Introduction

The ejaculates of most domestic animals contain more sperm than are needed for achieving pregnancies. Hence, by diluting the semen, it can potentially be used for several inseminations. In species such as the dog the horse, the whole sperm-rich fraction of the ejaculate Is diluted and child then either used for sequential inseminations of the same female over her extended estrus period or after various determinations of the fertile period ( CJEFCOATE and LINDSAY, 1989, BRINSKO and VARNER, 1993) in food animal species the ejaculate is generally diluted so that it can used to inseminate many females. In either case the maximum degree of dilution is determined from the minimum number of spermatozoa and the volume of inseminate that is required to acceptable pregnancy rate. These are themselves determined by the side of insemination the survival sperm in diluents and the idio syncrasies of individual species and sire. In general where an intrauterine insemination can be achieved the minimum number of sperm are one or two order of magnitude is lower than for an intracervical insemination. Hence where wide spread use of sires is required, get advantage exists in devising methods of achieving intrauterine insemination, even where, as in the ewe, this requires such a complex procedure as a laparoscopic insemination. (Watson, 1979) Objectives of extending semen: 1. To increase the volume of the ejaculate obtained from quality adult males, so that a large number of females may be mated from single does of his ejaculate. In natural mating one ejaculate is used to inseminate on female, where as by extention of the same volume of semen, several hundred( 500-1000) females can effectively be made pregnant. 2. Moreover , ejaculated sperm donot dervive for a long period and does to preserve fertilizing capacity of the spermatozoa for a long period various agents are added. The agents that comprise good extending media have the following functions: a. Provide nutrient as a source of energy. b. Provide lipoproteins and or lecithins to protect sperm cells against low temparature shock c. Then provide a buffer to prevent harmful shifts in PH as lactic acid is formed. d. Maintain the proper osmotic pressure and electrolytes balance e. Inhibit bacterial growth f. Provide a source of reducing substances to protect the sulfhydril containing cellular enzymes. g. Provide a buffer for a proper balance of minerals essential to the life of sperm cells.

h. Increase the volume of pure semen so that it can be used for multiple inseminations

Name of the different Extender:

1. Milk based extenders a. Whole milk b. Skim milk c. Reconstituted milk powder d. Milk whey 2. Egg, yolk extenders a. Egg, yolk citrate b. Egg-yolk- tries-fructose-citrate 3. Sodium citrate extender a. 2.9% sodium citrate dehydrate solution. b. Modified salisburys extender(EYGC ) c. Cornell university extender. d. Illini variable temperature extender(IVT) e. Tris buffer extender 4. Coconut milk based extenders 5. Commercial extenders a. Tomato broth b. Carro juice c. Caprogen d. Citrate acid whey 6. Anti-biotics 7. Glycerol 8. Fructose

Use of different extenders in case of liquid semen:

Glucose :- Provides energy Egg-yolk and milk :- Protect against cold shock of the sperm cells as they are cooled from body temperature to 5 C and providing lecethins, proteins, lipoproteins and similar compounds found in egg yolk or milk. These substances also contain nutrients utilized by sperm. Buffers :- A verity of buffers are used for maintaining a neutral pH and an osmotic of approximately 300 milliosmoles, which is equivalent to that of semen, blood plasma and milk. A satisfactory extender for one animal or one set of condition may not be acceptable for others. This has resulted a number of diluents of various species.

Antibiotics:- Penicillin, streptomycin, polymycin-B or other combination of antibiotics that cover a broad bacterial spectrum are used to enhance the keeping quality of semen. Procain penicillin is toxic to spermatozoa. Antibiotics will not completely eliminate Corynebacterium pyogenes, Bucella, Trichomonous foetus, Mycobacterium, Rickettsia and fungi. Addition of glyceraol further reduces the efficacy of antibiotics. Hence addition of antibiotics is not an extra precaution and can not be taken as an effective tool to permit use of semen from infective bulls or unhygienic semen processings. Glycerol:- Most widely used cryoprotective agent for bull spermatozoa particularly for frogen semen. The exact mechanism and the lake of activity is not clearly understood.The possible mods of action proposed are i. Modifies size and shape of ice crystals formed. ii. Binds Water and decreases frizzing point of solution and less ice is formed. iii. Prevents denaturant of proteins and rupture plasma membrane. iv. Reduces the soluble concentrations Fructose:- Provides glycolsable substrate for the sperm, prevent sperm agglutination, maintain required osmotic tension and electrolytes balance and gives added cryoprotection during deep frizzing

Use of different extenders in case of frozen semen:

The idea that the sperm cell will remain in living without expenditure of any significant metabolic energy for an indefinite period inspired the scientists to preserve living cell in the frozen state. Further it was the spectacular descovary of Polge in 1949, which showed that the death of spermatozoa on freezing could be avoided by suspending the cells in a medium containing glycerol. Thus a practical answer to freezing technique was evolved. At least 50 percent of the bull sperm from an unselected popilation ramins inactive or dead during freezing and thawing due to 1. internal ice-crystal formation which affects the structure of the spermatozoa 2. The increase in solute concentration as water is withdraw from the suspension medium 3. By interaction of these two physical factors, an ideal extender should quality for giving cryoptection during freezing. For bovine spermatozoa the basic components for an ideal extender are should quality for giving cryoprotection during freezing. For bovine spermatozoa the basic components for an ideal extender are:

