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Flashcards in sperm transport and storage Deck (36)
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in sheep how many sperm are in different parts of the female reproductive tract at 4 hours after insemination?

3, 000, 000, 000 sperm by natural mating
2,904,000 sperm in cervix after 4 hours
112,000 sperm in uterus
1680 sperm in oviducts

1 in 27,000 inseminated sperm passed the cervix
(1 in 10 million of the inseminate in the oviducts)


what amount of sperm reach the oviduct in sheep and pigs?

Sheep: 1 in 10 million sperm
Pig: 1 in 2.4 million sperm

they still need to transverse the oviduct


how can sperms ability to reach oviduct in vivo be predicted?

sperm migration through cervical mucus in capillary tubes.

can we replicate the natural sperm selection process?


what is migration efficiency?

distance (mm) travelled by the vanguards perm in 7 minutes.

high migration rate in vitro predicts migration to the oviducts.


what does high fertility require?

1)sufficient competent sperm for sufficient duration


what are causes pf poor sperm quality?

- 40-80% is due to reactive oxygen species
(ROS = free radicals with unpaired electrons)


what are ROS species physically important and damaging for sperm?

need for - capacitation, acrosome reaction, binding and traversing the ZP
however oxidative stress can cause severe damage: poor motility, morphological abnormalities, DNA damage and cell death.


can sperm DNA be repaired?

it cannot be repaired except by the oocyte itself.

sperm nuclear function is critical for fertility


what tests are commonly used to test sperms integrity?

- sperm chromatin structure assay - common
- nuclear protein composition (by protein separation)
- sperm nuclear maturity test (by nuclear staining)
- comet assay (by single cell gel electrophoresis) - common
- Tunel assay - common
- DNA oxidation


what are the measures and characteristics of sperm chromatin structure assay

measures: susceptibility of sperm DNA to denaturation
: objective, flow cytometry based, indirect assay, complex analysis, used clinically.


what are the measures and characteristics of nuclear protein composition (by protein separation)

measures: sperm histone and protamine levels
objective: gel electrophoresis assay, indirect assay, labour intensive


what are the measures and characteristics of sperm nuclear maturity test (bynuclear staining)

measures: chromatin, compaction. chromatin content
characteristics: simple, semi-quantitative, slide based, indirect assay


what are the measures and characteristics of comet assay (by single cell gel electrophoresis)

measures: double stranded DNA breaks (neutral assay)characteristics: objective, quantitative, direct assay, complex image analysis.


what are the measures and characteristics of TUNNEL assay?

measures: double stranded DNA breaks
characteristics: semi-quantitative, direct assay, quantitative if flow cytometry based.


what are the measures and characteristics of DNA oxidation?

measures: 8-hydroxy-2-deoxyguanosine
characteristics : quantitative, direct assey, labour intensive


what are the three steps of cryopreservation?

1) cooling
2) feezing
3) thawing


what does cooling do to sperm motility?

slows it down - eventually sperm stop moving


what steps are taken to protect the sperm when freezing them?

- cryoprotectant added some hours before freezing
- the sperm samples are cooled slowly (over 30 minutes to 2 hours) to about 5 degrees to avoid cold shock.
- samples are often left to equilibrate for a few hours
- samples are frozen according to optimal protocols


how do cryoprotectants change the solution?

they raise the solution osmolarity to high levels
cells initially respond by shrinking
they later re-equliberate and resume their originl volume.


what are frozen sperm stored in?

they are loaded into and stored in plastic straws or cryovials


what is required for successful cryopreservation

- majority of sperm should retain an intact plasma membrane; typically 50% do not
- cell functions in the live population should not be impaired (they usually have a shortened lifespan
- all organelles should be intact and functional
- sperm DNA should be intact and able to support development.


why does insemination timing using frozen sperm have to be very accurate?

be cause frozen sperm has a shortened life span.


what two effects does freezing have on sperm?

1) reduces the number of surviving spermatozoa
2)reduces competency of the survivors


what goes wrong with frozen sperm?

some cryoprotectants actually reduce the fertility of sperm
greater than 3% glycerol reduced fertility of pig sperm
glycerol is a contraceptive for chicken sperm
many of the survivor sperm demonstrate capacitation-like changes and have reduced survival time.


how long are fresh and frozen sperm viable for?

fresh - 2 days
frozen - 8 hours


what is cold shock?

fast cooling from body temp to about 5degrees Celsius

cold shock and even slow cooling cause a form of premature capacitation


what additives are used for sperm cryopreservation?

- egg yolk (whole or dries) is routinely used
- skimmed milk
soybean lecithin
- cocnut milk

- some use of sodium lauryl sulphate (detergent) to emulsify the egg yolk


what are the types of dilution?

1 step dilution
8 step dilution

if you add diluent in littles steps the amount of osmotic shock decreases and final motility is 95% where as if you add it all at once the volume of sperm increases a large amount and leaves motility at 30%.


what is vitrification?

means glass formation - but no crystals are formed
if solutions are cooled rapidly enough they do not form ice crystals
vitrification has typically involved such high cryoprotectant concentrations that sperm cannot survive initial dilution.
however it can be done without cryoprotectants


how is human sperm vitrification done without cryoprotectants?

1) drop 20 um droplets of sperm suspension directly into liquid nitrogen
2) 2 um volumes in closed or open straws plunged into liquid nitrogen
3) films of sperm suspension in cryoloops plunged into liquid nitrogen
3)post thaw motility and viability are good

there are a few reports of successful ICSO with vitrified sperm