COLLECTION AND PRESERVATION OF BULL SEMEN

By :-Alok SharanI.D No. V-2002 / 11

College of Veterinary and Animal SciencesSardar Vallabhbhai Patel University of Agriculture & Technology

Modipuram, Meerut

Introduction

Semen : • It is also known as seminal fluid, is

an organic fluid that may contain spermatozoa.

• It is secreted by the gonads (sexual glands) and can fertilize female ova.

• Seminal fluid contains several components besides spermatozoa: proteolytic and other enzymes as well as fructose are elements of seminal fluid which promote the survival of spermatozoa, and provide a medium through which they can move or "swim“.

Sperm : • Sperm is the male reproductive cell and is

derived from the Greek word sperma (meaning "seed").

• A uniflagellar sperm cell that is motile is referred to as a spermatozoon, whereas a nonmotile sperm cell is referred to as a spermatium.

• Sperm cells cannot divide and have a limited life span, but after fusion with egg cells during fertilization, a new organism begins developing.

Electron microbial observation of bull sperm

Above 12 months, healthy bull (vaccinated and free from diseases with good semen quality as well as quantity).

Teaser animal or dummy. Artificial vagina

(filled with hot water temp 42 ˚C - 45˚C, essential lubricants).

Diluters and extenders.

Sterile & clean test tubes and straws.

Liquid nitrogen tank.

Requirements

False mount method ( with the artificial vagina )

By dummy ( with the artificial vagina )

Electro ejaculation method (for those bulls which won’t or can’t

mount, get urine often)

Massage method :- -Seminal vesicles-Vas deferens

Methods of collection

False Mount

By dummy

Electro ejaculation method

Massage method

Semen should be evaluated grossly for abnormal appearance. The presence of small "clots" or blood can indicate such conditions as seminal vesiculitis.

Color Acceptable color ranges from milky to creamy .This indicates sperm per cubic millimeter of 500,000 or above.

Wave pattern determined by placing a thick drop of semen on a slide under a microscope on low power and with reduced light.

Progressive motility determined by putting a thin, diluted drop of semen on a slide under a microscope on low power, 100X.

Semen evaluation

a Very good 5 80-100% motile sperm cells

b Good 4 60- 80% motile sperm cells

c Fair 3 40- 60% motile sperm cells

d Poor 2 20- 40% motile sperm cells

e Very poor 1 0- 20% motile sperm cells

Semen evaluation

Abnormal Sperm Morphology Evaluation

Q R S

Normal bovine cell (a and n) and several abnormal cells appearing in bull semen. b-e, Head abnormalities; f-i. midpiece abnormalities; j. tail abnomality; k. proximal protoplasmic droplet; l. distal protoplasmic droplet and bent tail; m. bent tail; n. loose normal head; o. spermatid;

p. spermatogonium; Q. two tails; R. abnormal middle piece; S. two heads

Parameters Normal values

Ejaculate volume 5 ml (range 1-15 ml)

Sperm concentration 1200 million/ml (range 300-2500 million/ml)

Total sperm per ejaculate Typically 4-5 billion

Progressive motility Greater than 50%

Morphology Greater than 70% normal

Required parameters of normal semen

Semen preparation for preservation

Before preserving the semen diluters / extenders are added to collected semen are :-

Milk yolk glycerol (MYG)

Milk 75 ml

Egg yolk 20 ml

Glycerol 5 ml

Penicillin G 1000 I.U. / ml

Streptomycin 1 mg / ml

Lactose yolk glycerol (LYG)

11% lactose solution 75 ml

Egg yolk 20 ml

Glycerol 5 ml

Penicillin G 1000 I.U. / ml

Streptomycin 1 mg / ml

Lactose fructose yolk glycerol (LFGY)

11% lactose solution 56.25 ml

6 % fructose solution 18.75 ml

Glycerol 5 ml

Penicillin G 1000 I.U. / ml

Streptomycin 1mg / ml

Egg yolk 20 ml

Glucose yolk citrate glycerol (GYCG)

