eChapters

Collect the femur and tibia and clean them very well from blood, tendons, muscle, skin and other tissues (using scalpel)

Cut off epi-and metaphysis from the long bones with electric/manual bone saw
Transfer the open bones to a falcon tubes containing cell culture medium/DPBS without FBS but containing 1% PS (Penicillin-streptomycin)
Transfer the bones to a Petri dish containing 5 ml medium without serum (FBS) and without antibiotic (40-50 mm Petri dish)
Remove the pteri dish to the laminar cabinet
Collect the bone marrow by flushing out to a 50 ml falcon tube (use syringes and needles to flush out the bone marrow; flush out the bone marrow with complete (10% FBS, 1% PS, 90% DMEM-LG) culture medium
Centrifugation: 300rpm, 8 min (in the meanwhile prepare 5 ml of Ficoll into a 15ml falcon tube)
Aspire the medium

1.    Collect fat tissues from euthanized animals into a Falcon tube containing DPBS + 1% PS ( for transferring lab to lab)
2.    Wash 3 times with DPBS + 1% PS
3.    Inside a laminar cabinet, transfer the fat to a Petridis (use an sterile spatula to collect the fat)
4.    Cut the fat into small (mm) pieces using scalpel and forceps or scissors
5.    Transfer the fat into a Falcon tube (eg into a 50 ml Falcon tube you can add upto 10 ml fat tissues)
6.    Wash the fat by adding approx 40 ml DPBS; Centrifugation: 430g, 10 min (without break)
7.    Transfer the fat into a fresh 50 ml Falcon tube (with the help of sterile spatula); Repeat steps 5 to 7 for 2-3 times

Characterization of Oct-4/Sox-2 Transcription Factors by Immunostaining for Characterization of Stem Cells
Protocol
1) The cultured cells at a density of 70,000/well in a 6 well plate or 35,000/ well in a 12 well plate are used for immunostaining.
2) The cells are washed with Dulbecco’s phosphate buffered saline (DPBS) for two times and the cells are fixed using 4% paraformaldehyde for 20 minutes at room temperature.
3) After fixation, the spent Paraformaldehyde is removed and the fixed cells are washed with 1000μl of 1x DPBS for 2 times by clockwise and anti-clockwise rotation of the plate slowly.
4) About 400μl of 3% blocking buffer was added to the wells and plate was covered with aluminum foil to prevent the exposure of light and stored at room temperature for 90 minutes.
5) After blocking, the cells are again washed once with DPBS. About 500 μl of primary antibody Oct-4 in the dilution of 1:40 is added to the well and stored at 4°C over night.
6) After overnight staining with primary antibody, the antibody solution is removed from the wells and washed with DPBS for 2 times.
7) Take 500 μl of secondary antibody anti-rabbit-FITC diluted with antibody buffer solution is added in the ratio of 1:200 and cover with aluminium foil and incubated for 90 minutes.
8) Secondary antibody is removed and washed with DPBS.

1.    Remove the growth media
2.    Wash 2 times with warm PBS (w/o Ca and Mg)
3.    Add 5ml of trypsin /EDTA 0.35 % (Invitrogen), place at 370C in a CO2 incubator for 4-5 min
4.    Wait until cells are detached/lifted and make sure by examining on microscope
5.    Add equal volume (5ml-1:1) of growth medium as appropriate and mix with 5 strokes
6.    Transfer into 15 ml centrifuge tube
7.    Centrifuge at room temperature for 4 min in 980 rpm
8.    Remove supernatant complete but carefully without touching the cell aliquot
9.    Re-suspend in 10ml DPBS (with Ca and Mg)-5 strokes
10.    Immediately after mixing transfer 100µl of cell suspension into Casy vial, containing 10ml Casyton