Buffers: out of varieties of inorganic and organic buffer which have been tried for deep freezing of bovine semen, an organic buffer known as tris (hydroxymethyl aminomethane ) frist used by Graham ( 1972) at Ph 7 which has been found to most popular ven to day the buffer can be successfully used for bull, buffalo, ram, dog, poultry and even for human. It 1. Prolongs life of sperm at room temperature. 2. penetrates cells 3. acts as intracellular buffer 4. less toxic during freezing and 5. provides better clarity under microscope Fructose: addition of fructose provides 1. lycolysable substrate for the sperm 2. prevents sperm agglutination 3. maintains required osmotic tention and electrolyte balance 4. aids extra cryoprotection during deep freezing Glycerol: the possible use by which it functions are1. Modifies size and shape of ice crystal of the media 2. Binds water and decreases freezing point of solution resulting reduction of ice crystals 3. Acts trough salt buffering mechanism 4. reduces solute concentration 5. prevents denaturation of protein and thereby protects plasma membrane Egg yolk : 1. When Mammalian sperms are cooled to 5 C they are subjected to cold shock which causes leakage of intracellular enzymes, minerals, lipoproteins, ATP from inside the cell. Lecithin, proteins lipoproteins and other similar compounds present in yolk provides an effective means of guard against cold shock. 2. In addition to this the valuable nutrients of egg yolk are very slowly utilized by sperms. 3. Egg yolk also protect enzymes having disulphide bridges(SH group) as well as of anti-agglutinic factor present in seminal plasma. Antibiotics: Semen under normal condition generally get contaminated with both pathogenic and non pathogenic microorganisms. Organims which are contagious and can infect cow through pathogens are: Brucella abortus, Vibrio fetus, Tricomonous foetus, Leptospria Pomona, Mycobacterium tuberculosis, M. Paratuberculosis,( Johnes disease) and viral disease that cause infectious bovine rhinotracheitis (I.B.R), Foot and Mouth disease(F.M.D)

Refrigeration greately suppresses the multiplication of organisms but does not necessarily stop it. Organisms which survive freezing temparature of 190 C are Pseudomonous aeruginosa,Tricomonous foetus, Leptospria Pomona,Foot and Mouth virus and infectious Pastulla vulvovaginities virus. Penicillin and streptomycin are the two antibiotics widely used since 1950 and are still in used. They are found to be relatively hampless to sperm cells and perticullarly in combination, inhibit broad spectum microorganims. 500-1000 I.U. of crystalline penicillin-G and 500-1000 micrograms of dihydro-streptomycin per ml of extended semen is enough for routine use. Procin penicillin is toxic to the spermatozoa. Antibiotics will not completely eliminate Brucella abortus, Vibrio fetus, Tricomonous foetus, Leptospria Pomona, Mycobacterium tuberculosis, M. Paratuberculosis,( Johnes disease) and theyn will have no effect of viruses, rickettsia and on fungi. Hence addition of antibiotics is not a 100% safety from phathogenic contamination of semen. Semen from healty bulls managed under full hygienic environment and handled by an experienced hand is always preferred. SEMEN EXTENDERS FOR ARTIFICIAL INSEMINATION WITH FROGEN SEMEN Page= 325

COMPOSITION OR PREPARATION OF DIFFERENT EXTENDERS:

For many years the emphasis in A.I programs was on the use of liquid extended semen generally preserved at 5 C for 2-4 days for the purpose of increasing usefulness of superior sires. An average bull in commercial A.I service is currently being used to make pregnant to couple thousand cows annually in contrast with only 50-60 cows normally bred to bull under natural mating conditions annually. A number of media have been developed that provide adequate nutritional components( PH 6.5-6.7) , buffering capacity and protection against bacterial contaminants and tempareture shock. The semen and extender are initilally mixed together at the same temparature. While preparing extenders either for liquid or child semen or for frozen the following basic principles should be borne in mind: 1. |Accurate weighing of various recommended ingredients of the dilutor to provide exact osmotic tension, PH and electrolytes levels.

2. Exclusions of toxic materials and simultaneous inclusion of protective components. 3. Rigid control on pathogenic contamination. 4. Decide dilution grade so as to provide optimum number of viable spermatozoa per does at the time of insemination. 5. Maintain absolute sanitation of the room, technician, all metal and glass equipments and use quality reagents.

EXTENDERS FOR BULLS

Based on the various composition of a extenders used for bull semen they may be classified as below. What ever may be diluents, always add diluent to semen, never the reverse, as this may cause shock to the spermatozoa and reduce their motility. A. Milk or Skim milk based B. Egg-yolk besed C. Coconut milk based D. Other commercial extenders A.MILK BASED EXTENDERS: In recent years boiled whole milk, skim milk , reconstitute milk powder and milk whey diluents have been employed in commercial A.I. These diluents are economical, easy to prepare and provide to protection to spermatozoa. The foremost objection to whole milk as diluter is that the fat globules make microscopic examination difficult. Use of skim milk or even homogenized milk or milk of goats which is naturally homogenized can partially solve the problem. Fresh milk has got lactenin, and antibacterial substance present in milk albumin also exerts effects on spermatozoa which if heated to about 92 95 for 10 minutes will reduce sperm killing activity to a great extent. Greater success have been clamed with milk diluents from Pakistan. In India that diluents designed by Girish Mohan(1974) using low fat cow milk heated up to 93 C for 20 minutes in a water bath followed by over night cooling in a refregerator and removing fat particles by filtering through cotton plug gave improved results comparable with egg-yolk citrate dilutors. STUDIES AT TEXUS A. AND M UNIVERSITY indicates that evaporated milk can be used with glycerol as extender in freezing stallion semen. B. Egg yolk extenders : In 1934 Milovanov observed the benifial effects of adding egg yolk to extending fluids.

