eChapters

Natural Gene Flow in Plants

More than 70% of plant species, for example potato, oat, rape-seed, wheat, and kiwifruit have come from spontaneous interspecific or intergeneric hybridization although the extent of natural hybridization differs among the plant genera and families. It creates genetic variation and produce recombinant genotypes that may exhibit heterosis.The stabilization of these hybrids often through polyploidy (autopolyploidy or allopolyploidy) can result in the formation of new biological species (Masterson, 1994), thereby, having impact on both ecological and evolutionary processes (Martinsen et al., 2001). In addition, species genomes are often differentially permeable to introgression if they are under simple genetic control (unlike many genes contribute to hybrid fitness with linkages) and certain portion of the genome can readily incorporate alien alleles. Introgression is a selective process of gene flow that filters deleterious genes and takes only beneficial genes (incorporating the alleles from one taxon into other).

Introduction

The branch of science that deals with resolving the evolutionary relationships among organisms is phylogenetics. Phylogeny is the description of biological relationships, usually expressed as a tree. A statement of phylogeny among objects assumes homology and depends on classification. Phylogeny states a topology of the relationships based on classification according to similarity of one or more sets of characters, or on a model of evolutionary processes. In many cases, phylogenetic relationships based on different characters are consistent, and support one another. If different characters induce inconsistent phylogenetic relationships, they all are dubious. Conversely, note that the same similarity data may be consistent with different possible topologies or trees.
 
Molecular approaches to phylogeny developed against a background of traditional taxonomy, based on a variety of morphological characters, embryology, and from fossils, information about the geological context (stratigraphy).

Introduction

Microbes (bacteria, archea and eukarya) are single celled organisms that rule the planet. Microbes occupy every possible niche, from the high atmosphere to the deep ocean, from hot springs to artic ice, from hot deserts to deep forests including many regions that cannot yet be accessed for study. The biosphere contains between 1030 and 1031 microbial genomes, which is about 2-3 times more than the number of plant and animal cells combined (67). In fact, studies have shown that microbes associated with the human body are about ten times more than the number of human cells (5).  Microbes play a critical role in the life cycle of all living organism and environment in general.  Some microbes cause disease, but the most of them are either innocuous or play a role in physiology and cellular response including immune response, digestion and vitamin production of various organisms.

Abstract

DNA barcoding, an additional global standard taxonomic tool for identifying plant species helps the non-taxonomists also to identify the plant species in a more reliable way with the help of devices including nanotechnology. Researchers proposed various genes or gene portions as potential barcodes either singly or in combination, yet there is no standard/ universal barcode DNA region for plants. The more accurate plant DNA barcode region would have to be from highly variable markers in the nuclear genome preferably from multiple loci and in combination with variable DNA regions of chloroplast genome, to resolve the problems that arise due to hybridization, allopolyploidization and introgression. Nonetheless, the phylogenetically informative sites might not be the potential DNA barcode regions as the former requires synapomorphies unlike autapomorphies with high levels of informative characters in the latter DNA taxonomy, but could provide insights into genetic distances between species.

Abstract

Biological Pathways represent system level sophistication of organisms. An exclusive peek into the extent of sophistication of an organism may provide proof of its past evolutionary struggle and position in the developmental trend from pathway point of view. This knowledge may not align with the traditional universally acknowledged standard 16s rRNA trend. Analyzing the reasons behind such discrepancy can constitute an important research work. To map sophistication of one organism with respect to other organisms, phylogeny is required. Here, we are mapping the level of sophistication in terms of enzymes among a set of TCA cycles belonging to different species, arbitrarily chosen from the KEGG/PATHWAY database. For this purpose, we converted the metabolic pathway information into simplified enzyme-enzyme relational (E-E-R) graphs. Similarity scores obtained from inter-genus, intra-genus as well as inter-species comparison of these graphs are used for phylogenetic tree construction. These phylogenetic trees throw light on the trend of development of metabolic pathways among different species. But peculiarly, these trees have less similarity with the conventional evolutionary phylogenetic trees constructed from NCBI taxonomy data. In this paper, we have tried to find some justifications for this dissimilarity among phylogenetic trees, by considering their habitats.