1. Fix the organ or the animal in 5-10 % formalin preferably neutral buffered formalin. If the organ is an isolated thoracic limb, for example, then cannulate the axillary artery and perfuse the formalin. I find sometimes it is advantageous to pump some buffered saline solution to flush out any blood before pumping in the formalin. Organs, such as the heart, could be nicely flushed with water through major blood vessels, and immersed in formalin to fix. The same can be done with liver, kidney, etc. It takes a week for fixing a large structure (e.g., 10 kg) by immersing in formalin.
2. Dissect the specimen as needed. Remove all fat and clean as much as possible so the specimen looks nice.
3. If the specimen is dark brown, it can be lightened by leaving 1-2 days in 5% hydrogen peroxide. Check the specimen 1-2 times during the day to make sure desired light tone is achieved. Leaving it too long in the hydrogen peroxide may result in tissue degradation.
4. Drain the specimen well, and dehydrate in acetone. The dehydration should be thorough. To achieve this, use 100% acetone, at least 2-3 changes. The acetone can be recycled by repeated distillation. Acetone tends to evaporate, so use a container that has a tight lid. Acetone is also flammable, so use required precautions when handling acetone. If concentration of acetone remains up to 99% even after leaving organ for 4-5 days it is sure that dehydration is complete. If you are using any thin sheets of organ or Brain for dehydration keep slice between glass plates and then dehydrate.
5. Immerse the specimen in glycerin. Buy industrial glycerin, which may be cheaper. It may take 2-3 weeks for the glycerin to permeate into the tissues.

6.1 Designing primers

Designing correct primers are very important to get the expected result in PCR experiment. There are two primers (Forward & Reverse) required for the amplification of the desired DNA fragment. One primer should anneal to the sense strand (oriented to the 5'→3' direction) and another primer should complement the antisense strand (oriented in the 5'→3' direction).

There are various characteristics to be considered to design a good primer including Tm, length, GC content, secondary structure and specificity.

There are commercially available softwares besides free online tools at NCBI (primer blast) for designing PCR primers. In addition, primer sequences published in peer reviewed literature can also be used.

For RT-PCR, the extracted total RNA is first reverse transcribed to cDNA using reverse transcriptase enzyme. Following are the protocols for cDNA sysnthesis and RT-PCR.

Note: cDNA synthesis and RT-PCR can be performed according to the manufacturer protocol from where the specific reagents for the above methodologies are purchased.

8.1 Purpose

To lay down a uniform protocol for the synthesis of c-DNA from total RNA

8.2 Scope

The scope of this Standard Operating Procedure is to lay down a standard procedure for the synthesis of c-DNA from total RNA

Terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids. TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signaling cascades. The assay relies on the presence of nicks in DNA which can be identified by terminal deoxynucleotidyl transferase or TdT, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. Here we perform the apoptotic assay test by TUNEL assay kit to identify the apoptotic property of any agent or chemical after treatment of primary cells culture at different concentration as per protocol provided with TUNEL Assay Kit and apoptotic cells are identified by using fluorescent microscope. Detailed procedure of TUNEL assay is as follows:

1. Preparation of primary cells culture as described in earlier methods.

2. Induction of apoptosis in primary cells culture is done by treatment of primary cells culture with different concentration of xenobiotics and incubation in CO2 incubator for 12 h as mentioned in micronuclei assay method.

3. The positive and negative control cells provided with the kit are already f ixed. However, negative control cells are prepared from control sample with absence of inducing agent.

4. Cell Fixation: Cell fixation is a required step in the TUNEL assay and it is done according to the procedure mentioned in the supplied kit by using paraformaldehyde solution. Suspend 1–2 × 106 cells (treated) in 0.5 ml of phosphate-buffered saline (PBS) and the cell suspension into 5 ml of 1% (w/v) paraformaldehyde in PBS and place on ice for 15 minutes.

5. Centrifuge the cells for 5 minutes at 300 × g and discard the supernatant.

5.1 Purpose

To employ a standard protocol to perform agarose gel electrophoresis.

5.2 Principle

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb. Migration rate of DNA molecule depends on various factors like 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under UV light.

