eChapters

[Bray’s Method (Bray and Kurtz, 1945) – Acid Soils]

Introduction

The term available phosphorus (P) refers to the inorganic form, occurring in the soil solution, which is almost exclusively ‘orthophosphate’. This orthophosphate occurs in several forms and combinations, and only a small fraction of the total amount present may be available to plants, which is of direct relevance in assessing the P fertility level.

The phosphate concentration in solution is governed by heterogeneous equilibria in which it takes part. This situation can be represented as follows:

P adsorbed in solid phase ↔ P in the soil solution ↔ P precipitated

The phosphorus absorbed by plants from soil solution comes from the soil solution in which it exists as inorganic orthophosphate ions viz., H₂PO₄ ^–, HPO₄ ^2– and PO₄ ^3–. The most accessible ion is H₂PO₄ ^–, with the greatest activity coefficient, followed by HPO₄ ^2–. The quantity of P accessible to the plants is influenced by a series of soil properties. The relative abundance of these ions is, however, relatively dependent on the soil pH. For soils having a pH between 4.5 and 7.5, ions of H₂PO₄ ^– as well as HPO₄ ^2– exist in soil solution. At a pH of 7.2, H₂PO₄ ^–and HPO₄ ^2– ions have an equal activity, and, when the pH is strongly alkaline (>8.3), ions of HPO₄ ^2– predominate in solution. Above pH of 9.0, the trivalent ion (PO₄ ^3–) becomes more important than H₂PO₄ ^–, but even at a pH of 12, the HPO₄ ^2– concentration is still greater than that of PO₄ ^3–. Soluble phosphorus may be adsorptively retained at the surface of colloidal particles. This retention is more marked when the higher amounts of clay and sesquioxides are present. Relatively, the available inorganic P tends to accumulate in its most stable state under prevailing conditions, thus, in calcareous soils, the available inorganic P would be acid-soluble, whereas, in acid soils the adsorbed P would be more available. In flooded soils, with low oxidation potential, certain forms of P, normally considered as unavailable, for example, iron and phosphate, can be regarded as available. In most soils, the main source of orthophosphate is organic matter unless, of course, direct fertilization, with soluble phosphate, has been made.

(Neutral Normal Ammonium Acetate Method)

Introduction

The total potassium (K) content of a soil varies from 0.05 to 2.5 %. The total K is distributed in mineral form (lattice – K, 90-98 %), fixed non-exchangeable or temporarily retrograded K (1-10 %) and exchangeable plus water soluble K (1-2 %). Exchangeable K represents an average of 2 – 15 % of the sum of exchangeable bases and 1 – 3 % of the total capacity for cation exchange. The K concentration in the soil solution is on an average 0.08 – 3 meq/litre. Both water soluble and exchangeable K are most accessible to the plant. Available K can, thus be separated into that, immediately available, which is water soluble and exchangeable, and that, potentially available or fixed. The neutral normal ammonium acetate extract contains both water soluble and exchangeable K. K extraction by this extractant is considered as a suitable index of K availability in most soils, based on crop response correlation study. Thus, the K extracted by this method is equated as the available K.

[Williams and Steinbergs (1959) Method]

Introduction

Sulphur (S) occurs in numerous forms in soil, viz., sulphites, sulphates, sulphides and in organic compounds. It is considered that the most accessible form is ‘sulphate’ (SO₄ ^2–). The organic forms of S compounds become assimilable, especially following, microbiological transformation into SO₄ ^2–. The mineralization of organic S in a soil depends primarily upon the N:S ratio, and any SO₄ ^2– formed may be fixed against extraction, particularly if much Fe or Ba is present or if the soil is very acid. Despite the fact that the plants absorb S almost exclusively as SO₄ ^2–, mobility of SO₄ ^2– in soil may not always yield satisfactory results in accordance with the time of sampling, while assessing SO₄ ^2– availability.

Neem seed cake (NSC) despite of being rich in protein is not used as animal feed due to the presence of large number of toxic/bitter compounds in it. Studies have shown that NSC can be safely included the ration of animals after water washing / alkali/ urea treatment. These treatment methods appear to remove / reduce or largely inactivate the toxic principles present in NSC. In order to assess the quality/safety of the treated product, quantification of the residual toxic compounds is essential. One such compound is azadirachtin. It has been reported to be biologically most active.

One of the best techniques for the estimation of azadirachtin is by using high performance liquid chromatography (HPLC).

