eChapters

Brief History

1953: discovery of the molecular structure of DNA

1961-1965: decoding of the genetic code

1977: first method developed for DNA sequencing

1980: the principle of genetic mapping by anonymous molecular markers

1987: first “automatic” DNA sequencing machine developed

1990: publication of the BLAST algorithm for the comparison of DNA sequences

Bioinformatics is the study of analysis of biological data using computer programming, mathematics and statistics. Bioinformatics is the application of computer science and information technology to the field of biological sciences. Bioinformatics involves databases and information systems, web technologies, artificial intelligence and soft computing, information and computation theory, software engineering and statistics. The one of the most important achievement of bioinformatics is the human genome project which was mapped in 2001. Most of the work in this field is based on the research. Scientists make databases and develop different tools to analyze different gene expressions. Within last few decades, a number of research  in the field of molecular biology, along with advances in genomic technologies, have generated a huge number of biological information  Similar is the case in the field of animal production where many advances have occurred. The day to day advances in bioinformatics and computational science have led development in the field of animal as well as veterinary science. Bioinformatics generates new knowledge in the field of Animal Sciences, and improving & discovering new models of computation for the advanced livestock production.

Introduction
 
Animals used for experimental research purpose, which show results resembling to human beings are known as lab animals. They are mostly used because of their small size, lesser feed and space requirement. Furthermore, they are not natural host for most of the animal / human pathogens. The laboratory animal research is conducted at universities, medical colleges, pharmaceutical companies, farms, defense establishments and commercial facilities that provide animal-testing services to industry. It includes basic research in the area of genetics, developmental biology, behavioural science etc., as well as applied research in the area of biomedical science, xenotransplantation and drugs including cosmetics testing. Laboratory animals are also used for education, breeding and defense research. The common laboratory animals are mice, rats, hamsters, guinea pigs and rabbits. However, cats, dogs, monkeys, goats, sheep, cattle, pig, horses, fishes etc. can also be utilized as laboratory animals for experimental purpose in biomedical sciences.

Introduction

Sheep and goats have a vital economic importance in India, inhabitant in semi-arid region and mostly rose under harsh environmental conditions (high ambient temperature, scarcity of feed and water). The arid and semi-arid regions of India are drought prone due to low and uneven rainfall.  The vegetation of arid and semi-arid tract is more acute during summer when the quality of the pastures becomes vulnerable which in turn decreases the body condition score of animals and reproductive efficiency is being compromised (Maurya et al. 2009a & 2010).  The small ruminants are widely distributed in comparison of other domesticated species. These species of livestock is also a source of employment for marginal and land-less farmers particularly in the arid and semi-arid region of India. Most of the Indian farmers have limited resources and small ruminants production offers an opportunity to alleviate financial conditions and improve living standards.

Introduction
 
Agricultural activity in the arid and semi-arid divided into two main components are plant production and animal production. Animal production is associated with a component of a large proportion of workers in the agricultural sector, either individually or as part of agricultural activity Integrative. The livestock are plays major role in the lives of the rural population to the availability of several important products such as meat and dairy products.

Farm animals in the arid and semi-arid depend on their feeding mainly on crop residues and natural pastures that do not meet their needs due to scarcity of resources in these regions. Such scarcity is caused by a number of factors; the most important factor is limited agricultural land devoted to cultivation of fodder crops, low rainfall and the deterioration of natural pastures. However, there are many studies in different countries suggesting the possibility of widespread exploitation of cactus as a strategic resource, especially in the areas of low rainfall and scarce feed resources (FAO 2007). Those studies recommend the use Cactus to feed cows, goats, sheep and rabbits without causing any problems to the animals’ health. In addition, it can be used as medicine and cosmetics for humans and for many other uses.

As an engine requires fuel to run, similarly the animal body requires food to run its vital activities. Food, after its digestion, absorption and oxidation, provides energy to the body. The energy thus obtained is utilized by the animal for maintenance, growth, reproduction and production. Hence, a good quality of food should be provided to the animal so that it may fulfil all the requirements of the animal. Food contains water, carbohydrates fats, proteins, vitamins and minerals which are essential constituents of the body and indispensable for life. Water being an essential constituent of protoplasm carries out many other important and inevitable functions in the body. Water helps the body absorb nutrients from the food and throw out wastes from body in the form of sweat and urine. Carbohydrates, fats, and proteins provide energy to the body. Fat provides 2.25 times more energy than carbohydrate and proteins. Besides providing energy, proteins are also essential for body building, growth and repair of wear and tear of the body tissues. Vitamins protect the body against diseases and help in keeping eyes, gums, skin, teeth and bones healthy. Vitamins are required by the body in small quantities. Vitamins are essential for normal growth, health and reproduction. Prolonged deficiency of vitamins causes characteristic diseases. Minerals required in small quantities are essential for normal growth of body and maintaining good health. Skeleton system of the body contains principally Calcium and Phosphorus with small amount of Magnesium.

