eChapters

A. In vitro Rumen Fermentation Technique for Forage Quality Evaluation

The term in vitro means “in a glass” in contrast to in vivo which is defined as “within the living body”. In vitro implies a close relationship to an in vivo measurement an in vitro reaction is used as a substitute for a reaction which normally occurs and is observed within a living body. the terms  “artificial rumen” and “in vitro rumen fermentation” are often used interchangeably.

The main advantage of the in vitro techniques rests in the ability to use them to study activity of micro-organisms away form the control and influence imposed by the host animal.

Techniques to Conduct Digestion Trial Under Stall Conditions

Digestibilty definition. The digestibility of a food is most accurately defined as that  proportion which is not excreted in the faeces and which is , therefore, assumed to be absorbed by the animal. It is commonly expressed in terms of dry matter and as a percentage, the digestibility coefficient. For example, if a cow ate 11 kg of hay containing 10 kg of dry matter and excreted 4 kg dry matter in its faeces, the digestibility of the hay dry matter would be

The main groups of some diagnostically important enzymes are as below:

1. Oxido Reductases
Glucose 6-phosphate dehydrogenase, Isocitrate Dehydrogenase, Lactate dehydrogenase, Malate dehydrogenase, Sorbitol dehydrogenase.

2. Transferases
Alanine transaminase (GPT)    
Aspartate transaminase (GOT)
Creatin binase
Hexose-1 phosphate uridyltransferase

3. Hydrolases
Acetylcholinesterase
phosphatases
Amylases
Pepsin
Trypsin
Lipase

Celite Column Chromatography
(Method of Wiseman and Irvin, 1957)
Principle

Celite columns with an internal indicator have been developed for the quantitative separation of silage and rumen volatile fatty acids ranging from butyric to succinic. The aqueous phase employs minimal amounts of sulphuric acid to prevent retention of organic acids, sugar is added to the phase to increase elution resistance. Aqueous samples, 2 ml or less are added directly to a dry column cap. Eluting solvents are mixtures of acetone and petroleum ether. Single zone collections make possible a reduced number of titrations with increased accuracy. The column permits fairly wide separations of lactic acids, often difficult on silicic acid columns.

The method employs celite with alphamine red R as an internal indicator. A distinctive feature is the use of concentrated sugar solution as the stationary phase to resist leaching action of solvents.

Principle

The use of boric acid as an ammonia absorbent was first introduced by winkle  (1913-15). Conway and O’Malley (1942) used boric acid for ammonia absorption in their original micro diffusion technique.

The ammonia liberated in the outer chamber by alkali addition in the usual way is absorbed in boric acid mixture plus indicator in the central chamber (slightly reddish), the pH of this mixture being approximately 5 before absorption. During the ammonia absorption the pH of the mixture rises, and to upwards of 8.0 (the strength of the mixture being such as to prevent it rising further). After the required period, the fluid in the central chamber is titrated to a faint permanent reddish tint with standard acid. A mixture of methyl red and bromocresol green was found satisfactory  to work as an indicator to assist in titration job.

Steps in the Estimation of Trace Elements
Estimation of trace elements in feeds and fodders and other biological materials involves the following steps :

    1.    Production of an acidic solution of the inorganic elements in the biological samples after removal of the organic matter by dry ashing or wet oxidation.

    2.    Removal or masking of interfering substances.

    3.    Determination of the selected elements, usually by using colorimetry.

Results of trace elements determinations are expressed as parts per million (ppm), mg/kg or µg/g dry matter.

Unit of Length

A metre (m) is taken from the international prototype metre at the International Bureau of weights and measures; at 32ºF. (0ºC) = 39.37 inches (in.) = 3.280833 feet (ft.) = centimetres (Cm.). A centimetre (Cm.) = 10 millimetres (mm.) = 10,000 microns (µ) = 0.337 inch (in.) or 10-4cm. A millimetre (mm.) = 1,000 microns (µ) = 0.03937 inch. A micron (mµ) = 1,000 milli-microns (µ) = 3.937x10-5 inch. A milli-micron (mµ) = 0.001 micron or 10-7 cm. A foot (ft.) = 0.3048006 metre (m.) = 30.48006 centimetres (Cm.) = 12 inches (in.)., inch = 2.5 cm. An inch (in.) = 2.539998 centimetres (Cm.) = 25.39998 mm = 25,399.98 microns (µ).

