eChapters

ABSTRACT
Recent challenges for fossil fuels, associated environmental issues with disposal of organic garbage, and the rising costs for energy has encouraged researchers to search for alternate energy sources. Massive uncontrolled biogas from landfills, sludge and organic material degradation under anoxic conditions is an environmental management issue. Methane emission to atmosphere has a tremendous green house effect, but this can also be a valuable renewable energy resource. Biogas from kitchen wastes through anaerobic digestion by methanogens is such an alternate technology. Kitchen wastes are considered one of the best raw materials for biogasification. Biogas is mainly composed of 50-74% methane (CH₄), 25-28% carbon-di-oxide (CO₂), other volatile compounds including hydrogen sulphide (H₂S), water vapour and other trace gas compounds. The impurities in it, like Hydrogen sulphide (H₂S) which is corrosive, downgrade its quality. Purified biogas of higher calorific value attracts attention as a substitute fuel for additional applications. Its purification using various suitable scalable and economically-viable scrubbing technologies to enhance its quality and performance as an alternate fuel is an important step in this direction. Thus, a technology to generate and purify biogas from kitchen waste has significant prospects in the energy sector, and also as a promising enterprise to employ the youth.

ABSTRACT
Rice straw is one of the most abundant lignocellulosic agricultural wastes in India and can be used as a major resource for biogas production. The structure of rice straw is complex and is made up mainly of cellulose, hemicelluloses and lignin. The lignin physically shields the cellulose and hemicelluloses, the main components which can be converted to energy, and hinders the access of microbial enzymes to these carbohydrate moieties. To overcome this making the straw a potential resource of biogas production, effective pretreatment is necessary that can efficiently hydrolyse the straw and make it accessible to cellulolytic enzymes and methanogens. Various pretreatment methods which are employed currently in the market are discussed here. For a process to be economically feasible it is necessary that the pretreatment be efficient as well as cost effective. To gain some insights into this aspect, the techno-commercial feasibility of Biomethanation of rice straw has been discussed.

ABSTRACT
There is a quantum global growth in both number and size of dairy industries in recent times. These industries discharge thousands of liters of wastewater characterised by a heavy chemical oxygen demand (COD), biological oxygen demand (BOD), nutrients, inorganic salts, besides detergents and sanitisers each day (Kushwaha et al., 2011). High carbohydrates, fats and proteins contents in such waste streams originating from milk pose challenge to conventional municipal sewage treatment systems (Demirel et al., 2005). The easily biodegradable dairy effluent leads to precipitation of casein which is toxic and has a strong odour. Without adequate treatment, these effluents could pose serious threats to the receiving water bodies and disturb the ecosystem (Satpathy et al., 2017). For such heavily loaded organic effluents, biogasification through anaerobic digestion seems a promising strategy. This cost-effective energy generation process is also effective in environmental management compared to its other eco-friendly renewable source counterparts, such as, hydro, solar and wind (Satpathy, 2017). The Upflow Anaerobic Sludge Blanket (UASB) reactors are the most popular anaerobic systems to treat the fluctuating organic loads in dairy effluents. Anaerobic filter (AF), Upflow Anaerobic Filter (UPAF), Fixed-bed anaerobic reactor, Anaerobic Hybrid Reactor (AHR) etc. are few other systems that are demonstrably competent removing COD upto 90% and generating biogas. Successful anaerobic systems reported yielded (with upto about 85% CH₄ content) 0.32-0.34m3 CH₄/kg COD removed. Biomethanation process and reactors designs are constantly being optimised for effective treatment of the effluents while maximising the biogas production. The energy-rich gas thus obtained could further be utilised for heat and electricity at the domestic level, and also as a vehicular fuel. Residual digestate can also be used as organic manures (Angelidaki et al., 2007).

ABSTRACT
The annual accumulating organic wastes generated through milling, brewing and various other agro-industrial and agri-processing activities in India amount to about 500 MMT. Most of these contain three major structural biopolymers, viz., cellulose, hemicellulose and lignin, a large proportion of which is carbohydrate and phenolic in nature. Massive accumulations of this kind of recalcitrant biomass not only deteriorate the environment, but also generate huge amount of potentially useful materials. Biological transformation has globally become increasingly popular among researchers and academicians in treating agricultural, industrial, organic and toxic wastes these days. About 60% by weight of the original pineapple fruit in forms of peeled skin, core, crown end, etc. resulting from the pineapple processing remains unutilised being discharged as waste, causing disposal and pollution owes. Of the 10% dry matter content of such waste, about 96% is organic and 4% is inorganic. Being rich in cellulose and hemicellulose, it can be a valuable resource for numerous value-added products by bioconverting it to valuable products, including bioenergy. Biomethane is one such energy resource that can be produced by valorizing pineapple wastes by employing various bioconversion technologies. This chapter focuses on the bioconverstion technologies to produce biomethane from pineapple wastes.

