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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 The semen is withdrawn into glass catheter and used for AI.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.

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

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.

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
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