ARTIFICIAL INSEMINATION(For Veterinarians) 3) Introduction and Semen Quality(For Final Year Students) by Prof.Dr

1) Successful application of AI in cattle By Prof.Dr. Sayed Hattab Prof. of Theriogenology Alex Universityِ 2) ARTIFICIAL INSEMINATION(for veterinarians) 3) Introduction and Semen Quality(for final year students) By Prof.Dr. AHMED MAMDOUH OSMAN Department of Theriogenology Faculty of Veterinary Medicine Assiut University

4) ARTIFICIAL INSEMINATION By Prof.Dr.Afyfi El-Monofy Faculty of Veterinary Medicine Cairo University

1 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) ARTIFICIAL INSEMINATION (BY Prof.Dr.Sayed hattab) Definition Artificial insemination is a process by which sperm are collected from male, processed, stored and artificially introduced into the female reproductive tract for the purpose of conception.

History of A.I 1322, Arab Chieftain stole semen from stallion owned by an enemy 1677, Leeuwenhook used microscope to see sperm 1780, Spallanzani reported a successful AI in dog. 1900, Ivanov of Russia Pioneered AI research in livestock, he was the first to successfully inseminate cattle 1936, Denmark was the first to establish an AI cooperative association 1938 USA first AI cooperative association at New Jersey. 1960, 1970, become popular in cattle. 1990, expansion o horse and swine AI.

Objectives of A.I Genetic improvement Diseases control mechanism Possible to increase fertility Decrease breeding expense

Advantages of A.I Genetic improvement wide spread use and availability of genetically superior sires. One bull can breed 500,000 cows in a life time

2 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) After death , semen can be used coldest frozen semen 40-45 years old Rapid proof of sire (progeny testing

Control of venereal diseases Accurate breeding records Economic cost of very good sire reduced because extend semen cost to maintain sire reduced as do not need as many to breed all the females Safety

Disadvantages Estrus detection must be good Need well trained inseminators Use of poor male may increase if not tested well Technology to store cooled or frozen difficult to maintain Need to corral and restrain females

Process in A.I Centers Semen collection Methods of semen collection: Artificial vagina: Bull, stallion, buck, ram, dog sometimes poor Stimulate natural copulation Temperature at 45C ( worm water) Pressure (air and worm water) Lubrication (harmless to sperm and not excessive amount) Electro-ejaculation: used in bull and rams

3 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) increase Voltage in pulses (1-10v) low voltage --- accessory gland secretion and high voltage --- ejaculation semen volume higher and sperm concentration lower get urine often poorer quality ejaculate Massage method: Rectal massage of vesicular glands and ampullae

Process in A.I Centers Precautions During semen collection Precautions must be considered during collection of semen by AV to obtain good quality and quantity semen:

Adjust temperature (45c) AV must be clean, dry and sterilized Do not use any detergents or harmful chemicals to sperm Inner sleeve of AV must be free from any leakages Use of adequate amount of harmless lubricants Adjust the pressure inside AV to stimulate thrust Wash and clean perineal and perpetual regions Avoid the collection in dirty place Teasing the bull by false mount and foreplay Protect the semen collector from cold shock by hand or wool cotton piece

Process in A.I Centers Semen evaluation 1) Gross examination Volume Color Consistency PH

4 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) 2) Microscopic Examination Assessment of motility Mass motility Individual motility Assessment of sperm morphology Sperm abnormalities life and dead sperm ripe and unripe sperm -pathological cells Assessment of sperm concentration use of hemocytometer Automatic counter

5 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

6 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

7 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) Process in A.I Centers 3) Special laboratory tests Measurement of hygienic quality visual examination catalaze test Measurement of metabolic activity Methylene blue reduction test Electrical activity of semen Fructolysis index Resistant tests resistant to sod chloride sol.1% resistant to cold shock viability test