ABSTRACT
Biomethanation is the process of generating energy from anaerobic digestion (AD) of organic refuse. This helps reduce dependency on fossil fuels while reducing CO₂ emission. Organically-rich kitchen refuse (KR) generated daily from the households all over the world could be a good candidate to biogasify, though the quantity and quality of biogas depends on the KR proximate. To enhance biogasification, KR is subjected to physical, chemical and/or biological pretreatment. Physical pretreatments include chopping, grinding, ultrasonication and steam explosion, whereas chemical pretreatments could be by an alkali or a base. Biological treatment involves digestion of the organic matters by individual microbe or its consortium, although it takes place naturally. For biological treatment, standardising the substrate and inoculum ratio is essential. Candidate strains must be mutually compatible to formulate a microbial consortium. Bacillius seems to be a potential candidate as it is a facultative anaerobe exhibiting multiple essential enzyme activities (viz., amylase, cellulase and lipase). Maintaining an optimal pH of 7.0 and temperature of 37°C before and during anaerobic digestion is suggested. Other important factors in digestion include organic loading rate, retention time, C/N ratio, volatile fatty acids (VFA) concentration, etc. VFA content is vital as its accumulation during acidogenesis adversely affects the AD by dropping pH values to unfavourable levels. Acetogens convert VFA to acetate, CO₂ and H₂ in acetogenesis, finally converting it to CH4 in methanogenesis. For effective methanation,

ABSTRACT
The rapid industrialisation and speedy progress towards urbanisation across the world have created the huge demand for energy moreover creating enormous quantity of wastes. The latter is in charge for polluting the environment in an exponential manner, resulting out of the harmful and poisonous compounds released by them. In recent years, there has been a pattern change from ‘waste-to-wealth’, considering the value of high organic fraction accessible in the wastes. The best-accomplished methods are that of anaerobic digestion; contribute to the production of methane (CH₄). Similar biotransformation has partial net energy harvests. Recently, fermentation industries steering their interest towards the production of value added products, viz., biohydrogen (H₂), biopolymers, ethanol, vitamins, acetic acid, enzymes, butanediols, etc. from carbon rich organic waste like cellulosic substrates have been envisioned to flourish in a multi-step process, such as the ‘Biorefinery’. A contemporary development in fermentation technology holds the key to overcome the constraints for such processes that may lead to a sustainable economy. One related technology is electrofermentation that has attracted significant attention recently owing to its capacity to boost the metabolic activities of microbes by means of extracellular electron transfer.

ABSTRACT
The global energy demand is increasing day by day. Now-a-days, the supply of global energy is highly dependent on fossil fuels (viz., crude oil, lignite, hard coal, natural gas). But, on one hand the source of fossil fuel is decreasing at a rapid rate, and on the other hand the burning of fossil fuel is also degrading the environment due to huge Greenhouse Gas (GHG) emission (FAO/CMS, 2011). Hence, there is an urgent need of an alternate clean energy source that can sustain the future development undeterred while still protecting the environment. Biomass is considered as a sustainable source of clean energy that is useful in steam, fuel and chemicals production. It can be digested in controlled environment to produce biogas. The biogas is a clean, non-polluting and smoke-free fuel (Weiland, 2010) which can be used for applications that include cooking, space cooling/refrigeration, heating, electricity generation and gaseous fuel (CNG) for vehicles (Weiland, 2003). This paper analyses possible alternatives to process and utilise biogas for energy at industrial scale as a petroleum fuels replacement.

ABSTRACT
Nisargruna biogas technology has been developed in BARC for processing different types of biodegradable waste. This technology is based on biphasic separation of aerobic and anaerobic stages. Introduction of aerobic predigester has increased the efficiency of the process in terms of enhancing the quality and quantity of methane and also reducing the retention time of the process. Process parameters of the plant were studied and found that the modified design has proved very effective in faster digestion of complex waste. The technology has been commercialised and has spread across different parts of country for processing variety of wastes including kitchen waste, municipal waste, agriculture waste, textile waste and poultry waste. Microbial population from different stages of the plant was also studied and found their ability to convert waste to biogas, containing mainly methane.

