ASCI341 Lab Practicum Final Flashcards
What does a CL feel like when rectally palpating cattle?
Projection coming off of the ovary
What does the antral follicle feel like when rectally palpating cattle?
Fluid-filled, not protruding out
List the methods of reproductive technologies
- Semen sexing
- In vitro production (IVP) of embryos
- Trangenic animal production
- Gene Mapping
- Marker-assisted selection
What are the types of transgenic animal production?
Microinjection
Blastocyte injection of embryonic stem cells
Nuclear transfer / cloning
Spermatogonial germ cell transport
What are the applications for reproductive technologies?
- Propagate superior livestock animals
- Remove or add gene to improve a breed of livestock animals
- Select animals for specific traits very early in life
- Production of proteins which are essential to human life
- Organ transplantation
What is semen sexing?
Sexed semen is characterized by the presence of either X‐ or Y‐chromosome‐bearing sperm, allowing the production of offspring of the desired sex.
What is the main difference between x and y-chromosomes in sexed semen?
Main difference: the x-chromosome has an extra short arm that is absent on the y-chromosome.
Y-bearing chromosome: negative charge
X-bearing chromosome: positive charge
How does sexed semen work?
Pass sperm through a laser that excites vital dye. X-chromosome will light up more because they have more total DNA that will pick up more dye)
As sperm pass through the system individually. It gives sperm charge based on the light given off. It then passes through a magnetic field that separates them.
What is the advantage and disadvantage of semen sexing?
Advantage –> dead sperm pass through, which purifies sperm
Disadvantage –> expensive ($500K - 1 million)
What is the success rate of semen sexing?
95%
What is in vitro fertilization of embryos?
- Mix mature oocytes with sperm
- Develop embryos to specific stage and transplant into live animal
How do you superovulate a donor cow for in vitro embryo fertilization?
- Porcine FSH treatment –> promotes more follicle development
What are the steps of in vitro fertilization?
- Synchronization of recipients with donor
- Superovulation of donor
- Inseminate donor with semen from genetically superior bull
- Recovery and identification of embryos
- Transfer viable embryos into synchronized recipients
What stage of embryo is commonly collected during embryo flushing process?
Usually aiming for compact blastocyst stage (would be located in uterine horn)
What is transvaginal oocyte aspiration?
Newer technology –> long ultrasound probe inserted into the repro tract. Palpate through the rectum and pin the ovary against the vaginal wall. The probe has a needle pushing the needle through the vaginal wall into the ovary. Will be able to aspirate fluid out of follicles that contain eggs/oocytes. Connected to an ultrasound machine that has a targeting vector on it.
What is the advantage of transvaginal oocyte aspiration?
Separate oocytes out and can fertilize with sperm from different males
What is pronuclear injection/microinjection?
Foreign DNA is injected into pronuclei.
The oocyte is reduced to a haploid state; sperm has haploid complementary chromosomes. At this point, we perform a pronuclear injection.
What is syngamy?
Fusion of pronuclei
What order of membranes does the fine polled glass pipette pass through in microinjection?
Zona pellucida –> plasma membrane –> cytoplasm –> pronucleus (zygote)
Is pronuclear injection/microinjection more successful in mice or livestock species? Why?
Mouse = 10% success rate. Much less efficient in livestock.
Mouse embryo –> pronuclei are visible under light microscopy
Livestock embryos –> have much more lipid, so you can’t visualize pronuclei.
What are the advantages and disadvantages of pronuclear injection?
Advantage: if we want to over-express DNA, we can generally do it in one generation
Disadvantage: Gene incorporated into genome RANDOMLY and can cause damage. A lot less efficient, and end up killing a lot in the process.
When does blastocyst injection of embryonic stem cells occur?
It occurs at a much lower stage in the development (days 7-9 of embryo development)
Embryo classified with blastocoel (cells inside zona pellucida)
What do inner cell masses form?
Forms embryo
What do trophoblasts form?
Placenta = houses developing fetus during gestation
What are the steps of blastocyst injection of embryonic stem cells?
- get a donor embryo
- Isolate embryonic stem cells
- Gene targeting
- Transform embryonic stem cells
- Selection of transformed embryonic stem cells
- Isolation of transformed ES cells
- Expand transformed ES cells
- Inject transformed ES cells into recipient embryo (blastocyst)
What is a passage in cell culturing for isolating embryonic stem cells?
Let cells grow until they almost populate the whole plate. Then take a subpopulation of cells and split them up, and let them grow again to fill up another plate and so forth
What is gene targeting?
Add / replace / remove gene
What is homologous gene recombination?