There are six major classes of nutrients: carbohydrates, fats, fibre, proteins, minerals and vitamins. Water is not a nutrient but play important role in livestock industry. These nutrient classes can be categorized as either macronutrients (needed in relatively large amounts) or micronutrients (needed in smaller quantities). The macronutrients are carbohydrates, fats, fiber, and proteins. The micronutrients are minerals and vitamins.

The term minerals is used by nutritionists to refer to inorganic chemical elements, but is used by many other scientists to mean a homogeneous crystalline compounds. Mineral elements are not inert materials. Mineral elements may change valence states and are transferred from one chemical compound to another. Minerals are active participants in enzymatic reactions, have specificity of function, and are critical to life.

Introduction

A huge increase in the demand of animal production is expected in the next decades. Food and water security will be one of the other priorities for humankind in the 21st century. Over the same period the World will experience a change in the global climate that will cause shifts in local climate that will impact on local and global agriculture (Nardone et al. 2010). Global climate change is expected to alter temperature, precipitation, atmospheric carbon dioxide levels and water availability in ways that will affect the productivity of crop and livestock systems (Hatfield et al. 2008). For livestock systems, climate change could affect the costs and returns of production by altering the thermal environment of animals thereby affecting animal health, reproduction, and the efficiency by which livestock convert feed into retained products (especially meat and milk).

Deviation from normal well being may be for various kinds involving one or more organs and exhibiting array of symptoms. Examination of animals for diagnosis of disease is strenuous process because investigator has just got to know by several means of examination. The evidences produced by owner, animal house, feed being provided supported by prevailing environment and climate are immensely helpful in accurate diagnosis. Diagnosis, of production related diseases, is more difficult because of biasness in history revealed by the owner. At the same time diagnoses of poisonings are also not easy because owner’s description about possible cause and symptoms exhibited by sick animals are vague. So careful quizzing of owner about each and every aspect of disease and animal obviously seems to be first step towards diagnosis, followed by examination of animal. Examination of environment in relation to history revealed by owner and observations drawn from the examination of animal can infer some conclusion about cause of disease. Support of laboratory examinations such as fecal test, blood test and urinalysis are important tools in disease investigation. Following steps are essential in disease diagnosis.

Introduction

“Sustainable development as a concept represents the latest step in a long evolution of public concerns with respect to both natural resources and environment”. It is defined as “development that meets the needs for the present without compromising the ability of future generations to meet their own needs”. This definition indicates that to meet their needs, the present generations have no right to overuse natural resources and the environment, which will ultimately threat the living standards of the future generations. Therefore, stewardship of both natural and human resources is of prime importance (Freenstra 1997). Advocates of sustainable development perceive that the biosphere imposes limits on economic growth, express a lack of faith in either science or technology as leading to human betterment, are extremely averse to environmental risks, support redistributive justice and egalitarian ethics, profess concern over population growth and have faith in the wisdom of human capital development, and have survival of species and protection of the environment and of minority cultures, rather economic growth per se, as goals.

Several centuries ago, Father of medicine Hippocrates, has said “Let food be your medicine”. Our traditional therapies or alternative medicine effectively integrated this concept and include herbal remedies, nutritional interventions like vitamins/minerals supplementation specialized diets etc, and other techniques such as acupuncture, relaxation regimens, and homeopathic medicines. While, in modern medical science medicines are used to cure diseases which may have some inherited side effects. From the final decade of the last century, a renewed interest was seen in traditional therapies and people are thinking of integrated medicine with a focus on prevention of disease rather than its cure. In this context many terms like “dietary supplements”, “nutraceuticals” and “functional foods” were frequently used to address this issue. The term “nutraceutical” was coined from “nutrition” and “pharmaceutical” in 1989 by Stephen DeFelice, MD, founder and chairman of the Foundation for Innovation in Medicine (FIM). Nutraceutical can be defined as, “a food or part of a food, that provides health benefits, including the prevention and treatment of a disease.” However, the term nutraceutical as commonly used in marketing has no regulatory definition. There is confusion between the closely related terms nutraceuticals, functional food and dietary supplement. Kalra (2003) differentiated between these terms as: Functional food provides the body with the required amount of vitamins, fats, proteins, carbohydrates, etc, needed for its healthy survival. When functional food aids in the prevention and treatment of disease(s) and disorder(s) other than anemia, it is called a nutraceutical.