Blood Sampling

Blood samples shall be taken in the early morning hours before offering any diet to human subjects or ration to livestock. The syringe and needle should be autoclaved, needle should be free from any blockage etc. In the case of ruminants, blood sample is taken directly from jugular vein, while in swine, blood sample is drawn from ear vein, while in human subjects, blood is taken from prominent vein on the inner surface of the yellow usually the (medium basilic). The container (test tube etc.) should be dried and autoclaved. We should separate plasma (from anticoagulent mixed blood) and serum from ordinary blood by centrifugation immediately. We should remember by heart  that blood samples should not be kept in deep freezer, because there are chances of haemolysis as well as an abnormal distribution of certain ions and enzymes between the cells and the serum. We should never force the blood through the needle as such type of practice may result in extensive hemolysis. We should discard first few drops of blood, while taking sample. We should prepare protein free filtrate immediately. Protein-free filtrate may be kept in a much better way than whole blood or plasma.

Estimation of Calcium in Feeds, Fodders and Faeces
(Titrimetric method of AOAC, 1965)
Principle

When a solution containing calcium is treated with ammonium oxalate, all the calcium present is precipitated as calcium oxalate, The precipitate on treatment with sulphuric acid dissolves, forming calcium sulphate liberating free oxalic acid which is quantitatively estimated by titration against standard N/10 potassium permanganate solution to arrive at the calcium content present in the given solution. The reactions which take place are given below:

Estimation of Phosphorus in Feeds, Fodders and Faeces
(Titrimetric method of AOAC, 1965)
Principle

Metaphosphates and pyrophosphates are converted to the ortho-phosphate, which is treated at 65-70ºC with excess of molybdate in the presence of nitric acid. When acid solutions of phosphates are heated with ammonium molybdate, the phosphoric acid is precipitated as yellow ammonium phosphomolybdate (NH₄)₃ PO₄ (Mo03)12 2HNO₃.H₂O. Impurities like molybdic oxide, if happened to be formed any, goes into solution. The yellow precipitate formed reacts quantitatively with standard alkali, the excess of which is back titrated with acid.

Estimation of Magnesium in Feeds, Fodders and Faeces
(Gravimetric method - AOAC, 1965)
Principle

Evaporation of calcium free filtrate is carried out to drive off ammonium chloride and nitric acid is added to decompose ammonium salts, which is then subsequently driven off by evaporation. NH₄Cl + HNO₃ = HCl + NH₄NO₃ (driven off). Sodium citrate in solution yields citric acid which has got a tremendous effect on aluminium and iron in that the citric acid keeps these materials in solution.

At neutrality, first the normal magnesium phosphate is formed, which is then turned to magnesium ammonium phosphate, which on incineration gives rise to magnesium pyrophosphate (Mg₂P₂O₇).

TVFA Estimation in Blood
(Method of Friedemann, 1938 modified by Mc Anally, 1944)

A method for the determination of volatile fatty acids in various biological materials has been described by Friedemann (1938) which is said to be applicable to blood, then the blood is added to dilute sulphuric acid, then sodium tungstate solution and solid magnesium sulphate are added and the whole is steam distilled.

Reagents
    1.    Saturated magnesium sulphate solution to which has been added 2.5 per cent by volume of concentrated sulphuric acid.

Separation of Bacteria and Protozoa
(Method of Czerkawski, 1975)

Rumen contents are obtained just before and two hours after feeding from sheep that had been given an experimental ration. The samples of rumen contents are strained through gauze and fractioned by sedimentation and centrifugation to give preparations of mixed protozoa (PR), large bacteria (LB) and small bacteria (SB). Whole particulate matter is also isolated by centrifugation at 20000g. The detail steps are mentioned in the flow chart given below.

Total Protein in Tissue Extract or Blood, Serum or Plasma
(Method of Folin-Ciocalteu, 1927 modified by Lowry et al., 1951)
Principle

A deep blue dye is formed when protein is treated with the phenol reagent of Folin ciocalteu. The extraordinary sensitivity of the method is due to the fact that two colour reactions are taking place simultaneously.
    a.    The biuret reaction of peptide bonds with copper in alkaline solution.
    b.    A reduction of phosphomolybdic acid and phosphotungstic acid by the aromatic amino acids, tyrosine and tryptophan, present in the protein.

Pepsin Digestibility of Food
Principle

Solvent extracted samples are digested for 16 hour with warm acid solution of pepsin under constant agitation. Insoluble residue is centrifuged, dried and weighed and analysed for protein ; or filtered, washed and analysed for protein.

Apparatus
    1.    Centrifuge : Capable of at least 1750 rpm with conical bottom tubes of 150 ml capacity. If necessary, solution may be centrifuged in 50 ml tubes.
    2.    Agitator : or water bath with shaking device and having temperature controlling unit.
    3.    Glass fiber filter paper.