Gene swapped out with an altered sequence
Why do we add antibiotics when doing blastocyst embryonic stem cell transfer?
Add antibiotic resistant –> only vells that have taken up foreign DNA will acquire the antibiotic resistance. Only cells that survive antibiotic tx are the ones with genetic alteration
What is a chimeric animal?
An animal chimera is a single organism composed of two or more different populations of genetically distinct cells originating from different zygotes involved in sexual reproduction.
Comes from greek mythology –> aspects of 2 species (manatar)
Is a chimeric animal crossbred?
Not cross bread. Has purebred parts of each
What is a geep?
Sheep x Goat chimeric animal
Geep x Ewe
Lamb
Geep x Goat
Kids
What percent will be purebred Duroc when mixing male chimera with duroc female?
20%
1/5 of the time
What is the advantage and disadvantage of blastocyst injection of embryonic stem cells?
Advantage: Better efficiency than pronuclear injection. With this method, we can target alteration where it was previously random.
Disadvantage: takes 2 generations
What is cloning / nuclear transfer?
Nuclear transfer is a form of cloning. The step involves removing the DNA from an oocyte and injecting the nucleus, which contains the DNA to be cloned.
For cloning, where can donor cells be collected from?
- Skin / ear punch
- Liver cells
- Embryonic stem cells
These donor cells are then cultured
What was unique about Dolly the cloned sheep?
First cloned animal using somatic cells –> mammary gland cells.
Previously, frog eggs were not able to do this; would die at the tadpole stage
What animal is best for organ transplant and why?
Pigs - knocked out sugar that is important in organ transfer rejection
What are the steps of cloning?
- Collect donor cells and culture
- Enucleate egg (remove all nuclear material from cytoplasm)
- Combine with enucleated cytoplasm. Provide electric shock that will open pores in the cytoplasm. The donor cell will dump the nucleus into the cytoplasm of the enucleated oocyte.
- Let develop to a later stage –> freeze –> transfer surgically to a recipient
What are the advantages and disadvantages of cloning?
Advantage: make genetically engineered in one generation, more efficient
Disadvantage: Expensive, less efficient in livestock
How are glow-in-the-dark pigs from UNL made?
Using gene from sea coral
When would spermatogonial germ cell transplantation be used?
We would do this with 2 mammals
What are the steps of spermatogonial germ cell transplantation?
- Isolate primitive sperm cells from the donor testis (biopsy / whole organ)
- Stem cells into recipient male (cease sperm production
- Rete tubules = target locations. Set up shop in adluminal/basal compartment
What would the result of spermatogonial germ cell transplantation be?
Recipient produces mature sperm from donor
What type of damage(s) is done to the recipient in spermatogonial germ cell transplantation?
- Heat testis up
- Radiation kills sperm
- Antagonism GnRH
When would spermatogonial germ cell transplantation be used?
In cattle
Using genetically superior bull in range location (don’t want them to get injured)
What are the steps of rectal palpation in cattle?
- Approach cow to be palpated
- Arm covered with glove should be lubricated
- Hand is inserted by forming the thumb and fingers into a cone tip, with a slight rotary motion
- Rake fecal material out
- Push down on rectum to feel repro tract
Where should be genital tract be when palpating cows?
Should lie below the floor of the pelvis
What does the cervix feel like during rectal palpation of cows?
It appears as a firm rope-like structure
Why would we use estrous synchronization?
- Group females for parturition
- Reduce time required for estrous detection
- Eliminate estrous detection with timed insemination
What is the principle of using progestogens for estrous synchronization?
Maintain the cow under the influence of progesterone until CL regresses, remove progesterone - animal responds to decrease progesterone with estrus 2-5 days later
How can progesterones be administered for estrous synchronization?
Injection
Feed
Implant
CIDR
What is done for a two shot prostaglandin method for estrous synchronization?
Give first shot
Give 2nd shot 14 days after
Heat check 2 days after
AI window 3-5 days after 2nd shot
What is done for the AI EAZI-BREED CIDR protocol for estrous synchronization?
Insert CIDR
6 days after, inject PGF2a
Next day, remove CIDR
Next day, inject 2 mL CIDROL
Next day, heat check
Next day, AI window (3 days)
What is done for MGA feed administration for estrous synchronization?
Feed MGA 0.5 mg/hd/d for 14 days
17 days after, inject lutalyse
3 day AI window after that
How do you load an AI gun?
- Get semen ready, cover it from sunlight, and put it in water bath for 30-45 seconds
- Keep rod warm in jacket
- Load AI gun, lock with sheath on
- Keep warm until ready to AI cows
What are the three methods of estrous synchronization?