Historical background of dairy goats

Small ruminants sector plays an important role in the national economy. This enterprise is associated with social and cultural fabric of millions of resource poor farmers. Small ruminant especially goats gain importance mainly on account of their short generation intervals, higher rates of prolificacy and the ease with which they can be marketed. They are very useful in semi-arid and arid zone where they can sustain themselves on sparse vegetation and esteemed climatic conditions.
 
There are four main breeds of dairy goats commonly found in Western Country, namely, Alpine, Nubians, Saanens and Toggenburgs. They are considered to be the best milking breeds of the world. All these four breeds have recently been introduced in India for improvement of milk quality of local goats. These breeds have been improved by selection, upgrading, and the wise use of pedigree records and registration over a period of more than half a century in Europe and North America.

Introduction

The present world wide situation of food items calls for a broad range of poultry alternatives to meet increasing demand of quality protein. Besides, complication involved in managing chicken, emergence of new diseases, increase in feed cost and increasing demand of poultry meat bound us to search for alternative species of poultry. In this circumstance rearing of Guinea fowl framed for centuries to meet poultry production requirements of diversified agricultural milieu. The traditionally reared Guinea fowl could become a significant source of food and also a source for substantial supplement income.

History
 
Guinea fowl a bird of West African origin came to India during mediaeval age. Now guinea fowl are being raised in the districts of eastern Uttar Pradesh and in some parts of Tamilnadu. The birds are very popular with marginal farmers and poor classes and are being largely kept as foragers and insectivorous under semi-range and range conditions.

India has the highest livestock population in the world with 50% of the buffaloes and 20% of the world’s cattle population, most of which are milch cows and milch buffaloes. The dairy industry in our country is considered as one of the most successful development programmes in the post-Independence period. In the year 2006-07the total milk production in the country was over 94.6 million tonnes with a per capita availability of 229 gm. per day. The industry had been recording an annual growth of 4% during the period 1993-2005, which is almost 3 times the average growth rate of the dairy industry in the world. Milk processing in India is around 35%, of which the organized dairy industry account for 13% of the milk produced, while the rest of the milk is either consumed at farm level, or sold as fresh, non-pasteurized milk through unorganized channels. Dairy Cooperatives account for the major share of processed liquid milk marketed in the India. Milk is processed and marketed by 170 Milk Producers’ Cooperative Unions, which federate into 15 State Cooperative Milk Marketing Federations. Over the years, several brands have been created by cooperatives like Amul (GCMMF), Vijaya (AP), Verka (Punjab), Saras (Rajasthan). Nandini (Karnataka), Milma (Kerala) and Gokul (Kolhapur). Uttar Pradesh, Punjab, Haryana, Rajasthan, Gujarat, Maharashtra, Andhra Pradesh, Karnataka and Tamil Nadu are the milk surplus states in India.

To sustain the meat industry in profitable manner, efficient utilization of meat by-products is an important issue. In today’s competitive world the realization of old saying “Only the last cry of the animal is wasted” has become essential. The utilization of by-products not only ensures additional revenue but also reduce the cost of various mandatory disposal treatments. The live animals are becoming costlier over the dressed carcass which compels the utilization of by-products. Moreover, unused by-products is also a great threat to environment and public-health apart from loosing nutritional resources. According to FAO (2004) estimates 106.75 million livestock and more than 650 million poultry birds were slaughtered annually in India leading to production of 6.3 million MT meat and different by-products of appreciable value. Now a day’s various techniques are available for the utilization of by-products for different purposes still significant portion of the by-products are unutilized and poses huge economic losses.

Animal By-products are the portions of animal body which are not included in dressed carcass. Animal by-products can be estimated by subtracting the dressing percentage from 100. Dressing percentage is inversely proportional to the byproducts means lower the dressing percentage, higher will be the yield of by-products and vice versa. In normal cases dressing percentage usually ranges between 50-70 percent and by-products yield in between 30-50 percent. However, the actual percentage may be higher because it excludes the weight of bones in muscles which is a part of dressed carcass.

Introduction

After introduction  of Green Revolution and White  Revolution in our country, India had high growth in food and milk production but farmers of our country had  applying more chemical fertilizers, insecticides  pesticides, weedicides and various medicines  in injudicious way  to increase grain, vegetable, fruit, flower, milk, meat and egg production . Due to this reason soil fertilely and soil health is deteriorating continuously. The soil  of food bowl states of our country viz. Punjab, Haryana, Western Uttar Pradesh and plain area of Uttarakhand deficient in organic matter, macro and micro and trace elements. Now soil became non living due to death of earthworms and beneficial micro organism. Farmers are using mainly nitrogen, phosphorus and potash in crop production. However there are 15 essential plant nutrition’s for growing of crops. Due to more application of chemical fertilizers in crop production these days’ farmers are not properly using organic, green and compost manures in crop husbandry. Soil fertility and ground water level has been declined very fast by adopting intensive farming.

