Practical Exam

Question 1

P1: Method of semen collection for boars

Answer 1

“Gloved hand method”

Question 2

P1: Important points when collecting semen (3)

Answer 2

1. Avoid contamination of semen (clean the prepuce of the male).
2. Use clean, sterile, dry and warm semen collection vessels; avoid cold shock.
3. Ensure dummy sow is secure and non-slip mat is in place

Question 3

P1: Why should you work in groups of three when inseminating?

Answer 3

One inseminates
One applies back pressure
The other assists.

Question 4

P2: What temperature should ram ejaculate be kept at in water bath?

Answer 4

30 degrees

Question 5

P2: What are two ways of estimating concentration of semen?

Answer 5

1. Haemocytometer

2. Photocolorimeter

Question 6

P2: Types of abnormalities in sperm:

Answer 6

1. tailless (decapitated)
2. coiled tail
3. tapering head
4. broken at neck or midpiece
5. small or large head
6. enlarged midpiece
7. double head
8. double tail
9. protoplasmic droplet
10. abnormal arosome

Question 7

P2: Wave motion

Answer 7

5 - Dense, very rapid vigorous waves, changing direction very rapidly, individual spermatozoa cannot be seen; 80% or more of the spermatozoa are alive and active.
4 - Waves and eddies are not so rapid as for score 5; about 65 - 80% of spermatozoa are active.
3 - 45 - 65% of spermatozoa are active. Only a few small and slow waves, or no waves. Individual spermatozoa can be observed.
2 - 20 - 45% of spermatozoa are alive, motility is poor. No waves are formed.
1 - Very few sperm cells show signs of life with very weak and non- progressive movement.
0 - No motility. All spermatozoa are dead.

Question 8

P2: Motility of spermatozoa (Types of motion):

Answer 8

a. Progressive forward movement;
b. Rotary movement: individual sperm move in a circle;
c. Oscillatory, or convulsive movement, without progress and change in position.

Question 9

P2: When counting sperm heads using haemocytometer, how do you increase uniformity?

Answer 9

• Heads on top and right lights counted IN

- Heads on bottom and left lines counted OUT

Question 10

P2: When counting sperm heads using haemocytometer,, how many squares are counted?

Answer 10

5. 4 corners and a middle square.

Question 11

P2: What is the volume of 25 large squares on haemocytometer?

Answer 11

1/10,000 ml

Question 12

P2: What is the calculation to determine the number of sperm/ml in original sample from haemocytometer counts?

Answer 12

No. sperm/ml = No. sperm in (5 squares x 5) x dilution rate (200) x 1/volume of 25 squares (10,000)

Question 13

P3: Cryobiology

Answer 13

The study of the effect of sub zero temps on biological materials

Question 14

P3: Cryopreservation

Answer 14

The storage of biological materials at sub zero temps

Question 15

P3: Why cryopreserve

Answer 15

Increases the longevity of a sample

- preservation of tissue or cells e.g. sperm, oocytes, embryos

Question 16

P3: Role of cryoprotectant

Answer 16

Protects cells against freezing injury

• Decrease freezing point of a solution
• Maintain membrane fluidity

Question 17

P3: To allow cells to remain viable throughout freezing and thawing, it is important to:

Answer 17

• Minimise damage due to dehydration

- Minimise damahe due to ice formation

Question 18

P3: What effect does contamination of water (e.g. salt addition) have on it’s freezing/thawing point?

Answer 18

It DECREASES the freezing/thawing point

Question 19

P3: Define osmolarity

Answer 19

The concentration of a solution, often defined as osmols/L

Question 20

P3: Define tonicity

Answer 20

The osmotic pressure of a solution, often expressed as relative to an intercellular environment (e.g. hypo, iso, hypertonic)

Question 21

P3: Define pH buffer

Answer 21

A solution which reduces the change of pH upon addition of small amounts of acid or base.

Question 22

P3: Impact of freezing too slowly

Answer 22

= Dehydration

• Extracellular ice formation
• Slow increase in concentration of extracellular solution
• Water rushes out of cell

Question 23

P3: Impact of freezing too fast

Answer 23

= Explosion

• Intracellular ice formation
• Intracellular ice expansion
• Physical damage and cell death

Question 24

P3: What does the optimal freezing rate depend on?

Answer 24

• Cell size (larger cells like embryos have a slower cooling rate, smaller cells like sperm have a faster cooling rate)
• Extracellular solution (presence of cryoprotectant)

Question 25

P3: Role of egg yolk in cryopreservation

Answer 25

• Active ingredient = low density lipoproteins (LDL’s)

- LDL’s stabilise the structure and prevent loss of lipids from membrane during freezing

Question 26

P3: Vitrification

Answer 26

Freezing in the absence of ice

OR solidification of liquid without crystallisation

Question 27

P3: Pros and cons of vitrification

Answer 27

For
- Improved survival rates
- Application to whole tissue not just cells
Against
- Technically difficult 
- Questionable biosecurity

Question 28

P3: Seeding

Answer 28

= Facilitated ice formation or ice nucleation

Deliberate initiation of ice formation at predefined temperatures allows for more controllable and reproducible freezing conditions

Question 29

P3: DMSO and Glycerol as cryoprotectants

Answer 29

• Penetrating cryoprotectants
• DMSO: Permeates rapidly, embryo freezing
• Glycerol: Permeates slowly, sperm freezing

Question 30

P3: Sucrose and egg yolk as cryoprotectants

Answer 30

• Non-penetrating cryoprotectants
• Sucrose: Enhances effect of penetrating cryoprotectant
• Egg yolk: Improves membrane fluidity