- MGA
- CIDR
- 2-shot prostaglandins
How long is the estrous cycle in a sow?
21 days
What does a triangle indicate in sow estrous?
Indicated heat
What does the 2- -> 2+ mean in sow estrous detection?
How swollen vulva is
What are signs of a sow being in standing heat>
Lock up when pressure is applied, ears flick, swollen vulva +/- mucous.
What does it mean to superovulate cows?
Superovulation (SOV) is a necessary technique to produce large numbers of embryos for embryo transfer. In the conventional methods, follicular stimulating hormone (FSH) is administered to donor cattle twice daily for 3 to 4 days.
What was used to collect semen in the bull?
Electroejaculation
What direction do the probes go in electroejaculation, and why?
Probes go down
If they went up, they would hit the spinal cord. When they are facing down, they stimulate the accessory sex glands
What type of copulator is the bull?
Short
The bull mounts the female, does a couple of pelvic thrusts, and ejaculation occurs within a few seconds
What type of copulator is the stallion?
Intermediate
Mating takes between 30 seconds-1 minute. The volume of ejaculation is going to be much higher. Concentration will be lower than what we see in bull
What type of copulator is the boar?
Sustained
Mating in pigs takes between 5-20 minutes.
What is the volume/concentration of boar semen like?
Volume of ejaculate is significantly higher than other types of copulators, but sperm concentration is a lot lower
Characteristics of bull ejaculate
Creamy while, lower volume / higher concentration
Characteristics of boar ejaculate
Pre-sperm fraction = clear
Sperm-rich fraction = milky
Post-sperm fraction = gelatinous, tapioca-like
Higher volume, lower concentration
How did we prepare semen for evaluation?
- Invert the tube several times
- Submerge the tip of the capillary pipette into a sample of fresh semen and allow the tube to fill by capillary action
- Place diluted semen into ice for 4-5 minutes to induce irreversible loss of motility
- Place into hemacytometer
How do you use a hemacytometer?
Count the five large squares (80 small squares) on the diagonal from the upper left to lower right of the ruled area of the hemacytometer.
To avoid errors of omission or duplication, count only those cells entirely inside the triple-lines square and those on the top and left boundary lines. Do not sound those cells touching the right and bottom boundary lines.
What is the equation we use to count semen?
Dilution x constant (50) x # sperm x mL constant (1000_
Unit –> sperm/mL
How do you get the amount of sperm/ejaculate?
Take sperm/mL and multiply by volume
How does the live/dead sperm stain work?
Dead will take up the dye because there is a disruption of the acrosome and plasma membrane –> this allows uptake
What was the sperm diluted with before being placed in the hemacytometer?
Diluted with PBS
What are the types of sperm head abnormalities?
- Crater defect (nuclear vacuoles)
- Tappered heads
- Ruffled acrosome
- Knobbed acrosome
What head abnormality is this?
Crater defect
What are the types of sperm tail abnormalities?
- Coiled tail
- Double midpiece
- Folded tail
- Detatched head
What type of motion do we want sperm to have?
Forward progressive motolity
What is the key indicator of immature sperm?
Cytoplasmic droplet in proximal location, just below head
When we examine ejaculate, what do we look for?
- Concentration
- Dead/ alive
- Motility (progressively moving forward)
In general, what are stem cells?
Undifferentiated cells that replenish or repopulate either a specific tissue or organ OR multiple tissues and organs. This allows tissues and organs to maintain their viability and functionality
Totipotency
The ability of a single cell to differentiate and develop into a complete organism
How do you test for totipotency?
The only way to test for totipotency is to take a cell and perform nuclear transferring using a nuclear donor cell. If an entire animal is produced from that one cell, it can transform into every tissue cell type of the body.
Pluripotency
The ability of a single cell to differentiate into more than one cell type or tissue
What were the anesthetics used for surgical embryo retrieval and flushing in pigs?
T.A.R.K.
telazol // atropine // rompun // ketamine
What type of anesthesia were the pigs maintained under during surgery?
Isofluorine
What were the different tissue layers to be opened + methods?
What types of tubes are used to administer anesthesia during surgery?
Endonasal tubes are inserted into the nares of pigs. Pigs are obligate nose breathers, so they would rather suffocate than breathe out of their nose.
What type of flushing media is used during embryo flushing?
BECM
How did we isolate the embryos?
Where was the cannula inserted?
Once one ovary and oviduct are exposed, we will then pass the cannula into fimbriae.
Insertion of flushing cannula into the infundibulum of the oviduct.
The cannula must be placed in the infundibulum to ensure recovery of the embryo.