In its original concept, vaccination aims to mimic the development of naturally acquired immunity by inoculation of nonpathogenic but still immunogenic components of the pathogen in question, or closely related organisms. The term “vaccine” (from the Latin term “vacca,” meaning cow) was first coined by Edward Jenner to describe the inoculation of humans with the cowpox virus to confer protection against the related human smallpox virus and illustrates the close relationship between human and animal infectious disease sciences. The famous proverb saying ‘Prevention is better than cure’ implies perfectly to the vaccines. Vaccines have proven to be boon for animals which can be regard a potential weapons against infectious diseases caused mainly by bacteria and viruses. The major aims of veterinary vaccines are to improve the health and welfare of companion animals, increase production of livestock in a cost-effective manner, and prevent animal-to-human transmission from both domestic animals and wildlife. Vaccines have long been used to combat infectious disease; however the last decade has witnessed a revolution in the approach to vaccine design and development. Apart from improving animal health and productivity, veterinary vaccines have a significant impact on public health through reductions in the use of veterinary pharmaceuticals and hormones and their residues in the human food chain.

Introduction

Harshberger first used the term ethnobotany in 1895-96, for the plants used by the local tribes and people, for their basic needs and requirements. They also used plant, plant parts and plant products for animal healthcare and other practices. This indigenous or traditional animal health care practice is called ethnoveterinary medicine. Ethnoveterinary medicine is defined as a holistic comprehension of the indigenous systems of animal health, their interpretation through western medicine and the development of effective and appropriate technologies (McCorkle 1986). McCorkle et al. (1989) define Ethnoveterinary medicine (EVM) as dealing with the folk beliefs, knowledge, skills, methods and practices pertaining to the health care of animals. McCorkle et al. (1996) give a description of ethnoveterinary medicinal research as the holistic interdisciplinary study of the local knowledge and the socio-cultural structures and environment associated with animal health care and husbandry.
 
EVM consists of local people’s knowledge pertaining to animal health and production. According to Mathias (1996 & 2002), it includes:

The quality of medicinal plants is paramount in respect of its therapeutic value. The therapeutic value of a plant solely depends on its genuineness in respect of its identity and optimum quantity of therapeutically active phyto-constituents. The medicinal plants are resourced from wild and cultivated sources. The major sources of medicinal plants are wild sources viz. forest areas, open land, non-cultivated sources etc. The medicinal plants collected from wild sources remain questionable for their quality especially when they have been procured from trade channels owing to fair chances of adulteration, substitution and inappropriate storage condition which lead to deterioration in quality. All the medicinal plants which are available in dried form in the trade are termed as crude drugs and are always subject to quality check in a laboratory on the basis of pharmacognostical, physico-chemical, phyto-chemical, microbiological and other analytical specifications. These specifications are available in pharmacopoeial monographs under regulatory frame work.  The pharmacopoeial standards are experimentally established and are also approved standards incorporated in a pharmacopoeia. A pharmacopoeia is a book of standards applicable to drug substance and their dosage forms of common usage in a country. Pharmacopoeia is prepared by recognized authority appointed by the Government of a particular country. It is an essential requirement of a regulatory framework in the sphere of Drugs and Cosmetics Act 1940 and Rules there under (Anonymous 1940). Pharmacopoeial standards are experimentally established and approved standards.

Normal Solution in Analytical Chemistry

A normal solution is that one that contains one equivalent weight expressed  in grams (one gram equivalent weight) to be dissolved substance per litre of solution, or one gram-milliequivalent weight per millilitre.

In the case of acid a 1N solution contains 1.008 gram of replaceable hydrogen per litre of solution. A 1N solution of a base is one that contains 17.008 of hydroxyl per litre. A 1N solution of a precipitating agent contains a weight of precipitating ion equivalent to 1.008 hydrogen. The quantity of pure reagent necessary per litre of 1N solution of a precipitating solution is calculated by dividing the gram molecular weight by the valency of the precipitating ion. In case of normal solution of oxidising and reducing agents, the amount is calculated by dividing the gram molecular weight by the total valency change in the ion concerned.

The Metric System

At present metric system is followed to calculate final results in analysis work. This system is quite simple in nature as well as in principle. The units of the metric system are: (1) the  metre (linear measure), (2) the litre (measure of capacity), and (3) the gram (measure of mass.)

One litre is the volume of pure water at 4ºC and 760 mm pressure which weigh 1 kilogram (kg). One litre equals 1000.027 cubic centimetres (Cm3) for most work, the units “millilitre”, “cubic centimetre” and used interchangeably.