Introduction

Casein has been widely used as a substrate for measuring the trypsin inhibitor activity of natural trypsin inhibitors such as those which occur in Soybeans and other legume. The most common method employing casein is the one originally described by Kunitz (1947) which involves the spectrophotometric determination of the breakdown products produced by a given concentration of trypsin in the presence and absence of the inhibitor. On the other hand, the tryptic hydrolysis of a synthetic substrate such as benzoyl-DL-arginine-p-nitroanilide (BAPA), first introduced by Erlanger et al. (1961), does follow a zero order reaction, and one obtains, within broad limits, a linear relationship between the quantity of p-nitroaniline released and the concentration of the active enzyme. Although BAPA has been used for assaying the antitryptic activity of soybean fractions (Sambeth et al., 1967).

PEG Estimation
Introduction
Polyethylene glycol (PEG) is used as a marker for the estimation of rumen water volume and ruminant digestion studies (Hyden and Ekman, 1953). Long back, Hyden (1955) developed a turbidimetric method which is described below :

Principle
When trichloroacetic acid is added to a solution of PEG, a turbidity is produced, which can be measured photo-electrically. The intensity of the cloud, which consists of finely divided oily droplets, is increased in the presence of barium ions. Protein and sulphate must be completely removed from the sample before the addition of the trichloroacetic acid reagent. Sulphate is precipitated with barium chloride and the proteins are removed essentially according to Samogyi (1945). The method has been developed for determination of PEG in ruminal and abomasal fluids, but it has been tried with other biological materials too.

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.

I. Detection of Castor Husk in Oil Cakes and Oil Seed Meals (Method of ISI, (BIS), IS: 10165-1982)
Principle

The method is based on the fact that castor husk is not bleached under the conditions which cause the bleaching of almost all other materials of vegetable origin likely to be present in an oilcake. The method consists of the treatment of the material with dilute alkali and acid solutions followed by treatment with bleaching powder solution and the isolation of the unbleached castor husk.

(Method of Schram and Aines, 1959)

All the legume seeds and their byproducts are very rich in urease activity which may be responsible for an appreciable loss of urea nitrogen on long duration storage. Therefore it is necessary to estimate urease activity in legume seeds and their byproducts.

Reagents

    1.    Dimethyleaminobenzaldehyde solution (DMAB). Dissolve 16 g DMAB in 1 litre 95% ethyl alcohol and add 100 ml concentrated hydrochloric acid (Stable one month).
    2.    Pyrophosphate buffer. Dissolve 233 g Na4P2O7. 10H2O in approximately 980 ml distilled water. Add 3 ml of conc. Hcl and then dropwise further Hcl until the pH of the buffer is 7.7 to 7.8. Dilute to one litre.
    3.    Buffered urea solution. Dissolve 0.4 g urea in one litre pyrophosphate buffer (Stable one week).

(Colorimetric Method of Barker and Summerson, 1941)
I. Method for Blood / Plasma
Principle

In this method, glucose and other interfering material of the protein free blood filtrate is removed by the Van Slyke-Salkowski method of treatment with copper sulphate and calcium hydroxide. A part of the resulting solution is heated with concentrated sulphuric acid to convert lactic acid to acetaldehyde, which is then determined by reaction with p-hydroxydiphenyl in the presence of copper ions.

Preparation of Samples

Before withdrawing portions for analytical determinations, bring the sample to a temperature of 26 to 28ºC and mix throughly by pouring gently into a clean dry receptacle and back, until a homogenous mixture is assured. If lumps of cream do not completely disappear, warm the sample to about 40ºC, mix throughly, then cool to 26 to 28ºC. In case a measured volume is required in a determination, bring the temperature of the sample to about 27ºC before pipetting. All samples shall be allowed to stand for three to four minutes after mixing to allow air bubbles to rise ; the sample bottle shall then be inverted three or four times immediately prior to taking the milk for analysis. If a sample is curdled, excessively churned or decomposed, the sample should not be taken up for analysis.

Adulteration in animal and human foods has become common feature in the country, therefore we should be cautious while selecting animal/human feed ingredients.

The author of this compendium has developed. “Animal Feed Testing Kit” based on novel cud liquor enzyme complex (innovation certified by ISA, International Search Authority - Patent office, Vienna), registered with WIPO (Geneva). This method is described in detail in this chapter and Patent application is field at Indian Patent Office Delhi.

Now-a-days, animal feed is being adulterated with extraneous materials viz, Saw dust, Ric hulls, sand and Silica, Castor husk, oat hulls and urea (higher than 01 percent limit on dry matter basis).

Answer the followings
    Q.1.    What is mastitis?
    Ans.    It may be defined as inflammation of mammary parenchyma and characterized by physiochemical changes in the milk and pathological changes of the glandular tissue.
    Q.2.    What are the cardinal changes of milk and udder in mastitis?
    Ans.    Changes of milk includes discoloration, presence of clot, and presence of huge leukocytes whereas, mammary gland becomes hot painful swelling and induration.
    Q.3.    What is sub clinical mastitis?
    Ans.    In subclinical mastitis, normally mammary gland is normal by physical examination; however, cellular constitution only indicates presence of mastitis (i.e. increased somatic cell count).
    Q.4.    What is the basis of diagnosis of sub clinical mastitis?
    Ans.    Diagnosis sub clinical mastitis depends largely on indirect tests which are related to leukocyte content of milk.