Keywords: biogas, biphasic digestion, green energy, methane, microbial fermentation, Nisargruna, waste management

ABSTRACT
The ‘steel-drum type’ biogas plant installation programme in Punjab was initiated in the early seventies, whose construction works were entrusted on the Department of Agriculture. Because of their high cost and high failure rates then, only the affluent farmers could afford to install. In the meantime, a cheaper Chinese model, also known as Janta type (dome-type replacing the drum-type) plant, became popular but was still out of the reach of ordinary farmers. With dual objectives of cost reduction and efficient biogas production to make it affordable to small farmers, a coordinated research was initiated. Detailed extension surveys were carried out in Ludhiana district which inferred that the farmers did not install the gas-holding steel drum though they purchased it. A significantly low cost Katcha-Pucca plant model of 15m3/d capacity was developed and installed in Sangrur district. Similar digester was tried for Janta model in which the inlet, outlet and dome (gas holder) were masonry works but the digester was a Katcha pit, the dome of the plant being modified to a semi-circular dome. More than 80 such plants are functional in a single village, and more than 40–50% of the plants installed in Punjab and Haryana are these PAU designs. The chapter deals in greater details on the design aspects and the extensive field works carried out to achieve these successes, along with some relevant facts and figures. The write-up is written as a commentary, rather than a typical chapter, for the benefit and interest to the readers.

ABSTRACT
It is impossible to envision a world without energy provisions and has become inevitable and an integral part of a nation’s prosperity. It has unparallel contributions towards the socioeconomic empowerment, industrial growth, and technological advancements, as also in mitigating greenhouse gas (GHG) emissions. Judicious utilisation of energy sources without infringing upon the sustainable energy demand is the need of the hour. Taking India’s huge bovine strength into account, biogas technology has the potential to transform the standard of living of rural millions. Effectively implementing the Govt. of India’s National Biogas and Organic Manure Programme (NBOMP) could save precocious demand on electricity, fertiliser and firewood while promoting self-sustainable organic farming. In this chapter, the cost and benefit analyses of biogas technology (involving biogas plants of different capacities) are discussed along with the long-term approach at the grassroots level to promote digested slurry for organic farming. The commercialisation and carbon crediting of biodigester technology is also narrated. It is necessary to chalk out a revenue model to commercialise slurry at the grassroots level to economically empower the beneficiaries, from family-type to industrial-scale biogas plants. Small-scale biofertiliser hubs at district level may be organised to collect dry slurry from the beneficiaries and process them further to make it market-ready through entities operating at the national level developing manure for agricultural practices, as also feeding materials for animal husbandry practices.

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

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

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

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

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)

6.1    Copyrights
    
6.2    Trademarks
    
6.3    Geographical Indications
    
6.4    Trade Secrets

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

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.

2.1. INTRODUCTION

Many biological variables do not meet the assumptions of parametric statistical tests as they are not normally distributed, the variances are not homogeneous, or both. Objects in nature commonly exhibit one of three distributions:

3.1. INTRODUCTION

In this chapter, we will study the measures of central tendency which is a numerical method to explain the data in brief. We can see examples of summarizing a large set of data in day to day life like average marks obtained by students of a class in a test, average rainfall in an area, average production in a factory, average income of persons living in a locality or working in a firm etc.

One of the most important objectives of statistical analysis is to get one single value that describes the characteristic of the entire mass of data. Such a value is called the central value or an ‘average’ or the expected value of the variable. This single value is the point of location around which individual values cluster and, therefore, called the measure of location. Since this single value has a tendency to be somewhere at the center and within the range of all values it is also known as the measure of central tendency. The purpose of computing an average value for a set of observations is to obtain a single value which is representative of all the items.

4.1. INTRODUCTION

Dispersion is the extent to which values in a distribution differ from the average of the distribution. To quantify the extent of the variation, there are certain measures namely:

1. Range
2. Quartile Deviation
3. Mean Deviation
4. Standard Deviation

Range and Quartile deviation measure the dispersion by calculating the spread within which the values lie. Mean Deviation and Standard Deviation calculate the extent to which the values differ from the average.

