eChapters

Combining Ability Estimation
Crosses made in a definite fashion are a prerequisite for combining ability estimation. Estimated by half-sib (GCA) and full-sib (SCA) mating. Various steps like parent selection for crosses, performing crosses, their evaluation, and interpretation are involved while estimating of combining ability. For analysis of crosses, biometrical techniques like diallel analysis, partial diallel, and Line x tester analysis are used. If GCA variance is higher than SCA variance the importance of AGA & parent selection will be effective for the improvement of such traits. If SCA variance is higher than GCA variance there is importance of NAGA( dominance & epistasis) & Heterosis breeding may be rewarding. If GCA=SCA variance then AGA & NAGA are equally important in the expression of character, In this situation reciprocal recurrent selection may be adopted for population improvement.
Selection for GCA
General combining ability (GCA) is the average value of the inbred line based on its behavior in crosses with other lines. The mean performance of the line is expressed as deviation from the mean of all crosses.GCA is owing to the activity of genes which are largely additive in their effects as well as additive × additive interactions.

Principle

To retrieve and purify any specific DNA fragment from an agarose gel slice; the expected yield is from 50-75% of the amount in the gel slice.

Time required

Restriction digest - minimum of 2 hours

Electrophoresis - approx. 4 hours

Elution - 2-5 hours depending on fragment size

DNA Purification -2 hours

There is urgent need to increase the production in order to meet the demand for the exploding population. This could be achieved by applying various modern methodologies in selection and breeding of livestock. Increasing the productivity through genetic improvement requires adequate identification and intensive selection of genetically superior sires. About 93 per cent of the total herd improvement comes from breeding of young bulls from tested sires and only six per cent from selection of dams.
With the advances in artificial insemination and cryopreservation of semen, a sire has a potential of serving 3/4th million cows and producing 1/4th million progenies.
Thus, selection of bulls is of great importance in dairy herd improvement.
For maximising the genetic gain by sire selection, it is essential that the method of estimating breeding values of sires should be unbiased and efficient. The breeding value refers to the average genetic effect of the genes passed on by the individual to its offspring and is estimated to know whether the individual is genetically superior to other individuals or not for the trait concerned.

Poultry industry in India has a strong standing globally, being third highest egg producer with more than 50000 million eggs and fifth highest broiler producer with 2.25 million tones of broiler meat. Largely, Indian poultry industry is chicken dominated and the rearing of high yielding varieties under intensive rearing system is the backbone of its present face. Analysis of trend of egg production in last five years i.e. 2001-2006 revealed that annual growth rate in egg production is about 5-6 %, except a much lower rate of 3 % during 2003.

Principle

RNA is negatively charged at neutral pH and when an electric field is applied, it migrates towards the anode. RNA retains much of its secondary structure during electrophoresis unless it is first denatured. The addition of formaldehyde to the agarose gel maintains the RNA in its linear (denatured) form. The following protocol is based on Formaldehyde Agarose Gel Method.

Things required for cell culture

1. Lab – having negative pressure (HEPA filter)

2. Safety cabinet (Hood/laminar flow)

a) Biosafety level 1 (BSL-1) – Basic level of protection. Agents handled under this cabinet are NOT infectious.

b) BSL-2 – Appropriate for moderate risk agents. Most of the non infectious cell cultures are done under this facility.

This is an international convention governing the recognition of deposits in officially approved culture collections for the purpose of patent applications in any country that is a party to it. Because of the difficulties and on occasion of virtual impossibility of reproducing a microorganism from a description of it in a patent specification, it is essential to deposit a strain in a culture collection centre for testing and examination by others.

This was the year, 1974, the Director General of WIPO convened a committee of Experts to discuss the possibilities of international cooperation over the deposit of microorganisms for patent purposes

Livestock sector may be considered as a driving force for nutritional security and an important component of sustainable agriculture in India. Livestock sector besides providing milk and meat also provides a diverse range of output for agriculture (manure, draught power and transport) and for manufacturing sector (fiber and leather). According to estimates of the Central Statistics Office (CSO), the value of output from livestock sector at current prices was about 4,59,051 crore during 2011-12 which is about 24.8% of the value of output from total agricultural and allied sector at current price and 25.6% at constant prices (2004-05). The value of output of milk is 3,05,484 crore in 2011-12, which is higher than the value of output of paddy and wheat. The value of output from eggs and wool group is 17,803 crore and 318 crore respectively for 2011-12. Livestock agriculture is the only way to produce food in many of the world’s harshest environments. Locally adapted breeds enable these vast areas to be used in a sustainable manner.

