

The values of the ratio between dominant and recessive genes prevailed to dominant genes.

The coefficient of heritability could be estimated as medium (Hns) to high (Hbs) in both generations.

Number of seeds per pod Intercepting the regression line with

the vertical axis (Wr) below the beginning in both F1 and F2 indicated over dominance.

The regression coefficient was different from unity in both generations


52

Molecular Characterization of Hyalomma dromedarii from Bikaner, India

DOI: 10.7904/2068–4738–V(10)–52

HARISH K. CHANGAL1, G. NAGARAJAN2*, R. K. PUROHIT1, SHELESH KUMAR

SWAMI2, S.C. MEHTA2 and K.M.L. PATHAK3

1 Radiation Biology Laboratory, P.G. Department of Zoology, Govt. Dungar College, Bikaner, Rajasthan, INDIA

2 National Research Centre on Camel, Post Bag No. 7, Jorbeer, Bikaner–334 001, Rajasthan, India 3 Indian Council of Agricultural Research, Krishi Bhavan, New Delhi–110001, INDIA.

*Corresponding author: Dr. G. Nagarajan, M. V. Sc., Ph.D., Senior Scientist, Southern Regional Research Centre under Central Sheep and Wool Research Institute, (CSWRI), Mannavanur,

Kodaikanal, Tamil Nadu–624 103, INDIA; e–mail: [email protected]; [email protected]. Phone: +91–4542–276414; Fax: +91–4542–276413

Abstract. In this study, partial nucleotide sequences of cytochrome oxidase (COXI) gene and P–18 protein gene of Hyalomma dromedarii from one humped camels of National Research centre on Camel, Bikaner, India, were amplified by polymerase chain reaction from the salivary glands of the ticks. Sequence analysis revealed that COXI gene of H. dromedarii from Bikaner shared 99.1–99.2 % sequence identity at the nucleotide level with H .dromedarii isolates from Kenya and Ethiopia, respectively. With other species of Hyalomma, H. dromedarii from India exhibited 86.2–90.2 nucleotide identity. With other genera of the family Ixodidae, Hv. dromedarii from India showed 82.9–84.78 % nucleotide identity. But, salivary gland protein gene P–18 of H. dromedarii from Bikaner was having 90.4 % sequence identity with that of H. asiaticum.

Key words: Hyalomma dromedarii; Dromedary camel; National Research centre on Camel;

Bikaner; India

Introduction Hyalomma dromedarii can behave

as a three– or two host species, with the two–host life cycle seemingly the most common.

Camels are the main hosts of the adults, which also parasitize other domestic animals.

Nymphs and larvae can parasitize the same hosts as adults, especially camels, but rodents, hedgehogs, and birds can also serve as hosts [ANDERSON,

2002]. H. dromedarii is widely distributed

throughout North Africa, the northern regions of West, Central, and East Africa; Arabia, Asia Minor, the Middle East, and Central and South Asia [APANASKEVICH et al.,

2008]. H. dromedarii is a vector of many disease agents such as protozoa [d'OLIVEIRA

et al., 1997], bacteria [MONTASSER, 2005], virus [GUMES, 2006] and rickettsia [KERNIF et al., 2012].

Ticks species can be morphologically identified using

taxonomic keys for endemic species in several geographic regions [HUBALEK and

RUDOLF, 2012]. However, morphological

identification can be difficult because it requires some entomological expertise, and it is difficult to identify a specimen that is damaged or at an immature stage of its life cycle [HUBALEK and RUDOLF, 2012].

Molecular methods, such as the sequencing of the mitochondrial 12S [NORRIS et al., 1996], 18S [MANGOLD et al., 1998] and 16S rDNAs [NORRIS et al., 1996]; mitochondrial cytochrome oxidase subunit 1 (COX1); and nuclear internal transcribed spacer 2 (ITS2), have been developed to identify arthropods, including ticks [SONG et al., 2011].

The field of vector–host interaction has gained tremendously by high–throughput analysis of salivary gland transcripts and proteomes, collectively called the sialome [RIBEIRO and FRANCISCHETTI,

2003].



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Received: September 16, 2014 Article in Press: September 22, 2014



42

indicating presence of non–allelic interaction.

The additive (a) and dominance (b) components of variation were significant in both generations.

Figure 2. Diagram of climate in the vegetation period by years

The values of H1/D for F1 and F2 suggest that the dominance component was more important than the additive component for the expression of this trait.