Today, egg yolk sitrate is one of the commonly used semen extenders. Only the yolk portion is used because egg white contains a substance ( lysozyme ) toxic to spermatozoa. The eggs used to provid the yolk should not be more then 4 to 5 days old. The shell of the egg should be thoroughly washed with warm water, sterilized by wiping with a piece of cotton wool moistened with alcohol and dried. After cracking the egg and separating the white, the yolk is placed on a filter paper is such a way as not to rupture its membrane. All white can be removed from the yolk by gently rolling the yolk on the filter paper. The yolk is then run into a sterile beaker by folding and squeezing the filter paper. The membrane the yolk should remain of the filter paper and be discarded. Before adding it to the diluents, the egg yolk should be mixed in beaker with the aid of a sterile glass rod, specially when it has been obtained from more than one egg. The eggyolk tris fructose citrate diluent should be fressly prepared on each day of before use. a.a sodium citrate extender Salisbury et al. (1948) formulated the dilutor having 2.9% sodium citrate dihydrate solution with equal volume of egg yolk. The extender is very popular for use in bull semen. The medium maintains semen fit for insemination for 72 to 96 hours at 4 to 7 C. more over it gives clearer view of the microscopic examination and proved to be more efficient due to incorporation of antibiotics. Original egg yolk citrate extender has first been modified by Salisbury by adding glucose solution and later on by number of other scientists by in corporating glycine and other ingredients. Such modification which also include reduced proportion of egg yolk from 50% to 20% resulted into better keeping quality of semen, economy and efficiency even at higher dilution rate. a.bModified Salisburys diluent ( EYGC ) The dilutor is now known as egg yolk glucose sodium citrate diluent. The method of preparation is as follows a) Dissolve 2.9 g of crystalline sodium citrate dihydrate (Na2C6H5 O7 2H2 0) in 100 ml of glass distilled water--------50 parts b. Dissolve 5 g of Analar grade glucose in 100 ml of glass distilled water---30 parts c. Egg yolk --------------------------------------------------------20 parts d. Add 1000 I.U. of crystalline penicillin and 1000 micrograms of dihydrostreptomycin per ml just period to addition of semen to dilutor. To prepare the egg yolk for the egg yolk buffer extender, fresh yolk of the hens egg is always preferred. AT first fresh egg should be washed water and wiped with 70% alcohol. After allowing the egg to dry, the shell is broken in with a strile knife into two equal halves with one half holding the yolk, the excess albumin being collected in a beaker. Transfer of the yolk between the two halves of the shell will allow most of the albumin to be separated off and the yolk is then carefully transferred to a beaker

containing distilled sterile water at a temperature of about 50 degree C. The traces of albumin adhering to the yolk will coagulate, the water is poured out in the egg yolk transferred to a clean filter paper. Rolling back and forth on this paper dries the surrounding membrane which is then ruptured. The yolk is then allowed to flow in to a measuring cylinder, remaining the membrane on the filter paper. The egg yolk citrate buffer is usually prepared fresh each day before use. 3. Cornell university extender(CUE): in USA Cornell University workers , FOOTE* and BRATTON(1960) formulated a diluent termed Cornell university extender which permited longer storage at 5 C is narrated below: a.c Sodium citrate dihydrate (g)..14.5 Sodium bicarbonate (g)...2.1 Potassium chloride (g) 0.4 Glucose (g) ..3.0 Sulphanilamide (g) 3.0 Glycine (g) 9.37 Cytric acid (g) ..0.87 Distilled water (final volume) (ml) .1000.00 b. Add 20% (by volume ) the egg yolk and 80 % of buffer solution ( a) along with penicillin and dihydrostreptomycin at @ 100 I.U. and 1000 microgram per ml of final extender. The extender although proved a better type of extender but since it has many components and is requires careful weighing and adding and therefore found to be less popular for routine use. 4. ILLINI variable temperature diluent(IVT): Van Demark and Sharma (1957) developed the first successful techniques of preserving of diluted semen at room temparature and termed in the Illini varible temparature (IVT) technique and the medium, IVT medium. The diluent is made up of a. Sodium citrate dihydrate (g)..20 Sodium bicarbonate (g).2.1 Potassium chloride (g) 0.4 Glucose (g) ..3.0 Sulphanilamide (g) 3.0 Distilled water (final volume) (ml) .1000.00 b. Above buffer mixture is gassed with CO2 until the PH riches 6.3 c. add 1000 microgram of streptomycin and 1000 I.U. penicillin per ml. Along with 10% egg yolk. The semen diluted in this diluent is stored in one ml ampoules in dark at room temparature. As high as 75 % conception rate was reported from 111 insemination carried out with semen stored at room temparature for 7 days.

The IVT diluent has received wide attention in different parts of the world. The result are not consistent. Ulaganathn (1970) observed that the sperm survivability in the IVT diluent improved by the addition of ascorbic acid. Further investigation is necessary. 5. Tris buffer diluent: It is used as common extender for frozen semen. a. Tris hydroxy methyle amino methane3.97 g * professor R.H. FOOTE was the minor advisor of the author while he was persuing his Ph.D. studies at Cornell university during 1966 1970. Citric acid.1.72 g Fructose. 1.27 g Distilled water 99.13 ml (PH adjusted to 6.8 with citric acid or NaOH) b. Tries buffer, as in (a)74 ml -egg-yolk .20ml Glycerol.6 ml c. to a diluent at penicillin at a dose of 1000 I.U and streptomycin at 1000 microgram per ml. C.COCONUT MILK BASED EXTENDERS: The use of coconut milk (water) extender (CME) for the preservation of active sperms for 7 10 days at room temparature was first worked out by Norman et al. (1960, 1962). It is made up of sterilized coconut water and a 4.3% sodium citrate solution plus 100 mg percent CaCO3, 80 mg percent penicillin and 90 mg percent streptomycin. The PH is adjusted to 7.4 with 10 % percent solution of NaOH. The same workers in 1962 observed that CME to which 5% egg-yolk was added maintained motility better than EYC and skim milk extenders. Exposure to light produces photo oxidation wichi results into poor motility and depression of metabolic rate. TOMAR and SHARMA (1974) modified the CME by select the following components: 2% Sodium citrate dihydrate solution.50 ml coconut water .25 ml Egg yolk...12.5 ml Egg albumin.12.5 ml Limited availability of superior quality coconut water all round the year and all over the regions in India restricts its popularity. D.OTHER COMMERCIAL EXTENDERS: Fruit and vegetable juices have been used in some countries for extending bovine semen, with apparently acceptable results. Such juices as tomato broth and carot juice, which are abundant and economical have been used.

