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Animal Husbandry is making a significant contribution to the national economy and socio-economic development in the country. In rural India where over 15-20% families are landless and about 80% of the land holders belong to the category of small and marginal farmers, livestock is the main source of livelihood with the policy of the state Animal Husbandry Department to extend free breeding, vaccination and veterinary services, and permit free grazing on community lands, the farmers were encouraged to expand their herd size without any major financial burden. This has probably been the reason for the presence of the worlds largest livestock population in India. India rank first in buffalo population, second in cattle and goat, third in sheep and seventh in poultry (Table-1). Although the population of livestock during last 10 years has been stable around 485 million, the buffalo population during 1997-2003 has increased by 8.91%, while the cattle population has reduced by 6.89%. There has been a significant increase in population of goats during the last five decades, which is attributed to the decrease in the size of land holdings and persistent drought caused by erratic monsoon, forcing many small farmers to shift from large animals to small ruminants.

Dairy buffalo production has been a tradition in parts of the world like the Caucasian countries. Asia and Egypt, where fresh buffalo milk, dahi (cultured sour milk), ghee (butter oil) and yoghurt are popular. In Italy the dairy buffalo industry is flourishing thanks to the popularity of buffalo mozzarella cheese. Because of the market for Mozzarella, buffalo farming is a profitable enterprise and is carried out in an organized manner with modern equipment. In South American countries like Brazil and Argentina, buffalo reared for both milk and meat. In recent years buffalo milk and milk products, especially Mozzarella cheese, have become immensely popular and dairy buffalo production has founds its way into non traditional areas with the number of buffalo farms mushrooming.

Abstract

Performance records of 1085 crossbred cattle daughters of 57 sires maintained during 1974-2004 at Instructional Dairy Farm of G. B. Pant University of Agriculture & Technology, Pantnagar were used to evaluate sires for first lactation and life time performance traits. First lactation traits included were age at first calving (AFC), first lactation period (FLP),first dry period (FDP) and first calving interval (FCI) and life time traits were herd life (HL), total lactation length and life time milk yield. The data were analyzed to estimate the breeding values of sires using least squares methods (LSM), best linear unbiased prediction (BLUP) and derivative free restricted maximum likelihood (DFREML) method. The average breeding values of sires estimated by all three methods were similar and estimated for AFC, FLP, FDP and FCI as 1359.95 days, 356.56 days, 177.58 days and 534.15 days respectively and for HL, TLL and LTMY as 1641.30 days, 1320.20 days and 9110.30 kg respectively. The estimated breeding values (EBV’S) showed large genetic variation between sires for both first lactation and life time traits. The estimated breeding values (EBV’S) of sires had very high and significant product moment correlations and rank correlations among all first lactation and lifetime traits estimated by all three methods.

Abstract

The data for the present investigation were obtained from the performance records of 1081 crossbred cattle progeny of 65 sires maintained at Instructional Dairy Farm Nagla of G.B. Pant University of Agriculture & Technology Pantnagar, distributed over a period of 40 years from 1966-2005. A total of 65 sires were evaluated on the basis of single and multi traits of their progeny. For single trait sire evaluation, FLMY was considered as principal trait under univariate models viz- simple daughter’s average () method, least squares method (LSM), DFREML-I method and BLUP-I method. However, for evaluation of sires by multi traits FLMY+AFC, FLMY+FLP and FLMY+AFC+FLP, the multivariate models viz. DFREML-II, DFREML-III, DFREML-IV, BLUP-II, BLUP-III and BLUP-IV method were used. The lowest average breeding value (2616.25) for the trait was obtained by () method and the highest average breeding value (2660.42 kg) was obtained by the rest nine methods. The accuracy, efficiency and stability of different sire indices were compared to judge their effectiveness. The evaluation method that gave the lowest error variance was considered to be most efficient.

The state of Uttaranchal was carved out of Uttar Pradesh on 9^th November 2000 as the 27th state of the union of India and renamed as Uttarakhand. The state is located in western Himalayan region and is one of the geographically smallest states of the country. However, the state is enriched with livestock biodiversity represented by cattle, buffaloes, goats, pigs, horses, mules and poultry.  The climate of the state varies from subtropical in the valleys to temperate on higher slopes with a summer monsoon. The temperature ranges from -5.4ºC (Muketshwar, higher hills) to 40.2ºC (Pantnagar, plains) but it drops below freezing point in many parts of high mountainous areas of the region during winter. Preponderance of hilly terrains and forestland restricts the size of the agricultural land/ holding. The average rainfall recorded in the year 2008 was 1605 mm.  Higher rainfall, abundance of natural pastureland and suitable climatic conditions makes the state favorable for livestock development. The human population was 84.89 lacs with a density of 159 people per sq km during the year, 2001.