At present there are three different temperature scales in common use. These are: the Fahrenheit (F), used chiefly in English-speaking countries. the centigrade (C), used universally in scientific work and the Absolute (T) or Kelvin (K) scale, differing from the centigrade only in the position of the zero point, its divisions being of the same size. its zero point is -273ºC. The kelvin scale is used especially in dealing with very high or very low temperature and in computations involving the laws of gases. The following formulae can be used for converting one scale to another:

General Views about Weende’s System of Analysis

A team of scientists under the leardship of Henneberg and Stohmann of (University of Göttingen, Germany) developed proximate principles analysis system in the sixties of last century at Weende’s Experimental Station (Germany), located at the University of Göttingen. Main components of different fractions in the proximate analysis of foods are moisture, ash, crude protein, ether extract, crude fiber, and nitrogen-free-extract. These workers emphasized that carbohydrates could be grouped into: (1) the starches and the sugars, and (2) coarse fibrous fractions. Later on an insoluble residue after boiling the fat free food sample first with dilute acid and then with dilute alkali was obtained based on the acid stomach digestion and the subsequent alkaline intestinal digestion of consumed food. At the end of digestion insoluble organic residue was denoted as crude fiber.

Chemical changes occur during oven-drying because of the direct effect of high temperature, or of enzymic or bacterial changes during the early stages of drying, or of loss of volatile consitituents. Feeds containing molasses should be ground in a mortar.

Dried materials should be ground to pass 1 mm sieve. Samples expected to moisture during the treatments are usually predried. If wet samples are used directly in the assay, sufficient large quantity has to weighted out. In the first stage, roughages about 250-1000g are used, somewhat dependent upon homogeneity and moisture content of the wet material. These samples are weighted into trays and dried in draft oven at 60-70ºC. The dry samples are then again weighed, determined the moisture content. The trays are then left to equilibrate in air at room temperature for two days. before a second reweighing of the trays with samples. Then the sample is milled and filled into airtight containers and labelled as dry samples.

A biological sample received in the laboratory should be processed as below:

Fresh Green Sample: During metabolism trial period it is always advisable to take a sample of fresh green fodder daily so as to  avoid chemical composition variation from place-to-place in a field. Take about two and half kilo sample from the bulk, cut the whole plant including the stem and stalks with a pair of scissors into small pieces and mix thoroughly. Weigh about 100g accurately in the aluminium tray 23156cm approximately for estimation of moisture. It is always recommended to weigh the sample immediately in the laboratory so as to avoid the losses of moisture.

Dry matter is generally defined as the constant  weight a sample  attains when heated at 100ºC (occasionally 150ºC). This definition , suitable for inert materials such as sand, has three main lacunae when applied to biological materials.

    1.    In these materials, water is present in various states ranging from extraneous moisture  as rain or dew, through water present in cell sap, water present in various physico-chemical and chemical combinations.

    2.    Biological materials contain active respiratory enzymic systems which continue to function during the early stages of drying; in fact, as the material heats up, activity will be enhanced until it is stopped either  by denaturation of the enzymes or by desiccation. Besides altering the chemical composition of the material, such activity will  lead to loss of dry matter.

    3.    Most biological materials contain organic  compounds which are volatile at 100ºC and which are, therefore, lost on drying. This leads to an under-estimation of dry matter content, for example, in the case of faeces, silage and molasses.

The dry matter content of feed samples and other materials is expressed on three aspects as fed, partially dry, and dry.

As fed refers to the feed as it is consumed by the animal, the term as collected is used for materials which are not usually fed to the animal, that is, urine, faeces, etc. If the analysis on a sample is affected by partial drying the analyses are made on the as fed or as collected sample. Similar terms: Air dry, that is hay: as received: fresh; green and wet.

Partially dry, reports to a sample of “as fed” or “as collected” material that has been dried in an oven (usually with forced air) at a temperature usually about 60ºC of freeze-dried and has been equilibrated with the air; the sample after these processes would usually contain more than 88 percent dry matter (12 percent moisture); some materials are prepared in the way so that they may be sampled, chemically analysed and stored. This analysis is referred to as “partial dry matter” percent of “as fed” or “as collected” sample. The partially dry sample must be analysed for dry matter (determined in an oven at 105ºC) to correct subsequent chemical analyses of the samples to a “dry” basis. This analysis is referred to as “dry , percent of partial dry sample similar term - “Air dry” is also used.

Calorimetry

The process of measuring  the amount of heat generated by the combustion of a substance is known as calorimetry. The first successful calorimeter using oxygen under pressure for the combustion of substance in a closed vessel was devised by Berthelot in 1881. Subsequent development by Mahler in 1992, Atwater in 1899, and Parr in 1912, served to improve and to reduce the cost of the original Berthelot apparatus by variations in mechanical details.

