DSCI 330 Final

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DSCI 330 Final
2014-12-09 01:05:03
DSCI 330

DSCI 330, Artificial Insemination, Final Exam
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  1. Embryo candidates
    • Ideally, good to excellent quality embryos day 7 after AI (or IVF )
    • Should be frozen within three hours of recovery
  2. Two Methods of Freezing Embryos
    • Conventional—slow freezing with a programmable machine
    • -Conventional freezing— utilizing cryoprotectants and slow freezing rates 
    • Vitrification—fast freeze with no machine  
    • -Vitrification—utilizes high cryoprotectant concentration and fast freezing rates
  3. Cellular damage when freezing
    • Extra‐ and Intracellular ice formation
    • Osmotic stress
    • pH changes
    • Also, phase changes in the lipids of cell membranes are
    • altered with cooling (Edidin and Petit, 1977)
    • Countered with optimal freezing rates
    • Cooling rates above optimum causes death after formation of intracellular ice
    • Cooling rates slower than optimum expose cells to concentrated solutions of extracellular fluids for prolonged periods
  4. Ways of decreasing the damage when freezing
    • Cryoprotectants lower freezing points, decreasing the proportion of water frozen into ice at any particular temperature (antifreeze)
    • Cryoprotectants also have the effect of restoring equilibrium by permeating into cells and causing cellular dehydration (displace H2O)
    • Macromolecules (sugars) further facilitate the movement of cryoprotectant and water
    • (maintaining osmolar balance)
  5. Slow freezing/ conventonal vs. Vitrification
    • i.e. with GLYCEROL
    • expense of procedures (usually use a machine)
    • complexity of procedures (temperature gradient
    • issues pre and post freezing)
    • Usually higher cryodamage by intracellular ice crystal formation (lower concentrations of
    • cryoprotectants)
  6. Slow freezing/ conventonal vs. Vitrification
    • i.e. with ETHYLENE GLYCOL
    • Cheap to perform—
    • no slow freezing/seeding
    • required so no machine
    • Requires embryo handling in a timed format (skill required!!)—not popular
    • Low intracellular ice crystal formation but higher
    • cryoprotectant concentrations
    • Similar pregnancy rates vs. slow freezing embryos
  7. Splitting Embryos
    More Info
    • fine microsurgical blade
    • First true clones produced    
    • Each embryo has a different genotype and more than likely a different phenotypic expression due to its environment.
  8. Cloning
    • Asexual reproduction
    • Creating genetically identical organisms
    • Nuclear transplantation to ovum
    • Copying a animal genetically
  9. Methods of Cloning
    • Splitting of embryos 
    • Separation of blastomeres
    • Nuclear transplantation to ovum
    • Fusion of ovum and small cell
  10. Dopey Calf Syndrome
    • Usually large calves
    • Dystocia exacerbates
    • Slow to nurse
    • Recover completely with care
    • Incidence ~1%
  11. Nuclear Transplant Calves at Birth
    • Most morphologically normal
    • Most slow to nurse
    • Most metabolically abnormal
    • High incidence of neonatal death
    • 20-30% are oversized
    • Slight increase in congenital abnormalities
  12. Periods of Clone Loss
    • 1. Pre-implantation
    • -65% of blastocysts fail
    • 2. Day 30 to Day 60
    • -Death of >50% of embryos
    • 3. Second Trimester
    • -Spontaneous abortion
    • -Some abnormalities
    • -Abnormal placenta
    • -Low placentae count
    • -Thickened fetal membranes
    • 4. Third Trimester
    • -Hydroallantois
    • --Reduction in placentomes
    • -Edema of Umbilicus
    • 5. Postnatal
    • -Large birth size
    • -Inadequate Organ Development
    • --lung dysmaturity
    • --Respiratory distress
    • --Metabolic imbalance
  13. Take home points of cloning
    • efficiency not much more than 1-2%
    • Expect difficulty establishing pregnancy
    • and possibly abnormal calf
    • Cost to produce a clone (Viagen) $20,000+
  14. Fertility is a multi-factorial trait and its deterioration has been caused by a network of
    what 3 main factors?
    genetic, environmental and managerial factors.
  15. Genetic Factors specifically involve
    Selecting for non-production traits such as longevity(Single nucleotide polymorphisms, which are desirable variations in the gene sequence which can be a more predictive of performance exist for this trait)—selecting for Daughter Pregnancy Rate (SNPs) in bulls; and selection for SNP’s for growth hormone, which are associated with fertility
  16. Environmental Factors specifically involve
    • heat stress affects cow fertility at all levels from decreased estrus expression to early embryonic. 