Process in A.I Centers Semen dilution

Properties of semen extender: a- Isotonic -salts b- Buffered (PH 6.5) phosphate citrate Tris c- Cold shock protection Lecithin and lipoproteins find in egg yolk and milk d- Nutrients egg yolk and milk fructose and glucose e- Antibiotics Gentamycin Tylosin Lincospectin Penicillin + Streptomycin F-protection for freeze and thaw -glycerol g- preserve fertility -Known and unknown factors found by trial and error

8 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) Process in A.I Centers Processing

A) semen collection do not let temperature drop below 35c B) Antibiotic treatment 3-5 minutes before dilution C) pre-dilution start at 35c No glycerol yet up to half final volume D) cooling Cool to 5 c Slow cool over 2 to 4 hours E) Final dilution cooled extender + glycerol F) Equilibration - allow glycerol to act -minimum of 4 hours G) Packaging Plastic straws 0.5ml in US 0.25ml in Europe (French straws) Glass ampoules Old method Pellets No package Most success in swine H) Freezing use of liquid nitrogen vapors first then drop into the Nitrogen -196C I) Storage Dry ice and methanol (old) Liquid nitrogen is now used Do not let tanks dry.

9 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) Semen Tank management Avoid excessive movement or abuse of tank Monitor nitrogen levels routinely and keep a record of nitrogen loss Store the semen in an area with good light but out of direct sunlight. Observe tank daily. Once the tank fails, Nitrogen is lost very rapidly. Keep the tank elevated above the concrete floor or other wet and poorly ventilated surfaces 5-store only the amount of semen needed for six months

10 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Handling semen within the tank In the typical farm semen tank , dangerous temperatures (- 32 to 112 C) in the upper half of the neck of tank Exposure to these temperature can occur when the semen is transferred from tank to tank or when handling semen within the neck while trying to locate a specific unit of semen Thermal injury to sperm is permanent and can not be corrected by returning semen to liquid nitrogen.

Semen Handling practices to minimize thermal damage 1-Transfere of semen between tanks must be coordinated and rapid 2-Avoid unnecessary searching and exposure of semen to dangerously high temperature within neck region 3- Use of Tweezer to transfer the straw to the bath. Quickly lower the rack semen and canister into the tank body.

11 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Thawing of frozen semen Straws are thawed at 35 C for 12 seconds for 0 .5ml straw or 6 seconds for 0.25 ml straw Do not linger with the straw in the neck of LN storage unit or in the air Remove the straw from the thawing bath immediately after the proper thawing time has passed to prevent excessive worming of the semen after removal of straw carefully Dry the straws with a clean tissue Inspect the straws and discard any with cracks or defective seals

12 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) Assessment of quality of frozen semen Motility 20% of progressively motile spermatozoa are the minimal acceptable motility for frozen semen Number of motile spermatozoa per inseminate depend on breed and fertility level of each bull For most bulls each inseminate should contain at least 10 million motile sperms after thawing regardless of how semen is packaged The influence of the method of thawing on post-thaw motility will also be in the number of motile spermatozoa per inseminate. Acrosomal integrity Spermatozoa with deteriorated or damaged, acrosome unlikely to be capable of fertilization Examination of spermatozoa under differential interference phase contrast microscope The sample is incubated at 37 c and aliqutos are removed for evaluation after 0.2,4,8 hours For semen in egg yolk citrate extender, the semen diluted with two volumes of 0.2% glutaraldehyde in phosphate – buffer saline.

13 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) Insemination

Use recto-vaginal technique in cow

Time of insemination Cow in estrus in the morning must be inseminated in the evening Cow in estrus in evening must be inseminated in the morning

Cite of insemination In anterior half of the cervical canal or just anterior to internal os in the body of the uterus

14 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) ARTIFICIAL INSEMINATION BY Prof.Dr.A.M.Osman (for veterinarians) INTRODUCTION History (Arab Mare) Advantages Precautions to avoid disadvantages Bull Selection (Progeny Test)

TOOLS FOR ANIMAL IMPROVEMENTS

Selection Breeding Management Reproductive Care Artificial Insemination Liquid Frozen Embryo Transfer : Capacitated Sperm Mature Oocyte