Apparatus and Reagents

    1.    Soxhlet extraction asembly. This consists of 3 parts (a) condenser at the top, (b) the soxhlet or extractor in the middle, and (c) the receiver flask at the bottom. The 3 parts are assembled by means of their grounds glass interchangeable joints (Fig. 1).

    2.    Extraction thimble.

    3.    Petroleum ether (B.P. 40ºC to 60ºC)

    4.    Constant temperatue bath or six heater hot plate with temperature controlling device.

Principle

The nitrogen of protein and other compounds is transformed into ammonium sulphate by acid digestion with boiling sulphuric acid. the acid digest is cooled, dilute with water, and made strongly basic with sodium hydroxide. The released ammonia is distilled into a boric acid solution or standard sulphuric acid solution. When boric acid is used to collect ammonia then is the titrated with standard sulphuric acid or standard hydrochloric acid. When standard sulphuric acid is used to collect ammonia then it is titrated with standard sodium hydroxide solution.

True protein nitrogen is also termed a Albuminoid nitrogen in animal feeds and fodders and human foods. If material (Such as seeds, seed residue or oil cake) is rich in alkaline phosphates, 1-2 ml of 10 percent solution of potash alum is added to decompose them. If this is not done Cu3 (PO4) and free alkali may be formed and the protein-copper precipitate may partially dissolve in the alkaline liquid.

1. Ammonia Nitrogen by Distillation Method
Method I (For Animal Feeds)
Reagents
    1.    Mangnesium oxide (carbonate free)
    2.    Standard sodium hydroxide 0.25 N
    3.    Standard sulphuric acid 0.5 N
    4.    Methyl red indicator

Weigh accurately 2 to 4 g  of the prepared sample. Shake it with water and filter. Wash the residue thoroughly with water. Transfer the filtrate to the distillation flask and dilute to about 200 ml with water. Add about 5 g of the magnesium oxide, Connect the flask to the condenser by means of the connecting bulb tube and distill about 100 ml of liquid into the receiver containing standard sulphuric acid and methyl red indicator solution, Titrate the contents of the receiver with the standard sodium hydroxide solution. Carry out a blank determination using all reagents in the same quantities but without the material to be tested.

Definition
The residue of incineration at 550-600ºC is the crude ash.

Reagent
Dilute hydrochloric acid -approximately 5 N prepared from concentrated hydrochloric acid. Dissolve 445 ml conc. hydrochloric acid (about 36 percent HCl) in one litre distilled water. 

Procedure for Total Ash Estimation
Take a vitreosil crucible in oven at 100ºC for six hours, remove and keep it in a desiccator and weigh up to constant weight. About 5-10 g of sample is taken in the weighted vitreosil crucible. The material in the crucible is charred on a low flame and the crucible is then kept in muffle furnace and temperature is allowed to raise to 600ºC and kept constant for two hours. It is removed on cooling and kept in a desiccator  and weight of crucible is taken.

Introduction

Wet ashing is suitable for the determination of Ca, Cu, Fe, Mg, Mn, K, Na, Se and Zn in biological samples and may be applicable to the determination of other elements as well.

The organic matter of the sample is oxidized with concentrated nitric acid and perchloric acids. The acids are partially removed by volatilization and the solube mineral constituents remain dissolved in nitric acid. Any silica present is dehydrated and made insoluble.

Introduction

Wet ashing is suitable for the determination of Ca, Cu, Fe, Mg, Mn, K, Na, Se and Zn in biological samples and may be applicable to the determination of other elements as well.

The organic matter of the sample is oxidized with concentrated nitric acid and perchloric acids. The acids are partially removed by volatilization and the solube mineral constituents remain dissolved in nitric acid. Any silica present is dehydrated and made insoluble.

Introduction

As per statement of Dr. Henneberg of Weende Experiment Station (Göttingen, Germany) the carbohydrates are divided into two groups; crude fiber and nitrogen free extract. The nitrogen free extract, which  comprises the sugars, starch and a large part of the material classed as hemicellulose, is determined by difference. It is represented by the figure obtained when the sum of the water, ash, protein, fat and crude fiber of a feed is subtracted from 100. Since the figure is determined by difference instead of directly, it includes the cumulative errors of the other determinations and thus is not an exact value.

Limitation of Weende Method

The partition of carbohydrate into fiber (CF) and nitrogen free extract (NFE) is presumed to represent a separation of less digestible starch and sugars. However, in 20 to 30 percent feeds listed by Morrison, the NFE is less digestible than the CF. This comes about for several reasons, the most important of which is the CF method (successive boiling with dilute sulphuric acid and sodium hydroxide) does not recover all the fiber and large portions of fibrous constituents  are extracted into the NFE. The most  important of these fractions are lignin and hemicellulose are dissolved by both acid and alkali. The basic error of the NFE concept is the assumption that if constituents are soluble they are digestible. Lignin, the rigid component of wood, not only is indigestible but lowers the digestibility of substances with which it is associated.