In New Zealand an ambient temperature extender, known as Caprogen has been found very effective maintaining maximum fertility for 24 hours period. The extender includes caproix acid in an IVTCUE type of extender followed by gassing with Nitrigen. Recently at N.D.R.I. Karnal in Haryana , a diluent named as Citrate Acid Whey (CAW). At PH 6.8 found to be the best diluent for the preservsation of buffalo semen when CAW was compared with EYC, former could preserved the sperms for 5 days at refrigerated temparature (5 C) with 60 % motility as against 2 days in the case of the later diluent. The CAW is now available in packet containing cow skim milk powder and citric acid together. The entire content of the packet is dispersed in 100 ml distilled water . The curdled materil is filtered after 10 minutes through cotton plug and the PH is adjusted to 6.8 using 10% NaOH. The preparation is now ready for diluting semen. CAW packet can be preserved for about 8 months but ones is prepared, it can not be stored more than a day in arefrigerator as it losses its potency due to microbial fermentation of wheep. Similarly another successful buffalo extender has been developed in Pakistan where they suggested use of homogenized milk as diluent for buffalo semen.

COMPREHEBSIVE MORPHOLOGY OF SPERMATOZOA OF BUFFALO AND CATTLES PAGE 312

EXTENDERS FOR SHEEP AND GOAT: The diluent is commonly used for dilution of ram semen are 1.Tris or 2. Citrate as buffer, glycose or fructose as the energy source and egg-yolk protect the sperm cell membrane against cold stock. This diluents are also used for goat semen, but with a reduced egg-yolk content to avoid reaction which occurs due to egg yolk coagulating enzyme present in the seminal plasma of gaot ,bucks and it is higher when semen is collected by electro ejaculation. The problem can be over come by one of the following methods. a. Using a low concentration of egg yolk in the diluent. b. Using a medium containing no egg yolk( for example milk) , or c. Removal of seminal plasma and thereby the enzyme, by centrifugation. Composition of the two most common recommended diluents are given below: a. Egg-yolk-tris-fructose diluent Ram Goat buck Tris (hydrooxy 3.634 3.634 methyle) amino methane (g) Fructose 0.50 0.50

Citric acid/ 1.99 monohydrate (g) Egg-yolk (ml) 14.00 Glass distilled 100 water 3. Egg-yolk-glucose-citrate diluent: Ram citrate 3.634

1.99 2.5* 100

Sodium (2H2O) (g) Glucose (g) 0.8 0.8 Egg-yolk (ml) 20.00 2.50* Glass distilled 100 100 water To a diluent add penicillin at a dose of 1000 I.U. and streptomycin at 1000 microgram per ml when using above two diluents for buck semen, the coagulation reaction will not occur if the concentration of egg-yolk does not exceed. 2.0 percent after dilution. These should be considered during calculationg the dilution rate of the semen. With the above egg yolk concentration there will be no harmful effect provided in the buck semen is not diluted more than 1+3 (semen + diluent) if a greater dilution rate is required, the amount of egg yolk should be reduced accordingly. When preparing the above diluents , first the chemical should be weighed and dissolved in a 100 ml capacity measuring cylinder by adding 75 80 ml of glass distilled water (Ram) or 90-95 ml (buck). After addition of eggyolk and making at the final volume (100 ml) with distilled water the diluent is mixed by rocking the measuring cylinder several times backwards and forwards to obtain an even distribution of the egg yolk.

Goat buck 3.634

Extenders for Boar semen:

The following extenders are commonly used for extending boar semen A. Sodium citrate (g)..20 Sodium bicarbonate (g)...2 Potassium chloride (g) 0.4 Glucose-D (g) ..3.0 Sulphanilamide (g) 1.0 Penicillin (million) 1.00 I.U streptomycin (g) ..1.00 Distilled water (final volume) (ml) .1000.00 PH 8- 8.2 at 35C

B. Glucose D :..120 g Ethylenediamino acetate :7.4 g NaOH(Basic soln. 4%)..16 g Sodium citrate 35.5% 20 ml Penicillin (million) 100,0001 I.U. streptomycin (g) ..0.5 g Distilled water (final volume) (ml) .2000.00 C. Glucose D40g Sodium bicarbonate1 g Sodium citrate 3.8 g Cheloplex Helaton2.6g Egg-yolk 100 ml Combiotic2.5 ml Glass distilled water 1000ml D. Glucose D120 g EDT-A7.4 g Sodium bicarbonate 2.4 g Sodium citrate 7.5 g Penicillin100,000 I.U. Streptomycin 1 g Glass distilled water 2000 ml The above dilutors can preserve boar semen for 3-6 days at a temperature of +10C - +15 C EXTENDER FOR STALLION: It is very much practice to use undiluted stallion semen for insemination mares when the numbers of mares to a stallion is very limited.When large number of mares are assigned to one stallion, the semen has to be diluted and extended for use either for single or repeted insemination. On account of the sensitivity of the stallion sperm it is recommended that dilutor at body temparature should be added to semen by slow rotating movements as soon as collections is obtained. Before this operation the viscous fractions is seprated from the sperm bearing fraction Diluted semen is held in water bath at 20-27C and is used for insemination with in few hours of the extension. When egg-yolk dilutor is used the cooling process can be very rapid. The entire diluting semen is then placed in a refrigerator or into thermoflask containing ice water at a temperature of 5C. The compostion of a most effective diluter is as follows: Glucose..........................................................30g Lactose...........................................................20g Sodium potassium tartrate..............................10g

Glass distilled water ......................................1000ml Fresh egg yolk................................................200 g Para amino bengoic acid................................6 g The egg yolk is freed of albumin memebrane and chalazae. The dilution rate varied from 1:3 1:5. Farely good result obtained after 48 hours storage and long transport