ARTIFICIAL INSEMINATION IS ONE OF THE TOOL FOR ANIMAL IMPROVEMENTS AND FROZEN SEMEN USING STRAWS PROVED TO BE THE BEST ALL OVER THE WORLD

Requirements for Successful AI Programmes I.Personnels: Willing and Enterest Honest ,Sincere,Patient and Clever Disciplinarian and Faithful Hard worker Scintific background on animal Reproduction Believe in his Work

15 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) II. Selected Male

Genotype : Progeny or Sibling

Phynotype : Apparent Characters

Double Muscles : From Benilux Countries (Holland,Belgium and Luxumburg)

Selected Normandi and Buffalo bulls

III. Equipments

-Sterile glass wares -Sterile disposable plastic -Contiuous supply for liquid nitrogen - Tanks - Others

IV.Record System and Data Analysis: Conception Rate > 70% Calving Rate > 70 % Sire Index < 2 (Number of services per conception)

16 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) V. Females - Insemination during the last 6 hours of estrus

- Genitalia must be healthy with clear glassy mucoid secretion and contractile uterus

AI IN DIFFERENT COUNTRIES

- Large AI center in Budabest (Pellet Form)

- Private AI Unit in Munich (Ampule Form)

- Large Private Unit in Landshut – Germany, SPERMIX (Straws)

- Small Unit in Morocco ,School of Vet. Medicine (Straw)

SEMEN COLLECTION - ARTIFICIAL VAGINA (Temp 40-45 Cº) - ELECTROEJACULATOR - DUMMY COW - MASSAGE OF AMPULLAE AV Is the best and GOOD EJACULATE requires Teasing (False Mount)

SEMEN EVALUATION MACROSCOPIC EXAMINATION VOLUME : 3-11 ml COLOR : MILKY OPAQUE VESCOSTY : HIGH 3.7 CENTIPOISE PH : 6.75 AND LESS

MICROSCOPIC EXAMINATION MASS ACTIVITY > ++ INDIVIDUAL MOTILITY > 70 : SPERM ABNORMALITIES : PRIMARY < 3 % SECONDARY < 10 % PROTOPLASMIC DROPLETS < 3 %

17 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) ALIVE SPERM > 70 % SPERM CONCENTRATION > 1000,000 /cmm SPERM RESISTANCE > 300 ML NaCl (1%)

SPERM ABNORMALITIES

18 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

EXTENDRS FOR AI PURPOSES LIQUID SEMEN EGG-YOLK CITRATE BUFFER EGG - YOLK PHOSPHATE BUFFER EGG YOLK TRIS –PH 6.5 MILK – DILUENT : (SKIM , WHOLE, POWDER 9%) FROZEN SEMEN : ADD GLCEROL (7%)

ANTIBIOTICS PER ONE ML EXTENDER 500 I.U. PENICILLIN 1 mgm STREPTOMYCIN 3 mgm SULFANILAMIDE

EXTENDED SEMEN SHOULD BE USED AFTER 6 HOURS FROM ANTIOBIOTIC ADDITION

RATE OF SEMEN TO EXTENDERS LIQUID SEMEN (1 : 20 – 25 )

FROZEN SEMEN ( 1 : 5 – 10 )

FROZEN SEMEN PELLET FORM (0.2 ml) AMPOUL FORM ( 1 ml ) STRAW FORM (0.2- 0.5 ml)

EACH DOSE CONTAINS MORE THAN 15 MILLION MOTILE SPERM

FUNCTION OF GLYCEROL HYGROSCOPIC (ABSORB WATER) LOWERING FREEZING POINT (- 0.5 Cº ) BOTH CHARACTERS PREVENT ICE CRYSTAL FORMATION WITHIN SPERM CELL METABOLIZED BY SPERM RAISED FERTILITY RATE

19 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) STANDARD TECHNIQUE OF FREEZING SEMEN EXTENDED WITH EGG YOLK CITRATE AT ROOM TEMPERATURE , HALF THE REQUIRED FINAL DILUTION(A)