Answer the followings
    Q.1.    What is the relative incidence of  Streptococcus agalactiae, other Streptococci and Staphylococcus aureus
    Ans.    1:1: 2
    Q.2.    What are sources of contagious pathogen for mastitis?
    Ans.    Udder surfaces of infected quarters 
    Q.3.    What are sources of environmental pathogen for mastitis?
    Ans.    Bedding of cow’s environment
    Q.4.    When animal get infection from environmental pathogen?
    Ans.    Animal get infection at any time during the life such as milking time, between milking, during dry period and prior to first calving.
    Q.8.    What is the probable period of new infection from environmental pathogen?
    Ans.    Early part of dry period and first two months of lactation.
    Q.9.    What is the relation of growth of Corynebacterium bovis in milk and lactation?

Answer the followings
    Q.1.    What is the different stage of pathogenesis in mastitis?
    Ans.    There are three stages in pathogenesis 
These are as follows:
Invasion: here pathogens move from the exterior of teat to milk inside the teat canal.
Infection: Here pathogens multiply rapidly and invade the mammary gland.
Inflammation: In this stage clinical mastitis occurs with varying degree of clinical abnormalities of milk and udder.
    Q.2.    Of these phases, which phase is important to reduce the incidence of mastitis in control program?
    Ans.    Invasive stage is very important. The incidence of mastitis can be reduced by taking preventive measures at invasive stage by good management and good hygiene practices.
    Q.3.    What are the common abnormalities encountered in udder during mastitis?

Q.1.    What are the major clinical findings in mastitis?
    Ans.    These are abnormalities of secretions, abnormalities of udder related to size, consistency, temperature and sometimes systemic reactions.

Q.2.    How clinical forms of mastitis could be classified based of severity and duration?
    Ans.    Based on severity it is classified in four forms: Per acute mastitis, acute mastitis, subacute mastitis and subclinical mastitis. However, based on duration it is of three forms: Short term clinical or subclinical mastitis (Coliform mastitis), recurrent mastitis (Staphylococcus aureus mastitis, Streptococcal dysgalactiae mastitis) and persistent clinical or subclinical mastitis (Streptococcus agalactiae mastitis).

Q.3.    What is sub clinical mastitis?
    Ans.    In sub clinical mastitis there is evidence of inflammation which is evidenced by only raised SCC in milk without any visible abnormalities of milk and udder.

   Q.1.    How milk sample is collected for diagnosis of mastitis?
    Ans.    At first the end of teat is cleaned with a cotton swab dipped with 70% alcohol, then the dirt and wax are removed from the external sphincter by pressure. After this milk sample is collected in screw cap bottle.

    Q.2.    When milk sampling should be done in suspected cases of tubercular mastitis?
    Ans.    Last few streams of milking is crucial in cases of tubercular mastitis.

    Q.3.    What is the effect of freezing of milk sample on bacterial count?
    Ans.    On freezing of milk, Arcanobacter pyogenis and E. coli counts are decreased, coagulase negative bacteria counts increases whereas, Streptococcus and Staphylococcus spp. counts may be unaffected or increased.

    Q.4.    Which media is suitable for initial isolation of bacteria from milk samples?
    Ans.    Blood agar. However for isolation Streptococcus agalactiae selective media is required

Answer the followings.
    Q.1.    Which factors govern the efficacy of intramammary drug in mastitis therapy?
    Ans.    The important factors are spectrum of bacteria controlled, diffusibility through mammary tissue and cost of the drug.

    Q.2.    What should be line of emergency treatment when type of infection is not known?
    Ans.    Frequent removal of milk manually or by using oxytocin, broad spectrum antimicrobials parentrally as well as via intramammary route and non steroidal anti-inflammatory drug.

    Q.3.    Why therapy of mastitis often fails to respond in lactating cows in spite of intensive veterinary service?
    Ans.    The main reasons are as follows:
    a)    Ineffective drug diffusion
    b)    Inactivation of antibiotics by milk and tissue priteins
    c)    Formation of micro-abscesses which make inaccessible of drug to pathogens
    d)    Inefficient killing of bacteria and intracellular survival of bacteria

Answer the followings
    Q.1.    What is the international organizations associated for making outline the mastitis control programme?
    Ans.    National Institute for Research on Dairying (NIRD) and National Mastitis Council (NMC).

    Q.2.    What is the important objective of dairy industry as regards to mastitis control?
    Ans.    The important objective is to achieve an excellent udder health for production of high quality of milk.