5.1. INTRODUCTION

Mean and standard deviation summarizes the salient features of a data set. But they do not describe adequately the full characteristics of the data. For an adequate description of the characteristics of a data set, a convenient method is to plot a frequency distribution curve. Such curve is constructed by first drawing a frequency polygon, which may then be smoothened to a frequency curve, indicating the distribution. The frequency curve or distribution of specific biological variables generally has a specific form and plays a very important role in statistical theory and practice. It is customary to describe a curve in terms of its symmetry or assymmetry and its flatness or peakedness.

The following types of distribution are commonly seen in the biological world:

6.1. INTRODUCTION

All ecologist and biology researchers have to find out: (i) how reliable are the observed results? and (ii) what is the probability of difference between observed and expected results? The question of reliability is solved by estimating the confidence interval and the second question is addressed by the application of statistical tests for significance. Both these methods help us to draw conclusions on our observations, hence, may be grouped together as ‘inferential statistics’.

Referring back to the chapter on sampling, we may recall that it is usually not possible to calculate the ‘population parameter’ directly. Instead, we have to rely on estimates. Two types of estimates can be used, a point estimate and an interval estimate. A point estimate is a single numerical value of a sample statistic that is used to estimate the corresponding population parameter. For example, the sample mean, x , is used as an estimate of the population mean, μ Point estimates are not used widely because the value of some statistic, say mean, varies from sample to sample and we can’ t conclude with any degree of certainty that a single point estimate equals the population parameter. Therefore, an interval estimate is typically favoured. An interval estimate is a range of values within which the population parameter is likely to lie.

7.1. INTRODUCTION

The word ‘significance’ in statistics does not describe the strength of association. Statistical significance is simply related to the probability of the result being commonly or rarely encountered (If the probability of chance occurrence is low the result is likely to be determined by the set of circumstances under study and not by chance). If conclusions are to be drawn that a difference exists between two groups of subjects, then we have to ensure that they are equal in all respects except the one under test.

‘P’ values are used to assess the degree of dissimilarity between two (or more) sets of measurements. The ‘P’ value is the probability of obtaining a result as extreme as or more extreme than the one observed. The ‘P’ value is actually a measure of surprise and the smaller the value, the more surprising is the result. When ‘P’ value is between 0.05 and 0.01, the result is usually called statistically significant; when it is less than 0.01, it is usually called highly significant and if, values are lower than 0.005, then it is called very highly significant. Traditionally, the approach had been to indicate the level of significance by rounded off ‘P’ values e.g., P < 0.01. Now it has become a fashion to calculate the exact probabilities and thus arrive at a particular decision.

8.1. INTRODUCTION

The word ‘non-parametric’ means “not for any specific parameter”. For example, in the student’s t test we use the parameter ‘mean’. In the non-.parametric tests we check the randomness and trend of the pattern of distribution of a variable directly, without using a population parameter for comparing.

In contrast to the parametric tests, where many assumptions are made regarding the distribution of the parameter in the population (i.e. Gaussian features), here we do not make stringent assumptions about the population from which samples are drawn. These techniques are, therefore, also known as ‘distribution free’ tests.

We shall be discussing about 10 such non-parametric tests.

Introduction

Accumulation of excess soluble salts in the root zone of soils resulting in partial or complete loss of soil productivity is a worldwide phenomenon. The problems of soil salinity are widespread in the arid and semi-arid areas but salt-affected soils also occur extensively in the sub-humid and humid climates particularly in the coastal regions where the ingress of sea water through estuaries and rivers and through ground water movement causes large scale soil and water salinisation. Soil salinity is also a serious problem in areas where ground waters of high salt content are the only source of water available for irrigation. By far the most serious problems of salinity are being faced in the irrigated, arid and semi-arid regions of the world. Our ability to manage salt-affected soils and waters both in the irrigated and in the unirrigated regions will constitute a major effort in meeting potential food requirements. The magnitude of the problem can be appreciated from the following.