11.1 Principle

In situ hybridization techniques involve detection of specific nucleic acid sequences to in morphologically preserved chromosomes, cells or tissue sections. Along with immunocytochemistry, in situ hybridization can correlate microscopic topological information to gene activity at the level of DNA, mRNA or protein. The FISH protocol involves 1) denaturation and hybridisation performed on Day 1 and 2) washing and detection performed on Day 2. DNA of the chromosomes and the probes are denatured and allowed to hybridize overnight on the first day. On day 2, slides are washed to remove unbound DNA sequences followed by detection, counterstaining and mounting of the slides.

10.1 Purpose

To lay down a standard protocol to perform qPCR for quantitative analysis of target genomic sequence.

10.2 Principle

qPCR or quantitative polymerase chain reaction or real-time polymerase chain reaction, is a molecular biology technique to amplify and at a time quantify a targeted DNA molecule and based on the polymerase chain reaction (PCR). It is called real time as the amplified DNA is detected as the reaction progresses and holds an advantage over standard PCR where the reaction is detected at its end. There are two common methods in quantitative PCR to detect the products i) non-specific fluorescent dyes which intercalates with any double stranded DNA ii) sequence-specific DNA probes labeled with a fluorescent reporter consisting oligonucleotides. This reporters helps in detection of the complementary hybridization of the probe and quantifies amount of DNA in cells or tissues.

9.1 Purpose

To lay down a standard protocol to perform RT-PCR for quantitative analysis of target RNA.

9.2 Principle

RT-PCR technology is used to amplify a specific RNA sequence (target sequence) to measure its expression inside the cell. RT-PCR follows the same principle as of normal PCR, but instead of DNA here cDNA is used as template for amplification. Extracted RNA is reverse transcribed to cDNA with the help of reverse transcription enzyme. The cDNA is then subjected for PCR amplification with the help of DNA polymerase, target specific primers, nucleotides and reaction buffer. In molecular diagnosis, RT-PCR is used to detect the expression of oncogenes at the time of diagnosis. It is also used to detect minimal residual disease during treatment

Proto – first; Zoon – animal

Protozoa are single celled eukaryotic organisms, which lack cell wall and ability to move at some stage of their life cycle.

They are referred as the first animals.

There are about 65000 described species grouped into 7 phyla. Among them, 50 per cent are not available as living forms (as fossils) and out of remaining 50%, 22,000 species are free-living and 10,000 species are parasitic forms.

The protozoa are distinguished from other organisms by following characters

Introduction

The soil protozoa are unicellular and the simplest forms of animal life. Most of the protozoa are devoid of chlorophyll but transitional genera resemble the algae and possess chloroplasts containing chlorophyll pigments. The active life stage is commonly known as trophozoite phase where they feed and multiply but under adverse climatic conditions, they form resting stage or cyst, which develop a thick coating on the cell surface. Mostly, the protozoa multiply by longitudinal or transverse fission but some species reproduce sexually by fusion of two cells followed by exchange of genetic material between their nuclei and cell division.

Protozoa are single celled eukaryotes which are either parasitic or free living belonged to family apicomplexa. These protozoan affects the different birds like chickens, turkey, ducks, pigeons, game birds and also various pet birds. The infection caused by protozoan diseases may affect one system or more than one system of the body depending on the causative agents involved. The common protozoan diseases like leukocytozonosis, chochlosomiasis, cryptosporidiosis, coccidiosis, histoplasmosis causes infection in the ducks. The protozoan diseases cause severe economic loss to the poultry industry due to death of birds, expenditure on the treatment and prevention of disease and loss of egg and meat production.

Introduction

The protozoa (meaning proto- first and zoa mean animals) are considered to primitive and are unicellular organism. The protozoa are Eukaryotic organism having nucleus in which the genetic information is stored in chromosomes of nucleus. In poultry, the protozoan disease categorized into two main group

1. Intestinal protozoan parasites include Eimeria spp. (Coccidiosis), Cryptosporidia (Cryptosporidiosis) Histomoniasis (Histomonas meleagridis), Trichomoniasis (Trichomonas spp.) and Spironucleus (Hexamitiasis).

