In vitro Flashcards
(25 cards)
step of in vitro
-ovum pick up
compare the oocyte donors (OPU)
-calf, heifer, pregnant heifer, cow, problem cow
calfs and heifers need hormonal treatment
calfs need ovum pick up to be laparoscopically, while everyone else gets transvaginally
calfs produce the most embryos (3). cows 2-3 heifers 0.5-1
effect of cow breed on the number of oocytes recovered via OPU
- beef cattle produce more oocytes (bos indicos 251 per OPU)
- Anti-Mullerian hormone (AMH) significantly increase the antral follicle count
- able to collect many more embryos via OPU IVF than MOET
- again in vitro had many more embryos produced than in vivo and a greater amount of viable embryos per procedure
- same percentage of pregnancies obtained from in vitro and in vivo ie no difference if the embryo was in vivo or in vitro
problems with IVP derived embryos
lower embryo development (40-50% in bovine)
ie oocytes derived from immature follicles, apoptotic follicles, embryos are less resistant to cryopreservation, higher embryo/fetal loss
How do we improve the efficiency of IVP?
- produce/select developmentally competent oocytes
- produce better and more resistant embryos ie competent for implantation and resistance to cryopreservation
- methods to select developmentally competent embryos
- methods to identify and prepare recipient females with a higher potential to support pregnancy
advantages of IVP over IVD
• More offspring per female are produced.
• Can be performed in different reproductive stages: prepubertal
heifers; estrus-cycling or noncycling cows;
pregnant (3-4 months).
• Hormonal treatments (superovulation) are not required.
• Less interference with the reproductive cycle.
• Less effect on productive performance.
• Extends the reproductive life of the cows.
• Increases genetic gain and variability (oocytes can be
separated and fertilized with semen from different bulls).
• Better use of expensive semen and sexed-semen.
what are the steps to pre-attachment for a bovine embryo?
fertilization, cleavage, compaction, blastulation (day 6), expansion, hatching, elongation (day12)
what are the steps to pre-attachment for a bovine embryo?
fertilization, cleavage, compaction, blastulation (day 6), expansion, hatching, elongation (day12)
cumulus cells
special accessory cells that surround the developing oocyte and help isolate and nourish it
a mature oocyte has
condensed chromatin, polar body, and expanded cumulus cells
what are the parts of an oocyte (from outside in)
zona pellucida, cytoplasm, nucleus, nucleolus
what triggers the meiotic maturation of the oocyte?
LH (which also triggers ovulation). fully grown oocytes that are removed from the follicle and placed in culture will mature without LH
oocyte growth - purpose, size, time
accumulate size to undergo divisions, accumulate proteins, ribosomes, mRNA, mitochondria that will support the early embryo. It will grow 100-200 fold in volume and it takes 3-4 months
what are gonadotropins?
hormones that regulate reproduction ie LH FSH
maturation of the oocyte - stages
- arrested in prophase 1 - oocyte
- added gonadotropins (LH) to stimulate meiotic division #1
- spindle reassembly, spindle relocation, polar body produced
- the cell is arrested in metaphase 2 and is now an egg cell
how is meiotic maturation regulated?
protein kinases
the immature oocyte will not mature without an active CDK1 (with 1 P) bound to cyclin B (inactive form has 3P on the CDK1)
role of active CDK1
nuclear membrane breakdown
chromosome condensation
spindle formation
CPEB phosphorylation (translational activation)
-basically anything that will help prepare the cell for meiosis I
acrosome reaction
- this is how the sperm penetrates the zona pellucida
1. sperm penetrate the cumulus cells to reach the ZP
2. sperm binds with the ZP
3. sperm undergoes exocytosis and releases acrosomal contents (enzymes)
4. side of sperm binds to the egg membrane and then fuses with the membrane
what is the purpose of sperm capacitation?
so the sperm can bind to the zona pellucida and perform the acrosome reaction
- detaches sperm from oviductal cells
- penetrates the oviductal mucus
how is sperm capacitation attained in vitro?
• In vitro capacitation requires cholesterol removal - fluidize the plasma membrane
• Heparin, bicarbonate and calcium activate the kinase pathways that drive capacitation - increased cAMP
production
• Frozen-thawed sperm from many species appear to be
capacitated
what does sperm capacitation mean?
- change in motility
- NO change in sperm morphology
sperm prep for IVF
• Semen Type: frozen, fresh, refrigerated
• Semen enrichment: percoll gradient, swim-up,
filters
• Chemical capacitation: heparin 10 microg/mL
-want a fertilization between low fertilization and polyspermy
-
what factors can affect sperm capacitation?
- Semen: male, batch, concentration, time of exposure
* Capacitation: agent, dose, time of exposure
cortical granules
located in the cytoplasm of the egg, they are released when a sperm and oocyte fuse to prevent polyspermy. block the acrosome reaction