The data for h2/H2 (0.59 for F1 and 0.84 for F2) showed that one group of genes controlled this trait.

The alleles in each polymorphic locus showed dominance and a non–uniform distribution between the parents, since Н2/4Н1 for F1 was 0.20, while for F2 it was 0.19.

The values of the ratio between dominant and recessive genes prevailed on recessive ones in F1 and on dominant genes in F2. In F1, low narrow sense heritability was observed, while in F2 a moderate heritability was found.

As in previous cases, broad sense heritability was high in both hybrid generations.

Simple progeny selection and search for better segregants in later generations may be followed as a procedure for the improvement of this trait.

Nodes per plant The regression line intercepted Wr

axis below the beginning, revealing

overdominance in F1 and in F2. The regression coefficient was different from zero and close to unity, indicating the absence of non–allelic interaction in both hybrid generations.

The components of variation indicated that the additive (a) and dominance (b) components were significant in both F1 and F2.

The values of H1/D > 1 and F > 0 suggested over dominance in both populations.

According to the proportion h2/H2 (1.99 for F1 and 1.64 for F2) this trait is controlled by one or two groups of genes.

The ratio Н2/4Н1 in both populations (0.22) showed that the selection for decreasing or increasing number of nodes per plant have identical chance.

The ratio (4DH1)½+F/(4DH1)½–F suggested a prevalence of dominant genes. Heritability in the broad sense was higher than the one in narrow sense in F1 and in F2.

Internode distance The regression line in both

generations intercepted Wr below the beginning, indicating that this trait was influenced by overdominance.



43

The estimates of components of variation revealed higher values for dominance genetic components (H1) than additive components (D) in F1 and F2 (H1/D > 1).

The ratio h2/H2 (1.46 for F1 and 1.99 for F2) indicated that this trait was under the oversight of two groups of genes. In F1 (0.20) and F2 (0.22), the ratio Н2/4Н1 showed that dominant alleles in the polymorphic loci are distributed almost uniformly between the parental forms.

The coefficient of heritability in narrow sense was much lower than the one in broad sense.

Seed weight per plant The regression line intercepted Wr

axis in it the negative part in F1 and F2. Non–significant was the additive

and dominant genetic parameters b and b1 only in F2. In both generations, the coefficients of regression were close to unity, indicating the absence of non–allelic interaction.

The value of the H1/D is very much over unit as in F1 (5.16) and F2 (6.86), being an evidence for over dominance.

This trait was controlled from two–three groups of genes h2/H2 (2.56 for F1 and 2.50 for F2). In this case, it is not possible to obtain rapid selection results on seed weight per plant since Н2/4Н1 was 0.23 in F1 and 0.25 in F2.

The values of the ratio between dominant and recessive genes showed a small prevailing on dominant genes toward the recessive ones. Heritability in the narrow sense of this trait was the lowest in comparison with all the others.

1000–seed weight The regression line intercepted Wr

axis in its positive part, depicting a partial dominance in F1 and F2. The regression coefficient (b) was close to unity and was highly different from zero, indicating the absence of non–allelic interaction.

The coefficient of the regression equation was positive and H1/D < 1, being an indication of the influence of additive genes actions. The obtained data for h2/H2 (0.69 for F1 and 0.34 for F2) indicated that 10000–seed weight was influenced by one group of genes.

The value of the Н2/4Н1 (0.18) in both hybrid generations showed that dominant alleles in the polymorphic locuses were distributed non–uniformly between the parents.

The heritability (Hns and Hbs) was high in F1 and F2.

Comparatively low difference between the coefficient of heritability in narrow and broad sense probably indicated that the genetic diversity for this trait was strongly inheritable and that the selection by phenotype might be effective.

Number of fertile nodes per plant The regression line in both

generations intercepted Wr below the beginning, indicating that this trait was influenced by over dominance.

The regression coefficient was different from zero indicating absence of non–allelic interactions.

The estimates of genetic components showed that the dominance variation (H1) was higher than additive (D) genetic, that is, H1/D > 1. The ratio h2/H2 (1.95 in F1 and 1.71 in F2) indicated that one–two groups of genes controlled this trait and exhibited dominance.

There were observed very high estimates for broad sense heritability in comparison with that in narrow sense.