India is very rich in biodiversity especially in animal genetic resources and animals have been part and parcel of agricultural heritage. There are about 30 well defined breeds of cattle found in different parts of the country. To name a few are Sahiwal, Red Sindhi, Gir, Kankrej, Deoni, Tharparkar, Hariana, Ongle, Nagori, Amritmahal and Hallikar. The cattle breeds are classified into three types i.e. dairy, dual purpose and draft/drought animals. The number of the animals of majority of indigenous breeds has declined over the years. Further, their number is very low and majority of cattle population (80%) in India is non-descript. The introduction of systematic crossbreeding program of our native cattle with exotic breeds in seventies have further led to the depletion of prized germplasm of all the indigenous breeds under farm and field conditions. However, considering the importance of indigenous cattle, some of the breed improvement programs have been initiated to conserve and multiply these breeds. The associated herd progeny testing program has been going on at NDRI Karnal for the genetic improvement of Sahiwal cattle. All India Coordinated Research Project on Hariana and Ongole cattle has been undertaken at different centres by Project Directorate on Cattle, Meerut. Lately, three more breeds namely Sahiwal, Gir and Kankarej have been included in the indigenous breed improvement program (IBP) and different centres have been identified as germplasm units and data recording units for different breeds under the program.

Precautions to be taken while working in the laboratory for personal safety and to prevent any hazards.
    1.    Be vaccinated against dangerous viruses viz., rabies and hepatitis B as persons can contract infection while working with these organisms.
    2.    Use eye protection viz., goggles, spectacles etc to avoid splashing microbes into the eyes. 
    3.    Do not rub eyes with contaminated hands or objects.
    4.    Do not eat or drink anything in the microbiology laboratory as it will contaminate the laboratory.

Early and accurate diagnosis of disease is of prime importance for both the successful treatment of the patient and in limiting the spread of disease in the community. It is in this context that immunodiagnostic methods are now being used extensively since they can be extremely sensitive and highly specific. Among the most successful methods are those utilizing labelled antibodies or antigens. Examples of these are immunofluorescence in which antibodies labelled with fluorescent dyes are utilized and radioimmunoassay in which antigens or antibodies are labeled with isotopes. Both of these procedures have proved to be of great value for particular purposes.

Principle

The infectivity of a virus may be neutralized by specific antibody when they are mixed and incubated. In some cases, the addition of homologous or guinea pig complement to the mixture enhances neutralization, particularly when early antiserum is used. Serum must be inactivated by heating at 560C for 30 minutes to inactivate complement or to remove non-specific virus inhibitors. Serum-virus mixtures are inoculated into appropriate cell cultures, which are then incubated until the only virus controls develop cytopathic effects. Antibody by neutralizing the infectivity of the virus, protects the cells against virus destruction.

Processing

On arrival in the laboratory, the specimens are processed immediately or refrigerated until processed. For inoculation into animal/cell culture/embryonated eggs (EE), swabs are shaken in fluid medium, faeces are dispersed in fluid medium and tissue specimens are homogenized in a high speed blender/pestle and mortar with the help of an abrasive like powdered glass or sand. Good quality hard sand should be properly washed in distilled water, treated with HCl, washed thoroughly, dried and sterilized before use. Generally the infective tissue is homogenized in a few ml of buffer usually having a pH of 7.2 to 7.3 to make about 10% tissue suspension. The suspension is centrifuged at about 1000g for 10-15 minutes to sediment the tissue debris and coarse materials.

Bovine
 
1.    PPR    Eye, mouth, rectal swab in PBS on ice, 10 ml blood  in anti-coagulant at height of temperature, Prescapular lymph node, spleen, piece of intestine on ice, lung, liver, spleen, tonsil in 10% neutral formol saline solution.

The tentative diagnosis of viral diseases of livestock can be made by clinical symptoms alone. The clinical diagnosis is substantiated by the laboratory to obtain a precise, correct and prompt diagnosis. The success however depends on the collection of suitable materials in proper transport medium at a particular stage of disease condition and its dispatch to the laboratory in a manner that little or no alterations occurs in the specimen. This would facilitate the isolation and identification of the causative viral agents easily.

The most important property of a virus is its infectivity or ability to invade a cell and parasitize that cell to replicate itself. To measure infectivity, a titre is defined as the given number of infectious virus units/unit volume and an infectious unit is the smallest amount of virus that produces some recognizable effect in the host system. There are two basic types of infectivity assays (a) Quantal assays and (b) Quantitative assays.

Quantal assays do not measure the number of infectious virus particles present in the inoculum but denote a value for the virus titre where measurement is based on an all or none principle, CPE + or -, dead or alive etc. In the quantitative assays, the number of virus particles present in the suspension can be quantified.