    • Heat stress also exacerbates nutritional stresses by decreasing dry matter intake and increasing metabolic demand. Maximum shade, fanning and misting
    • use of IVF produced embryos  
    • Transferring frozen embryos    
    • Poor or inappropriate management is one of the leading causes of infertility
    • Nutrition can not be underestimated. High levels of crude protein can be fatal to embryos
    • vaccination protocols must be implemented
  17. Regarding pinpointing causes, why is studying infertility so difficult in the modern dairy cow?
    • Complex interactions
    • of these factors (environmental, genetic, and managerial) make it difficult to determine the exact reason for this decline
    • Early embryo loss is also difficult to study since most pregnancy detection can not be performed until at least day 26 and not routinely performed until after 30 days
  18. What are the some of the significant contributing factors to the decreasing pregnancy rates in Figures 1 and 2?
    • Negative energy balance, uterine infections, hormonal imbalances
    • Influences...poor timing to fertilization due to improper insemination times/lack of
    • accurate heat detection, sociological stress, immune suppression in part due to
    • metabolic stress and mineral imbalance
  19. Considering these factors, how can we practically recognize (propose methods) and
    test for (propose methods) possible causes of these decreases at varying time points
    from fertilization to birth to assess not only the base fertility level of the herd but those
    factors affecting it?
    • looking at heat detection/conception/pregnancy rates,    
    • include ultrasound to detect number of problem cows in the herd at a given time (i.e. cystic ovarian structures and uterine infections), progesterone levels could be tested  
    • Assessing BCS (ideally 3 at calving) and changes thereof (should be less than 0.5 between calving and first service)
  20. Before we concentrate too hard on abnormalities, let’s define the normal post calving dairy cow. How do the authors define a “normal” post-partum dairy cow (section 3.4)?
    • A ‘normal’ post partum dairy cow can be defined as one which has resolved uterine involution, resumed ovarian follicular development, ovulated a healthy dominant
    • follicle early postpartum and continues to have normal oestrous cycles at regular intervals of approximately 21 days, coupled with homeostatic concentrations of insulin, IGF-I and glucose
  21. What is one obvious though underutilized physical characteristic that we use to
    measure the nutritional well-being of a cow?
    • Body condition score is an internationally accepted, subjective visual and tactile
    • measure of body condition and temporal changes in BCS are used to monitor nutritional and health status of high producing cows during their productive cycle
  22. We shouldn’t think of fertility in a “here and now window.” Why are some insults (i.e.
    heat stress, negative energy balance, metabolic disease) in the cow able to affect her
    fertility up to months later
    • Exposure of ovarian oocytes to unfavorable physiological events during follicle development from primordial to pre-ovulatory stage may result in the ovulation of defective oocytes up to 3 months after the insult. Therefore, early postpartum disorders such as NEB, abnormal
    • gonadotropin secretion, uterine infection and other disorders discussed earlier IN ADDITION TO HEAT STRESS could have subsequent
    • deleterious effects on oocyte development and competence, ultimately affecting conception/pregnancy rates as well as pregnancy loss rates.
  23. What can be concluded from studies collecting embryos at different stages from lactating versus non-lactating cows?
    • Embryos recovered at day 7 from non-lactating Holstein-Friesian heifers and beef heifers were of higher quality compared to lactating Holstein-Friesian cows  
    • one approach is to help ensure adequate levels of progesterone which may include administering hCG (LH analog) that would cause ANOTHER CL to form (added progesterone to
    • benefit early pregnancy) between days 5 and 7 post AI. ALTERNATIVELY, a CIDR
    • could be inserted to supplement progesterone levels.
  24. Do you think that feeding moldy hay to dry
    cows is a good idea? Why or why not?
    • runs risks of increasing pregnancy loss/abortions due to spreading of fungal spores
    • mold decreases palatability and may decrease overall DMI and prevent cows from reaching ideal BCS prior to calving.
  25. What are the major approaches to improving fertility and how can these be practically implemented in an on-farm approach?
    • high producing dairy cows have reduced immune competence that can lead to increased incidence of lameness, mastitis and endometritis when compared to low producing dairy cows.
    • increasing dry matter intake during the transition period, minimising NEB, decreasing BCS loss early postpartum and resolution of uterine infection.
  26. The procedures and troubleshooting for the AI procedure including semen handling (starting with ID'ing your semen straw)
    I.D BullSet StrawThaw—45 sec. in 95° F water (i.d. bull again onstraw) 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-ringLock sheath and gun together with snug twist of the o-ring
  27. How do in-vivo and in-vitro conditions differ in their effect on embryos?
    • In vivo (made in the“living” cow)
    • In vitro (made in the “glass”)