When the estimates of heritability (Hns and Hbs) were fairly medium to high, thus suggesting that simple selection procedures like progeny selection may be opted for improvement of this trait.

Similar gene actions for this trait were reported earlier KUMAR, 1976 .

Singh and Ram SINGH and RAM 1988 , observed that additive and non–additive gene action predominated for 100 green pod weight, pod length, number node at which appear of first flower, primary branches per plant and plant height green pod yield in diallel analysis of garden pea. Genetic components of variation analysis supported these conclusions.

Rastogi RASTOGI, 1988 reported the presence of high non–additive genetic variance (H1 and H2) as compared to additive genetic variance (D) in a diallel analysis of ten parents for some traits of garden pea seed in F1 generation.


50

pH places a major role for the extraction of Chebulinic acid.

To optimize the pH for this process different pH extract samples viz., 4, 5, 6, 7 and 8. It was observed that the extraction of Chebulinic acid of Terminalia chebula was found to be optimum pH at 6.0 [LOKESWARI et al., 2006; BOSTN et al.; 2013] and optimum concentration was 3.4 mg/mL.

The Amla fruit and Longan seeds shows optimum pH at 5.0 and optimum concentrations were found to be 2.7 mg/mL and 3.0 mg/mL.

The results were shown in figure 7.

Figure 7. Effect of pH for the Extraction of

Chebulinic acid (CBA)

h) Effect of Weight Dosages for the Extraction of Chebulinic acid (CBA):


Figure 8. Effect of Weight Dosages for the

Extraction of Chebulinic acid Different weight dosages were used

to find out the optimum concentrations of Chebulinic acid of these plants.

The present investigation suggests that the extraction of Chebulinic acid at different weight dosages indicates that the optimum weight dosages were 7.2 gm of Terminalia chebula fruit, 8.0 gm of Amla fruit and 7.4 gm of Longan seeds.

The optimum concentrations were 6.2 mg/mL, 4.8 mg/mL and 5.7 mg/mL.

Conclusion Experiments were performed for the

optimization of extraction of Chebulinic acid from medicinal herbs like dry fruits of Terminalia chebula, Amla and seeds of Longan fruit. Chebulinic acid estimation and optimize the physico–chemical parameters by using FD reagent method.

The parameters like effects of different solvents, soaking time, and extraction time with hexane, particle size, different solvent percentages, and different volumes of hexane with ethanol and methanol as solvents, pH and different weight dosages for the extraction of Chebulinic acid concentrations were observed from the experimental work.

The highest chebulinic acid concentration for optimized conditions of Terminalia chebula fruit, Amla fruit and Longan seeds were 6.2 mg/mL, 4.8 mg/mL and 5.7 mg/mL respectively.

References 1. Anil, D. Mahajan; Nandini, R. Pai;

Development and validation of HPLC method for quantification of phytoconstituents in Haritaki Churna. International journal of ChemTech research, 2011, 3(1): 329–336.

2. Asmawi, M.Z.; Kankaanranta, H.; Moilanen E.; Vapaatalo, H.; Anti–inflammatory activities of Emblica officinalis Gaertn leaf extracts. Journal of Pharmacy and Pharmacology, 1993, 45(6): 581–4.

3. Atita, Panyathepa; Teera, Chewonarina; Khanittha, Taneyhillb; Usanee, Vinitketkumnuen; Antioxidant and anti–matrix metalloproteinases activities of dried longan (Euphoria longana) seed extract, Journal of Science Asia, 2013, 39: 12–18.

4. Avani, Patel; Amit, Patel; Patel, Dr. N.M.; Estimation of Flavonoid,



49

d) Effect of Different Particle size for the Extraction of Chebulinic acid (CBA):

Different particle size viz., 354, 328, 250, 205, 149 and 125 microns were used to find out the optimum concentrations of Chebulinic acid from these plants.

The present investigation suggests that the extraction of Chebulinic acid at different particle sizes indicates that the optimum particle size [ANIL D. MAHAJAN et al.,

2011; BUTNRIU and BOSTAN, 2011] was 125 microns of Terminalia chebula, Amla fruits and Longan seeds for extraction of Chebulinic acid. The optimum concentrations were 2.6 mg/mL, 2.0 mg/mL and 2.2 mg/mL.


Figure 4. Effect of Different Particle size

for the Extraction of Chebulinic acid

e) Effect of Different Solvent percentages for the Extraction of Chebulinic acid (CBA):

Percentage of the solvent is also plays a vital role for the extraction of components.