The Patent Cooperation Treaty provides an overview of an International Treaty which is administered by the World Intellectual Property Organization. The Patent Cooperation Treaty is an international treaty with more than 157 Contracting States by 2022. India deposited its instrument of accession on 7 September 1998 to both international treaties of WIPO in Geneva i.e. Paris convention for the protection of Industrial Property and Patent Cooperation treaty. PCT provides a system for filing a patent application and allow us to obtain patents in multiple countries around the world on the basis of a single patent application.

It is known that the phenotype of an individual is a combination of genotype and environment. Animal breeding analysis is mainly done to partition the genetic part from the environmental part so as to assess the actual genetic worth of the animal. In this process correction for the known environmental part is integral to any animal breeding analysis. In this chapter, a list of common factors affecting important economic traits are described. The analytical part for these effects will be explained in later chapters.

Correction or standardization of data is very important in any animal breeding analysis. Unless the candidates under selection are placed on a common platform, various factors affecting the trait will lead to bias in assessing the performance of the individual with respect to a particular trait. For eg. a lamb born during a favorable season, a lactating cow in its third parity, first lactation yield of a cow with longer age at first calving.

Cell counting is the fundamental techniques in various fields of biology, medicine and biotechnology.

Importance of cell counting

1. Research: Cell counting is a critical component of biological and medical research. It helps scientists understand the behavior of cells in various contexts, such as studying cell proliferation, differentiation, and response to stimuli.

2. Disease diagnosis: Monitoring various medical conditions, including infections, autoimmune diseases, cancer and blood disorders. Complete blood counts (CBCs) are routinely used to assess the levels of different blood cell types and assess disease conditions.

3. Stem Cell Research: Cell counting plays a significant role in stem cell research, where precise quantification of stem cells is necessary for various applications, including regenerative medicine and tissue engineering.

4. Cell Culture: Cell counting is integral part to determine seeding density. Seeding of cells to available surface area for growth is important consideration for successful culture.

5. Cell-Based Assay: The cell-based assays are used for drug screening, toxicity testing, and evaluating the efficacy of new compounds. Accurate cell counts are essential to ensure the reliability of assay results.

Biological Diversity Act, 2002 was passed by Indian government in pursuance of Convention on Biological Diversity, 1993 The Convention on Biological Diversity (CBD) is a legally binding multilateral environmental agreement that has 194 contracting Parties (Countries) as its members with three objectives mentioned below:

1. Conservation of biological diversity
2. Sustainable use of the diversity and
3. Ensuring fair and equitable sharing of benefits of such use.

Introduction

Peripheral blood mononuclear cells (PBMCs) consist of chiefly of lymphocytes and monocytes. Purified PBMCs are used in vitro to evaluate a variety of functions of lymphocytes viz; a) proliferation to mitogenic (PHA, Con-A) stimulation, b) monitoring of prior sensitization in antigen recall assays by scoring lymphocyte proliferation, c) immunophenotyping for surface markers as well as intracellular molecules in monocytes and lymphocytes etc. Activation of monocytes/macrophages by small molecules, cytokines and pathogen components can also be monitored. PBMCs can also be used for a variety of structural and functional studies for addressing issues in human immunology such as scoring for apoptosis and production of cytokines as well as other mediators in vitro.

Background information

Blood is a liquid mixture comprised of plasma, cells and proteins. The liquid portion, plasma, makes up half of the blood volume, and cells the other half. Isolation of lymphocytes is a common procedure in immunology and cell biology that involves separating and collecting lymphocytes.

This protocol is designed for potato stem internodes on ZIG medium, but might work for leaf pieces as well.

Plant Material

Cut stem internode explants from 5-6-week-old in vitro plants (or leaf explants from 3-4-week-old in vitro plants) grown on 15 ml PROP in 25 x 150 mm test tubes.

Day 1

Streak out desired Agrobacterium strain on YEP + antibiotic to obtain a single colony. Grow at 28 C for 2 days. Alternatively, skip this step and use frozen stock for next step.

Day 3

Use a single Agrobacterium colony to inoculate 20 ml YEP + antibiotic in 250-ml flask. Grow overnight at 28 C with vigorous shaking (~200 rpm). Some Agrobacterium strains require an additional day to reach sufficient density.

