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Significant advancement in high throughput technologies (microarrays, automated sequencing and mass spectrometry), transcriptomics, the global study of transcription, together with genomics and proteomics, has undoubtedly contributed to a systems biology approach. These technologies have generated vast amount of sequence data from plants, animals and microorganisms. Fortunately, efficient computational tools (intelligent data networks, query, retrieval, analysis and visualization tools) have now optimized data mining, accelerating the process of discovery. The analysis of sequence variation is of major importance in genetic studies. In this context, molecular markers represent one of the most powerful tools for the analysis of variations in the genome and the association of heritable traits with underlying genetic variation. The development of high-throughput methods for the detection of simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) has led to a revolution in their use as molecular markers. The availability of large sequence data sets permits mining for these molecular markers, which may then be used for numerous applications such as genetic mapping, association mapping, evolutionary and phylogenic studies.

Introduction

The proteome is often described as the set of all expressed proteins in a cell, tissue or organism. It is the systematic analysis of cellular proteins, that is, their identity, quantity and function. One advantage of proteomics over transcriptome is that mRNA represents an early stage of gene expression whereas the study at the proteome level describes the state of protein which is the final functional molecule of the cell. Thus, it gives a clear picture of the cell function that may not be concluded from mRNA study. In fact we can say that proteins are more than just information carriers so it gives more information with respect to only nucleic acid based studies.  Proteomics covers two-dimensional gel electrophoresis (2-DE), image analysis, mass spectrometry, amino acid sequencing, and bio-informatics to resolve comprehensively, to quantify, and to characterize proteins. Biomarker discovery as an important aspect of proteomics technology is highly appreciable when it comes to diseases. Today,  a better understanding of the disease mechanisms leading to discovery of new disease specific biomarkers are very much possible due to the sequencing of the human genome. It has accelerated the proteomic technologies for accepting the challenges so as to meet the diseased related investigations.

1. Introduction
The era of genome sequencing was exploded by the upcoming of Human Genome Project (1989), coordinated by the U.S. Department of Energy and the National Institute of Health to identify all the approximately 20,000-25,000 genes, 3 billion chemical base pairs, store this information in databases, improve the tools for data analysis, transfer related technologies to the private sector, and address the ethical, legal, and social issues (ELSI) that may arise from the project. The dawn of genome sequencing began with Fredric Sanger’s sequencing almost twenty-five years ago. The time consuming and labor intensive gel preparation and their running, as well as the cost of such experiments replaced the conventional sequencing by “shotgun” sequencing.

Abstract
In-silico analysis of nucleic acids and proteins from fishes has enriched our understanding to several fundamental problems of classical fish biology and genetics. Mining of data which is analogous to information discovery is the process of automating knowledge discovery. Analysis of data in all possible manner and recognition of a pattern which has a scientific basis by an efficient algorithm is the fundamental principle of Bioinformatics. Ever increasing number of Bioinformatics tools as computational packages are now a days used in tandem to analyze sequence of a loci and to model a novel gene product. High efficiency algorithms with excellent homology searching functions have lead to this advancement. Cat fishes, a group of diverse bony fishes have been studied with respect to genetic analysis in several species and several peptides. Tracking of the evolutionary foot prints using genomic libraries and between expressed sequences has been generating much interest.

Introduction

Saffron is a naturally derived plant product from the dried stigma of the Crocus sativus flower  (Gresta et al., 2009).The saffron (Crocus sativus L.) is an Iridaceae family member, herbal perennial, sterile (2n=3x=24) geophyte, traditionally propagated by vegetative corms. Its cultivation dates back to late Bronze Age in the Mediterranean region (Negbi, 1999) in Anatolia since the ancient times (Arslan et al., 2007) and for at least 3500 years in Egypt and Middle East. Saffron contains more than 150 volatile and aroma yielding compounds. The chemical components mainly include carotenoids, crocin, picrocrocin, safranal etc (Abdullaev, 1993, Tarantalis et al., 1995, Karaoglu 
et al., 2007). Saffron is mostly used as spice, food colorant, dye or perfume. The dried stigmas introduce unique color, taste and fragrance and thus making it the world’s most expensive spice (Baghlian et al., 2010). Saffron has been used in folklore herbal medicines for centuries (Basker and Negbi, 1983).