The study on different solvent (ethanol, methanol) percentages like 0%, 20%, 40%, 50%, 60%, 80% and 100% shows significant variations.

Figure 5 shows that the optimum solvent percentages were found to be at 50% ethanol of both Terminalia chebula fruit & Longan seeds and 80% methanol of Amla fruit for the extraction of chebulinic acid.

The optimum concentrations of 50% ethanol were 3.0 mg/mL & 2.6 mg/mL and 80% methanol was 2.3 mg/mL.


Figure 5. Effect of Different Solvent percentages for the Extraction of

Chebulinic acid

f) Effect of Different volumes of Hexane for the Extraction of Chebulinic acid (CBA):

To determine the volume of hexane for the extraction of Chebulinic acid at the different volumes of hexane with solvent (ethanol, methanol) were considered such as 1:1, 1:2 and 2:1.

The optimum extraction of Chebulinic acid was achieved at 1:1 with ethanol as a solvent of Terminalia chebula fruit & Longan seeds and the optimum concentrations were 3.2 mg/mL and 2.8 mg/mL. The optimum extraction was also recorded at 1:1 with methanol of Amla and the concentration was 2.5 mg/mL. The observed results were shown figure 6.

Figure 6. Effect of Different Volumes of Hexane for the Extraction of Chebulinic

acid g) Effect of pH for the Extraction of

Chebulinic acid (CBA):


44

The role of additive, non–additive and both additive and dominance gene action may be attributed to the variable genetic material used by researchers in their studies NARAYANET et al., 1998; SINGH and

SHARMA, 2001 .

Sarawat SARAWAT et al., 1994 found that both additive and non–additive gene effects were important in the expression of branches per plant, pods per plant, seeds per pod and plant height

The dominance variance was higher for all the traits except pod length and seeds per pod. The degree of dominance indicated over–dominance for all the traits except pod length and seeds per pod.

The distributions of genes in the parents were asymmetrical for all the variables. Kumar and Bal KUMAR and BAL, 1995 carried out graphical analysis and predicted over dominance for yield, number of pods per plant, 100 seed weight and partial dominance for other.

The knowledge of gene action is very useful to a plant breeder in the selection of parents for hybridization SHARMA et al., 2013 , the estimation of some other genetic parameters and choice of breeding procedures for the genetic improvement of various quantitative characters.

Conclusions Prevalence of overdominance was

observed for most of the traits in both hybrid generations (F1 and F2) except for 1000–seed weight.

Non–additive gene action appeared to be more predominant for the inheritance of most the traits studied.

The fraction of h2/H2 indicated that at least one group of genes controlled dominance for all the traits, namely 6 for seed weight per plant and 3 for number of seeds per pod.

The Н2/4Н1 ratio was close to the expected value of 0.25 for number of seeds per plant and seed weight per plant, indicating symmetrical distribution of dominant and recessive genes.

Dominant alleles were more frequent in parental forms for the inheritance of most of the traits except

number of seeds per pod in F1. For number of seeds per pod (F1) and 1000–seed weight (F1 and F2) there was established a comparatively low difference between the coefficient of heritability in narrow and broad sense, indicating that the selection in early generations might be effective.

None one of the cultivars did not have only dominant or only recessive alleles.

Exhibited dominant alleles were found in EFB33 for plant height, first pod height, internode distance and number of fertile nodes per plant, in Mir for number of pods per plant, number of nodes per plant and 1000–seed weight and in Pleven 10 for number of seeds per plant and seed weight per plant.

The cultivars Kerpo exhibited recessive alleles for all the traits except number of seeds per pod. References 1. Bourion, V. Fouilloux, G. Le Signor, C.

Lejeune–Henaut, I. Genetic studies of Genetic studies of selection for productive and stable peas. Euphytica, 2002. 127:261–273.

2. Csizmadia, L. Inheritance of earliness in a diallel cross of garden pea varieties. Zoldsegtermesztesi Kutato Intezet Bulletinje, 1994. 26:19–32.

