Assisted Reproductive Technologies in Sheep - semen cryopreservation Flashcards
Ways to collect semen from ram
- 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
Advantages and disadvantages of electroejaculation (+ about)
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
advantages and disadvanatges of artificial vagina
adv ~ natural collection ~ often good quality ejaculate ~ no contamination ~ inexpensive dis ~ 2 week training period for rams ~ need teaser ewe
reasons for semen collection
- 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
Semen assessment - what to look for
- 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
semen assessment - motility
~ score 5
= dense rapidly moving waves
- over 90% sperm motile
semen assessment - motility
~ score 4
= Vigorous movement
- 70-85% sperm cells active
semen assessment - motility
~ score 3
= Small slow moving waves
- 45-65% active
semen assessment - motility
~ score 2
= No waves, some movement of spermatozoa
- 20-40% motile
semen assessment - motility
~ score 1
= Weak movement, no waves
- 10% sperm motile
semen assessment - motility
~ score 0
= Dead: all spermatozoa motionless
semen assessment -concentration
~ score 5
~ typical ram semen conc and volume
- 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)
semen assessment -concentration
~ score 4
- cream/pale cream
- 3.5-4.5 (x109) /ml
semen assessment -concentration
~ score 3
- thin creamy/ cream to milky white
- 2.5-3.5 (x109) /ml
semen assessment -concentration
~ score 2
- milky/milk white
- 1.0-2.5 (x109) /ml
semen assessment -concentration
~ score 1
- cloudy/ transparent white
- 0.3-1.0 (x109) /ml
semen assessment -concentration
~ score 0
- insignificant no. sperm
- watery (clear)
semen assessment - morphology
- 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
semen assessment - visability
- 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
what is the ideal ejaculate
- 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
basic components of cryodiluent
- 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)
sperm cryopreservation protocol
- 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
What is cold shock and the impact it has on sperm
- Rapid cooling from 30C to 0C
- 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
Role of low density lipoproteins
- 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
alternatives to ldls from egg yolk
- 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
Reactive oxygen species - about
- 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)
reactive oxygen species - affect on sperm func and when it occurs
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
ROS and antioxidants in sperm
- 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
antioxidants in seminal plasma - enzymatic
- 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
antioxidants in seminal plasma - non-enzymatic
- 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
Heat shock proteins
- 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
HSP90 in sperm - fresh compared to cryopreserved semen
- 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
