Artificial reproductive technology 1 Flashcards
Define artificial insemination
Placing semen into the female reproductive tract
What kind of semen can be used for AI?
- Fresh
- Fresh-extended
- Extended-chilled
- Extended-frozen-thawed
What is an extender in AI?
A formulation that keeps sperm alive over a longer period of time
What are the benefits of AI?
- Allows greater access to superior genetics and maximises genetic improvement
- Reduces mating costs and risks
- controls reproductive disease
- Allows use of dead or injured sires (if collected before death or injuery)
- Can be used as part of embryo transfer regimes
When might semen analysis be undertaken?
- Lowered fertility suspected
- Abnormal sexual behaviour
- Before sale
- Before breeding season
- If pathogenic infection is suspected (to enable culture/isolation of pathogens)
What information can be gained from semen analysis?
- Fertilising potential of the ejaculate
- Appearance
- Motility
- Live:dead ratio
- Morphology
Why is semen analysis a poor discrimiinator between moderate and high fertility of an ejaculate?
- Would require sperm function tests
- Not done routinely
- Temp, pH, ATP content, penetration of cervical mucus
Outline the features of a gross abnormality assessment of semen
- Colour compared to normal
- Normal fractionation
- Normal gel component
What is the normal appearance of the sperm rich fraction of semen?
Creamy with some density
- Can be graded 1-5 and used to estimate sperm density
What is important when using a semen sample to assess motility?
- Spermatozoa very sensitive to cooling
- Must be maintained around body temperature (37)
- slides and dilutents must be warmed
What are the 2 methods of motility assessment?
- Subjective assessment (standard)
- Objective assessment (computer aided)
Describe a subjective assessment of sperm motility
- Placed on warmed slide on heated stage, evaluate immediately
- Estimate percentage of sperm with different motility types
- need progressive (forward) movement
What is the difference between motility and progression?
- Motility can be good i.e. lots are moving in one way or another
- But may have poor progression i.e. very few are moving forwards
- Poor progression means lower fertilising ability
Describe the objective assessment of sperm motility
- Computer Assisted Sperm Evaluation
- Measures mean sperm velocity, mean sperm linearity, lateral head deviation, mean curvilinear velocity
How can sperm viability and morphology be assessed?
- Staining of sperm with dyes and observation under phase microscope
- Observation of fixed sperm by differential interference contrast (DIC)
- Either need to highlight the sperm by background staining, or stain the sperm themselves
What is differential interference contrast?
- A method of assessing sperm viability and morphology
- Objectives mounted in microscope
- Observation of fixed sperm with contrast
How is membrane function of a sperm assessed?
- Staining
- Using nigrosin-eosin staining
- Poor membrane will be stained by eosin and will appear pink
- Good membrane will exclude eosin and appear pink
How can fertility of sperm be assessed?
- Not by conventional evaluation
- Rely on sufficient number of normal and motile sperm to assume normal fertility
- Can do fertilisation assays (not routine)
Name the fertilisation assays for sperm
- Zona binding test
- Oocyte penetration test
List the methods for semen collection.
- Teasing followed by one of these below
- Artificial vagina with teaser or dummer female
- Teaser or dummy female and manual manipulation of penis
- Electro-ejaculation
What is the semen collection method usually used in bulls and stallions?
Artificial vagina with teaser or dummer female
What is the semen collection method usually used in boars and dogs?
Teaser or dummer female with manual manipulation of the penis
What is the semen collection method commonly used in rams?
- Teaser or dummy female with artificial vagina
- Electroejaculation
How is bull semen usually stored?
Freezing
How is ram semen usually stored?
Frozen or fresh
How is boar semen usually stored?
- Poor freezing ability
- Extended at ambient temperature
How is stallion semen usually stored?
- Poor freezing outcome and significant individual variation
- Fresh, chilled
- Frozen in straws
How is dog semen usually stored?
- Poor freezing outcome and significant individual variation
- Chilled
- Frozen
- Rare
What are the 3 types of sperm abnomality than can occur?
- Primary: defect of spermatogenesis
- Secondary: epididymis (maturation)
- Tertiary: post-ejaculation (trauma)
Give examples of commonly seen sperm abnormalities
- Pyriform head
- Knobbed acrosome
- detached or swollen acrosome
- Nuclear vacuoles
- Detached heads
- Distal midpiece reflex (kink in tail)
- Looped tail
- Coiled tail
- Cytoplasmic droplets
What species have fractionated semen?
- Stallion
- Boar
- Dog
How can semen be preserved?
- Fresh-extended (room temp)
- Diluted/cooled (5degreesC)
- Diluted/frozen (-196degrees C)
What is the process for storing semen as diluted/frozen?
Frozen using liquid nitrogen
What is the life span of semen that has been fresh-extended at room temperature?
Up to 8 hours
What is the life span of diluted/cooled (5degreesC) semen?
Up to 4 days for most species, 10 days for boars
What is the life span of diluted/frozen (-196degreesC) semen?
Indefinite - only thawed when eneded, can be frozen forever and deep freezing preserves structure and viability
How do extenders carry out their function?
- Protect sperm during cooling/freezing/warming
- Supply an energy (glucose and protein) source to sperm
- maintain pH, osmolarity and ionic strength (contain bicarb as buffer)
- Prevent bacterial growth
What factors are important in semen preservation?
- Protective agents (milk or egg protein/glycerol)
- sugars
- pH, osmolarity, ionic strength
- Antibacterial agents
Describe freeze-thawing of semen
- Can cause significant sperm damage
- Additional cryoprotectants required
- Controlled freezing rate and thawing rate required
Give examples of cryoprotectants
- Penetrating (glycerol, DMSO)
- Non-penetrating (proteins, large molecular weight sugars)
Describe what happens to the spermatocytes if the freezing rate is slow enough and permeable to water
- During freezing, water leaves cell which progressively dehydrates
- Small ice crystals form inside cell
- On thawing, ice crystals melt and cell rehydrates and is functional
Describe what happens to spermatocytes if the freezing rate is too quick, or the cell is impermeable to water
- During freezing, cell does not dehydrate, large ice crystal s form inside cell
- Cell is damaged
- On thawing, the cell is non-functional
Describe what happens to spermatocytes if the freezing rate is too slow
- During freezing, cells dehydrate and solute concentration increases
- Solutes tend to precipitate
- Cells shrink beyong minimum compatible with survival
- Cell is damaged
- On thawing is non-functional
Describe sperm counting using a haemocytometer
- Mutliply semen volume by sperm concentration
- Place semen into haemocytometer coutning chamber after dilution
- Count central square of counting chamber, by counting the 4 corner squares and the middle square of this
- Multiply by 5 to get average, then by the volume of the counting chamber, then by the dilution factor
What is semen sexing?
- Separation of sperm based on DNA content in order to determine/choose whether the conceptus will be female or male
What is the significace of semen sexing?
Economic value e.g. if cows are to produce milk or meat
How is semen sexing carried out?
- X chromosomes have more DNA than Y
- X larger
- Separate by labelling live sperm with DNA fluorescent dye and sorting by flow cytometry
- More fluorescence from X, sorted into different containers