Traditional knowledge is a type of Intellectual property. It is also known as indigenous knowledge generated from the local communities and tradition of regional technology areas. This knowledge is orally passed over generation from person to person. According to world intellectual property organization, Traditional knowledge is an indigenous, cultural and intellectual property. The biodiversity of a country, the uses and applications of biological resources and prevalent practices are examples of traditional knowledge associated with life science. TK has the tremendous potential of being transformed into commercial opportunity by the development of products and processes. Hence, a share of benefits must accrue to creators and holders of Traditional Knowledge.

The aim of the breeder is to improve on various economic traits through selection. However, due to the lesser number of parents used, over a period of time, there creeps in the effect of inbreeding and loss of genetic variability which in turn causes depression and loss of fitness. This is more prominent in finite endangered populations and evaluation for genetic variability also forms an integral part of the animal breeding analysis.

Genetic variability is an integral part of domestic animal populations as fitness is directly related to the variability.

The large volume, type and nature of animal breeding data and the complex nature of analytical requirement warrants additional tools for handling, storage, retrieval and solving complex equations. Accuracy, speed, computational requirement, applicability, etc., has resulted in the development of numerous software exclusive for animal breeding data analysis. The database management system is already explained in the chapter on ‘Nature of animal breeding data and database management’. This chapter gives a note on the commonly used animal breeding software.

Principle

The quantification of infectious virus particles is commonly achieved through the utilisation of the Median Tissue Culture Infectious Dose (TCID50) assay. The assay functions through the introduction of a serial dilution of the virus sample into cells in a 96 well plate format. The chosen cell type is specifically intended to exhibit a cytopathic effect (CPE), which refers to observable changes in morphology or cell death resulting from viral infection. Following a period of incubation, the cells are examined for cytopathic effects or cell death, and each well is categorised as either infected or uninfected. The concentration at which 50% of the wells exhibit a cytopathic effect (CPE) is employed to determine the 50% tissue culture infectious dose (TCID50 ) of the viral sample. The end point is the dose or dilution that infects or kills or shows CPE of the cells. The calculation can be performed using various mathematical techniques, such as the Spearman-Karber method or the Reed Muench method. The virus titer is quantified as the number of tissue culture infectious dose 50 (TCID50 ) per millilitre (ml). The non-cytopathic virus can be quantified by employing the fluorescent antibody test, immune-peroxidase assay, or other appropriate methodologies.

Principle

Cell viability refers to the quantity of healthy cells in a sample, while cell proliferation is a crucial indicator for comprehending the mechanisms and pathways involved in the survival of cells or death following exposure to toxic substances. The methods used to assess viability are typically the same as those used to detect cell proliferation. Cell cytotoxicity and proliferation assays are commonly employed in drug evaluations to determine if the test compounds impact cell proliferation or exhibit direct cytotoxic effects. Cell viability can be determined by calculating the ratio of total live cells to the total number of cells (live and dead). Staining also helps to observe the general cell structure. Cell proliferation assay by MTT. Metabolically active cells reduce the yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) through dehydrogenase enzymes to produce NADH and NADPH. The purple formazan produced inside the cell can be dissolved and measured using spectrophotometry. The MTT Cell Proliferation Assay quantifies cell proliferation rate and detects decreases in cell viability caused by metabolic events leading to apoptosis or necrosis. The MTT Reagent produces minimal absorbance values when no cells are present.

Cell culture refers to removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been establwashed.

Primary culture refers to the stage of culture after the cells are isolated from tissue and proliferated under appropriate conditions until they occupy all of the available substrate (i.e., reach confluence). At this stage, the cells have to be subcultured (i.e passaged) by transferring them to a new vessel with fresh growth medium to provide more room for continued growth.

Pharmaceutical and chemical industries depend largely on toxicological evaluations to provide information regarding safety of their commercial products. Toxicologists so far have been relying on animal studies to investigate potentially beneficial or harmful effects of drugs and chemicals prior to testing them in humans. Traditionally, the safety assessment process during drug development has been based on screening indiscriminately a plethora of new chemical entities (NCEs) using large number of animals as testing models in order to identify new therapeutic agents with the expectation that one or a few of these NCEs have some competitive advantages over the existing therapeutic agents. That is why they are more efficacious, bioavailable, selective, and safer. This approach is costly, time-consuming, and requires large quantities of test material. Currently, it takes about 10-12 years and a huge expenditure for a drug to move from research laboratory to patent. Thus alternative cell culture models in the early phases of drug discovery process can save time and money both by eliminating least beneficial NCEs.