How were each tissue layers sutured?
Size/type of suture material, type of needle, stitch
Suturing body wall –>
Needle: Use an atraumatic needle (only has a sharp edge on the very end of the needle). Use a larger, curved needle, so it doesn’t dive under and damage organs.
Suture: #2 CATGUT (absorbable type of suture derived from sheep intestine.)
Stitch: Each suture will be a double knot (interrupted suture)
Suturing skin –>
Needle: Cutting edge needle (sharp end and sides)
Suture: #1 or #0 CATGUT (thinner)
Stitch: Continous stitch
How did they superovulate sows for embryo flushing?
PG600 and hcG
Where is the incision made for embryo retrieval?
The 3-inch skin incision is made on abdomen between the umbilicus and the last pair of teats
What part of the blade is used to cut into the abdomen and why?
Curved edge of the blade
We never use the pointed part because we could accidentally puncture organ
What method is used to dissect through fat and peritoneum?
Use hands to bluntly dissect because we don’t want straight cuts on blood vessels. It will narrow and clot off to avoid too much bleeding.
Where was the blunt needle inserted?
Penetrate the uterine lumen and move through the utero-tubular junction into the oviduct with a blunt-end needle (onto the other side of the isthmus)
One cell location and day
Oviduct
0-2
Two cell location and day
Oviduct
1-3
Four cell location and day
Oviduct or uterus
3-5
Eight cell location and day
Uterus
3-5
Sixteen cell location and day
Uterus
4-5
Morula cell location and day
Uterus
5-8
How does embryo develope
1 cell –> 2 cell –> 4 cell –> 8 cell –> 16 cell –> morula –> compact morula –> early blastocyst –> blastocyst –> expanding blastocyst –> hatching blastocyst `
What are the two types of cells in a blastocyst? What do they eventually develop into?
Inner cell mass –> forms embryo/fetus
Trophoblast –> forms placenta
Hematopoietic stem cells
Form blood and immune cells, responsible for constant renewal of blood, isolated from blood or bone marrow
Umbilical cord stem cells
Isolated from the umbilical cord, a perfect match for the donor, and differentiate into a variety of cell types
What day does cell become ovum –> embryo?
Day 13
What happens on day 21 od development?
Heart beat begins
Lungs, liver, and pancreas begin to develop in foregut
When do limb buds change to legs and toes, the tail grows, and eyes and nostrils become apparent?
day 30
When does the allantoic sac fill extend the full length of the chorionic sac?
Day 36
When do bones begin to ossify?
Day 38
What is the yolk sac?
Origin –> early entodermal layer
Function –> vestigial
What is the amnion?
Origin –> cavitation from inner cell mass
Function –> encloses fetus in a fluid-filled cavity
What is the allantois?
Origin –> diverticulum of hindgut
Function –> blood vessels connect fetal with placental circulation; fuses with chorion to form the chorioallantoic placenta
What is the chorion?
Origin –> trophoblastic capsule of blastocyst
Function –> Encloses embryo and other fetal membranes; intimately associated with lining of uterus to form placenta
What is the umbilical cord?
Origin –> amnion wraps around the yolk sac
Function –> encloses the allantoic vessels and acts as the vascular link between mother and fetus
What are the layers of the placenta?
The innermost placental layer surrounding the fetus is called the amnion.
The allantois is the middle layer of the placenta.
The outermost layer of the placenta, the chorion, comes into contact with the endometrium.
What are the 4 types of placentations?
- Diffuse
- Cotyledonary
- Zonary
- Discoid
What species have a diffuse placenta?
Horse / sow
What animals have a cotyledonary placenta?
Cows / rumonants
What animals have a zonary placenta?
Dogs / cats
What animals have a discoid placenta?
Humans / rodents
Diffuse placenta
This type of placenta occurs over the entire surface of the uterine luminal epithelium with the formation of folds/villi and is found in horses and pigs.
Cotyledonary placenta
Multiple, discrete areas of attachment called cotyledons are formed by the interaction of patches of allantochorion with endometrium. The fetal portions of this type of placenta are called cotyledons, the maternal contact sites (caruncles), and the cotyledon-caruncle complex a placentome
Zonary placenta
The true villi occur in a girdle-like band in the middle of the chorionic sac while the ends are smooth chorion
Discoid placenta
The true villi are limited to one to two disc-shaped areas
What side are caruncles on?
Uterine side
What side are cotyledons on?
Placenta side
What type of caruncles do cows have?
Convex
What were the four fetal things observed during the lab?
- Crown –> rump length
- Gender
- Weight
- Age/stage time point