Cryopreservation
Longer-term storage of semen is achieved through cryopreservation. Cryopreservation maintains the fertile life of semen virtually indefinitely, although a large proportion of individual spermatozoa fail to survive the considerable stresses of freezing and thwing. For sperm to survive freezing, they need to be extended in a diluent that not only contains substances that protect them against cold shock, but also cryopreservation, such as glycerol, which protect them from the deleerious consequences of freezing. The general responses of cells to freezing (reviewed by Farrant, 1980, Watson, 1990) were not understood until long after empirical methods of cryopreservation had become widely adopted. Imitially , as the temperature of the external medium falls below its freezing point, crystals of pure water start to form. The concentration of solutes in the unfrozen part of the medium therefore rises as, in consequence , does its osmotic pressure. Thus, the intracellular contents undergo a period of supercooling, during which thecells loses water to the unfrozen part of the extracellular medium by osmosis. A variable degree of cell dehydration follows, which is terminated by the formation of intracellular ice crystal. Thus , damage can occur to cells in one of two ways.Where a substantial degree of cellular dehydration occurs intracellular water can be damaging whereas, if only slight dehydration occurs, large ice crystals can form within the cell, which cause physical damage to its internal and bounding membranes. The degree to which each affects the cell is determined by the rate of cooling the slower the rate the more dehydration the faster the rate the greater the damage by ice formationare the size of the cell, such that the larger the cell the slower its inherent rate of dehydration. Cryopreservation agents may either penetrate or remain outside the cell, but both act by binding water outside the cell, but both act by binding water and therefore alter the availability of water either for dehydratative loss or for ice crystal formation. Penetrating cryopreservation, such as glycerol or dimethyl sulfoxide(DMSI) appear to not only reduce the loss of water from the cell, therby reducing solute damage, but also bind it in a form that renders it unavailable for crystal formation thereby reducing the effects of intracellular ice. Non penetrating cryopreservation, such as disaccharides or proteins, may hasten dehydration during vary rapid cooling, thereby minimizing intracellular ice formation. Notwithstanding the aforegoing, precise understanding of the mode of action of cryopreservation remains elusive, and much information relating to their paractical use remins empirical.

Glycerol(Polge et al, 1949) is the main primary cryopreservation usded in preparing mammalians semen for freezing (Watson,1990), despite the fact that it has some directly toxic effects upon sperm (Watson, 1979). The concentrations used depend upon speceies and the other components of the diluents. For expample, diluents for bovine semen that contain disaccharides can utilize lower percentages (3-4%) of glycerol than diluents that lack such disaccharides, which have a final glycerol concentration of at least 7% (Unal et al. 1978). Whether the toxic effects of glycerol are exacerbated at high temperatures has been a matter of debate. Certainly Polge(1953) considered that the addition of glycerol was more damaging to bovine sperm at 28C than at 4C, although Salisbury et al (1978) reviewing the (by then ) copious literature concluded that the effects of temparature pf glycerolization were equivocal. Nevertheless, the practice in commercial bovine. AI centers is, in general, that where the final concentration of glycerol contrations is required to be high, a primary dilution of the semen is made with a diluent containing little or no glycerol, with glycerolization being carried out after reducing the temperature to 4C, whereas diluents which utilize lower final concentrations are added in one step, at 30C. With boar semen, by contrast, the toxicity of glycerol at high temperatures is much less equivocal, and low temparatures glycerolization is desirable (Paquignon, 1985) Originaly, diluted semen was palaced in galss ampoules for freezing in a mixture of alcohol and solid carbon dioxide at 79C, or drops of diluted semen were placed directly onto the surface of a block of solid carbon dioxide where they in pellet form (Salisbury et al, 1978). Lond-term storage at 79C was not satisfactoroy, however, as deterioration occurred at that remperature (Pickett el al, 1961; Sewart, 1964). Storage in liquid nitrogen at 196C has sub-sequently become established as the standard medium for long-term preservation of semen and , over the 40 years for which it has been practiced, has maintained sperm fertility unscathed. At the present time, semen is frozen is one of two main ways. Diluted semen is packed into thin, plastic tubes of 0.25 or 0.5 ml capacity, then in the simpler techniques, these tubes then are suspended in the vapour from liquid nitrogen, which is at about 120C, for about 10 minutes (Cassou, 1964), Jondet, 1964). The straws are then pluged into the liquid nitrogen . More recently , a greater degree of control of freezing rate has been exercised by the use of microprocessors-controlled freezers which improvedc sperm survial justifying the increased cause of the processing( Eg:Landa and Almquist, 1979; Parkinson and Whitfield, 1987). Drawing of the semen needs to the rapid:- slow thawing allows recrystalization of ice with in the cells, causing membrane damage. In practise the rate of drawing is rarely critical. The surface area -to -volume ration of the 0.25ml tubes being so great that any temparature of the thowing water between 0-40 C will thaw adequately. Of greater importance is the temparature control of the thawed semen. This should not be allowed to cooled below the final temparature achieved thawing, other wise substantial sperm losses can occur. Rethawed spermatozoa are as sensitive to fluctuation in temparature as are their unfrozens counter parts( Roberts, 1986)

DEEP FREEZING OF SEMEN

In India artificial insemination (AI) utilizing liquid(chilled) semen from exutic dairy breeds has been playing vital rules for nearly 4 decades. The event of Forzeen semen technology has by this time become reamarkable by any measure in turning this century to the great way of white revolution. As early as 1938 a german scientist, JAHNEL found that by deep freezing testicular tissue of rabit, sperm could survive freezing to 192C. During 1949 Polge, Smith and Parkes accidentally added 15-20 % glycerol to a fowl sperm solution and observed very good motility after freezing to 79C. Thus the remarkable peroperty of glycerol in maintaining life of deep frozen sperm were subllished. Later on it was observed during the resistance against deep freezing is quite different in different species. It is known that bull semen can not with stand a sudden drop of temparature so called temparature shock. For that region Polge and Smith (1950) cooled the glycerol treated bull semen slowly to 79 C and got a much better survival of the semen than that by rapid cooling. The first insemination with deep frozen were performed by Steward(1951) in cooperation with Polge and Rowson. Out of 5 inseiminated cows one become pragnent. Subsequenlt developments have been the introduction of new freezing techniques, improved semen dilution and additives.

