DSCI 330 Test 2

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  1. IVF Procedure
    • Oocyte collection
    • Cow restraint, epidural    
    • Ultrasound guided follicular aspiration 
    • Vaginal wall puncture into ovary and retrieve oocytes
    • Oocyte searching/processing  
    • Rinse—sterile saline
    • Pack in sterile sleeve with saline
    • Surround with room temperature (18°C) saline/fluid bags
    • Send to lab within 12 hours 
    • Sperm: Sperm must be separated from seminal plasma proteins. sperm Wash: to remove freezing extender/cryoprotectants 
    • Add drop* of sperm and cook for 18h**    
    • Shake vigorously: Fertilized oocytes, vortexed 1 min to remove cumulus cells 
    • Cleavage...24hours
    • 44‐48h: 4 cells  
    • Day 6 or 7:blastocyst stage reached 
    • Day 8: Delayed embryos (no good for transfer)
  2. Animals you can use IVF on
    • OPU: 
    • Cows/heifers (anything w/ follicular waves)
    • Dry (preferrable)
    • Milking (not during peak lactation)
    • Pre‐pubertal?
    • pregnant heifers/cows
    • DECEASED (recently: <12 hours)
  3. IVF rates of success/expectations/relevant statistics
    • 8‐20 oocytes per collection, depending on cow/breed and protocol response
    • 4‐5 transferrable embryos per session (a good cow)
    • preg rate is about 50% (individual variability)
    • The best protocols (ovary stimulation with FSH) utilize
    • every‐2‐week sessions (hoping for 10 follicles/ovary)
    • a good expectation is 2‐3 pregnancies/ session
    • So average* is about 5 pregnancies per month
  4. Factors that influence success of IVF
    • depends on quantity AND quality of the retrieved oocytes and quality of embryos transferred
    • OPU technician
    • OPU interval, hormonal treatment and response
    • Bull, especially if sexed semen
    • Recipient quality (heifers always better
    • than multiparous cows)
  5. ET Procedure (extracting from Donor)
    • Superovulation - usually 4days of FSH TWICE DAILY (Am/Pm injections) 
    • Ova randomly released from superovulated ovaries over a period of 6‐12h
    • First AI 12h after onset of standing heat
    • Second AI 12h after first insemination
    • If donor still in heat after second AI: inject GnRh
    • breed donor a third time 12 h after GnRh injection
    • Embryo collection is done on day 7 after 1st AI
    • Donor is palpated for superovulated ovaries and CLs  
    • Equipment: Y‐junction tubing Foley Catheter and stylet Embryo collection cup/filter 
    • Cuff distended with air/fluid occludes cervix or horn to allow uterine distention with fluid 
    • About 1 L flushing media, with uterine massage
  6. ET Procedure (Recipient)
    • Epidural injection (5ml lidocaine)
    • The vulva/rectal area must be cleaned
    • Part the vulva and insert ET gun 
    • Position in the uterine horn ipsilateral to the CL
    • Very gently slide the rod as far into the uterine horn until resistance is encountered (possible PGF2 liberation)
    • Flunixin Meglumine 10 min previous to transfer (Schrick et al.,2001)
    • The embryo must be expelled at this point
  7. Animals you can use ET on
    • Improves efficiency in repeat breeders (poor pregnancy rates)
    • Donor:genetically superior animal,Must pass the health program, At least 50 days postpartum Positive energy balance
  8. ET rates of success/expectations/relevant statistics
    • Probabilities of success from transferring 4 embryos if the true pregnancy rate is 50% 
    • Pregnancy rates for most technicians are in the 50 to 70 %
  9. Factors that influence success of ET
    • Innate fertility and age of donor cow
    • Response to superovulation protocol
    • Recipient quality (can decrease 10‐20% if using cows)
    • Semen quality (sexed semen lower success)
    • Embryo grade—inf luences pregnancy rate
    • Dependent on technician skill—recovery and transfer
    • Pregnancy rates for most technicians are 50 to 70 %
  10. How is it different from conventional semen and how can it best be used?
    • HERD REPLACEMENTS! rapid herd expansion 
    • Need to breed fewer for replacements and so
    • increase selection intensity; 
    • Decreasing dystocia (bull calves average about
    • 5 lb heavier BW)
  11. How is Semen sorted for sex? How is it different from conventional semen and how can it best be used?
    • Semen is collected by either mounting with AV or with electro-ejaculator
    • H03342 binds to DNA x-sperm have more DnA Aim laser light at sperm H033842 fluoresces. Measure fluorescence. Computer analysis
    • Flow cytometer
    • -lasers and detectors sort based on change
  12. What are the reasons for implementing a synchronization program? (ideally to increase pregnancy rates but what are causing those poor pregnancy rates to begin with?)