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

Introduction

Sugars are commonly found in the form of biopolymers. Cellulose, a biopolymer of glucose is the predominant constituent of plant tissues, and may be shortly described as the structural bases of vegetable world. In this case, the glucose moieties are strung together by P-glycosidase linkages, which form the linear chain, making it resistant to chemical attack. Hemicellulose consists of short, highly branched, chains of sugars. It contains five carbon sugars (usually D-Xylose and L-Arabinose) and six carbon sugars (D-Galactose, D-Glucose and D-Mannose) and uronic acid. Its branched nature renders hemicellulose amorphous and relatively easy to hydrolyse to its constituent sugars. Lignin is a biopolymer rich in phenolic components, which provide structural integrity to plants. Acid and neutral detergent fraction methods (ADF and NDF) involve the gravimetric determination of residue previously treated with acid and neutral detergent solutions. These methods determine the insoluble fraction and their individual components. NDF represents the total plant fibre or cell wall including hemicellulose

1. Introduction

Insects, like many other organisms, rely heavily on their diet for essential nutrients that regulate their growth and development. The quality of food intake significantly impacts various aspects of an insect’s life cycle. Research, such as that conducted by Lee (2015) highlights that consuming a diet lacking in essential nutrients can lead to premature aging, a process known as senescence, in insects. Furthermore, such inadequate nutrition can hasten the rate of mortality as the insect ages. This underscores the critical role that proper nutrition plays in the longevity and overall health of insect populations.

Carbohydrates are constituents of all living cells. Animal as well as plant cells use carbohydrates as source of energy. Blood glucose is a useful indicator of energy metabolism and fertility status. Under certain clinical circumstances, estimation of carbohydrates becomes essential. Sugar levels (particularly glucose) in blood or urine are used as an important parameter for diagnosis of ketosis and diabetes mellitus.

Urea is the detoxification product of the ammonia derived from the deamination of amino acids. Urea is therefore the most common nitrogen-containing end product of protein catabolism. Its synthesis from ammonia occurs in the liver (via the urea cycle), and it is then excreted by the kidneys. The concentration of urea inside red cells is slightly less than in plasma due to the presence of large amounts of hemoglobin inside the cells. Whole blood urea concentration is therefore slightly less than plasma (or serum) urea. Therefore, usually serum is used instead of whole blood for the determination of urea. The familiar term BUN (blood urea nitrogen) persists in spite of the fact that it is serum urea nitrogen which is usually measured.

Estimation of calcium and phosphorus in feeds: Calcium is precipitated as calcium oxalate by adding ammonium oxalate to acid soluble extract prepared after running a sample for ash. Precipitates of calcium oxalate are dissolved in sulfuric acid and titrated with potassium permanganate to know the content of calcium.
Using N1V1 ≡ N2V2, 1.0 ml of 0.1 KMnO4 = 2.004 mg Calcium.
Calcium, g % = Burette reading difference X 2.004 X dilution factor X 100 / weight of the sample used for preparation of acid soluble mineral extract X 1000.
[dilution factor, (for example) = 250 / 25 (ml aliquot taken) = 10]
Phosphorous present in the sample is precipitated with ammonium molybdate and the precipitates are dissolved in NaOH solution. Excess sodium hydroxide is titrated with nitric acid to calculate the actual volume of NaOH used up which is direct proportion to the Phosphorous content.
1 ml of 0.1 N NaOH = 0.1347 mg Phosphorus
Phosphorus % = Actual volume of 0.1N NaOH solution used to dissolve precipitate X 0.1347 X dilution factor X100 / weight of sample used for acid soluble extract X 1000
[Here, dilution factor is 250 / 50 (aliquot taken) = 5]

Principle

When a solution containing calcium is treated with ammonium oxalate, all the calcium present is precipitated as calcium oxalate. The precipitate when dissolves in sulphuric acid, forming calcium sulphate and liberating free oxalic acid which is quantitatively estimated by titration against standard N/10 KMnO₄ to arrive at the calcium content present in the given solution. Following chemical reactions takes place in the estimation of calcium:

Theory

The energy available in food is expressed in unit is called joules or calories. One kilocalorie is equal to 4.184 kilojoules. The kilocalorie is defined as the amount of heat required to raise the temperature of 1 kg of water by 1°C. The calorific/ energy value of foods is usually determined using the instrument called bomb calorimeter. It is working based on the principles of calorimetry. Basically, this device burns a food sample and transfers the heat into a known mass of water. It measures the heat produced when the food sample is ignited by an electric spark in the presence of oxygen and platinum which acts as a catalyst. The quantity of food sample to be tested is placed inner part and known volume of water is taken in outer part of the bomb calorimeter. When the food sample is electrically ignited, the surrounding water absorbs the heat produced. The energy value of the food is calculated by measuring the rise in temperature of water.

I. Titrimetric Method of Estimating Salt in Fish Meal
(Modified AOAC, 1965 method)
Principle

The salt content is calculated as sodium chloride from the content of water solubles chlorides. The chlorides are dissolved in water and directly precipitated by titration with a silver nitrate solution using potassium chromate as an indicator.

Principle

When a sample of moisture and fat free feedstuff is boiled with dilute acid and then with dilute alkali, the soluble carbohydrates and proteins go into the solution and are extracted. The undissolved residue left behind represents cellulose, hemicellulose, lignin and mineral matter. The cellulose, hemicellulose and lignin put together are called crude fibre (CF). When the residue after drying and weighing is ignited, the fibre being organic in nature is burnt whereas the ash or mineral matter is left over. The fibre is obtained by deducting the weight of ash from the weight of the dried residue. In simple way it can also be explained that the crude fibre is determined gravimetrically as part of the substance which is left when protein, soluble carbohydrate, fat, ash and water have been removed.