Principles of deep freezing:

The general principles of deep freeing of semen is to dilute the semen with an extender containing glycerol. After equilibration the suspension is frozen according to special rules and thereafter stored in liquid nitrogen (-196C) as the cooling medium. As an intra and extra cellular water of media of crystallized as ice, the salt get concentrated in the recidula fluid which becomes progressively m ore hypertonic thus the sperm cell become subject to severe osmotic stress and in particular to Increase concentration of electrolytes when the water is freezing out. Glycerol has the ability to attach itself a considerable quantilty of water which Is then not available to form ice but can still act as solvent. The use of glycerol therefore enables freezing to be carried out slowly enough to avoid thermal stock without exposing the sperm cell to lethal concentration of electrolytes.

MARITS AND LIMITATIONS OF FROZEN SEMEN:

Merits: 1. Frozen semen permits maximum utilization of semen form a given sire. No semen need to be discarded because of age, as white liquid semen.

2. Long term preservation removes barrier on time and distance in AL international semen transport has become into existence today. 3. Can be undertaken for: a. Selective breed b. Grading c. Cross breeding d. Pregnancy testing of bull. 4. Transportation costs of semen are minimized compared to liquid semen usage, as large consignments can be delivered at infrequent intervals and full utilization can be maded each bull semen producing capacity. 5. minimizes requirement of breeding bulls to be maintain at breeding farm. 6. provide round the clock bredding facility even at the vilagges 7. frozen can safely be used in an out breack of disease( food and mouth ).without dislocation in semen transport. 8. complised with hight hygienic standard in artificial breeding. 9. comperative studies on cost factor between liquid and frozen semen has been done at National Dairy Research Institute , Bangalore. It has been observed that the average cost of A.I with liquid semen came to Rs 60.85 as copared to frozen semen which is Rs. 18.63 . further the cost involve per calf born was Rs 235.47 and Rs 53.13 respectively in using liquid and frozen semen. 10.frozen semen is extremely valuable in carring on the influence of superior sires even after 50 100 years of their death. Limitation of using frozen semen 1) about 30% of bulls semen does not with stands the rigor of freezing. The cost involved initially is high. 3) Frozen semen limits number of sires used, if our selection method are not perfect.this may lead to wider damage among future progenies. 4) Requires sound technical experts for handling the frozen semen. METHODOLOGY FOR FREEZING SEMEN 1. Preparation of diluent: Duluent always prepared fresh, henerally an hour before collection of semen so that the medium gets stabilized. As has been mentioned earlier, Tris yolk glycerol dilutor is the choice of preference for frozen semen.. It is prepared by mixing: Fresh egg yolk 1 part or Tris buffer * parts or Glycerol 7% or * Compostion of Tris buffer Tris (Hydroxy methyl amino methaneh) 20 cc 80 cc 7 cc 30.481 g

Citric acid Fructose Glass distilled water Penicillin Streptomycin

17.000g 12.500 g 850 ml 10 bcs 1g

The diluent is kept ready for in water bath at 30C for further dilution. As far as possible egg should be fresh(not more than 7 days old) and stored in refregerator. Eggs should never be water washed since egg is covered by a thin protective membrane which disappears on washing and thereby allows microbes to enter into egg through the micro pores on the shell. 2. Collection of semen The bulls center should have a schedule to prescribe days for collection from each bull. The usual practise is to collect semen from a healthy bull ones a week and twice from young bulls all of which is should be sound, having good libido and sexual behaviour. Semen collection should be made during early morning hours before feeding is done. The teaser cows should be well cleaned before she is brought to the service crate. It is better to tie her tail with a rope on one side so that swinging of the tail does not cause any obstruction in proper mounting. The dummy should not be taller than the bull to be collected. The artificial vagina prewarmed with hot water and refilled to bring the inner temperature of 40 C 46C when ready for use. Half to two third capacity of the A.V. should be filled with warmed water. The inflation of air should be done in such a way that the opening of the A.V. should on the graduated galss tube to avoid temparature shock. During collection , A.V should be held at 45 angle to the groud, so that the long axis parallels the line of the penis. Excessive bending of the penis before or during the thrust can distruct and injured the bull. The A.V. should be handled in such a way that the penis just touch the lining. The condition inside A.V. should equal to that of inside the natural vagina and this condition stimulates the ejaculation. The vagina must never be pressed on the penis. Generally 15 minutes of teasing and one or two false mounts will suffice. 3. Evaluation of semen: Immediately after collection of semen is evaluated for volume, colour, consistency and presence of foreign matter. One drop of semen is placed on glass slide and is evaluated under phage contrast microscope for initial forward motality. Samples showing more than +3 are taken for further processing . From this , 0.1 ml of semen is taken for estimating concentration of spermatozoa either by photoelectric colorimeter methods or by haemocytometer counts. Till the final dilution rate is decided semen is prediluted to a known low volume of extender and stored in water bath. The individual motility of the

sperm is assessed with the diluted semen using phase contrast microscope. Sample showing more than 60 % of motility is taken for further processing.

4. Dilution of semen: The basic idea of dilution of semen is 1. To preserve fertilizing capacity of the spermatozoa for a long period and 2. to increase servies to number of females. The dilution rate is decided based on the motility rete, sperm concentration and capacity of straw used. In general a minimum of 30 million motile sperms in each dose is recommended. As soon as the final volume of diluted semen has been determined the remainder of the diluent is added and the flask containing diluted semen is gradually cooled to +5o C in a refrigerator over a period of 45 to 60 minutes. 5. Equilibration of Semen Before storing the diluted semen at frozen state (-196oC), the practice is to preserve it at much higher temperature (+5oC) for about 6 hours. This pre-freeze storage of diluted semen is known as 'Equilibration' of diluted semen. This is so done for the glycerol to bring about its beneficial action on the spermatozoa. Glycerol permeates sperms and gives better resistance to withstand freezing stress. During this equilibration period, filling of straws with diluted semen is carried out inside a cold handling cabinet maintained at +5oC. Equilibration period of 6 hours is generally practiced. At the end of equilibration period, the per-freeze motility is again recorded. Sample showing more than 60 per cent motility are taken for "Test freezing". This is done by filling processed diluted semen in 2 to 3 straws for each sample which are initially kept either in big semen containers or in mini-freezer firstly in liquid nitrogen vapour having a temperature between -120 to -130oC and then plunged in liquid nitrogen. The entire treatment will take about 10-15 minutes. After this the revival rate is determined and satisfactory samples are taken up for filling and further processings. 6. Different Packaging System of Frozen Semen Packaging frozen semen in various single dose containers for storage and delivery systems started in U.S.A. since 1954 where use of glass ampoules was first developed. Japanese scientists developed a technique for freezing semen in pellet. French, Danish and German were responsible for introduction of straw technique. Other packing techniques such as pipettes, bulk sausages, gelatin capsule etc. have been reported for frozen semen without any acceptance due to high cost and lower fertility. (i) The ampoule Method