    Q.3.    What is the basic principles of mastitis control programme?
    Ans.    There three basic principles of mastitis control programme:
    a)    Elimination of existing infection
    b)    Prevention of new infection 
    c)    Monitoring of udder health status

Q1.    What is the etiology of Staphylococcus aureus mastitis?
    Ans.    Coagulase positive Staphylococcus aureus.

Q.2.    It causes contagious or environmental mastitis?
    Ans.    Contagious mastitis

Q3.    What are the important animal risk factors of S. aureus mastitis?
    Ans.
    (i)    Presence of coalgulase –ve Staphylococci and Corynebacterium bovis in the mammary gland act as protective factor for S. aureus mastitis.
    (ii)    Abrasion of teat orifice epithelium is a major risk factor of S. aureus mastitis.
    (iii)    Cows with increased parity are more prone to clinical and subclinical S. aureus mastitis.
    (iv)    Presence of different periparturient diseases like parturient peresis, retained placenta, dystocia, ketosis and ulcers in different organs are also risk    factors for S. aureus mastitis.
    (v)    Animals having very low SCC in milk are more prone to S. aureus mastitis.

Fill in the blanks

    Q1.    …………… is a contagious pathogen in bovine mastitis
    Ans.    Streptococcus agalactiae

    Q.2.    Streptococcus agalactiae causes ……… (acute/chronic) mastitis if control procedure of mastitis is not used.
    Ans.    Chronic

Answer the following
    Q.1.    Mention the strategy to reduce the prevalence of Streptococcus agalactiae mastitis at herd level.
    Ans.    Good hygienic measures, proper disinfection and efficient treatment of clinical cases.

    Q.2.    What is the relation between SCC and Streptococcus agalactiae infection?
    Ans.    The possibility of Streptococcus agalactiae infection is high with high bulk milk somatic cell count.

    Q.3.    What are the common sources of Streptococcus agalactiae infection in a dairy herd?

    Q.1.    How many species of Mycoplasma are responsible for causing mastitis?
    Ans.    At least seven species like Mycoplasma bovis, Mycoplasma Canadensis, Mycoplasma bovigenitalium, Mycoplasma alkalensis, Mycoplasma capricolium, Mycoplasma californicum, Mycoplasma disper and Ureaplasma sp. are responsible for causing mastitis

    Q.2.    Which herds are more prone to such type of mastitis?
    Ans.    The herds where milking hygiene is very poor and when the cows are imported from other farms or from public sale yards.

    Q.3.    Which organs act as common habitat of the Mycoplasma organisms?

Q.1.    Enumerate the etiological agents responsible for Coliform mastitis.
    Ans.    Different serotypes of E. coli, Klebsiella sp. and Enterobacter aerogens.

Q.2.    What is the primary reservoir of Coliform mastitis?
    Ans.    Primary reservoir of Coliform is the dairy cow’s environment as it is an environmental pathogen, however, infected mammary gland acts as reservoir of Coliform mastitis

Q.3.    What is the important factors of E. coli that affect the normal bactericidal activity of leukocytes?
    Ans.    Lipopolysaccharide-endotoxin, somatic and capsular factors. Similarly, fibronectin binding property of E. coli is also an important virulence factor which help to adhere to ductular epithelium.

    Q.1.    What are the most common isolated environmental Streptococci from mastitis cases?
    Ans.    Streptococcus uberis and Streptococcus dysgalactiae.

    Q.2.    What are the common sources of environmental Streptococcus uberis in a herd?
    Ans.    The common sources are bedding materials, lips, nostrils and abdominal skin of cows.

    Q.3.    What are the common sources of environmental Streptococcus dysgalactiae?
    Ans.    The common sources are tonsils, mouth, vagina and mammary gland of cows.

    Q.4.    Why incidence of Streptococcus uberis is highest just prior to calving?
    Ans.    The important reasons are the excessive accumulation of milk due to lack of milking, lack of keratin plugs from the streak canal and immunosuppression at periparturient period.

    Q.5.    The cattle fly sometimes involves in persistent bacterial contamination on teat of a cow. Mention the name of the cattle fly.

    Q.1.    The mastitis-metritis-agalactia (MMA) is a disease syndrome of which animal species? 
    Ans.    Swine

    Q.2.    What is MMA syndrome?
    Ans.    MMA syndrome is post parturient disease of sows and manifested as fever, anorexia, lethargy, agalactia, mastitis, swollen udder and appearance of vulvar discharge due to development of metritis.
 
    Q.3.    What is the critical time of occurrence of MMA in sows?
    Ans.    The mastitis-metritis-agalactia (MMA) syndrome occurs between 12-48 hrs of farrowing in sows.

    Q.4.    What is the probable causes for the development of MMA syndrome?
    Ans.    The probable causes of MMA syndrome are infectious mastitis, metritis, more feeding at pregnant period, nutritional deficiency, endocrine disorders and constipation.