EXTENDER INCLUDED 14% GYCEROL , EQUAL VOLUME (B) IS PREPARED

BOTH (A) AND (B) SOLUTIONS WERE KEPT IN REFREGERATOR AT 5 Cº

AFTER GRADUAL ADDITION OF (B) TO (A) , KEEP THE FINAL MIXTURE CONTAINING 7% GLYCEROL FOR EQUILIBRIUM PERIOD (AT 5 Cº) FOR 4-18 HOURS

PACKAGE IN EITHER VIALS,PELLETS OR STRAWS AT THE SAME TEMPERATURE

FREEZE AT A COOLING RATE OF ONE Cº PER minute FROM + 4 TO -15 Cº

THE RATE ,THEN INCREASED TO 3-4 Cº PER MINUTE UPTO –79 Cº (CARBON DIOXDE ICE AND ALCOHOL) OR UPTO –196 (LIQUID NOTROGEN) KEEP UNTIL USE

OTHER METHODS OF FREEZING SHORTER EQUILIBRIUM PERIOD 2-4 HOURS PACKAGE AT ROOM TEMPERATURE KEEP PACKAGE SEMEN ABOVE THE SURFACE OF LIQUID NITROGEN FOR 10 MINUTE THEN IMMERSE DEEPLY INSIDE THE CONTAINER AND KEPT UNTIL USE NEVER LEAVE THE CONTAINER WITHOUT REPLACEMENT OF THE EVAPORATED LIQUID NITROGEN (REFIL EVERY TWO WEEKS) KEEP THE CONTAINER IN COOL ROOMS

THAWING IN PELLET FORM : SINGLE PELLET SHOULD BE THAWED IN VIAL CNTAINED I ML BUFFER CITRATE AT ROOM TEMPERATURE. INSEMINATING CATHETER FITTED WITH SYRINGE IS USED FOR INSEMINATION IN AMPOUL FORM : SINGLE FROZEN AMPOUL THAWED AT +5 Cº FOR FEW MINUTES BEFORE BEING USED FOR INSEMINATION AS ABOVE

20 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) STRAW FORM PUT THE STRAW IN WATER BATH AT 37-40 Cº FOR 20-30 SECOND OR PUT THE STRAW AT ROOM TEMPERATURE FOR 1-2 MINUTS OR PUT THE STRAW IN WATER BATH AT 80 Cº FOR FEW SECONDS

TECHNIQUE OF INSEMINATION RECTOVIGINAL : (APPLIED WITH ALL KIND OF INSEMINATION) STRAWS : INSEMINATING GUN MANUFACTURED FOR USE WITH EACH TYPE OF STRAW CONSISTS OF : METAL GUN PLASTIC SHEATH

21 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) ITE OF SEMEN DEPOSIT

IN THE ANTERIOR THIRD OF THE CERVIX

22 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

23 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

CORONA RADIATA Granulosa Cells Extra Cellular Matrex Zona Pellucida Perivitelline Space Vitellus Germinal Vesicle (Nucleus)

24 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

INTRACYTOPLASMIC SPERM FERTILIZATION ICSI Using the PIEZO NEEDLE

25 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) ARTIFICIAL INSEMINATION IN CATTLE BY Prof.Dr.A.M.Osman (for final year students) Introduction and Semen Quality ARTIFICIAL INSEMINATION MEANS THE INTRODUCTION OF SEMEN IN THE FEMALE GENITALTRACT USING SPECIAL INSTRUMENTS HISTORY 1400 Arab mare in Saudi Arabia 1677 Van Loenhork,Duch Scientist, Discover sperm 1780 Lazaro Splanzani in Italy with Dogs 1922 Evanof ,Russia,Devoloped AV in cattle 1937 Sorenson,Danish Verinarian, Developed Recto-vaginal T.I. (Liquid Semen) 1949 Polge,British Scientist,developed frozen semen . 1960 Cassou ,France Veterinarian,developed