This method was mainly used in U.S.A Semen is placed in glass ampoules, sealed, frozen and preserved in liquid nitrogen. For insemination the ampoule is thawed in warm water, ampoule is cut, semen drawn into glass catheter and used as liquid semen.

Merits
(1)Since the semen is sealed inside ampoule, contamination during storage is avoided.
(2) Identification of sample is possible as the bull no. etc. and be marked on the ampoule.

Demerits
(1) As the semen is frozen in a larger volume the freezability and fertility are less. (2) It occupies more storage space in frozen semen containers. (3) About 8-10 per cent semen is lost while handling semen at the time of thawing and insemination. (4) Use of glass catheters for Artificial Insemination has several dawbacks as experienced with liquid semen. Hence this method is not popular (ii) The pellet Method Freezing is done by depositing 0.1 to 0.2 ml of semen on the depressions created on the dry ice (solid CO2). The semen gets frozen and after 10 minutes the pellets (like tablets) are collected in a goblet and stored in liquid nitrogen a -196oC. At insemination a pellet is dissolved in 0.9 ml of warm diluent and used just like liquid semen. Merits: (1) Economical method (2) Occupies less storage space. Demerits :

1. Identification is difficult. 2. As the semen is stored uncovered they may get contaminated with organisms if present in liquid nitrogen or other pellets.
3. When the pellets are handled by forceps they break and get attached to forceps and results in loss of sperms. 4. The freezability is moderate. 5. Cumbersome procedure involving the necessity of separate thawing solution, glass rod for Artificial Insemination etc. Thus the potential for spread of disease is high and the pregnancy from wrong bull is not uncommon with this technique. (iii) The Straw Technique

Plastic straws were introduced in Denmark in 1940 for packing liquid semen. The first technique forfreezing of semen in straws using liquid nitrogen vapour was developed by Adler in 1960. These techniques were later modified

and refined by Cassou in 1965 and most of the procedures currently employed with 'French straw' made of polyvinyl chloride was introduced. In the year 1972 a plastic straw called 'mini tube' or German straws or ' Lanshut system' was developed in West Germany. A straw known as 'U.S.' or Continental straws made of polypropylene was developed in United States.
Straw technique has got several advantages over pellet or ampoule methods and hence it is popular all over the world. Merits: 1. Semen is processed in thin film, with its greater surface area to volume ratio, there is rapid heat exchange and better revival rate. 2. Reduced volume of semen is better tolerated by uterus. 3. The diameter of A.I. catheter is small and hence passing cervix in heifers and cows in late heat is easy. 4. Complete delivery of semen into the uterus is possible. 5. Identification of sample by colour of straw, colour of PVA sealing powder or coloured plastic beads is possible. 6. Use of steel gun is safer compared to glass catheter. 7. Use of disposable plastic sheath for each A.I. excludes spread of disease during Artificial Insemination. 8. This occupies less storage space compared to ampoules. 9. With full automation for filling and sealing, it meets hygienic standards in semen processing. The dimensions of French and German Straws are given below: Length Diameter Volume French Medium 135 2.8m.m. 0.5 ml French Mini m.m. 2.0 m.m. 0.25 ml German 135 2.8 m.m 0.25 ml m.m. 65 m.m. 7. Printing of straws Before the diluted semen is filled into straws, the straws have to be lebelled by printing the breed, name or no. of bull, date of collection, etc. by using and automatic straw printing machine. The printing must become dry before the straws are taken up for filling. 8. Filling of Straws

Filling of straws can be done manually as well as by automatic machine. Manual method is cheaper and easy, as such the method is described hereunder. It requires vacuum pump, filling comb, rubber tube, straw clips and polyvinyl alcohol (PVA) power. The straws (15 Medium or 20 mini) are clipped together by using straw clips. The clipped straws are cooled to +5oC in the cold handling cabinet. The straws in the clamps are fitted on the filling comb, which in turn is fixed to vacuum pump through the rubber tube. Semen is taken in the bath of the bubbler and filling is done by operating vacuum pump. Due to negative pressure semen is drawn into the straws. Appearance of two distinct bands in the factory seal indicates complete filling of straws. An uniform air space is created in all the filled straws by pushing the open ends of the straws on to the teeth of the bubbler. The bubbler and the bath are discarded after use is required in order to allow for expansion of the semen during freezing. In its absence the sealing will be pushed out by the frozen semen. Further this air space also preventing possible contamination. 9. Sealing of Straws The sealing powder, PVA is available in 10 different colours. The French straws are also available in 16 different colours. The combination of both assures 160 positive identifications. The powder is spread on a clean glass dish to provide a thickness of 4-5 mm. The open ends of filled straws are dipped into the powder and when the powder penetrates 4-5mm into the straw a satisfactory seal is made. This is known as 'laboratory seal' which will appear as a single band. Immediately after sealing, the straws are placed in water bath at +5oC for the equilibration period to be completed. At the same time this allows the sealing to become firmer and the excess powder on the ends falls to the bottom of water bath. The entire operation of filling and sealing has to be done at +5oC within the cold handling cabinet. At end the tubes are rolled and dried carefully with pre-cooled towels since ice will from on damp straws and this will reduce freezability and storage space. 10. Freezing the Straws in Liquid Nitrogen Vapour Liquid Nitrogen vapour can be obtained from wide mouthed stainless steel container (LR 320, LR 250) where the straws are also finally stored. For the