    • Controlling the timing of: Ovulation,estrus, breeding/AI
    • Knowing cycle stages/repro status of herd
    • OVERALL increase repro Efficiency! 
    • BUT we operate under energy, time and money
    • constraints, so have to treat the entire HERD as a INDIVIDUAL
  13. The procedures and troubleshooting for the AI procedure including semen handling (starting with ID'ing your semen straw)
    • I.D Bull
    • Set Straw
    • Thaw—45 sec. in 95° F water (i.d. bull again on
    • straw) 
    • Wipe the straw COMPLETELY DRY and warm your gun 
    • Make sure the plunger is back ~ 6 in. to allow for insertion of the straw.
    • Place the cotton plug end of the straw in the gun. 
    • Cut straws 1⁄4" below lab seal at 90o angle OR use a straw cutter 
    • Slide on sheath over the gun and underneath o-ring
    • Lock sheath and gun together with snug twist of the o-ring
  14. What happens to a cell undergoing freezing? Why do we use cryoprotectants and the abuses that sperm undergo in the process?
    • cell undergoes a freezing rate too quickly=death; ice crystals form in and out of the cell and a high degree of cellular damage occurs.
    • freezing occurs too slowly, the cell will be dehydrated beyond revival
    • combine cryoprotectants and optimal freezing rate to mitigate damaging effects by balancing cellular dehydration (some degree is needed to prevent high intracellular ice crystal formation) and the rate of intra and extracellular ice crystal formation
    • The pre‐freezing cooling rate (occuring before this slide at around 40°F/4C has to do with reducing sperm metabolism prior to the freezing process and glycerol is usually added last to the extender since it does have some
    • toxic effects on the cell
  15. The process of freezing semen (what happens to a cell undergoing freezing), why do we use cryoprotectants and the abuses that sperm undergo in the process?
    • Collect 2-3 times per week with 2-3 ejaculates per day
    • When semen arrives it is visually inspected
    • Determine the number of motile sperm per dose of semen to be inseminated.  
    • Target concentration to freeze: ie 20 million live, motile sperm per dose.
    • After collection and initial assessment,
    • semen is transferred to a cold room (40oF)
    • After cooling for two hours semen is packaged into straws 
    • Addition of cyroprotective agent; Extender provides a protective environment for the sperm during freezing and thawing 
    • semen is packaged in 0.5 and 0.25 milliliter
    • straws 
    • Each straw is printed with the:Company logo, bull’s full registered name, country of registration, registration number 
    • Fast‐working machines in the cold room fill
    • and ultrasonically seal the straws 
    • inserted into specially designed straw racks 
    • A computer controlled freezing process allows consistent freeze rate
  16. Procedure of collecting semen and requirements and circumstances surrounding it
    • 1) Artificial vagina (AV): temp: 42‐48°C/107 to 118°F and pressure (provided by water)
    • typically collected 2 or 3 times/ wk with 2 or3 ejaculates per collection day
    • has to be sexually stimulated prior to mounting  
    • 2) Electroejaculation: electroejaculation (“quick and dirty” method): stimulation of smooth musculature and accessory sex glands
    • Requires proper amplitude and timing which may have to be adjusted per bull  
    • 3) If you’re really desperate—from the epididymis (dead or castrated) 
    • Semen is packaged in 0.5 and 0.25 milliliter
    • straws
    • Each straw is printed with the:Company logo, bull’s full registered name, country of registration, registration number
  17. Problems and solutions with heat expression and detection and possible solutions
  18. How do we define fertility?
    • Heat Detection Rate
    • Conception Rate
  19. What affects fertility success on a typical dairy?
    • days to first service
    • days to conception 
    • calving interval
    • services per conception
    • conception rate
    • estrus detection rate
    • pregnancy rate 
  20. 21 Day heat detection rate, conception rate, and pregnancy rate
    How are they all related and what are goals/reasonable rates?
    • Heat Detection Rate: cows detected in estrus and inseminated/total cows eligible to be inseminated (i.e. open cows) 
    • Conception Rate: cows diagnosed pregnant/ cows actually detected in estrus and inseminated
    • Pregnancy Rate: cows diagnosed pregnant/total cows eligible to be inseminated (i.e. open cows)