Frozen Straws. Used ADVANTAGES -Selected males improved herd quality and genetic constitution - Reproductive improvements with increased fertility rate , population and production - Control the spread of coital diseases - Low costs with maximum economic gain - Decreased numbers of males - A.I Centers provide much facilities beyond the limit of small herds (Vaccination, lab. diagnosis .. etc ) for participants -Frozen semen extends useful life of proven sires even after their death (more than 10 years)

26 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) DISADVANTAGES Unselected , genetically bad quality bulls * Infected males with specific coital or any other genital diseases * Bad handling and shipping of semen * Poor knowledge of animal reproduction * Unhygienic measure during semen handling * Increased incidence of silent heat * Inseminate cows not in estrus or pregnant

SELECTED BULL

Selected Bulls

27 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Selected Bulls

SEMEN Consists of : Sperm : Originated in Testes through Spermatogenesis Seminal Plasma : Originated from Accessory Glands (Seminal glands, Prostate and Bulbo-Urethral glands)

SPERM MORPHOLOGY Sperm consists of : Head:Ovoid(8x4x1 µ),occluded with nucleus (mainly deoxyribonucleoprotein) The anterior part is covered with a cap-like acrosome (polysaccharide) and the perforatorium The posterior part covered with the post-nuclear cap and proximal centriol The equatorial plate lies in between The whole sperm is covered with plasma

28 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) Neck : The weakest point of the sperm Middle Piece : 14-16 µ in length , contained the axial filament complex (2+9+9 fibrils) which is surrounded with mitochondrial sheath or broad helix.The 20 fibrils are elastic and responsible for the whip–like movements of the sperm Main tail Piece : 38-42 µ in length, contained the axial filament complex and a thinner sheath only The velocity of sperm reached 130 µ/second

29 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

30 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

*Bull sperm needs 6-8 hours incubation in female genitalia to be fully capable for the act of fertilization in the Oviduct(Ampulla) *Removal of decapacitation factors *Activation of enzymes (hyalouronidase and corona radiate penetrating enzyme ) from acrozome *Release proteolytic enzyme from the perforatorium In the lab.heparin can induce capacitation

31 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

32 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

SEMINAL PLASMA Alkaline and characterized by the presence of high concentrations of the following: Fructose and Citric acid Prostatic phosphatase and prostaglandin Phosphorylcholine,glycerylphosphorylcholine

33 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) Inositol and ergothionin Secretory granules and globules Exfoliated epithelial cells Macrophages,microphages (spermiophages

FUNCTION OF SEMINAL PLASMA * Act as a vehicle for sperm activity * Supply energy from fructose and others * Supply energy to sperm in the female tract from the glyceryl- phosphoryl-choline * Supply suitable buffering media * Contained decapacitation factors which prevent prematuration of sperm and preserving the fertilizing capacity *Contained prostaglandin which induce negative uterine contractility

SEMEN COLLECTION I. ARTIFICIAL VAGINA

II. Dummy Cow

III . Electro ejaculator

IV. Massage of Ampullae

34 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) Probes Electroejaculator

ARTIFICIAL VAGINA - Outer hard rubber sleeve (40 cm length) - Inner latex neutral longer sleeve (50 cm) - Rubber cone and collecting centrifuge tube – Protective bag for the collecting tube - Bandages for fixation - Vaseline or buffer solution as lubricant - Thermometer and short hand glove - Water warmed at 65 C°

PREPARATION OF THE A. V. - Fix the inner sleeve to the outer sleeve

35 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) - Put the water in between and close the screw then fix the cone and put the tube - Wait till the temperature of the inner lining or sleeve descend to 45 C° then put the lubricant and apply the protective bag - Put the prepared AV in an inclined position with the lubricated opening down

COLLECTION OF SEMEN * Preparation of the bull : Clean , Dry and excited with teasing Preparation of the cow or Teaser : * Clean, dry and of compatible size * Preparation of the ground and stanchion : Clean and Not slippery * The temperature of the inner sleeve just before collection should be ranged between 42-45 C°

SEMEN EVALUATION GROSS AND MICROSCOPICAL EVALUATION

GROSS EVALUATION Color : Creamy white rarely yellowish Volume : 3-11 ml , average 5-6 ml Viscosity and density : 3.7 centipoises