The analysis of chromosome behaviour at meiosis can provide valuable information about the genomic composition of both species and hybrids. The basis for this analysis is the idea that chromosomes that pair and undergo chiasma formation are homologous. Where two genomes are present, as in diploids, the expectation is that only bivalents will be observed and where there are multiple genomes, as in polyploids, two general patterns of pairing are possible. In autopolyploids, configurations of multiple chromosomes are to be expected since all the component genomes are homologous and have the possibility of pairing with each other, whereas in allopolyploids, with different genomes, bivalent formation is the expectation. Thus, meiotic analysis can provide an oversight for genome analysis to investigate genetic relationships between the species. Further, chromosomal rearrangements are correlated with pollen fertility in species and F₁ hybrids.  Observations on pollen formation, size and viability can provide useful information, as low pollen viability is often indicative of meiotic irregularities and large sized pollen can indicate the formation of unreduced gametes. In addition, the occurrence of nucleolar dominance has been recorded in many interspecific hybrids.

Introduction

The recent revolution in genomics and bioinformatics has caught the world by storm. The issues surrounding the human genome, including the analysis of genetic variation, access to genetic information and the privacy of the individual have fuelled public debate and extended way beyond the scientific and technical literature. During the past few years, bioinformatics, defined as the computational handling and processing of genetic information, has become one of the most highly visible fields of modern science. A flood of data means that many of the challenges in biology are now challenges in computing. Bioinformatics, the application of computational techniques to analyse the information associated with bio-molecules on a large-scale, has now firmly established itself as a discipline in molecular biology, and encompasses a wide range of subject areas from structural biology, genomics to gene expression studies. In this chapter we provide an introduction and overview of the current state of the field.

Introduction

Functional genomics is a field of molecular biology that attempts to make use of the vast wealth of data produced by genomic projects (such as genome sequencing projects) to describe gene (and protein) functions and interactions. Unlike genomics and proteomics, functional genomics focuses on the dynamic aspects such as gene transcription, translation, and protein-protein interactions, as opposed to the static aspects of the genomic information such as DNA sequence or structures. Functional genomics includes function-related aspects of the genome itself such as mutation and polymorphism (such as SNP) analysis, as well as measurement of molecular activities. The latter comprise a number of “-omics” such as transcriptomics (gene expression), proteomics (protein expression), phosphoproteomics and metabolomics. Together these measurement modalities quantifies the various biological processes and powers the understanding of gene and protein functions and interactions. Functional genomics uses mostly high-throughput techniques to characterize the abundance gene products such as mRNA and proteins.

The equipments used in artificial insemination should be clean and sterilized. The use of unsterilized equipments in AI causes the spread of infection in the herd and infertility. Rinse the equipments with tap water immediately after use and then lukewarm detergent solution. The detergent used for washing the AI equipments should be non corrosive. Use of corrosive detergents will reduce half-life of the plastic and rubber wares. Wash thoroughly with tap water till the equipments become grease free and perfectly clean. Then rinse in distilled water at least thrice. Use of distilled water does not leave any salt residues on articles after drying.

21.1 Liquid Nitrogen Container/ Cryocan

To store liquid nitrogen a special type of container is used and is known as a LIQUID NITROGEN CONTAI NER OR CRYOCAN. Frozen semen doses are always preserved in liquid nitrogen which is an inert liquid having a boiling point of -1960C and a freezing point of -212°C. Liquid Nitrogen Containers are doubled walled metal (Stainless Steel/Aluminium) vacuum vessels with a very efficient insulation system. Due to highly efficient insulation system the temperature inside the container can remain -196 oC as long as atleast two inches of Liquid Nitrogen is present in the container. It has got an inner chamber which is suspended in outer chamber through neck tube which is a non metal and bad conductor of heat. Walls of the inner chamber are coated with a high quality of insulation material. The space between the inner and outer chamber is also filled with high quality insulation material. Vacuum is created in between inner and outer chamber. Loss of vacuum will lead to the boiling of the liquid nitrogen gas and hence rapid loss of this gas from the inner chamber.