Expression of genetic potential for growth and yield of a crop variety is directly dependent upon the environment comprising both climate (weather) and soil components. As discussed in the preceding chapter, the term ‘salt-affected soils’ encompasses a broad group of adverse but distinct soil conditions, namely, saline, alkali (sodic) and acid soils with further variations such as coastal and inland locations as well as arid and water-logged situations (Swaminathan 1977; Bresler et al. 1982). Factors limiting growth and yield of crop plants in various categories of problem soils are different and specific. Likewise, adaptive strategies evolved by plants to cope with the prevailing edaphic stresses, such as salt stress, also vary a great deal as indicated in Table 1. It is not unexpected therefore, that even varieties within a crop often show notable differences regarding tolerance to adverse soil conditions (Epstein et al. 1980; Devine 1982; Shannon 1984; Sayed 1985). Hence, it is essential that the breeder must understand the inherent charaderistics of the problem soil with which he is concerned and should also simultaneously appreciate that basically it is the sensitivity of crop variety, rather than the soil parameters per se, that essentially determines the occurrence as well as the magnitude of actual soil problem (Rana 1977). Since the degree of tolerance (adaptation) of crop varieties to different kinds of salt affected soils is genetically controlled, it is axiomatic that it can be improved through genetic manipulations by adopting suitable breeding procedures.

Introduction

Drought is the most common adverse environmental factor which limits crop production in different parts of the world. Often drought is accompanied by relatively high temperatures, which promote evapotranspiration, and hence could accentuate the effects of drought and thereby further reduce crop yields. Since these events occur more frequently in tropical and semitropical regions where most of the developing countries are situated, droughts are often associated with food shortages and an overall setback to developmental activities. Therefore, raising of drought-resistant crops is common sense to achieve stability in production and to enhance the possibility of self-suficiency in food. Consequently, breeding for drought resistance is a major objective of many research programmes in the international and national institutions. This has been so for the past several decades. But, it is disappointing that, there has not been any measurable success in these programmes compared with the success achieved in breeding for yield; or breeding for disease resistance or breeding for quality attributes. In fact, this was amply expressed by Arnon (1980) when he stated “Breeding for drought resistance has been a consistent theme for as long as I remember and probably the greatest source of wasted breeding efforts in the whole field of plant breeding.” However, the fact remains that we do hear of drought resistant varieties in many crops. For example, C-306 in wheat, Lalnakanda in rice and M 35-1 in Sorghum are some of the better known examples in India. It is also claimed that a large number of cultures among the Assam Rice Collection exhibit a considerably high degree of drought tolerance. Many land races and wild relatives of several crop species are said to contain drought resistant traits, which could be profitably utilised in breeding programmes. This situation is not unique to India alone but is a common experience in different parts of the world. However, to afirm a conviction a short questionnaire to assess the current state-of-art on breeding for drought resistance was distributed to 75 distinguished plant breeders and plant physiologists all over the world. The questionnaire and a summary based on the responses is as follows:

Introduction

Man’s intervention in the evolution of plants, to adapt them to suit his needs, constitutes the practice of plant breeding. Historically regarded as an art and still too empirical to be accepted universally as a science, it has recently been described as a technology (Riley 1978). Whether art, science or technology, it certainly draws on a wide range of disciplines and embodies all those principles of organisation and management that determine success in any production-oriented programme.

Perhaps the biggest contribution that the plant breeder has made to increased production during the past 50 years has been in helping to alleviate the effects of natural hazards such as lodging, pests and diseases on crop production. While most of this progress has been through the manipulation of biotic stresses, much is still to be achieved under non-biotic stresses such as drought, salinity, temperature, etc. This argument is strengthened by the fact that in spite of the spectacular achievements made possible by the ‘Green Revolution’, the productivity level of most crop plants, including wheat and rice, has remained both low and static under stress conditions. Concerted efforts are needed through an approach of interdisciplinary research, since not much has been done to define criteria for stress assessment and evolving breeding approaches for incorporating stress tolerance in crop plants.

Introduction

Drought is one of the primary factors responsible for depressing rice yield in chronic areas and in destabilising rice production in drought-prone areas. It is a production constraint common to all rainfed rice cultures: wetland, upland, and deepwater (O’Toole and Chang 1979). Breeding for drought resistance, a major component of the International Rice Research Institute’s (IRRI) Genetic Evaluation and Utilization (GEU) Program since the early 1970s, aims at countering the adverse effects of drought in unfavoured rice production areas.

Rice germplasm has such remarkably rich diversity that a continuous spectrum of genotypes differing in the various physiological mechanisms can be found and is available for genetic manipulation (Chang et al. 1982a). However, effective evaluation methods are essential for unravelling the specific mechanism(s) involved and for devising eficient selection criteria in a breeding program. Moreover, the broad spread in growth duration among rice cultivars makes it impractical to obtain uniform evaluation of a large number of cultivars when they differ in physiological responses that are specific to certain growth stages. Methods that can accommodate large numbers are essential to breeding program.

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.

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:

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

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.

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 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.

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:

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.