Other Laboratory Tests

most important part in predicting fertility of domestic animals

semen quality evaluation

effective harvesting of the semen involves

obtaining the maximum number of sperms of highest possible quality in each ejaculate

ultimate objective of semen collection

make maximum use of superior sires

semen collection is a complex procedure involving

coordinated efforts between the animal handler and the collector

age when semen can be collected in bull

12 months

age when semen can be collected in boar

6-8 months

age when semen can be collected in ram

6-9 months

age when semn can be collected in stallion

20-24 months

age when semen can be collected in dog

8-12 months

why is bull preparation essential?

• decrease microbial load

• increase semen volume, sperm concentration

• decrease reaction time

bull preparation involves

• clipping of preputial hairs

• preputial washing with warm, normal saline

• cleaning and washing the bull

• ordinary washing soap and mild brush

• paper towel or damp towel

preputial hairs must be clipped leaving a tuft of 2cm all around

a tuft of 2cm all around

preputial washing with warm normal saline may be used to

decrease microbial load

clean and wash the bull, especially the ______________, _______ before the semen collection

• hind quarter

• 1 hour before

during bull preparation, special attention must be given on

lower abdomen area and prepuce

paper towel or damp towel may be used to

remove excess water

a teaser/dummy is a

female animal in estrus

teaser/dummy preparation

• cleaned the same way as the bull

during teaser/dummy preparation, special attention should be given to

the back and perineum area

washing area should not be more than _____ away from _________________

• 20m away

• collection area/serving area

washing area should be made of

rough concrete

the floor of the washing area should be

slanting with drainage facility of water, dung, etc.

washing area should have an

adequate facility of water with reasonable pressure

collection area should be away from

noisy places

collection area should not be more than _____ away from ____________________

• 3om away

• semen evaluation laboratory

the ground of the collection area must not beor unstable, as it may pose safety hazards during the semen collection process.

slippery/muddy

collection area should be fenced with _______________ to avoid ____________

strong metallic bars to avoid escape of bull

this is needed in the collection area for the restraint of the teaser

one slanting metal stanchion fitted with wooden arms

there should be sufficient space in the collection area for

free movement of bulls and the semen collector

methods of semen collection

1. pan collection method

2. vaginal spoon method

3. sponge method

4. breeder’s bag method

5. sperm collector method

6. artificial vagina (AV) method

7. massage method

8. electro ejaculation

9. fistula method

10. dummy female

what is this called?

artificial vagina

most commonly used and best method of semen collection

artificial vagina (AV) method

used for semen collection from lame bulls and in dogs

massage method

used for semen collection from lame bulls

• electro ejaculation

• massage method

fastest and most sanitary method of semen collection

artificial vagina (AV) method

why is artificial vagina (AV) method done?

• semen is collected in natural status

• semen is collected in hygienic condition

frequency of semen collection

2-3x a week

more frequent semen collection results in

poor semen quality

advantages of artificial vagina method

• Practically the whole ejaculate is collected in uncontaminated and natural stage.

• It is free from the extraneous secretions.

• Sterile conditions of the apparatus ensure disease control.

• The viability of the sperm is better.

• No female is needed if dummy is a success.

disadvantages of artificial vagina method

• Occasionally it is difficult to get the males to serve the artificial vagina.

• The apparatus involved is slightly costly and requires technical hands.

why is semen evaluation essential?

• to investigate infertility problems

• for breeding soundness evaluation of bulls

• for semen extension and processing

methods of semen evaluation

1. macroscopic/physical

2. microscopic

3. biochemical

macroscopic evaluation of semen

1. color

2. volume

3. consistency

4. pH

color of semen

creamy to milky white color

pH of semen

6.6 to 6.9

microscopic evaluation of semen

1. mass motility

2. individual motility

3. sperm concentration

4. live sperm count

5. abnormal sperm count

biochemical evaluation of semen

1. fructolysis index

2. methylene blue reduction test

3. resazurin reduction test

normal values of semen in cows

normal sperm morphology

why do we need to check for sperm abnormalities?

only morphologically normal sperm participate in fertilization process which is prerequisite to fertilization

sperm abnormality is detected by

Nigrosin staining technique

edit

classification of sperm abnormalities

• primary

• secondary

abnormality caused by aberration during spermatogenesis

primary

abnormality caused by aberrations at the subsequent developmental stages

secondary

i.e. after spermatogenesis is complete and sperm has left the seminiferous tubules

secondary sperm abnormalities could be due to

dysfunction of epididymis, seminal vesicles, and genital diseases

primary sperm abnormalities include abnormalities in the

• head

• middle piece

• tail

sperm head abnormalities

• mega

• micro

• pyriform

• elongated

• double and abnormal

• detached

sperm middle piece abnormalities

• abaxial attachment

• thick short

sperm tail abnormalities

• double

• stumpy

• ring form

secondary sperm abnormalities include

• normal detached heads

• loose heads

• coiled midpiece

• presence of cytoplasmic droplets

chemical medium used for preservation, extension and protection of sperm cells against various shocks during processing, storage and transportation used for artificial insemination