  21. Methods of heat detection, what they measure, troubleshooting poor heat detection rates using these methods.
    • Tail chalk: combines the primary and most reliable sign of
    • estrus (standing to be mounted by a herdmate)
    • with a quick visual inspection for secondary signs 
    • Mount Detector: “HeatWatch uses patented computer based, remote sensing technology that detects and records over 95 percent of all standing mounts ‐ 24 hours a day, seven days a week” 
    • Visual Observations: at least 3xs daily but 4xs ideal (estimated 50% of cows in heat for <8hrs) At least 15 min. observation times (20-30 min better)
    • Chinball markers:
    • Pedometers: High thresholds-> false negatives:
    • under breeding. Low thresholds-> false positives (biggest drawback) overbreeding
  22. Factors affecting heat expression (Problems and Solutions)
    • 1. Housing/overcrowding
    • -reduce number of cows in pen
    • -build more pens
    • 2. Footing
    • -put grooves in concrete
    • -have outside pens
    • 3. Lameness
    • -foot baths
    • -have hoof trimmer come out regularly
    • 4. Loss of Condition, problem cows
    • -investigate nutrition/adjust rations
    • -make sure they have access to feed
    • 5. Heat stress
    • -covered areas
    • -plenty of water
    • -misters and fans
  23. Two Methods of Oocyte collection
    • Ovum/oocytepick‐up(OPU)—involving transvaginal
    • follicular aspiration in a live cow
    • Ovary harvest—follicular aspiration
    • slaughter‐house ovaries...
    • recently deceased cows/heifers
  24. Ovsynch 48/56
    • Controlling the CL and follicles
    • Recommended for timed insemination
    • Also can inseminate animals exhibiting estrus
    • following first GnRH injection 
    • The longer you can wait to give GnRH the better
    • usually starting around 60 days (VWP)after
    • calving
    • Nearly 100% AI rate (vs that with 50% HDR) 
    • Downside: Cows may ovulate abnormal follicles—too young or too old‐>infertile cycles; Cows with varying numbers of follicular waves
    • GnRH,7 days-> PGF,56h->GnRH,16h->TAI
    • GnRH,7 days-> PGF,48h->GnRH,16h->TAI
  25. Cosynch 72
    • AI same time as GnRH—much more practical
    • All cows (n=20) ovulated within 24 to 32h after the second GnRH injection of Ovsynch 
    • GnRH,7 days->PGF,72h->GnRH and TAI
  26. 5 day Cosynch
    GnRH,5 days->PGF,72h->GnRH and TAI
  27. Double ovsynch
    • Another pre-synch method
    • Better follicular wave synchrony and better preg rates  
    • GnRH,7days->PGF,3days->GnRH,7days->start TAI
  28. 2 Pgf presynchs
    • presynch methods used before ovsynch
    • used with ovsynch programs to increase pregnancies per AI
    • programs can be used with or without estrous detection 
    • ensures a more complete luteolysis of herd 
    • synchronizes the follicular waves
    • Chosen for fresh cows     
    • Shots can start 38 days prior to end of voluntary waiting period. 
    • PGF,14 days->PGF,11-14 days->ovsycnch
  29. Re-synch
    • 1. Start Ovsynch method after PD  
    • 2. Start timed AI method before PD
    • 3. 1xPGF/Ovsynch
    • 4. GnRH/Ovsynch           
    • Day 0: Timed AI
    • Day 33: Preg check and inject GnRH only to open cows
    • Day 40: Inject PGF2a to resynch cows on day 33
    • Day 42: Inject GnRH and TAI according to the program selected
  30. Factors affecting estrus expression
    • Low BCS
    • Uterine Infection
    • Cystic ovaries
  31. GnRH
    • Follicular control
    • LH surge-> ovulation within 24 to 32h
  32. PGF2a (Prostaglandin)
    • CL lifespan control
    • Decreases progesterone
    • Brings cow to heat 2-5 days later
  33. CIDR
    • Maintains high progesterone levels
    • mimics luteal phase
    • controls follicular waves and prevents heat
    • ovulation
    • GnRH and insert CIDR,7days->PGF and remove CIDR,2days->GnRH and TAI
  34. How can cows be synchronized?
    • Estrus Synchronization: prostaglandin shots, CIDR insertion and removal
    • Ovulation synchronization: prostaglandin and GnRH combinations
  35. Synchronization strategy: TIMING!
    • PGF2a (Prostaglandin) combined with GnRH shot, Ensures better response from cows that otherwise would have a CL at the time of GnRH
    • A GnRH shot BEFORE the PGF2a; Ensures more cows will have a CL (for lysis) and dominant
    • follicle (forovulation) 
    • Step 1: GnRH—ovulation or follicular wave boost
    • Step 2: PGF2a after GnRH (5 to 7 days)— luteolysis
    • Step 3: GnRH 48h later causes ovulation
    • Step 4: AI (no estrus detection needed) 16 hrs
    • later
  36. Dairy Heifer Synchronization Protocols
    • AI after detection of estrus
    • -2xPGF
    • -CIDR-PGF (7 days in between)
    • Programs for Timed AI (TAI)
    • -5dCIDR_Cosynch72
Card Set:
DSCI 330 Test 2
2014-11-17 05:17:56
DSCI 330

Dairy Science 330 AI
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