3. Dixit, G.P. Genetic analysis of certain quantitative characters in field pea. Indian J. Genet, 1999. 59: 113–115.

4. Hayman, B.I. The analysis of variance of diallel tables. Biometrics, 1954a.10:235–244.

5. Hayman, B.I. The theory and analysis of diallel crosses. Genet. 1954b.39: 789–809.

6. Kumar, H.A. Genetic study of plant height in garden pea. Egyptian J Genet Cytol. 1976. 5:257–26.

7. Kumar, J.C. Bal S.S. Inheritance of economic traits in garden pea (Pisium sativum L.). Haryana J. Hort. Sci. 1995. 24(3–4):251–255.

8. Kumar, S. Singh, K.P. Panda, P.K. Combining ability analysis for green pod yield and its components in garden pea (Pisum sativum L.). Orissa J. Hort. 1996. 24: 21–25.

9. Narsinghani, V.G. Rao, U.S.N. Singh, S.P. Diallel cross analysis for quantitative



45

traits in mutant pea types. Indian J Agric Sci. 1982. 52:364–367.

10. Panda, P.K. Singh, K.P. Kar, R.M. Singh, A.K. Genetic components for yield and its contributing traits in pea (Pisum sativum L.). J. Appl.. Biol, 1996. 4: 9–12.

11. Parmar, B.S. Godawat S.L. Heterosis and combining ability in pea (Pisum sativum L.). Intl. J. Tropic Agric. 1990. 8:19–22.

12. Raj, Narayan. K, Rastogi B. Dogra B.S. Genetics of yield and its components in garden pea. Veg. Sci. 1998. 25: 18–21.

13. Rastogi, K.B. Genetics analysis of vitamin C content in green seed of pea (Pisum sativum L.). Haryana J. Hort. Sci. 1988. 16(3–4):241–247.

14. Rathore, P.K. Gupta, V.P. Plaha, P. Genetics of seed yield and some other traits in pea using NCIII and triple cross analysis. Indian J Pulse Res, 1995. 8:119–122.

15. Sarawat, P. Stoddard, F.L. Marsall, D.R. Ali, S.M. Heterosis for yield and related characters in pea. Euphytica, 1994. 80:39–48.

16. Sharma, B.B. Sharma, V.K. Dhakar, M.K. Punetha, S. Combining ability and gene action studies for horticultural traits in garden pea: A review. African Journal of Agricultural Research, 2013. 8(38):4718–4725.

17. Sharma, D. Adarsh–Bala, S. Chaudhary, D. Bala, A. Studies on combining ability and gene action in pea (Pisum sativum L.). Indian Journal of Hill Farniing, 1999. 12(1–2):32–36.

18. Singh, K.N. Santoshi, U.S. Singh, H.G. Genetic analysis of yield

components and protein content in pea. Indian J .Agric. Sci. 1986. 56:757–764.

19. Singh, M.N. Singh, R.B. Genetics of seed number and seed weight in pea. Indian J. Pulse Res. 1991. 4:165–167.

20. Singh, T.H. Sharma, R.R. Gene action for yield and its components in three crosses of pea (Pisum sativum L.). Indian J Genet, 2001. 61:174–175.

21. Singh, Y.V. Ram, H.H. F2 diallel analysis of some quantitative traits in garden pea. Narendra Deva J. Agric. Res. 1988. 3:83–89.

22. Sood, M. Kalia, P. Gene action of yield–related traits in garden pea (Pisum sativum Linn.). SABRAO J. Breed. Genet. 2006.38:1–17.

23. Srivastava, C. Tyagi, M. Agrawal, R. Rai, B. Combining ability analysis for seed yield an related traits in peas of Indian and exotic origin. Madras Agricultural Journal. 2000. 86(7–9):366–370.

24. Ukai, Y.A. Microcomputer program DIALL for diallel analysis of quantitative characters. Jpn. J. Breed. 1989. 39:107–109.

Received: July 1, 2014



48

Results and discussion a) Effect of Different Solvents for

Extraction of Chebulinic acid (CBA): Different organic solvents such as

methanol, ethanol, ethyl acetate and water were used to extract the optimum yield of chebulinic acid from these plants.

The solvent, ethanol of Terminalia chebula fruit and Longan seeds were shows best results of chebulinic acid and its concentrations were 1.5 mg/mL and 0.35 mg/mL respectively.

The solvent, methanol of Amla fruit was shows best results of chebulinic acid and it concentration was 0.6 mg/mL.