extender or diluent

advantages of a good extender

• provide energy for metabolic activities within sperm cell

• maintain osmotic pressure and pH of the medium

• keeps a check on the contamination of the medium to protect semen from microbial growth

basic components of a semen extender

1. egg yolk

2. buffer

3. antibiotics

egg yolk function on semen extender

membrane stabilizer, cryoprotectant

buffer function on semen extender

provides buffering capacity to semen

antibiotics function on semen extender

to check microbial growth

extenders used for chilled semen

• egg yolk-citrate extender

• tris-yolk extender

egg yolk-citrate extender is used in

cow bull and buffalo bull

tris-yolk extender is used in

buffalo bull

antibiotics added to the extender

• penicillin - 1000 IU/ml

• streptomycin - 1000 μg/ml

composition of citrate buffer

• Sodium citrate dihydrate :2.9 g

• Penicillin G sodium :1 lac Unit

• Dihydrostreptomycin sulphate :100 mg

• DDW :100 ml

composition of tris buffer

• Tris buffer :3.028 g

• Citric acid monohydrate :1.675 g

• Fructose (Anhydrous) :1.250 g

• Penicillin G sodium :1 lac Unit

• Dihydrostreptomycin sulphate :100 mg

• DDW :100 ml

semen preservation

• Refrigeration method at 4-6°C

• Ultra low temperature at bovine sperm frozen at -80°C , in dry ice or liquid nitrogen

• Semen at room temp at 18-25 °C

advantages of frozen semen

1. selective mating

2. early progeny testing

3. maximum utilization of sires

4. long storage

5. reduced transportation cost

6. disease control

7. reduced feeding and management cost of bulls

8. progeny even after death of the bull

9. no wastage of semen

10. no need for sterilization of AI gun

11. no fear of breakage of AI gun

disadvantages of frozen semen

• • Semen from about 10 to 20 percent of bulls will not withstand freezing. These

  are often bulls of low fertility.

• The ampouling, freezing and storage equipment are very costly.

• In the freezing process about 40 percent spermatozoa are killed. Hence, increased number of spermatozoa per insemination are required.

• If proper bull health is not maintained, frozen semen has great potential for the

  spread of viral and bacterial diseases.

bulls with _______________ have semen that will not withstand freezing

how many percent of bulls?

low fertility bulls, around 10-20% of bulls

Packing Methods for Freezing

• straw method

• medium straws

• mini straws

• French straws

• glass ampoules

• German straws - mini tubes-landshut method

this was introduced by a scientist of Denmark in 1940 for packing of liquid semen

straw method

Adler in 1960 froze semen packed in straws using

liquid nitrogen vapor

Cassou in 1965 modified Adler’s technique and was then called as

medium straws

Cassou in 1968 brought further improvement in semen packing by __________________ which is then called as

• reducing diameter for better freezing

• mini straws

Van Denmark and Kinney (1954) packing method

glass ampoules

Simmet (1972) packing method; also known as; capacity?

• German straws

• mini tubes-Landshut method

• 65mmx2.8mm - 0.25ml capacity

dimensions of French and German straws

the French straws have two ends -

• factory seal end

• laboratory seal end

the seal end that was formed while manufacturing the semen straw at the factory

factory seal end

the seal end that is open when purchased and is formed at the semen processing laboratory

laboratory seal end

the laboratory seal has to be created in the semen processing laboratory after __________________________ before _____________

• filling the straw with diluted semen

• freezing

factory seal has two cotton plugs with ____________________

PVA powder in between them

laboratory seal formed with ____________________ or ___________________ after ____________________

• PVA powder

• sealing machine

• filling of semen

best temp & time in thawing frozen semen

warm water, 35-40 degrees Celsius temp, 30 seconds

once semen is stored at lower temperature, there should

never be a change in the temperature of stored semen

semen should also be transferred to other places ______________________ at which it is stored

at the same temp

the vial containing chilled semen should be wrapped in? reason?

wrapped in paper before it is placed in thermos with ice to avoid cold shock to sperms coming directly in contract with the ice in the thermos

frozen semen should always be transported in this container

liquid nitrogen containers

usual sample submissions from small animal clinics

• blood samples for CBC and blood chemistry

• urine for urinalysis and culture (if suspected with bacterial infection)

• smears for cytology examination

• tissues for bacterial culture and isolation

usual sample submissions from farms, especially in outbreak investigations

• blood samples for serology

• diarrheic feces for scour work-ups include parasitology work-ups, microbiology work-ups, and virology work-ups

diarrhea is also known as

scour

a laboratory test or kit designed to identify the cause of diarrhea (scour) in animals, particularly calves and adult animals, by testing for various pathogens and parasites in fecal samples

scour package