36 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) in comparison with water. High density with high viscosity PH : 6.7 (6.4-6.9) High density with high acidity PH indicator paper or medium green color with bromothymol blue (yellow 6 to blue 7.6) . Alkaline semen is abnormal

MICROSCOPICAL EVALUATION Sperm Motility : Required warm mechanical stage and slide (at 37 C°)

Mass activity : Drop of fresh semen and low power of microscope(Score:+++/+)

Individual motility : Fresh diluted semen (1:10) with saline or buffer citrate and a cover slide with high power of microscope (70% or more forward sperm motility)

Sperm Abnormalities Primary :Originated from testes Secondary : Originated from excurrent duct Tertiary : Originated through handling

Total sperm abnormalities should not exceed 12 % in normal ejaculates

STAINS USED FOR SPERM

EVALUATION Alkaline Methyl Violet :(Sperm morphology) 9 parts from 1% aqueous sol of the stain + 1 part Sod Carbonate 1% (freshly prepared) Prepare smear from diluted semen and fixed with hot flame or dry air Wet the smear with Dist.water

37 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) Apply the stain for 3-5 minutes then wash with dist.water and plot with filter paper Examine under high power of microscope (total 200 sperm should be examined)

Sperm abnormalities Dwarf, coiled ,giant and bent

Ruffled acrosome Tapered Diadem (Craters) microcephalic

38 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

39 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

40 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) DAG defect Coiled tail

41 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Eosin-Nigrosin Stain:(Alive/Dead sperm) 1% Eosin in buffer phosphate (PH 7.4) 10 % Nigrosin in Dist.water * One drop fresh semen mixed gently with one drop Eosin and 4 drops Nigrosin * Smears were made from the final mixture and fixed as usual then examined under the high power of microscope * Dead sperm stained red,Alive sperm stay unstained Normal semen with more than 70%

Indian Ink or Opal Blue: (Ripe and Unripe Sperm) One drop of semen + Two drops of the stain (any one of them) After gentle mixing , smears were made and left to dry as usual Examine under high power of microscope for the determination of sperm with protoplasmic droplets (proximal or distal) Normal semen usually with less than 3 % sperm with protoplasmic droplet

42 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Sperm Abnormalities Alive / Dead Protoplasmic droplet

SPERM CONCENTRATION 1-Cytometer : (Improved Neubaur Thoma-rulling) (With 5 large squares in a diagonal position) Number of sperm per c.mm = Total n X rate of dilution X 50 The normal sperm concentration is : one million / c.mm 2- Spectrophotometer 3- Calorimeter 4- Brown`s Opacity tube 5-CMT Reagent

Procedure : 10 ml sod chloride 1% or dist.water contained drops of sod hydroxide (to stop sperm motility)is prepared in dry test tube 0.05 ml semen(using micropipette) is added to get a dilution rate of 1:200 After gentle mixing,allow small drop of the mixture to flow in the counting cambers of the Cytometer.Wait for 10 minutes then count sperm in 5 large squares as usual

43 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

SPERM RESISTANCE Resistance to 1% Sod.chloride : The amount required to stop the sperm motility should be more than 1 : 300

Add 10 ml sod chloride 1% to 0.02 ml fresh semen in a successive lots , till the sperm motility completely stopped

SPERM METABOLIC ACTIVITIES Oxygen uptake: Good semen diluted 1:4 with buffer phosphate, PH 5.8, has a Respiratory Quotient of 0.79 R.Q. is the quantity Co2 produced by motile sperm divided by the Oxygen consumed in a physiological reaction condition at 37 C° using Varburg Manometer

SPERM METABOLIC ACTIVITIES Methylene Blue Reduction Time: 0.9 Fresh semen diluted 1:4 in small vial 0.1 ml M.B.(5mg/10 ml buffer citrate) Cover the mixture with few drops of paraffin