Distil water 100 ml

Glucose 58 mg

Sodium citrate 5 g

Egg yolk 20 ml

Glycerol 5 ml

Penicillin G 1000 I.U. / ml

Streptomycin 1 mg / ml

Tris citrate fructose

Tris 3.028 gm

Citric acid 1.675 gm

fructose 1.250 gm

Egg yolk 20 ml

Glycerol 7 ml

Penicillin G sodium 1 lac unit

Dihydrostreptomycintreptomycin 10 mg

Distill water 80 ml

Processing and Preservation

The collected semen sample is placed in a water bath at 30-34˚C and examined microscopically under low power for wave pattern and gross motility.

Stains are made using eosin nigrosin for sperm morphological examination thereafter.

The IMV electronic photometer is used to determine the concentration.

At 30˚C, the semen is diluted to 50% of the final volume of the final volume of diluents using the 3% glycerol diluents half of the final volume without any glycerol.

Semen is transferred into the cold cabinet at +5˚C and allowed to cool and stabilize for about 30 min.

The second part 11% glycerol diluents or half of the final volume with 14 ml (14%) glycerol content is added to the semen to make up the total volume which will yield 7% glycerol content.

Contd…..

The diluted semen is held at +5˚C for at least 2 h equilibration time.

Semen is sucked by automatic filling machine into the straws and on meeting the polyvinyl alcohol powder plug at one end and impervious seal is made.

The other end is pinched together and cold sealed.

The semen is being checked for individual motility while the straws are held at 5˚C for another 2 h equilibration.

After equilibration, a minimum of 4 h, the straws are then ready for freezing by one of the two methods:

a) horizontal method b) vertical method

Horizontal method :- Straws are racked horizontally and are frozen in liquid nitrogen vapour at –120 to ̶ 130˚C

4 cm above the liquid nitrogen level in the freezing tank for 9 min. The straws are transferred to the container with liquid nitrogen at –196˚C after which

the motility of primary freezing is checked and recovery rate assessed. Vertical method :- Straws are racked vertically and frozen in liquid nitrogen vapour at –120 to ̶ 130˚C 0.5

cm above the liquid nitrogen level for 18 min. Straws are transferred into a container with liquid nitrogen at –196˚C after which the

motility of primary freezing is checked and recovery rate assessed.

Packaging :- Semen is packaged in 3 ways:-1. Glass ampoules of 0.5 to 1 ml of extended semen.2. Glyvinyl chloride straws of 0.25 to 0.5 ml of extended semen.3. Pelleted semen of approximately 0.1 ml. Freezing :- Packaged semen has been freezed through the following methods: Mechanical

freezing, dry ice, liquid air, liquid oxygen, liquid nitrogen. Storage :- Frozen semen are stored at -196˚C in liquid nitrogen for transportation

anywhere AI is desired.

Evaluation of fertility of frozen semen post thawing

A variation in the number of services per conception in different lactations has been reported to be due to difference in fertility of frozen semen.

Methods of checking the quality (fertility) of diluted or frozen semen :-a. Laboratory method :- 1) Motility2) Sperm cell morphology3) Sephadex filter test4) Glutamic oxaloacetic transminase (GOT) use to essay sperm cell membrane

damage during preservation.5) Amplified restriction fragments length polymorphism (AFPL) technique to assess

the semen motility.6) Live ability at 37˚Cb. Field :- Fertility is the only accurate determinant of the quality of frozen semen.

Advantages

Collected semen can be diluted and extended to create hundreds of doses from a single ejaculate.

Semen can be stored for long periods of time, meaning that males can produce offspring long after their natural reproductive lives end.

The collection process allows for the screening of disease agents.

Collected semen is also routinely checked for quality, which can help avoid problems associated with male infertility.

Easy transportation of good germplasm in the form of frozen semen.