Assisted Reproductive Technologies in Sheep - semen cryopreservation

• 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

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

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

• 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

• 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

= dense rapidly moving waves - over 90% sperm motile

= Vigorous movement - 70-85% sperm cells active

= Small slow moving waves - 45-65% active

= No waves, some movement of spermatozoa - 20-40% motile

= Weak movement, no waves - 10% sperm motile

= Dead: all spermatozoa motionless

• 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)

• cream/pale cream - 3.5-4.5 (x109) /ml

• thin creamy/ cream to milky white - 2.5-3.5 (x109) /ml

• milky/milk white - 1.0-2.5 (x109) /ml

• cloudy/ transparent white - 0.3-1.0 (x109) /ml

• insignificant no. sperm - watery (clear)

• 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

• 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

• 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

• 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 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

• 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

• 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