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Advances In Animal Production Science > Assisted Reproductive Technologies in Sheep - semen cryopreservation > Flashcards

Assisted Reproductive Technologies in Sheep - semen cryopreservation Flashcards

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1
Q

Ways to collect semen from ram

A
  • teaser female
  • artificial vagina
    ~ mimic vagina conditions by altering temperature/pressure
    ~ 40-45 degC ideal
    ~ 30-35 degC = too cold by the time ram jumps
  • ram trained to mount = 30 seconds
  • electroejaculation
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2
Q

Advantages and disadvantages of electroejaculation (+ about)

A 
adv ~ quick
~ almost guaranteed sample
~ dont need teaser ewes
~ dont need to train ram
Dis ~ lower quality ejaculate (low conc, less motile) = no teaser female
~ welfare concens
~ high chance urine/blood contamination
~ only used by rejestered vet
- low stimulus initially, increase strength gradually until collection semen 
- 2-4 secs stimulus, 2-8 sec break
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3
Q

advantages and disadvanatges of artificial vagina

A 
adv ~ natural collection
~ often good quality ejaculate
~ no contamination
~ inexpensive
dis ~ 2 week training period for rams
~ need teaser ewe
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4
Q

reasons for semen collection

A
  • sex sorting
  • test fertility
  • in vitro fertilisation
  • MOET (multiple ovulation embryo transfer)
  • ICSI (introcytoplasmic sperm injection)
  • artificial insemination
    ~ rapid rate genetic gain
    ~ incapacitated males
    ~ control of disease
  • cryopresevation
    ~ patients with impaired fertility
    ~ conservation
    ~ storage and transport of genetics
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5
Q

Semen assessment - what to look for

A
  • motility ~ wave motion, sperm motility
  • concentration ~ colour, consistancy, cell number
  • morphology ~ what sperm looks like, abnormalities
  • computer assisted sperm analysis ~ straight line motility, acrosomal integrity, dna integrity
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6
Q

semen assessment - motility

~ score 5

A

= dense rapidly moving waves

- over 90% sperm motile

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7
Q

semen assessment - motility

~ score 4

A

= Vigorous movement

- 70-85% sperm cells active

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8
Q

semen assessment - motility

~ score 3

A

= Small slow moving waves

- 45-65% active

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9
Q

semen assessment - motility

~ score 2

A

= No waves, some movement of spermatozoa

- 20-40% motile

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10
Q

semen assessment - motility

~ score 1

A

= Weak movement, no waves

- 10% sperm motile

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11
Q

semen assessment - motility

~ score 0

A

= Dead: all spermatozoa motionless

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12
Q

semen assessment -concentration
~ score 5
~ typical ram semen conc and volume

A
  • Thick creamy /pale cream
  • 4.5-6.0 (x109) /ml
  • 2 – 6 x 109 (2-6 billion) sperm/ml
  • 1-2ml in volume
  • sperm creamy colout = more present
  • small volume (as too much would not go through vagina)
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13
Q

semen assessment -concentration

~ score 4

A
  • cream/pale cream

- 3.5-4.5 (x109) /ml

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14
Q

semen assessment -concentration

~ score 3

A
  • thin creamy/ cream to milky white

- 2.5-3.5 (x109) /ml

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15
Q

semen assessment -concentration

~ score 2

A
  • milky/milk white

- 1.0-2.5 (x109) /ml

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16
Q

semen assessment -concentration

~ score 1

A
  • cloudy/ transparent white

- 0.3-1.0 (x109) /ml

17
Q

semen assessment -concentration

~ score 0

A
  • insignificant no. sperm

- watery (clear)

18
Q

semen assessment - morphology

A
  • A relatively detailed test- rarely used in the field
  • Useful for rams introduced to an AI programme
  • > 15% abnormal sperm should not be used for semen freezing or AI
  • 30% motile sperm after thawing = if valuble ram
    But need to be 40% or above idealy = dont want to breed from sub fertile males
19
Q

semen assessment - visability

A
  • Sperm with impaired membranes are considered “non-viable” (not dead!)
  • Eosin-Nigrosin Stain
    ~ Stain enters sperm heads with impaired membrane
    ~ stain binds to dna
    ~ Non-viable sperm are stained pink
    ~ Viable sperm are not stained – appear white
    ~ Nigrosin (pink) acts as a background stain to help visualisation
20
Q

what is the ideal ejaculate

A
  • 1-2ml in volume
  • Creamy – high concentration (at least 3.5 x 109 sperm/ml)
  • Wave score of 4 or 5 (at least 70% motile)
  • Less than 15% of sperm in ejaculate abnormal
21
Q

basic components of cryodiluent

A
  • Buffer (to maintain pH = 7-7.8 ram, and osmolarity)
  • Energy source (glucose/fructose)
  • Protective cooling agent
    ~ To protect against cold shock
    ~ Traditionally egg yolk
    ~ Other alternatives researched
  • Cryoprotective agent (glycerol)
  • Antibiotics (penicillin and streptomycin)
22
Q

sperm cryopreservation protocol

A
  • sperm in tube in 30 degC water bath
  • as there is a risk in cold shock, cooled in fridge at 5 degC for 2 hours
  • then frozen into pellets (-80 degC dry ice block) or straws (liquid nitrogen gradually)
  • straws used as can identify each, no need for dry ice and there is less user variability
23
Q