Fill in the blanks

    Q.1.    ---------- and -------------- are the synonyms of mastitis of sheep.
    Ans.    Bluebag, garget
    Q.2.    In gangrenous mastitis, besides Staphylococcus aureus, ----------- organism may also be involved.
    Ans.    Clostridium perfringens A
Answer the followings

    Q.1.    What are the characteristic symptoms of mastitis in sheep?
    Ans.    The important symptoms include severe necrotizing inflammation of mammary gland, severe systemic reaction and reduced milk production
    Q.2.    Classify the sheep mastitis based on etiology and clinical symptoms.
    Ans.    a) Gangrenous mastitis by Staphylococcus aurcus mastitis b) Herd mastitis by Pasteurella hemolytica
    Q.3.    When the risk of mastitis in sheep is high?
    Ans.    During summer month between parturition and weaning

Fill in the blanks
    Q.1.    ………… organisms are the common isolates from clinically affected quarters.
    Ans.    Coagulase-negative staphylococci
    Q.2.    ………….is the most common cause of mastitis in goats.
    Ans.    Staphylococcus aureus
    Q.3.    Streptococcal zooepidermicus causes …………. in does.
    Ans.    Chronic suppurative mastitis.
 
Write True/ False about the statements
    Q.1.    Pseudomonas organism causes an acute mastitis characterized by extensive necrosis and fatal septicemia.
    Ans.    True.
    Q.2.    In summer mastitis, non-lactating goats develop severe mastitis whereas lactating goats suffer from moderate degree.
    Ans.    False

    Q.1.    What is the cause of mastitis in non-lactating mare?
    Ans.    Mastitis may develop in a non-lactating mare due to abnormally high estrogen levels in her system. This excess female hormone can result from a pituitary gland tumor or consumption of high estrogen containing hay. High estrogen concentrations can cause mammary gland enlargement and trigger the production and secretion of milk, even though the mare is not pregnant and has not foaled. If the non-lactating mare begins to secrete milk due to excess estrogen, she may become a candidate for mastitis.

    Q.2.    Why equine mammary glands are less prone to mastitis than bovines?
    Ans.    The possible reasons are as follows:
    a)    Mammary glands are relatively smaller in size
    b)    Concealed position of the glands
    c)    Small milk capacity of the glands
    d)    Greater resistance to infection in equine mammary glands because of differences in endocrine environment and local immunity

    Q.1.    How mastitis occurs in dogs? 
    Ans.    Mastitis in dogs initiates with the infection in the milk ducts of the dogs, where in the dog’s breast is enlarged as well as milk duct is blocked. The skin around the nipple might get cracked which might give a way to bacteria. In neglected cases it leads to cause gangrenous mastitis which is a severe form of mastitis in dogs.

    Q.2.    What are common pathogens responsible for mastitis in bitch?
    Ans.    Staphylococcus spp., Streptococcus spp. and Escherichia coli.
 
    Q.3.    What are the characteristic features of milk cytology during mastitis in bitch?
    Ans.    Milk cytology will show the presence of erythrocytes, degenerated neutrophils and bacterial agents.

    Q.4.    What characteristic physical changes of milk are seen during mastitis in bitch?
    Ans.    The viscosity of milk is increased and the colour of milk will be yellow to brown depending on the amount of blood and purulent exudates present.

    Q.1.    What are the common infectious diseases affect teat skin of mammary gland?
    Ans.    The common infectious diseases that affect teat skin of mammary gland are cowpox, buffalopox, pseudocowpox, bovine ulcerative mammillitis, black pox, foot and mouth disease, vesicular exanthema, swine vesicular diseases, contagious exanthyma, Staphylococcus sp., Pasteurella sp etc.

    Q.2.    What is thelitis?
    Ans.    Thelitis is the inflammation of tissues of teat wall caused by traumatic injury to the teat cistern. It is generally manifested with gangrenous mastitis.

    Q.3.    What is the main etiology of gangrenous mastitis?
    Ans.    Staphylococcus aureus

    Q.4.    What are the causes of coming blood in milk and mentions its treatment?

1. California mastitis test

The California mastitis test (CMT) is conducted during milking of lactating cows. The CMT paddle is used to collect about three to four ml of fore milk from each quarter of cows after discarding few streams to which an equal volume of CMT reagent (composition mentioned below) is mixed immediately by circular motion (Fig. 1). The reaction is graded by intensity of gel formation and color changes as follows:

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Bennett, R.H. and Jasper, D.E. 1977. Bovine mycoplasma mastitis from intramammary inoculation of small numbers of Mycoplasma bovis. I. Microbiology and pathology. Vet. Microbiol., 2(4): 341–355.