Evolution is a self-occurring natural phenomenon by which new organisms develop gradually as a result of change in genetic pool of a population. It is a very widely and historically studied phenomenon which still surprises scientists due to its underlying complexity. The three processes which contribute most to evolution are genetic drift (also known as random drift or allelic drift), natural selection and mutations. Genetic drift and natural selection act at a population level, whereas mutations act at individual level. For evolution to occur, the changes these processes bring about must be heritable, which they are.
 
Genes are present on chromosomes inside nucleus of the cell and the collection of all the genes in a cell is known as the genome of that cell. Genome size and complexity varies from species to species. Simple organisms, such as bacteria and protozoans have a very simple genome, with very few genes, whereas complex higher organisms such as humans have a large genome, with thousands of genes. The position of a gene on a chromosome is known as a locus. Organisms may have more than one copy of a chromosome in the cell.

Chromosome number and nuclear DNA amount are important biodiversity characters together provide a very powerful method for determining evolutionary relationships. Furthermore provide an estimate of relatedness between species that in turn suggest reasons underlying the success or failure of crosses, even for interploidy hybridization. It is expected that species that are cytogenetically most similar will be easiest to hybridize and produce viable interspecific offspring (Thomas, 1993). Hence, it is important to study karyotypes and other characters of the genome of the species involved in a breeding programme.

According to Jackson (1971) and Stebbins (1950) the karyotype is the phenotypic appearance of the somatic chromosomes in contrast to their genic content.

Introduction

Zymomonas mobilis, a gram negative, anaerobic, micro aerotolerant, ethanologenic bacterium metabolizes glucose, fructose, and sucrose. One of the major factor that limits the usage of Z. mobilis is the narrow substrate range. Moreover, there are no obvious genes reported for using lactose, maltose or cellobiose. Our preliminary sequence analysis of the Zymomonas mobilis ZM4 genome indicated the presence of a putative beta-galactosidase (bga) gene. However, the mechanism of beta-galactosidase (Bga) function is still unknown. Hence, Bioinformatics analysis was made to predict the physicochemical, biochemical and biophysical properties, domains, secondary and tertiary structures of the putative beta-galactosidase. These studies suggested that the putative beta-galactosidase gene was poorly expressed from its native promoter in Z. mobilis. The absence of beta-galactoside transacetylase and permease genes in Z. mobilis led to its inability to metabolize lactose. Hence, function predictions of Bga are performed because of the fact that it has a homologous partner.

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.

Biology is in the middle of a major paradigm shift driven by computing technology. Due to the impact of information technology, biological sciences have been rapidly becoming much more computational and analytical. Rapid progress in research in the field of genetics and related field combined with the tools provided by modern biotechnology has generated massive volumes of genetic and protein sequence data over last two decades. Compilation, storage, analysis for extraction of information and knowledge from this data becomes a challenging task as usual analytical procedures are not directly applicable to these data sets.
 
Bioinformatics has been defined as a means for analysing, comparing, graphically displaying, modeling, storing, systemising, searching, and ultimately distributing biological information, which includes sequences, structures, functions, and phylogeny. Thus, bioinformatics may be defined as a discipline that generates computational tools, databases, and methods to support genomic and post-genomic research. It comprises the study of DNA structure and function, gene and protein expression, protein production, structure and function, genetic regulatory systems, and clinical applications. Bioinformatics applications need knowledge from computer science, mathematics, statistics, medicine, chemistry and biology.

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.

A microarray, as the name suggests, is a two dimensional array of microscopic probes which captures certain molecules in an environment by a process known as hybridization, and are used to detect quantities of these molecules in the environment. The probes can vary according to requirement of experiment, and so can the environment, which in technical terms is an extract of tissue under study.

Systematic recording is vital for smooth running of any artificial insemination enterprise. All the information in the center should be so arranged so that it can be readily be traced out and used for analysis of performance of the center. The records should be critically analyzed for selecting better animals / personnel and for improving reproductive management. The objective of record keeping is as follows.

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

While collecting and processing semen the following points needs to be considered:

1.    Good quality Artificial Vagina (AV) should be used for semen collection. Strict hygiene should be maintained while collecting semen.