What is cold shock and the impact it has on sperm

A
  • Rapid cooling from 30C to 0C
  • Protected by cooling agents such as egg yolk
  • Induces changes in sperm membrane structure (lipids) = colesterol removed, start capacitating, mem fluid
  • Alters membrane permeability to calcium
    ~ Calcium influx
    ~ Capacitation
    ~ Acrosome reaction
    ~ Reduced sperm longevity
24
Q

Role of low density lipoproteins

A
  • LDLs associate with the sperm membrane to stabilise it.
  • Phospholipids in LDLs coat sperm membrane and form a protective film in sperm surface and/or replace phospholipids lost during cryopreservation.
  • LDLs compete with detrimental sperm proteins in binding to sperm membrane to protect sperm
  • LDLs bind directly to seminal plasma proteins that typically initiate capacitation thus preventing capacitation
25
Q

alternatives to ldls from egg yolk

A
  • A potential biosecurity/microbial risk = contamination disease spread
  • Egg yolk can cause sperm agglutination
  • Variation in egg-yolk compositions cause inconsistencies
    ~ nutrition impact composition of ldls
    ~ more variation in free range eggs than caged
    ~ need to use cage to reduce variation but welfare issue
  • Milk: whey proteins and casein have similar effect to LDL and can scavenge seminal plasma proteins
  • Soy Lecithin (phosphatidyl choline) – a common fraction of soy and egg yolk
26
Q

Reactive oxygen species - about

A
  • During normal respiration oxygen reduced to produce water
  • Incomplete reduction of oxygen forms Reactive Oxygen Species (ROS)
  • 1-4% of oxygen consumed in metabolism diverted into generation of ROS
  • Oxygen is a free radical as it has 2 unpaired electrons
  • As oxygen scavenges electrons it creates ROS (O2- H202 and OH)
27
Q

reactive oxygen species - affect on sperm func and when it occurs

A

oxidative stress results in
- damage sperm mem through oxidative damage to FAs and PUFAs
- induce sperm capacitation and acrosome reaction
reduce sperm motility and viability
- oxidative damage to dna, proteins and enzymes
- alter biochemical sperm properties
- decrease fertilising ability of sperm

~ oxidative stress occurs when there is imbalance of ROS and antioxidants
~ over production ROS
~ insufficient antioxidants

28
Q

ROS and antioxidants in sperm

A
  • Low concentrations of ROS stimulate capacitation, hyperactivation, and the acrosome reaction (they are needed)
  • Excess ROS are detrimental to sperm function and integrity
  • Antioxidants are vitamins and minerals which, together with enzymes, neutralise free radicals
  • Seminal plasma contains antioxidants – freezing dilutes this out AND reduces antioxidant enzyme activity
    ~ lower than 10% seminal plasma = antioxidants reduced, sperm cant ptorect themselves
29
Q

antioxidants in seminal plasma - enzymatic

A 
- superoxide dimustase (SOD)
~ converts H2O2 to O2 and OH
~ Major natural enzymatic Ax in semen
- Catalase 
~ converts OH to H20
- Glutathione Peroxidase (GPx)
~ converts OH to H20
- Glutathione Reductase 
~ converts reduced oxidised GPx to active GPx
30
Q

antioxidants in seminal plasma - non-enzymatic

A
  • vitamins, minerals and amino acids
    ~ immediately reduce free radicals
    ~ donate electrons
    ~ e.g. vits A,C,E (common)
  • Cystine
    ~ amino acid
    ~ enters sperm and increases gluthathione
    ~ thiol groups on cystine enter sperm and donate electrons
  • Taurine
    ~ main free amino acid of seminal plasma
    ~ direct detoxification of ROS e.g. H2O2
31
Q

Heat shock proteins

A
  • Intracellular proteins present in all cells
  • Protect against damage caused by cellular stresses such as:
    ~ Temperature change
    ~ Oxidative stress
  • Many families including HSP60, HSP70 and HSP90
    HSP90 knock-out mice are infertile
  • Treatment of sperm with HSP90 inhibitor reduces motility and increases reactive oxygen species
  • HSP90 expression reduced with semen storage
  • HSP90 protein expressed in the sheep cervix
32
Q

HSP90 in sperm - fresh compared to cryopreserved semen

A
  • fresh = HSP90 expression at the acrosome and mid piece
  • cryopreserved = HSP90 expression at post-acrosomal region and tail
    ~ translocated to tail = maintain motility?
    ~ detrimental to sprem head for fertilisation

Advances In Animal Production Science (13 decks)

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