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Bostedt, H., Lehmann ,B. and Peip, D. 1988. The problems of mastitis in mares. Tierarztl. Prax.. 16(4):367-71.

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Buchanan, A. G. 1982. Clinical laboratory evaluation of a reverse CAMP test for presumptive identification of Clostridium perfringens. J. Clin. Microbiol. 14:761–762.

Bushnell, R. B. 1984. Mycoplasma mastitis. Vet. Clin. North Am. Large Anim. Pract., 6(2):301-12.

Chareonsirisuthigul, T., Kirpal, G., Amtsberg, G. 1979. Biochemistry, serology, pathogenicity and antibiotic resistance of an Escherichia coli strain from healthy sows and from animals with mastitismetritis-agalactia syndrome. Berl. Munch Tierarztl. Wochenschr. 92(21):409-16.

Christie, N. E., Atkins, N. E. and Munch-Petersen, E. 1944. A note on a lytic phenomenon shown by group B Streptococcus. Aust. J. Exp. Biol. Med. Sci. 22:193–195.

Claxton, P.D. and Ryan, D. 1980. Bovine mastitis bacteriology. Australian Standard Diagnostic Techniques for Animal Diseases. Pp. 1-8

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De, U. K. and Mukherjee, R. 2005.Status of nitric oxide production by polymorphonuclear cells during clinical mastitis in lactating cows. Indian J. Comp. Microbiol. Immunol. Infec. Dis., 26(1): 61-62.

De, U. K. and Mukherjee, R. 2008. Anti-inflammatory and respiratory burst activity of Azadirachta indica during acute bovine mastitis Indian J. Anim. Sci., 79(2): 32-34.

De, U. K. and Mukherjee, R. 2009. Expression of cytokines and respiratory burst activity of milk cells in response to Azadirachta indica during bovine mastitis. Trop. Anim. Health Prod., 41(2): 189-197.

De, U. K. and Mukherjee, R. 2009. Inhibitory response of Azadirachta indica extract on nitric oxide production by milk leukocytes during clinical mastitis. Vet. Arhiv, 79(1), 41-50.

De, U. K. and Mukherjee, R. 2009.Prevalence of mastitis in crossbred cows. Indian Vet. J., 86:858-859.

De, U. K. and Mukherjee, R. 2011. Potential of Indigenous Enzymatic Activities, Nitric Oxide and Ceruloplasmin in Bovine Milk to Diagnose Subclinical Mastitis. J Veterinar Sci Technol 2:109. doi:10.4172/2157-7579.1000109

Aquaculture is the culture of aquatic organisms per unit water area per unit time for maximum profit. The economically important finfishes and shellfishes, whose body physiology is adjustable to varied environmental conditions, are considered as aquaculture candidate species. So, an overall knowledge on the physiology of such species is required to improve the culture practices.

1.1. Finfish

A fish may be defined as a vertebrate, adapted for a purely aquatic life, propelling and balancing itself by means of fins, and obtaining oxygen  (O2) from the water for survival and life processes. As the fishes have fins, they are also known as finfishes. Fish describes a life from rather than a taxonomic group. They share certain features with other vertebrates. These are gill slits, a notochord, a dorsal hollow nerve cord and tail. In number of individuals, and may be also in number of species, fishes are presently superior to other vertebrates, including mammals. There are 20,000 different species of fishes. Fishes form largest group of vertebrates, Pisces. The various types of fishes differ so much in shape, color and size that it is difficult to believe they all belong to the same group of animals. Fish in general are cold-blooded and adjusted either to freshwater, brackishwater or marine environment.

The knowledge of food, feeding habit, anatomical adaptations and the physiology of digestion of the candidate aquaculture species is highly essential to develop cost-effective artificial feed for successful culture practices. The digestion of poikilothermic aquatic fishes and shellfishes is influenced by many factors, and is different from the digestion of terrestrial animals.

2.1.    Anatomy and Physiology of Fish Digestive System
2.1.1.  Feeding Behavior of Fishes

Based on the feeding habit, the fishes are generally classified as herbivores, omnivores, carnivores and detritivores. Within these categories, the fishes further categorized as euryphagous, having a mixed diet, stenophagous, consuming a limited combination of food types and monophagous, accepting only one type of food. A majority of fishes are euryphagous carnivores, and their feeding mode and food types are associated with the body form and digestive system. Fish generally change their feeding habits depending upon the availability of food. Fishes may be classified as predators, grazers, strainers etc. Plankton is a collective term for variety of marine and freshwater plant or organisms that drift on or near the surface of the water. Most fishes eat the material that is smaller than them. The smallest fishes eat these planktons, which act as their main food and all smaller fishes are the food of the bigger fishes. Largest shark feed on plankton by straining these tiny marine plants and animals from the water. The zoo-planktons, comprise protozoa, small crustaceans, jelly fish, and mollusks, together with the eggs and larvae of many species of marine and fresh water.