In feeding of simple stomached animals, the analysis of crude fibre content is very essential because the amount of crude fibre predicts the digestibility of the feedstuff. Higher the fibre content lesser will be the digestibility.

Principle

Crude protein constitutes a mixture of true protein and non protein nitrogenous substances and is determined by estimating nitrogen and then multiplying the nitrogen content of feed by the factor 6.25.

The factor 6.25 is derived on the basis that all proteins contain 16% nitrogen. The total nitrogen content in the feedstuff is determined by Kjeldahl method which generally consists of three steps, i.e. digestion, distillation and titration. The sample is digested with an excess quantity of concentrated sulphuric acid which hydrolyses the protein, decomposes to ammonia and finally the ammonia combines with the excess of sulphuric acid forming ammonium sulphate. The acid digest is cooled, diluted with distilled water and distilled with strong alkali (sodium hydroxide, 40 per cent), the ammonia released during distillation is taken up into a boric acid solution forming ammonium borate complex and such ammonia is titrated with standard sulphuric acid. While estimating the nitrogen content, following reactions take place:

Objective

To determine the electrical conductivity (EC) of soil, which is an indicator of the soil’s salinity and its ability to conduct electricity. This provides valuable information about the nutrient availability and potential toxicity to plants.

Materials Required

• Soil sample

• Distilled water

• Electrical conductivity meter (EC meter)

• Beakers (100 mL)

• Measuring cylinder (50 mL)

• Stirring rod

• Filter paper and funnel

• Mortar and pestle (for soil grinding, if necessary)

The Electrical Conductivity (EC) of the soil is a measure of soluble salts present in the soil and is expressed as millimhos/cm or dSm^-1.

Principle

As the amount of the soluble salts in a solution increases, the electrical conductivity also increases. This electrical conductivity is measured in terms of resistance offered to the flow of current using a conductivity bridge.

It is known that solutions offer some resistance to the passage of current through them, depending upon the concentration of the soluble salts present. Hence, EC is measured in terms of electrical Resistance between parallel electrodes immersed in the soil water suspension.

In such a system, the solution between the electrodes becomes the electrical conductor to which the physical laws relating to resistance are applicable. The electrical resistance ‘R’ is directly proportional to the distance ‘L’ between the electrodes and inversely proportional to the cross sectional area ‘A’ of the conductor.

Accurate estimation of embryonic/foetal age play vital role in the developmental anatomy research related to morphogenesis and histogenesis of an organ or system. The observations and recordings of normal developmental features of embryos/ foetuses at successive stages of gestationare very important for studying functional development of an organ in anatomical research. The documentation of normal embryonic and fetal growth can serve as a guide for understanding the consequences of harmful influences at various stages of gestation (Evans and Sack, 1973). Post-mortem studies have provided valuable information on fetal development, identifying stages such as implantation, appearance of forelimb bud, tail development and hind limb development (Alberto et al., 2013). The normal development of external features of animals has been described by Evans and sack (1973), however, subjective assessment of foetal age from the external features alone is not much reliable, although it may be useful adjunct to more precise measurements (Richardson et al., 1990). For accurate estimation of developmental age, fetal measurements are needed which show a consistently rapid rate of growth and which are least variable at a given age (Richardson et al., 1990), however, these parameters are affected by individual variation and by breed differences within a species (Greenwood et al., 2005).
A number of methods have been discussed by many workers for estimation of age of foetus in animals. The method mainly focus on foetal biometry recorded either inside uterus during normal gestation process or after taking out foetus during post-mortem examination. In-utero foetal biometry allows estimating gestational age and time of parturition by ultrasonographic methods (Manning

(Method of Visek et al., 1953; Lueker and Lofgreen, 1961)

By using isotope dilution technique, It is possible to separate faecal calcium into endogenous and exogenous fractions. The true digestibility can be measured independently by making correction for faecal endogenous loss. The isotope dilution method described by Visek et al., (1953) involves daily intravenous injection of 45Ca for 15 days, followed by a collection of the serum and the faeces for 15 days. However, these numerous veinpunctures result in considerable discomfort to the animal. Lueker and Lofgreen (1961) modified this method in sheep by giving a single subcutaneous injection of 45Ca, followed by a collection of blood and the faeces for 7 days only.

The endogenous faecal calcium and true digestibility of calcium is calculated by the formula of Lofgreen and Kleiber (1954) as given below:

Principle
When a sample of feed, which is free from moisture, is extracted continuously with fat solvent like petroleum ether, the vapour from the ether boiling in the flask passes into the condenser where it condenses and drops back on the sample which dissolves ether soluble materials such as fat, sterols, lecithin, resins and volatile oils in the extraction flask. The extract is termed as crude fat or ether extract (EE) because it also contains other fat like substances in addition to true fat.

Equipment/glass wares
    1.     Analytical balance
    2.     Beaker
    3.     Cotton wool
    4.     Dessicator
    5.     Extraction thimble
    6.     Hot air oven
    7.     Oil flask (150-250 ml)