Another portion of the indigestible part of NFE arises from an artifact in the calculation of faecal NFE, where it is presumed that faecal nitrogen is protein (N x 6.25). Actually, 80-90 percent faecal nitrogen is NPN and is composed of bacterial residue in which the ratio of organic matter to nitrogen is about 1:4. Thus, part of the faecal NFE arises by an underestimation of the organic matter associated with faecal nitrogen.

Principle

The residue remaining from the hemicellulose determination is digested with 72 percent sulphuric acid at room temperature. The lignin and acid insoluble  ash are retained in the filter crucible while the digested carbohydrate passes through. The lignin is separated from acid insoluble ash by ashing the residue.

Equipment

    1.    Acid digestion apparatus. A glass tray about 18 cm x 30 cm, 4 cm deep will hold twelve, 100 ml Griffen beakers. Another tray of the same size can be inverted over the beaker.
    2.    Filter crucibles. Same as described for cell wall determination.
    3.    Filtering apparatus. Same as described for cell wall determination.

Principle

The residue from the cell walls determination is extracted with boiling 8 percent sulphuric acid for one hour to hydrolyse the hemicellulose without dissolving cellulose, lignin or acid insoluble ash.

Equipment
    1.    Refluxing apparatus.
    2.     Filtering apparatus: same as described for cell wall determination
    3.    Filtering crucibles: same as described for cell wall determination.

Reagents

    1.    Sulphuric acid (8%) : Standardize Reagent grade sulphuric acid to specific gravity of 1.052 at 20ºC.
    2.    Bromocresol green indicator solution: Same as used for lignin determination.

Estimation of Cellulose in Feeds and Fodders
Principle

The feed well ground is digested with acetic acid which dissolves all other constituents like protein, disaccharides, fatty acids, etc., and cellulose and mineral contents are unaffected. The residue is made acid and alcohol free by washings. Alcohol removes water, benzene removes pigments and fat and at the end the sample is dried and ashed. The difference between the dried weight before ashing and weight after ashing gives cellulose content of the feed sample.

Principle

Dried milled samples are extracted successively with diethyl ether, 0.5 percent ammonium oxalate solution, NH₂SO₄ and 72 percent sulphuric acid. Soluble carbohydrate in the ammonium oxalate extract determined with anthrone and hydrolysed pentosans and hexosans in the combined N H₂SO₄. Seventy two percent sulphuric acid extract are determined with aniline acetate and chromotropic acid respectively. The method gives a maximum variation of 4 percent from the mean of duplicates with each carbohydrate component. The residual material is ashed to obtain crude lignin and a nitrogen correction applied.

Soluble carbohydrate - this group, which includes sugars, oligosaccharides, and the fructosans of grasses, provides a source of nutrients  readily available to livestock.

Principle

Dried milled samples are extracted successively with diethyl ether, 0.5 percent ammonium oxalate solution, NH₂SO₄ and 72 percent sulphuric acid. Soluble carbohydrate in the ammonium oxalate extract determined with anthrone and hydrolysed pentosans and hexosans in the combined N H₂SO₄. Seventy two percent sulphuric acid extract are determined with aniline acetate and chromotropic acid respectively. The method gives a maximum variation of 4 percent from the mean of duplicates with each carbohydrate component. The residual material is ashed to obtain crude lignin and a nitrogen correction applied.

Soluble carbohydrate - this group, which includes sugars, oligosaccharides, and the fructosans of grasses, provides a source of nutrients  readily available to livestock.

Method I
Reagents
    1.    20% sodium carbonate.
    2.    Conc. hydrochloric acid.
    3.    4% sodium hydroxide
    4.    10% calcium chloride
    5.    Ammonium hydroxide
    6.    Potassium permanganate (0.05 N)

Procedure
Take 2-5 g of powdered sample in a beaker without spout, and add 40 ml of water and boil for half an hour. In case of faeces, add 40 ml of 4 percent NaOH instead of 40 ml of water to form silicates. Now boil, for half an hour with cold water flask over the beaker, then add 10 ml of 20 percent sodium carbonate solution and boil for  another half an hour. During these two cooking processes, the following changes take place.

Introduction

Substances found in many plants are generally related to one of the phenols, pyrogallol or catechol. Tannins are solids, usually extracted by hot water insoluble in ether, chloroform, carbon disulphide, benzene; soluble in alcohol ether mixture, alcohol, water and in ethyl acetate, possessing a bitter astringent taste.