Embryologically, the circulatory system is the first organ system to develop in the living animals. The form and function of this system vary considerably among the animals and are shaped by a variety of environmental factors i.e. phenotypic plasticity. Oxygen (O₂) acquisition and carbon dioxide (CO2) elimination occur simultaneously at the gill site of fish and shrimp. The gases are transported around the body by the circulation of the blood.

3.1. Circulatory System of Fishes

In fishes, heart is simply a muscular enlargement and modification of a part of the main blood vessels, lying between the hepatic capillaries and the gills. Fishes have a closed circulatory system with a heart that pumps blood in a single loop throughout the body. From the heart, blood goes to the gill capillary bed. Then via the systemic capillaries it returns back to the heart. Usually the gills account for approximately 30% of the total resistance to blood flow, the rest being in the visceral and somatic vasculature. Tuna requires more O₂ and the gill area is large with gill resistance up to 57%.

Various activities of the body in different external environmental conditions are co-ordinated by the appropriate message from the neurons due to the stimuli from the environment. These messages pass in the form of impulses through the co-ordinated links between a number of neurons or nerve cells. The constant passage of messages require a very little energy but high level of O₂ .There is interaction between the nervous and endocrine system with the areas of interchange among the two systems. Some neurons show the action of both the systems and thus termed as neurosecretory or neuro-endocrine. Fishes have a well-developed nervous system. Broadly the nervous system can be divided into central nervous system (CNS) and peripheral nervous system (PNS).The brain and spinal cord are the parts of the CNS. The motor and sensory nerves link the receptor and effector organs. All nerve tissues other than brain and spinal cord are grouped under PNS. Peripheral nervous system consists of nerves, ganglia and receptors. The nerves carry sensory information from special receptor organs like eyes, internal ear etc. to the integrating centers of the brain and spinal cord. The PNS also carries information via different nerve cells from the integrating centers of the brain and spinal cord. This coded information is carried to the various organs and body systems, such as the skeletal muscle system for appropriate action in response to the original external and internal stimulus. The PNS can be divided into somatic and visceral. The term visceral is sometimes used in referring to the autonomic or involuntary nervous system (ANS), controlling visceral functions. The ANS helps to co-ordinate the activities of many glands and organs, and it is closely connected to the integrating centers of the brain.

The respiration of animals includes the exchange of gases either at the surface of the body or through the internal processes involving circulatory system. The gases that are exchanged are oxygen (O₂), carbon dioxide (CO₂) and the inert gas like nitrogen. When the organism respires, the O₂ that is taken up from the external environment is transported by the vascular system to individual cells within the tissues. At the same time this system picks up CO₂, an end product of cellular metabolism, and releases it to the environment. Thus respiration is a term applicable either to a whole organism or one of its cells. The mechanical process, by which respiration is achieved, is called ventilation.  This ventilation rate of aquatic organisms is controlled directly by the concentration of dissolve oxygen (DO₂) in water.

Respiration in aquatic medium is produced by a water current passing in a single direction. The area of respiratory surface is thus an important limiting factor in the movement and growth of the animal. For aquatic animals, if the water is at all deficient in O₂, the rate of breathing is naturally accelerated, and the animal appears to respire hurriedly. Other factors, which lead to an increased rate of respiration, are the stresses.

Excretion is the process of elimination of metabolic wastes that include toxic nitrogenous end products of the protein metabolism and the gas such as carbon dioxide (CO₂). These nitrogenous wastes are excreted through urine. Fecal matter is not included in the excretory products as it is simply the undigested food materials, and are not metabolic end products. The membranes of aquatic animals, that is permeable to gases, pass water and solutes between environment and body. Most aquatic animals maintain in their bodies a reasonably constant proportion of water and solutes. When it is different from the surrounding water, it requires some mechanism for regulation, which is known as osmoregulation. Most invertebrates in the sea have nearly the same salt concentration (isosmotic or isotonic) as the environment. Sharks and rays (elasmobranches) tend to have a slightly higher concentration of salts and urea (excretory end products) than seawater but the body fluids of most teleosts (bony fishes) have much lower salt concentration than seawater. Freshwater fishes, which live in a medium of lower salt concentration (hyposmotic or hypotonic), have the threat of hydration, so they have special mechanisms to get rid of excess body water. Marine fishes that live in an environment of high salt concentration (hyperosmotic or hypertonic) have the regulatory mechanisms by which excessive loss of water from the body is prevented and body osmolarity (proper osmotic concentration) is maintained by retaining less toxic nitrogenous excretory products. Marine fishes, which lose water osmotically across the gills and must get rid of salt, tend to drink large volume of seawater and produce very small quantities of urine (Fig-6.1).