Figure 1. Effect of Different Solvents for Extraction of Chebulinic acid

b) Effect of Soaking Time for

Extraction of Chebulinic acid (CBA): The samples were incubated under

proper conditions at different time intervals viz., 1, 2, 3 and 4 days to investigate the influence on extraction of Chebulinic acid.

It was observed that Terminalia chebula fruit was shows first day was the best soaking time for the extraction of chebulinic acid and the concentration was 2.0 mg/mL.

Amla fruit and Longan seeds were shows second day was the best soaking time and its concentrations were 1.3 mg/mL and 0.9 mg/mL respectively.


Figure 2. Effect of Soaking Timefor

Extraction of Chebulinic acid

c) Effect of Extraction Time with hexane for extraction of Chebulinic acid (CBA):

To investigate the influence of hexane on extraction of Chebulinic acid different time intervals were taken viz., 1, 2, 3 and 4 hr.

Solvent–Solvent extraction was done with hexane as one of the solvent.

The fruits of Terminalia chebula, Amla and Longan seeds were shows that optimum concentrations were observed at first hour extraction time with hexane for extraction of chebulinic acid.

The concentrations were 2.2 mg/mL, 1.6 mg/mL and 1.4 mg/mL respectively.


Figure 3. Effect of Extraction time with

hexane for Extraction of Chebulinic acid



47

The dried longan pulps are used as a tonic in traditional Chinese medicine to nurture heart and spleen, nourish blood, calm mind, add luster and beauty to the skin, and have therapeutic effects on heart palpitation, insomnia, amnesia, and anxiety.

Longan seeds have long been used as a folk medicine in China for treatment of pains, hernia, and skin diseases, acariasis, hernia, wound hemorrhages, eczema and scrofula etc.

This seeds have been found to be a rich source of poly phenolic compounds [SOONG and BARLOW, 2005] such as gallic acid, corilagin, chebulinic acid, chebulagic acid, ellagic acid flavonoids like quercetin etc.

Longan seeds [ATITA PANYATHEPA et al.,

2013] extract showed strong scavenging activities of free radicals, inhibition on the proliferation of human colorectal carcinoma cells, antifatigue effect, hypoglycemic effects etc.

Chebulinic acid ((2R)–2–[(3S)–3–carboxy–5,6,7–trihydroxy–1–oxo–3,4–ihydroisochromen–4–yl]butanedioic acid) is a phenolic compound [MANOSROI et al., 2010]

found in the fruits of Terminalia chebula, Phyllanthus emblica and seeds of Dimocarpus longan species etc.

It helps to remove toxins and unwanted fat from the body. It showed many pharmacological activities including inhibition of cancer cell growth like human leukemia K562 cells [Yi et al., 2004], colon adenocarcinoma HT–29 cell lines, anti–neisseria gonorrhoeae activity, inhibiting the contractile responses of cardiovascular muscles activities etc.

The present paper Optimization of Physico–Chemical Parameters for the Extraction of Chebulinic acid (C14H12O11) from fruits of Terminalia chebula, Phyllanthus emblica and seeds of Dimocarpus longan species.

Material and methods Chemicals and reagents Folin Denis reagent, Sodium

carbonate (Na2CO3), Methanol, Ethanol, Ethyl acetate, Hexane, Distilled water.

Collection of Plant material The dry fruits of Terminalia chebula,

Amla and seeds of Longan collected from

local market in Visakhapatnam, Andhra Pradesh, India.

Processing of the Plant material These fruits and seeds were cut into

small pieces and powdered. The total powder done in to different

mesh sizes from 44 to 120. The different size powders were

stored in the air tight small covers. Extract preparation Weigh the different amounts of 7.2

gm of Terminalia chebula fruit, 8.0 gm of Amla fruit and 7.4 gm of Longan seeds of powders and add ethanol (50%) and methanol (80%), in different flasks and makeup this solution up to 50 mL.

Soak the solution for 1 day and 2 days respectively.

After the soaking time filtrate the solution by using Whatman No. 1 filter paper and heat the filtrate solution at 78 C and 65 C respectively.

So that the solvent which is taken in the glass wear is evaporated and make up this solution up to 25 mL with distilled water to this solution add 25 mL of hexane solvent [HARPREET WALIA et al., 2011] mix the solution thoroughly.

Pour the entire mixture in the separating funnel by using glass funnel.

Incubate the solutions of ethanolic and methanolic extract for 1hr.