Tannic acid is Pentadigalloyl glucose, on hydrolysis yields digallic acid and glucose. Tannins are water soluble glucosides of polydepsides, polymeric ester of gallic acid. It is hydrolysed enzymatically and some efforts have, therefore, been made to preserve tannic, if it is to exert its activity in the duodenum. On the basis of physical or chemical principles which they employ, the methods of tannin analysis  can be grouped as follows.

Principle

Cyanide is distilled from chloroform and water soultion into KOH solution, forming potassium cyanide. KCN is titrated with silver nitrate; 2 KCN + AgNO₃ = KCN. AgCN + KNO₃. An excess of AgNO₃ produces insoluble AgCN, which is the end point of titration.

Nitrates and nitrites are known to have injurious effects on animals when excess amounts are ingested. Nitrites are much more toxic but nitrates are reduced to nitrites by bacterial action in the digestive tract

Nitrites (NO₂) are rarely found in plants  in appreciable amounts except under very unusual conditions such as severe disease infections; therefore, the results are usually reported in terms of nitrates (NO₃). The usual method is to determine the total nitrate and nitrite in terms of nitrate. However, the method can also be used to determine the nitrite content of a feed separately if a nitrite problem is suspected.

Introduction

Aflatoxins are a group of highly subsituted coumarine, of which aflatoxin B1 and G1 are  most  highly hepatotoxic (toxic to liver) to animals. The toxins are produced by some strains of Aspergillus flavus and other species of Aspergillus which develop in many food stuffs, particularly groundnuts. cottonseed, and their cake and flour. Protein concentrates meant for babies as well as adult are now a day  made from groundnut flour and the cakes are used as feed for poultry and livestock. It has been found that even fodgrains such as maize, rice and wheat are attacked by the fungus producing the toxins.

The method described here gives an approximate thioglucoside and isothiocyanates contents in rapeseed meal.

Reagents and Apparatus
    1.    Barium chloride (5% solution)
    2.    Volumetric flask, 600 ml
    3.    Steam bath

Method

To 10 g meal (defatted by soxhlet extraction) add 250 ml distilled water, hydrolyse at 54ºC for one hour and then boil for two hours, keeping volume constant. Filter, retaining filtrate and wash residue three times with 50 ml hot water. Add washings to initial filtrate and make up volume to 600 ml. Precipitate barium sulphate by heating and adding excess barium chloride solution. Leave on a steam bath for a few hours and then filter. Ash in muffle furnace and then weigh precipitate.

Two procedure are described for the determination of free gosssypol . The first for normal meals and the second for meals which have been chemically treated and so contain dianilinogossypol.

Reagents
 
    1.    Aqueous acetone:  7 parts acetone, 3 parts distilled water (V/V).
    2.    Aqueous acetone-aniline solution: To 700 ml acetone and 300 ml distilled water, add 0.5 ml redistilled aniline. Prepare solution daily.
    3.    Aqueous isopropyl alcohol solution: 8 parts isopropyl alcohol. 2 parts distilled water (V/V).
    4.    Aniline Distill reagent grade aniline over a small quantity of zinc, dust discarding the first and last 10 percent of the distillate. Store refrigerated in a brown glass stoppered bottle. Solution is stable for several months.

Isolation of RNA from Rat Liver
(Method of Schmidt and Thannhauser, 1945)
Principle

Most of the RNA (ribouncleic acid) of cells is found in the nucleoli and microsomes.These fractions of cells may be isolated by differential centrifugation of cellular homogenates and thus provide an enriched starting material for RNA extraction. Such methods require high speed  centrifugation. An alternate procedure used in the present method is to disrupt the whole cells by homogenization and isolate the nucleic acid of entire homogenate. This procedure consists of the following sequential steps.

Principle
(Method of Patterson 1963)

The albumin bound long-chain fatty acids of plasma are usually referred to as unesterified or non-esterified fatty acids (NEFA) to distinguish them from the long-chain fatty acids present in the esterified form in neutral fats and phospholipid. In the case of ruminant plasma, it is also important to distinguish these “free fatty acids” from the small molecular weight steam volatile fatty acids of dietary origin which are found in the free state as the carboxylate ion.

Basically, the method of Dole (1959a) has been used to estimate plasma NEFA employed by Annison (1960), which is to evaporate the heptane extract of plasma to dryness before titration. This is conveniently done in a stream of nitrogen on a warm hot plate rather than by evaporation in vacua. In place of the 0.05 M- phosphate buffer (pH 6.0) of Annison or the 0.02 N-H₂SO₄ used by Dole (1956a), in the present method 0.02 N-HCI is used as an extraction medium. This is preferred because owing to its volatility, it is less likely to cause serious accidental contamination of the supernatant heptane layer.