Coccidiosis is important disease of poultry caused by protozoa of the genus Eimeria. The disease occurs by ingestion of sporulated oocyst ehcih upon action of trypsin rupture in gizzard and proventriculus relealeses sporozoites. The sprozoites penetrate mucosa of intestine and two generation of asexual reproduction occurs (Schizogony) and releases merozoites which mature and produces gamonts. Sexyual reproduction occurs (Gametogony) and produses zygot which mature to form oocysts. The oocysts are passed in faeces. The high moisture (exceeding 30%), immunosuppressive diseses and environmental and managemental stress lead to occurrence of disease. The birds affected with coccidiosis show depression, ruffled feathers, blood tinged diarrhoea and pale comb and wattle. Specific part of intestine is affected by each species of Eimeria. Diagnosis of coccidiosis is done by finding oocyst in faeces under microscope.

1 Professor (Retired) Department of Agricultural Extension and Rural Sociology, Tamil Nadu Agricultural University, Coimbatore-641 003, Tamil Nadu

2 Assistant Professor, Department of Agricultural Extension and Rural Sociology, Agricultural College & Research Institute, Madurai-625 104

Introduction

The word ‘Extension’ was first coined in U.S.A. in the mid-part of the nineteenth century, to connote extension knowledge of improved practices in agriculture and home economics from Land Grant Colleges to the farmers and their families in their fields and homes. The literary meaning of Extension is ‘Ex’-out; Tensio’-to stretch i.e. Extension means stretching out. Extension, in the context of rural community development, means educating rural people and it implies production of changes in human behaviour -in terms of attitude, knowledge and skill. Extension is often misconceived as disbursement of subsidies and providing certain material incentives; it is certainly not.

There are several conventional extension techniques approaches and tools for transfer of any farm -related technologies, be it any enterprise, crop or their mix. Extension teaching aids are broadly classified as:

The condition is characterized by proventriculitis and dilatation of proventriculus.

Etiology

The cause of transmissible viral proventriculitis (TVP) is a birnavirus referred as Chicken proventricular necrosis virus (CPNV). TVP is a disease of 3-8 week-old broiler.

Agricultural research in India has contributed new varieties of crops and production technologies for increased production at the national level. These modern technologies benefited only a particular section but farm youth in rainfed areas are facing many problems. One of the problems identified in the diffusion of technologies to farm youth in sustainable agriculture was the lack of appropriate technologies suitable to the farm and social situations. When farmers in rain fed areas are exposed to innovations, they react to the practices/innovations from different viewpoints. A practice appropriate to farm youth in rain fed areas must be less in cost, high in profits, less risky,  highly compatible with socio-cultural values and beliefs, less in complexity, highly trialable on a small scale and the results of the same should be easily observed and communicated. These dimensions are perceived by farmers differently and the degree to which they perceive them favourably are likely to adopt the same in rain fed areas. Many a time, several past investigations have only indicated whether the recommended package of technologies has been adopted or not by farm youth. This gives percentage of farm youth adopting the practices but not the percentage of recommended technologies adopted by them and the problems encountered by them in adopting the recommended technologies in rain fed areas.

Definition

Wound with infection with 6 hours of occurrence can be brought to non infected stage by flushing.

Procedure

The infected wound at fracture site is cleaned / flushed thoroughly using sufficient quantity with antiseptic solution.

The site is applied with topical antiseptic / antibiotic and the size of the wound is reduced by placing interrupted stitches for skin closure.

For drainage of inflammatory fluid, the counter opening is made as the distal part of the swelling of the dependent part of flushing catheter is placed inside to carry out regular flushing of the wound without removing the immobilizer.

The fracture site is immobilized as per the conventional procedure using POP cast leaving two window over the opening of counter opening site.