  28. What have been the trends regarding fertility and the modern day dairy cow?
    As milk production goes up, fertility rates go down
  29. What defines a "problem cow" vs. a normal cow from the ovarian and uterine standpoint?
    • reduced fertility
    • Poor conception rates to estrus bred cows
    • poor timing, breeding pregnant cows
    • Anovular/ cystic cows
    • prolongs cycle, complicates breeding and increases days open
  30. What infectious disease processes are at work against the dairy cow's fertility?
  31. Anovulatory/cystic follicles
    • Oversized follicles that do not ovulate
    • persist for a variable period in the absence of a CL
    • Follicles normally ovulateat 18‐22mm
    • Disruption of GnRH/LH hormone axis and or receptor number/function 
    • Usually fail to ovulate
    • Some still may produce estrogen and inhibin
  32. Follicular cyst treatment
    • GnRH->ovulation/luteinization
    • Or LH analog (human chorionic gonadotropin hCG)-> ovulation/luteinization (not as well documented)
    • Return to normal cyclic ovarian activity occurs in 72% to 85% of the cows treated
  33. Luteal cysts (less common)
    • Usually over 2.2 cm
    • Thick wall >3mm
    • Clear or trabeculated centers
    • Progesterone present
  34. Luteal cyst treatment
    • Prostaglandin
    • IF sufficient luteal tissue is present
    • May require a prior dose of GnRH or hCG (to get full
    • luteinization before PGF will respond) and second PGF shot
  35. How NOT to treat cysts
    • DO NOT “POP”  
    • may result in trauma and hemorrhage causing
    • adhesions and contributing to fertility reduction
  36. Can cysts be addressed with timed AI/Synchronization Programs
    • Resolve cysts (since both GnRH and PGF2 used)
    • Some support a PGF shot before first GnRH (modified ovsynch)
    • BUT also part of the reason protocols may fail
    • Thus, these cows deserve ultrasound/palpation re‐checks and CID
  37. Addressing cysts with CIDR’s
    restoring the responsiveness of the hypothalamus to the positive feedback of estradiol, resulting in normal estrus and ovulation within 7 d after the implant is removed.
  38. Uterine diseases of post‐calving cows
    • Metritis: inflammation of the uterine lining
    • and deeper layers 
    • Endometritis: inflammation of the endometrium (lining of the uterus) 
    • -Clinical: (20% (5‐>30%)—signs evident (discharge and can see it on ultrasound)
    • -Subclinical: (30% (11‐>70%)—signs inobvious (no discharge/not detected on ultrasound)
    • Pyometra
  39. Endometritis on ultrasound
    • Abnormal uterine fluid of varying echogenicity 
    • Sometimes palpable if enough fluid present
  40. Endometritis
    • Inflammation of the uterine lining (superficial)
    • Usually transient, bacteria eliminated after one or so cycles 
    • Cow is not sick
    • -Appears as abnormal uterine
    • -fluid if visible at all
    • -Requires little intervention (as usually occuring before VWP)
    • -Prostaglandin +/‐antibiotics
  41. Metritis
    • Inflammation of ALL layers of the uterine wall
    • the cow often systemically sick (off‐feed, fever, downer, decreased milk yield, laminitis)
    • Appears as thickened uterine wall, with abnormal fluid
    • Requires intervention
    • Anti‐inflammatories (Banamine) and systemic
    • antibiotics
    • Discharge may or may not be present 
    • Usually earlier on within 2 weeks post calving
  42. Treatment of Endometritis/pyometra
    • Caution with antibiotic infusion in uterus!
    • Drug withdrawal times!
    • Changes uterine pH, irritating, can cause a chemical irritation->adhesions
    • Bacterial antibiotic resistance! 
    • Often respond with oxytocin (if no CL)/prostaglandin (if CL) treatment
    • Better treated with prevention!
    • Ensuring adequate mineral balance and nutrition
    • Clean calving areas and sterile obstretrics
    • Decreasing stress->adequate bunk/housing space
    • If incidence of uterine infection in a herd exceeds 20%, clean up your act!
  43. How can we incorporate synchronization programs and ultrasound to help better diagnose and manage problem cows in the herd? Specifically understanding the troubleshooting diagram we covered in lecture
    • Resolve cysts (since both GnRH and PGF2 used)
    • Some support a PGF shot before first GnRH (modified ovsynch) 
    • BUT also part of the reason protocols may fail
    • Thus, these cows deserve ultrasound/palpation re‐checks and CIDRS
  44. Addressing cysts with CIDR’s
    • Progesterone “priming” may induce cyclicity and
    • help resolve cysts 
    • restoring the responsiveness of the hypothalamus to the positive feedback of estradiol, resulting in normal estrus and ovulation within 7 d after the implant is
    • removed.
  45. Diagram
  46. Factors that attribute to fertility loss (30 day loss)
    • uterine contamination
    • not giving shots correctly
    • negative energy balance and body condition score
    • ovarian cysts
    • scar tissue around the infidibulum
    • inappropriate management of high milk producing dairy cows.
  47. Factors that attribute to fertility loss (30-60 day drop)
    • accidentally give a shot of PGF
    • Body stress/environmental stressors
    • Heat stress
    • Infections
    • not having the proper nutrition
    • AI technician bringing in bacteria when AI'ing the cow
    • Managerial: probably the most significant contributing cause