sake of economy and easy to work, the equilibrated straws after withdrawing from water bath at +5oC (which were placed in a freezing rack) are now placed in a mini freezer by holding in a horizontal position inside the straw racks at 4 to 5 cm above the level of liquid nitrogen, thereby exposing to the vapours of liquid nitrogen. The temperature of semen reaches -130oC to -150oC by about 10-15 minutes. The main advantage of horizontal freezing is the efficiency and simplicity of the method. The racks hold the straws at a constant level and the straws are frozen at even rate all along the length of the straw. 11. Storage of Frozen Semen Frozen semen may be stored at: (i) -79oC by using solid CO2 (dry ice) and alcohol. (ii) -190oC by using liquid air (iii) -196oC by using liquid nitrogen (iv) -296oC by using liquid helium Storage of frozen semen in liquid nitrogen is the most convenient and accepted method all over the world. Soon after the temperature of straws reaches about -140oC in Mini Freezer, the straws are collected by pre-cooled forceps and finally transferred into per-cooled goblets (a wide mouthed polythene tube like container without handles to hold the straws filled-in with semen). Goblets available in different capacities and sizes, e.g., bigger goblets hold 360 medium straws, small goblets 100 straws and even smaller holds out 25 straws. These goblets are then immersed in liquid nitrogen and kept in already identified canisters, stainless steel container (with long handle) to hold goblets deep inside liquid nitrogen of highly insulated double wall stainless steel wide mouthed semen containers (LR 320, LR 250). 12. Thawing of Frozen semen (Melting). Thawing means to melt or become liquid. Thawing of frozen semen should be done immediately before use and as quick as possible to prevent recrystallisation of the water into bigger crystals. After thawing, frozen spermatozoa do not survive long as unfrozen semen and also do not withstand re-freezing. Therefore, one must be certain that the semen is going to be used soon once it has been thawed.

The time and temperature to be allowed for thawing depends upon size of the packaging used. A universally adopted system dose not exist. The most widely practiced temperature of 37-40oC for 30 seconds is suitable to get optimum survival of spermatozoa. Following steps should be taken while thawing the semen. 1. The straw should be removed with the forceps from the liquid nitrogen. 2. The straw should be shaken vigorously once or twice to remove liquid nitrogen from the cotton plug. 3. Immediately after this procedure straw should be placed in warm water (37-40oC) for only 30 seconds. 4. Dry the straw with a tissue paper; also wipe the scissor. 5. Warm the chamber of the insemination gun by rubbing vigorously. 6. Hold the straw vertically with the cotton plug or ball downwards. 7. Cut the straw at right angles to remove the powder plug or sealed plug through the air space. 8. Withdraw the piston of the syringe. 9. Place the straw in the warmed up chamber of syringe. 10. Take the sheath from the container and fix the sheath over the straw to ensure the firm union between the straw and sheath. 13. Procedure for Loading the A.I. Gun 1. Identify the canister from which the desired semen is to be taken. Ascertain the colour of the straw by reading of identification gag. 2. Remove the lid from the container, lift the proper canister upto the level of the frost line. Never lift the canister above the neck level. 3. With a pair of tweezers grasp an individual straw and remove it, at the same time lower the canister immediately back into the container. 4. With the wrist movement give one or two jerks to the straw to expel liquid nitrogen trapped at end of factory seal. 5. Dip the straw into a clean water bath at 37-40oC for 30 seconds. During this period the entire straw must be completely submerged in water bath. 6. Remove the straw from bath, dry the straw with a clean tissue paper or cotton. Inspect the straw carefully and discard straw with cracks or defective seals. Semen must never come in contact with water. 7. Place the straw in the chamber of insemination gun. To obtain a perfect fit with medium straw, it is essential that the laboratory seal is removed by cutting at right angle through middle of the air space. The air space

could be brought to the top by gently tapping of straw. Make sure that the clipped end of straw has a straight clean cut with no jagged edges. Straws cut at other angles or cut too short will result in back flow and wastage of semen at the time of insemination. 8. Fit a sheath over the straw and the chamber of the insemination gun and obtain a perfect fit between the extremity of the straw and the cone of the sheath. Move the sheath about 1" up and check whether the straw follows the sheath, if it is not correctly fixed it will not move upwards with the sheath. Fix the sheath by locking the plastic "O" ring at the flange of the gun with a rotating motion. Do not touch the sheath in the tip or middle portion, handle only the base portion. Sheaths are sterilized and packed in 50 or 40 numbers in a plastic packet. The base of the sheath packet should be cut open by means of a small cut made at an angle. The sheath packet should be kept inside a sheath container made of aluminum and closed with rubber stopper on either side. After removing each sheath, the container should be tightly closed to keep it free from dust and contamination. 9. Extrude a tiny drop of semen to remove air bubble, to move the factory seal and to ensure good fitting of the sheath. 10. The post thaw survival of spermatozoa is poor. For maximum reproductive efficiency, thawed semen should be used immediately. So, do not thaw more than one straw simultaneously.
Table 8.11

Artificial insemination of Domestic Animals with Frozen Semen

Cattle
Volume of inseminate (ml) Number of motile sperm per inseminate (105) Time of insemination Site of semen deposition 0.2-1 30 8-16 hr after estrus Uterus-Cervix

Sheep
0.05-0.2 120-150 10-24h after onset of estrus Cervix

Goats
0.02-0.2 100-150 12-30 h after onset of estrus Cervix or uterus

Swine
50 5000 after onset of estrus Uterus

Horses
20-50 1500 Every 2 days during of estrus Uterus

Turkeys
0.025-0.05 100 Weekly Vagina