2.    Highly scientific and flawless collection technique should be used for semen collection.   

3.    Neat semen should be maintained at close to body temperature.

4.    Semen should not come in contact with any contaminant such as lubricant, dust, infection etc. in the collection yard or in any stage of semen processing.

5.    Direct exposure of semen to sunlight or cool white fluorescent light should be avoided as it may cause chromosomal damage.

6.    Enclose all cool white light sources e.g. laminar flow hood, cold room etc. in gold or orange sleeves that absorb ultraviolet and low wavelength visible light (< 475 to 500 nm).

Early research in field of semen storage and delivery systems was conducted in USA. The straw technique for the storage of frozen semen was evolved by work of French, Danish and German workers whereas the pellet system of storage of semen was devised by Japanese scientists. The different packing systems of frozen semen are:

    9.1    Ampoule method
    9.2    Pellet method
    9.3    Straw method

9.1 Ampoule Method
In this method the semen is placed in a glass ampoule and is subsequently sealed and frozen in liquid nitrogen. The ampoule is thawed just before insemination and is then cut open. The semen is withdrawn into glass catheter and used for AI. Since the ampoule is sealed there is no risk of contamination during storage. The relevant details can be marked on the ampoule.

Deep freezing or cryopreservation of semen refers to freezing and storage of semen at –79ºC using dry ice and alcohol or at -196ºC using LN2 as refrigerant. Cryopreservation of semen was first attempted by Parkes in 1945. But cryopreservation of semen received a boost by discovery of glycerol a cryoprotectant by Polge in 1949.  Bull semen was first successfully frozen by Stewart in 1951. Cooling rate is a vital factor determining the post thaw survival of the sperm cells. Cell death occurs in rapid freezing due to formation of intracellular and possibly extracellular ice. In contrast slow cooling rates cause cell death due to diffusion of intracellular fluid into the extender, which then become increasingly hypertonic as the extender’s water freezes causing intracellular osmotic damage as the cell dehydrates. Hence, the cooling rate needs to be balanced in a way that it is neither too fast to cause cell death due to cold shock nor too slow to result the same due to osmotic shock. Ultra rapid freezing gives excellent morphological preservation but with poor retention of functions on thawing due to large number of minute crystal formation.

The Tris citric acid egg yolk glycerol extender is the most frequently used extender in the cryopreservation of cattle and buffalo semen. Preparation of the extender plays a very important role in determining the quality of the processed semen. Therefore, care must be taken to ensure that the extender is prepared aseptically and it contains all the ingredients in appropriate concentration. Extender should always be prepared under a laminar air flow. It is also important to check that the pH and osmolarity of the semen extender for a particular species corresponds to the prescribed limits for that particular species. The Tris citric acid egg yolk glycerol contains the following ingredients:

Semen dilution/ extension is done to meet the following objectives

1.    To preserve the fertilizing potential of semen for a long period of time

2.    To increase the number of services per ejaculate. Ideally approximately 500 cows/ buffaloes can be inseminated from diluted/ extended semen of  a single ejaculate.

3.    Extenders provide the spermatozoa with a medium in which they are able to remain viable and fertile for a prolonged time period.

In order to be an ideal extender, it should be able to provide conducive environment to prolong sperm survival and modify the sperm structure to prepare them for freezing through the following properties:

1.    It should be isotonic with blood and should maintain this during preservation.

For the diagnosis of clinical reproductive problems it is essential to collect blood, penile swab, Prepucial swab, prepucial washing, semen, smegma etc from breeding bulls. These samples are dispatched to the nearest laboratory for clinical diagnosis. 

5.1 Collection of Blood Serum

The bull is properly restrained and jugular vein area is cleaned and sterilized with 70% alcohol. An sterile hypodermic needle is used to collect 5-10 ml blood aseptically in a sterile test tube. This test tube is then kept in a slanting position. Serum is separated after the formation of blood clot and dispatched to the laboratory.

5.2 Collection of Seminal Plasma

Semen sample is collected as per the standard procedure. Carbolic acid or 1 % sodium acid is added to preserve seminal plasma at the rate of 2 drops in 1 ml ejaculate in the centrifuge tube. It is then centrifuged at 3000 rpm for 20 minutes. The supernatant is collected and stored in deep freeze till further analysis.