44 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) Mix gently and incubate at 43 C° . Count the time till the blue color disappear Normal ejaculate with high motility ,viability and metabolic activities and with expected high fertility takes a period less than 5 minutes. Liberated hydrogen from sperm metabolic activities transfer M.B. to Leuco M.B.( colorless)

SPERM METABOLIC ACTIVITIES Fructolysis Index: It is the amount of fructose consumed by 10 billion sperm for one hour at 37 C° It is 1.4-2 mg fructose in good semen Fructose is secreted mainly from seminal glands and to some extent from the ampulla Its higher value in fresh semen reflects high androgen levels and good accessory glands function

HYGIENIC CONDITION OF SEMEN Catalase Test: Clean semen has has a slight catalase activity to liberate oxygen from H2O2. In a special catalase tube put 0.5 ml semen then add H2O2 till the first ring. Put the stopper containing the capillary tube and agitate vigorously every 5 minutes till 20 minutes with inverted position for the tube. Measure the liberated oxygen . Unpolluted semen should not exceed 300-400 logarithmic scale

45 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Artificial Insemination Prof.Dr.Afyfi El-Monofy

Semen Collection Methods 1- Sponge 4- Urethral Fistula

2- Spooning 5- Insertion of Insulating Bag in Vagina

3-Pipetting 6- Condom

The Most Suitable Methods are: 1- A.V. (Advantages) 2- Electro-ejaculation 3- Digital Manipulation (of the penis in dogs) 4- Massage of Accessory Glands in Bulls

Collection of Semen from Bulls Collection of Semen from Bulls by the Artificial Vagina Preparation of the Bull

46 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) Grooming

Washing

47 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) Apron

The Teaser Female : Estrous Female Non-Estrous Female Advantages Disadvantages

Male Advantages Disadvantages

Dummy : Fixed Mobile Advantages Disadvantages

48 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Teaser Male in the Collection Chute

Automatic Mechanical Dummy

49 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Mobile Dummy

How to Prepare the Teaser ?

Artificial Vagina

50 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Outer Jacket Inner Sleeve Fixing Rings Air Valve Connecting rubber or Polyethylene Funnel Collecting Cup (Tube)

Preparation of A.V.

The Day Before Collection Fixing Inner Sleeve Washing (2% Sod. Bicarbonate) Adding Hot Water (Amount, Temp.) Maintaining Temperature

51 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Preparation of A.V. Fixing Inner Sleeve

Washing (2% Sodium Bicarbonate) Maintaining Temperature

52 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Adding Hot Water (Amount, Temp.)

On The Day of Collection Lubrication Connecting the Collecting Cup Adjusting Air Pressure Adjusting Temperature

Lubrication Connecting the Collecting Cup

53 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Well Assembled A.V. Adjusting Air Pressure

Adjusting Temperature

54 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Preparing the Site of Collection Site: Closed, Open

Collection Chute

Floor Cleaning Ventilation Avoiding Stress (Direct Sun Light, Cold,…)

55 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Time: Early in the Morning Proper Handling of A.V. False Mount Correct Holding of the Erected Penis and Directing it into the A.V. Ejaculatory Thrust Quick Removal of A.V. off the Penis Holding A.V. in a Vertical Position Protection of the Semen-Containing Cup

56 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

57 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

False mounting to arouse bull

An erect penis indicates arousal Collector ready with AV

Collecto r diverting penis to the AV Electroejaculation

58 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Handling of Freshly Collected Semen Protection of Freshly Collected Semen from Cold Shock At Collection Using Insulating Bag Using Double-Wall Cup filled with Warm Water Holding the Collecting Tube

In the Laboratory Transfer Semen Quickly to a water bath at 20-25 oC Until Evaluation

Evaluation of Physical Characteristics Ejaculate Volume Colour Consistency Homogeneity

59 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Determination of the Per Cent Live Sperm Determination of the sperm cell abnormalities Semen Dilution Role of seminal plasma?

Aims of Dilution Extending the ejaculate volume.

Protection of the spermatozoa during preservation.

