sperm transport and storage

Question 1

in sheep how many sperm are in different parts of the female reproductive tract at 4 hours after insemination?

Answer 1

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)

Question 2

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

Answer 2

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

they still need to transverse the oviduct

Question 3

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

Answer 3

sperm migration through cervical mucus in capillary tubes.

can we replicate the natural sperm selection process?

Question 4

what is migration efficiency?

Answer 4

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

high migration rate in vitro predicts migration to the oviducts.

Question 5

what does high fertility require?

Answer 5

1)sufficient competent sperm for sufficient duration

Question 6

what are causes pf poor sperm quality?

Answer 6

• 40-80% is due to reactive oxygen species

ROS = free radicals with unpaired electrons

Question 7

what are ROS species physically important and damaging for sperm?

Answer 7

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.

Question 8

can sperm DNA be repaired?

Answer 8

it cannot be repaired except by the oocyte itself.

sperm nuclear function is critical for fertility

Question 9

what tests are commonly used to test sperms integrity?

Answer 9

• 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

Question 10

what are the measures and characteristics of sperm chromatin structure assay

Answer 10

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

Question 11

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

Answer 11

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

Question 12

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

Answer 12

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

Question 13

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

Answer 13

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

Question 14

what are the measures and characteristics of TUNNEL assay?

Answer 14

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

Question 15

what are the measures and characteristics of DNA oxidation?

Answer 15

measures: 8-hydroxy-2-deoxyguanosine

characteristics : quantitative, direct assey, labour intensive

Question 16

what are the three steps of cryopreservation?

Answer 16

1) cooling
2) feezing
3) thawing

Question 17

what does cooling do to sperm motility?

Answer 17

slows it down - eventually sperm stop moving

Question 18

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

Answer 18

• 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

Question 19

how do cryoprotectants change the solution?

Answer 19

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

Question 20

what are frozen sperm stored in?

Answer 20

they are loaded into and stored in plastic straws or cryovials

Question 21

what is required for successful cryopreservation

Answer 21

• 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.

Question 22

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

Answer 22

be cause frozen sperm has a shortened life span.

Question 23

what two effects does freezing have on sperm?

Answer 23

1) reduces the number of surviving spermatozoa

2) reduces competency of the survivors

Question 24

what goes wrong with frozen sperm?

Answer 24

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.

Question 25

how long are fresh and frozen sperm viable for?

Answer 25

fresh - 2 days

frozen - 8 hours

Question 26

what is cold shock?

Answer 26

fast cooling from body temp to about 5degrees Celsius

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

Question 27

what additives are used for sperm cryopreservation?

Answer 27

• 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

Question 28

what are the types of dilution?

Answer 28

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%.

Question 29

what is vitrification?

Answer 29

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

Question 30

how is human sperm vitrification done without cryoprotectants?

Answer 30

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

Question 31

what is a key problem of vitrification?

Answer 31

liquid nitrogen is not sterile
semen is not sterile

micro-organisms are preserved by freezing together with sperm
therefore they are a potential source of infection

liquid nitrogen tanks can be contaminated with bacteria and viruses.

Question 32

how many sperm will meet the egg?

Answer 32

1 millionth

the female reproductive tract is very selective

Question 33

what happens to pig and human sperm when chilled?

Answer 33

in humans after 10 hours around 50% DNA fragmentation

pigs - basically indestructible - after 96 hours only 30% of DNA fragmentation

Question 34

when sperm is frozen why does the temperature increase after 2 minutes?

Answer 34

because when ice crystals form they release heat

the temp stays at this higher level for about 3 minutes and the continues to cool

Question 35

what causes channels to form in frozen sperm?

Answer 35

salts come out of the crystals and channels form

glycerol stops these channels forming

Question 36

why do you have to compromise the cooling rate?

Answer 36

• when you cool too fast ice crystals form inside the cell

- when you cool too slow - too much salt exposure which is damaging to the cell.