Semen collection is the most important component of any frozen semen production programme as a high quality end product is not possible unless the raw material is of excellent quality. Therefore, all care should be taken to collect semen aseptically with the aim to harvest the best quality neat semen for processing. The person collecting semen should understand bull’s behaviour and should be friendly with him. Professional bull management results in increasing sperm output and enhancing productive life of the bulls. One must know potentials and limitations of the reproductive capacity of bulls. A highly professional and dedicated team of bull handlers and semen collectors is required for this purpose. The persons involved in bull management should be familiar with bull’s sexual behavior and male reproductive physiology. Preferably a veterinarian should collect semen.

3.1 Selection of Breeding Bulls

A sire is called half the herd and the breeding bulls to be used for semen production should be carefully selected. Selection of superior sire for breeding purpose is very important for the success any livestock breeding programme. Therefore, breeding sire must be carefully selected and maintained in scientific way so that its breeding life is prolonged and maximum number of frozen semen doses can be produced from it. 
The selection criteria for future bulls should be meticulously designed keeping into account all the factors/parameters which affects the breeding programme. The selection of breeding bulls is based on three broad parameters-

3.1.1 Pedigree Examination
3.1.2 Gross Physical Examination
3.1.3 Laboratory Examination

Success of AI programme depends on fertility of bulls used for semen production. If infertile bulls are selected in semen station for production of semen doses, large population of cows inseminated with such doses is affected. Therefore, the breeding bulls or bulls to be used for frozen semen production should be selected carefully keeping in view genetic potential, disease condition, reproductive health, libido and semen production potential of the bull. The emphasis should not only be on elimination of bulls with questionable or unsatisfactory potential, but also on induction of bulls with good semen production potential into the semen station. 

The following aspects should be taken into consideration while performing a breeding soundness examination of bulls:

Knowledge of male reproductive system and its physiological functions is essential for understanding male reproductive behavior and for effective reproductive management of breeding bulls. Therefore, personnel involved in management of breeding bulls, semen collection, handling and artificial insemination must have sound knowledge of male reproductive anatomy and physiology of breeding bulls. This section provides a recapitulation of the basic physiology and anatomy of male genitalia.
 

Functions of Male Genital System

    (a)    Primary function of the male genital system is to monitor reproduction by providing cutaneous and glandular system of male gonads

    (b)    Secondary function of male genital organs is to provide glandular, ductular, copulatory and auxillary system associated with male reproduction.

1.1. INTRODUCTION

In this chapter, we will know how to classify raw data. The purpose of classifying raw data is to bring order in them so that they can be statistically analysed easily. Classification of objects or things saves our valuable time and effort. It is done in a logical manner and similar things are arranged into a group or class.

Appendix A: Diagrammatic Presentation of Processes Involved in Obtaining an Indian Patent

6.1    Copyrights
    
6.2    Trademarks
    
6.3    Geographical Indications
    
6.4    Trade Secrets

5.1    TRIPS
    
5.2    WTO
    
5.3    WIPO
    
5.4    Paris Convention
    
5.5    Berne Convention
    
5.6    Budapest Treaty
    
5.7    Madrid Agreement Marks (MAM)
    
5.8    The Patent Law Treaty (PLT)

4.1    Provisional and Complete Specifications
    
4.2    Publication
    
4.3    Request of Examination
    
4.4    PCT Application: Gateway to Foreign Patents

3.1    Competitive and Collaborative Research
    
3.2    The Strategy to Protecting Plants : Plant Variety Protection Act
    
3.3    IPR Issues in Biotechnological Patents Haldi, Neem and Basmati Rice
    
3.4    Patent Litigation

2.1    Specification Drafting
    
2.2    Methods and Precautions in Drafting Patent Specification
    
2.3    Disclosure Requirements
    
2.4    Patent Claims
    
2.5    Filing of A Patent Application

1.1    General Introduction, History, Ownership of Tangible and Intellectual Property
    
1.2    Patentable Subject Matter
    
1.3    Basic Requirements of Patentability
    
1.4    Novelty, Public Domain, Obviousness and Non Obviousness
    
1.5    Inventive Step (Non-Obviousness) and Industrial Application