Prolonging of the sperm life span. Do You Know the Properties of a Good Semen Diluent ? Types of Synthetic Media

1. Extenders 2. Protectors 3. Implementors

Requirements of Synthetic Media

Isotonic solution of a non-electrolyte e.g. Sugar or a week electrolyte e.g. Glycine

Isotonic Salt solution With: multivalent anion and monovalent cation e.g 2.9 % sodium citrate

A Phospholipid e.g egg yolk

60 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Antibiotics in Semen Diluents Common Diluents EggYolk Citrate diluent Tris buffered-yolk diluent Milk Diluent Egg-yolk Lactose diluent Semen Preservation

Preservation of Semen at 2-5 OC

Process of Dilution Important Tips

Semen Diluent

Diluent Semen

Package Semen in 1ml Vials (Ampules) Close Vials Tightly with Rubber Corks Put Ampules in a Water Bath at 25 – 30 o Vials should be Labeled with Bull Number Date of Processing Production Center

61 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Transfer the Water Bath to a Refrigerator at 4 OC.

Leave Diluted Semen for about 6 hrs before use. Why?

Freezing of Semen

Methods of Freezing Freezing in Straws (French Straws, Minitubes) (0.25, 0.3, 0.5, 1.0 ml) Freezing in Ampules Freezing Pellets (0.1 – 0.2 ml)

Each Method Has Its Advantages and Disadvantages Don’t You Know This? Procedural Steps Dilution Cooling Packaging Freezing Thawing All Procedural Steps Vary with the Technique Used

A Cold Cabinet for Cooling of Semen Prior to Freezing

62 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) Automated French Straw Printing Machine

Manual French Straw Printing Device

Sealing

63 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) Machines for Sealing Straws

Straws are Sealed either Hermitically or using Polyvenyl Chloride Powder Freezing LN (-196 OC) is the Freezing Agent for Straws

CO2 is the Primary Freezing Agent for Ampules Ampules May Also Be Frozen Directly Using LN

Horizontal Freezig of Semen

64 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Straws in Goblets and Canisters Ready Automatic Programmable to Store in LN F

Bovine Semen Stored in LN Container

65 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Identification Straws are Available in 20 Different Colours The Sealing Powder Coloured Tubes

66 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Thawing The Golden Advice: Freeze Rapidly, Thaw Rapidly Transfer Straws Rapidly into a Thawing Bath at 38 – 40 OC for 30- 60 Sec

Freezing of Semen in Ampules Slow Freezing Regime for Ampules: From +5 OC to -15 OC at the Rate of 1OC/ min (20 min) From -15 OC to -80 OC at the Rate of 5OC/ min (13 min)

Ampules are Thawed in Ice Water (+ 5 OC) for 15 min

Freezing Semen in Pellets Dilute Semen 1:1 to 1:3 Using Lactose- Egg Yolk Diluent Containing Glycerol (According to the Pellet Size) Cool to 5 OC in 30 -60 min

Pellet Thawing: Thaw a Pellet in 1ml Pre-warmed (38 OC) Sodium Citrate Solution (2.9%)

67 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) Artificial Insemination Technique

Load the Insemination Gun

After Thawing, Remove Excess Moisture From Cut the End of The Straw From Straw By Using a Paper Towl the Previously Sealed Side

68 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM) Review of Anatomical Relationships Important for A.I

Locating the cervix

Palpate inner surface of the pelvis

Palpate from one side to the other

Cervix should be intersecting the path of palpation

69 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

Feeling the Cervix with Left Grasping the Cervix

Hand in Rectum

Proper Holding of the Cervix Wrong Fixation of the Cervix

Wrong Fixation of the Cervix Catheter Introduced where the Semen Should Be Deposited into the Anterior Third of Cervix (Liquid Semen) or Uterine Body (Frozen Semen

70 copyright@ E V E T C -O59-I0 Prof.Dr.M.El Naggar (PM)

DDoo NNoott FFoorrggeett !!

Proper Restraint of the Animal to be Inseminated in a Holding Gate

Washing Hands Before Leaving Washing and Disinfecting Before Leaving