Cell culture is the most convenient and widely used system for the isolation and propagation of many viruses. Either primary cell culture or established cell lines can be used to isolate the suspected viruses. One can normally get 10-100 virus particles per cell inoculated. Many details of the infection process are controllable to a high degree (including cell density, age, multiplicity of infection (M.O.I.), medium composition, cell type and length and temperature of incubation). Many cell types from different species can be susceptible to a particular virus and vice versa. For replication to occur, the cell type used must be permissive for a particular virus under study.

Enumeration of cells is essential to determine the multiplicity of infection, efficiency of plating and to obtain the specific number of cells for an experiment. The concentration of cells of a suspension can be counted using a haemocytometer. Each chamber is divided into a grid of nine large squares. Each large square is 1mmX1mm (area 1 mm²) and the depth is o.1 mm. The volume of each square is 0.1 mm³ or 10^-4 cm³. Since 1 cm³ is equal to 1 ml, the number of cells per ml of suspension can be obtained by multiply the average count per large square by 10⁴ and reciprocal of dilution factor. Dilution is made in such a way that one large square contain about 20-60 cells. The haemocytometer chambers should be filled by capillary action and cell clump are counted as one cell. A total count does not distinguish between living and dead cells. The number of viable cells can be determined by staining the cells with trypan blue or erythrosine B. In trypan blue staining cells appear as unstained whereas dead cells appear as blue.

Cell culture contamination continues to be a major problem and a subject of great concern. Contamination may enter the cell culture system as physical, chemical or biological component of the environment and biological contamination represent the greatest threat as living organism metabolize and replicate. Replication increases the titre of the contaminant.
 
Physical contamination : Physical components of cell culture systems such as radiation, irradiation (UV light and fluorescent light), temperature extremes can cause reduced cell growth and cell death. To minimize the problems, incubators should be located where ambient temperature is relatively constant and no vibration (near centrifuge). Media and media components should not be stored in glass door refrigerators, next to radioisotopes, bench tops or outside the refrigerator or in the hood for prolonged period.

Early passage cell lines are relatively unstable and go through a period of adaptation to culture. However, between 5^th and 35^th generation (human diploid fibroblasts), the culture is fairly consistent although it varies from one cell to another cell. Finite cell lines should be preserved after adaptation but before senescence (getting older). Cells should be preserved to protect cell line instability, availability in case of contamination, incubator failure or accidental loss.

There are a number of different cell lines derived from human and animals. With the dramatic increase in numbers of cell lines, the risk of intraspecies and interspecies cross contamination rises proportionally. It is particularly a major problem in laboratories where many different cell lines of human and animal origin are handled at a time. The purity of the primary cell or continuous cell lines and species of its origin should be checked on a regular basis to authenticate the cell line identity. In the absence of such monitoring, inter and intra species cell line contamination are likely to occur in the laboratory resulting in loss of investigator’s time effort and resources. The advantages of working with a well defined cell line free from contaminating organisms would appear obvious.

Small volume cultures are useful for experiments such as studying cell morphology, titration of viruses, plaque assay, pock assay, SNT and to compare the effect of agents on growth and metabolism. However, there are other application where there is a need of large number of cells such as extraction of DNA/RNA from cells, production of viruses for vaccine production and a number of products namely interferons, interleukins, hormones, enzymes, antibodies, glycoproteins etc. The large quantity of cells can be obtained by using a large number of flasks/bottles but it is tedius, labour intensive, expensive and there is a risk of contiamination. Although the unit processes are more cost effective and efficient, it needs some modification to overcome limiting factors such as oxygen depletion, shear damage and metabolic toxicity. There are a number of aberrations available to scale up cell culture described below.

If the concentration of cells in suspension cell culture has reached to 7X10⁵ cells/ml, it is to be subcultured. Suspension cells are diluted to concentration of approximately 1X10⁵ cells/ml although it varies from cell line to cell line. So, to subculture into a 3 litre glass flask using 500 ml of suspension of cells, there is a need of 5X10⁷ cells. As the concentration of cells is 7X10⁵ cells/ml, there is a need of 71.4 ml of cells. Adherent cells are put in the culture flasks at a concentration of 5X10⁴ cells in a 25 cm² flask. For example, after trypsinization, the cells have been resuspended in 5 ml of medium and cell concentration is 1X10⁵ cells/ml or total number is 5X10⁵ cells. If one wants to put cells into two 25 cm² flasks each containing 5X10⁴ cells in 5 ml volume, there is a need of 1X10⁵ cells, which can be obtained from one ml of cell suspension (1X10⁵ cells/ml).