Artificial insemination (AI) is the technique of placing sperm into a female’s uterus (intrauterine), or cervix (intracervical) using artificial means rather than by natural copulation. Modern techniques for artificial insemination were first developed for the dairy cattle industry to allow many cows to be impregnated with the sperm of a bull with traits for improved milk production. It is the first generation reproductive biotechnology which has revolutionized the animal productivity. It has now been established as cheapest and fasted mode of genetic improvement of livestock. AI can be done using fresh semen, semen preserved at room temperature, refrigerated (4ºC) and cryopreserved (-196ºC) semen. The use of AI with frozen semen has become very popular and is being successfully used throughout the country.

The process of converting the frozen semen to liquid state while retaining maximum semen quality is called thawing. The process of thawing is as important as freezing as there is equal probability of loss of semen quality in freezing and thawing. Therefore, great care should be taken while thawing to insure that it is done with minimum loss of sperm quality. There are two methods of thawing:

1.    Rapid thawing

2.    Slow thawing

The effectiveness of each method of thawing depends upon the extender composition and freezing procedure. With any thawing procedure, the recommendations of the semen supplier should be followed. The thumb rule is that rapid freezing rates require rapid thawing.

18.1 Estrus Detection
18.1.1 Estrus Detection on the Basis of External Signs

The following signs are suggestive estrous period in cattle and buffaloes

1.    Partial inappetance due to excitement

2.    Drop in milk yield

3.    Restless ness of varying degree

4.    Frequent micturition with raised tail and crouching the back and lumbar region

5.    Marked bellowing

6.    Swollen vulvar lips- Due to swelling the wrinkles on vulvar lips disappear. The vulvar lips appear turgid and stand prominently.

7.    Congestion of vulvar lips 

8.    A clear, shiny mucus discharge seen at the vulvar lips extending down to perineal region and smearing the hind quarters. The discharge is stingy, having good consistency and no odour.

The bacterial load in the semen samples is estimated by standard plate count method. The bacterial contaminants of semen have been a major concern for most of the frozen semen production laboratories as it adversely affects semen quality and hence subsequent fertility. There exists a significant negative correlation of bacterial load and sperm livability, motility and in vitro fertility tests. Macrophages and polymorphonuclear granulocytes, which form the first line of defense against these microorganisms, produce reactive oxygen species (ROS) to kill these microorganisms. ROS, however, is also released outside these cells and may react with molecules and cells such as spermatozoa in their vicinity. The cellular antioxidants, present mostly in the cytoplasm, are scanty and inadequate to counteract this ROS as the sperm cell cytoplasm is very small (20 µm3) and mostly distributed in the midpiece. Therefore, semen samples with minimum bacterial load are expect to give better results on insemination.

The following tests are discussed under this section:
    16.1    Estimation of ascorbic acid concentration
    16.2    Effect of cold shock
    16.3    Mitochondrial activity
    16.4    Capacitation of spermatozoa and calcium influx 
    16.5    Morphometric analysis
    16.6    Flow cytometry

16.1 Estimation of Ascorbic Acid Concentration

Ascorbic acid concentration in semen is beneficial for its antioxidant action. It protects the spermatozoa against oxidative damage of free radical produced in the semen.

Materials Required

    1.    Test tubes with stand
    2.    Burrette with stand
    3.    Centrifuge tube with stand

Semen metabolic activity can be assessed with the help of the following tests:
    
15.1    Incubation test
    
15.2    Methylene Blue Reduction Test
    
15.3    Resazurine Reduction Test
    
15.4    Millovonoo’s Resistance Test (‘r’ test)
    
15.5    Fructolysis Index
    
15.6    Oxygen utilization test
    
15.7     Glutamic oxaloacetic transaminase activity

15.1 Incubation Test

The longevity and viability of spermatozoa during storage is a good index of fertilizing ability of sperm. In this test assessment of motility is carried out over a period time to obtain an idea about the length of time over which acceptable level of motility is maintained in the semen sample. Therefore, this test serves as a mean of determining storage probability within a fairly short period of time.

