British Society of Animal Science , 2007-02-13

Title : ( Microscopic Studies and Molecular Identification of Ruminal Zygomycytes Fungi in Sheep )

Authors: Mohammad Hadi Sekhavati , Mohammadreza Nassiri , Mohsen Danesh Mesgaran , A. Mohammadi , Ali Javadmanesh , H. Esmaeilzadeh ,

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Introduction Rumen is an area with high potential for growing of diverse living organisms including anaerobic fungi. The most studies on rumen fungi focused on anaerobic fungi classified as Chytridiomycetes, family Neocallimasticaceas, while few studies have done on determination of Zygomycetes that can be native in the rumen. The most fungi within the subkingdom Zygomycotina belong to the class Zygomycetes. Fungi in this class possess some distinctive properties that contain rapid growth, nonseptate mycelium, reproduction by sporangiospores (Pitt et al., 1985) and production of some hydrolytic enzymes (Vinogradova et al.,2003). The aim of this study was microscopic examination and molecular analysis of nuclear small subunit (SSU; 18S rDNA) of ruminal fungi, Zygomycete, and its reproductive manner in sheep. Materials and methods Rumen fluid from a rumen-fistulated sheep, fed a 50:50 concentration:hay ration, was filtered through a layer of muslin and clarified by centrifugation at 10,000 × g for 30 min and used as a source of fungi. Inoculum was cultured anaerobically in reduced medium and incubated at 39 °C for 3 days as described by Joblin (1981). 100 μl of each cultured samples was transferred on sterilized glass slide which was into a sterilized petri dish, and then by potting a cover glass on it and incubated under a CO2 atmosphere at 39 °C for 24 h. Fungal mycelium and their sporangiospores were photographed using a bright- field microscopy. In an attempt to isolate specific fungi, colonies were picked and transferred into sisal broth medium. Genomic DNA was extracted from biomass harvested by centrifugation (2000 Rpm, 10 min) from pure culture grown on sisal broth medium (Fliegerova et al., 2004) by Guanidine Thiocyanate-Silica Gel method (Boom et al., 1990). Primers NS1 (5 ́–GTAGTCATATGCTTGTCTC-3 ́) and NS2 (5-́GGCTGCTGGCACCAGACTTGC-3 ́) amplified a 550-bp fragment from SSU 18S rDNA (Fliegerova et al 2006). In a volume of 20 μl PCR reactions contained: 50 ng of template DNA, 2μl 10-X PCR buffer, 2.5 mM MgCl2, 200 μM each dNTPs, 10 pM of each primer, and 1 U Taq DNA polymerase. Thermal conditions for 35 cycles were 95 °C (40 sec), 45 °C (40 sec), and 72 °C (1 min) followed by a final extension at 72 °C (5 min). PCR products were visualized by electrophoresis on 1.3% agarose gel stained with etithium bromide. Results Anaerobically culture of the fungi showed that these fungi are able to grow in rumen condition with sexual reproduction (Figure 1). A 550 bp fragment of the SSU 18S rDNA was amplified successfully from extracted DNA (Figure 2).


, Rumen, Fungal, PCR
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author = {Sekhavati, Mohammad Hadi and Nassiri, Mohammadreza and Danesh Mesgaran, Mohsen and A. Mohammadi and Javadmanesh, Ali and H. Esmaeilzadeh},
title = {Microscopic Studies and Molecular Identification of Ruminal Zygomycytes Fungi in Sheep},
booktitle = {British Society of Animal Science},
year = {2007},
location = {londan, ENGLAND},
keywords = {Rumen; Fungal; PCR},


%0 Conference Proceedings
%T Microscopic Studies and Molecular Identification of Ruminal Zygomycytes Fungi in Sheep
%A Sekhavati, Mohammad Hadi
%A Nassiri, Mohammadreza
%A Danesh Mesgaran, Mohsen
%A A. Mohammadi
%A Javadmanesh, Ali
%A H. Esmaeilzadeh
%J British Society of Animal Science
%D 2007