The present experiment was aimed to detect osteopontin gene transcript from bull spermatozoa. Fresh semen samples from 12 Jersey cross breed bulls were collected using artificial vagina. The volume and concentration of the individual samples were recorded. The spermatozoa were separated from bull semen samples by swim up protocol using sperm TALP. The initial concentration of semen sample was checked immediately before proceeding for RNA isolation and normalization of initial concentration was done. So, initial amount of every sample was made equal. Total RNA from the bull spermatozoa were extracted by the RNeasy® Mini Kit, Qiagen as per manufacturer's protocol. The purity and concentration of the samples were measured at A280/260 by using Nano Drop TM 1000 spectrophotometer. The first strand cDNA was synthesized from 1μg total RNA using M-Mu LV Reverse Transcriptase Revert Aid TM H minus first strand cDNA synthesis kit (Thermo Scientific) as per manufacturer's protocol. PCR product of about 267 bp fragment length of OPN gene was confirmed by conventional PCR. The bulk PCR product of about 100μl was purified by Gen Elute™ PCR Clean-Up Kit Sigma-Aldrich as per manufacturer's protocol. The concentration and purity of PCR product were 64ng/μl and 1.6 respectively. The cloning and expression of OPN gene was carried out by using TA cloning kit. The ligated product was then transformed into E. coli DH5α cells. The plate was incubated for overnight at 37 ˚C for selection of the colonies and stock cultures were maintained in LB media at -80˚ C. The clones which showed the amplification of 400 bp DNA fragment were considered as positive clone carrying desired insert. Plasmid isolation was done as per the protocol followed in Hi Yield TM Plasmid Mini Kit (RBC Cat. NO. YPD100) and the eluted DNA were stored at -20 ˚C. The concentration and purity of PCR purified product were 67ng/μl and 1.6 respectively. The eluted plasmid DNA was sequenced commercially (Amnion, Bangalore) using M13 primers. The cloned partial sequence of OPN gene isolated from bovine spermatozoa was submitted at DDBJ (Accession No. AB983656). On blast analysis using NCBI nucleotide blast (Online tool), 99 per cent identity was found with reported consensus sequences except single nucleotide variation in DNA sequence at 234 th nucleotide. There was substitution of adenine instead of cytosine.
Acute Phase Proteins are blood proteins primarily synthesized by hepatocytes as part of the acute phase response (APR). APR to disease is accompanied by an increase in the circulating concentration of a number of plasma proteins which are collectively known as the Acute Phase Proteins. According to the concentration, APPs are classified in positive APP, if they increase or negative APP, if they decrease. During mastitis, APPs move from the systemic circulation to mammary gland or there could be de-novo synthesis of the APP within mammary gland tissues. Among the APPs, Serum Amyloid A (SAA), Haptoglobin (Hp), Alpha 1-acid glycoprotein and Lipopolysaccharide binding protein play a major role during mastitis in cattle and their concentration will increase with the severity of the infection, inflammation or trauma. SAA is involved in defense against Gram positive and Gram negative pathogens. SAA acts by modulating innate immune system and by acting as an opsonin. Among the SAA isoforms, mammary associated SAA3 (m-SAA3) is important. The blood concentration of SAA and Hp increases dramatically after LPS infusion, with SAA (acute and middle phase of APR) appearing before Hp (Late phase of APR). The herd level APP might be useful for determining the prevalence of clinical and subclinical infections indicated by the high serum concentration of selected APP and by serving as the prognostic tool. APPs, used as markers of animal health may possibly be influenced by environmental factors, handling procedures and other types of stress in the absence of disease.
This review analyses the use of molecular techniques in rumen microbial identification. The use of regular methods like roll tube and most probable number resulted in under estimation of rumen microbial growth. So molecular biology acts as an advanced tool for rumen microbial culture and identification of various new species. These techniques give complete descriptions of individual ruminal populations. Use of molecular techniques like PCR, DDGE and FISH, which also pave pathway for genetic manipulation of rumen microbes in the field of rumen manipulation. So the combination of traditional and molecular assays gives accurate and satisfactory results.
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