Only few single sperm viability parameters show a significant correlation with fertility of semen samples. Therefore, need to explore functional in vitro fertility tests having capability to disclose the ability of spermatozoa to undergo complicated processes such as capacitation, binding of zona pellucida, acrosome reaction, fertilization of Oocyte and induction of embryo development invitro have been designed and explored for their correlation with fertility achieved after artificial insemination.

In vitro fertility tests used in evaluation of semen includes: 

14.1 Estimation of membrane integrity
        
    14.1.1 Morphological integrity
        
    14.1.2 Functional integrity
        
    14.1.2.1 Hypoosmotic swelling test
        
    14.1.2.2 Acrosome integrity

14.2     Cervical Mucus Penetration Test

13.1 The Normal Sperm Morphology

A mature spermatozoa is an elongated cell consisting of a flattened head containing the nucleus, a neck and a tail containing the apparatus necessary for cell motility. The sperm cell consists of the following parts covered by the plasmalemma or plasma membrane: 
    
13.1.1    Head
    
13.1.2    Neck
    
13.1.3    Tail / flagellum – The sperm tail consist of     
    
a.    Mid piece
    
b.     Main piece / Principal piece
    
c.     End piece

Sperm Plasma Membrane

The sperm plasma membrane is three layered membrane. It lies in close contact with the underlying cytoplasm and attached at three places i.e. over anterior outline of the sperm head, around the base of the head, in circle around the distal end of the midpiece (Jensen’s ring). This attachment causes creation of three compartments with probably no communication with each other. The sperm plasma membrane is invisible under normal light or phase contrast microscopy but can be visualized after addition of substances with light refractory index e.g. bovine plasma albumin or synthetic polysaccharides. It is very easily permeable to water but there is low permeability for inorganic cations (Na, K, Ca etc.) and even lower permeability for anions (NO3, Cl). Exposure of sperm cells to hypertonic media will cause the water to go from sperm to the surrounding media leading to shrinkage of sperm cell whereas exposure of sperm cells to hypotonic media will induce influx of water causing swelling of the sperm cell. The more water that goes into the tail the more it changes its shape and becomes rounded to give room for increasing amount of water. As the length of the tail decreases as it becomes round, the tail fibrils have less space and tail bends and coils within the membrane vesicle. Tail fibrils are not damaged initially and will continue to contract till the critical volume of bull sperm is 3.8-3.9 times of its volume in an isotonic solution. The swelling and change in shape of the spermatozoa are momentary processes and can be corrected if hypotonicity of the media is corrected before the sperm cells reach the critical volume for rupture.

Sperm membrane is responsible for maintenance of the intracellular biochemical environment. Damaged plasma membrane is responsible for the leakage of electrolytes and organic substances such as enzymes and may also lead to damage of sperm cells by foreign substances from the surrounding media.

The following tests are used for microscopic evaluation of semen: 
    
12.1    Sperm motility
12.2     Per cent Live sperm
12.3     Sperm concentration

12.1 Sperm Motility

The following two methods of estimation of sperm motility are followed in routine practice:
 
12.1.1    Mass activity
12.1.2    Progressive sperm motility
12.1.1 Mass Activity

Mass activity can be defined as ‘en-mass activity of the spermatozoa in the semen, immediately after collection’. Mass activity is generally assessed by vigour of swirls and waves motion in undiluted semen under 20 X magnification and expressed on 0 to 5 scale.

Gross evaluation is generally done to get a rough idea of the quality of semen and to assess the semen producing capacity of the bull. This also gives a fairly good idea about the reproductive health and any pathological conditions of the semen donor. The following are recorded under gross evaluation of semen:

    11.1    Color and appearance of semen
    11.2     Semen Volume
    11.3    pH of semen
    11.4     Semen consistency/ Semen density
    11.5     Presence of foreign matter
    11.6     Gross motility