Determination of Chebulinic acid

(CBA) by Colorimeter By Folin–Denis Method: 1mL of

ethanolic and methanolic extracts was withdrawn in a 10 mL volumetric flask separately.

To each flask 0.5mL of Folin Denis reagent [AVANI PATEL et al., 2010; BUTNRIU et al., 2011] and 1mL of Sodium carbonate were added and volume is made up to 10 mL with distilled water.

The mixtute was allowed to stand for 30 min at room temperature.

The absorbance of the reaction mixture was measured at 700 nm using colorimeter.

The Chebulinic acid was determined by using calibration curve.


46

OPTIMIZATION OF PHYSICO–CHEMICAL PARAMETERS FOR THE

EXTRACTION OF CHEBULINIC ACID FROM MEDICINAL HERBS

DOI: 10.7904/2068–4738–V(10)–46

Surya Prakash DV1, Meena V2 1Centre for Biotechnology, Department of Chemical Engineering, Andhra University, Visakhapatnam–530003, Andhra Pradesh, INDIA. e–mail: [email protected] 2Centre for Biotechnology, Department of Chemical Engineering, Andhra University, Visakhapatnam–530003, Andhra Pradesh, INDIA. e–mail: [email protected]

Abstract. Chebulinic acid is a phenolic compound found in the fruits of Terminalia chebula

(Haritaki), Phyllanthus emblica (Amla) and seeds of Dimocarpus longan (Longan) species etc. It showed many pharmacological activities including inhibition of cancer cell growth like human leukemia K562 cells, colon adenocarcinoma HT–29 cell lines, anti–neisseria gonorrhoeae activity etc. The present studies on optimization of physico–chemical parameters like effects of different solvents, soaking time, extraction time with hexane, particle size, different solvent percentages, different volumes of hexane with ethanol and methanol as solvents, pH and different weight dosages for the extraction of Chebulinic acid from Medicinal herbs. The highest chebulinic acid concentration for optimized conditions of Terminalia chebula fruit, Amla fruit and Longan seeds were 6.2 mg/mL, 4.8 mg/mL and 5.7 mg/mL respectively.

Key words: Chebulinic acid, Terminalia chebula fruit, Amla fruit, Longan seed, Folin–Denis reagent.

Introduction Terminalia chebula species

commonly called as Black myrobalan, Ink tree or Chebulic myrobalan.

It is belongs to the family combretaceae. It is used for the treatment of number of diseases like cardio vascular diseases, arthritis, paralysis, cancer, gout, ulcers, epilepsy etc. contains tannins up to 30%, chebulic acid 3–5%, chebulinic acid 30% [KAREL D. KLIKA et al., 2004], tannic acid 20–40%, ellagic acid, 2,4–chebulyi–β–D–gluco pyranose, gallic acid, ethyl gallate, flavonoids like luteolin, rutins, and quercetin etc.

It shows several pharmacological activities [SURYA PRAKASH and MEENA, 2014] like anti–oxidant, anti–cancer [SALEEM et al., 2002], anti–diabetic, anti–microbial etc.

Phyllanthus emblica, also known as Emblic, Emblic myrobalan or Indian gooseberry (Amla).

It is belongs to the family Phyllanthaceae. These fruits fruits are reputed to contain high amounts of ascorbic acid (vitamin C), ellagitannins such as emblicanin A (37%), emblicanin B (33%), punigluconin (12%) and pedunculagin (14%).

It also contains punicafolin, phyllanemblinin, gallic acid, ellagic acid, chebulinic acid [EKTA SINGH et al., 2011], flavonoids like quercetin and kaempferol etc.

It used as a rasayana (rejuvenative) to promote longevity, and traditionally to enhance digestion, treat constipation, reduce fever, purify blood, reduce cough, alleviate asthma, strengthen the heart, benefit the eyes, stimulate hair growth, enliven the body, and enhance intellect.

It shows antitumor activity, anti–inflammatory activity [ASMAWI et al, 1993], antimicrobial activity, cytoprotective activity, antioxidant activity [JUREE

CHAROENTEERABOON et al., 2010] etc. Dimocarpus longan, commonly

known as the Longan and belongs to the family Sapindaceae.

It is a tropical tree that produces edible fruit.

This fruit used as a traditional Chinese medicine for different treatments, such as soothing nerves, relieving insomnia and promoting blood metabolism.

Longan pulps are tasty and rich in nutritious ingredients.

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