Laboratory Tests Used in Reproduction

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kris10leejmu
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Laboratory Tests Used in Reproduction
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2012-11-25 07:36:28
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Lab Tech ll
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  1. What is the purpose to do reproductive lab testing?
    • to facilitate breeding
    • to assist in diagnosis of reproductive disorders
    • to assist in diagnosis of the causes of infertility
    • theriogenology
  2. What is theriogenology?
    veterinary specialist for reproduction
  3. How long is a dog's estrous cycle?  When is it considered abnormal?
    • normal:  averages 6 months
    • abnormal:  < 4 or > 12 months
  4. What are the different stages of the estrous cycle?
    • proestrus
    • estrus
    • diestrus
    • anestrus
  5. How long does a dog's proestrus last?
    3 - 17 days
  6. What are the signs of proestrus for a dog?
    • vulva swollen
    • vaginal bleeding
  7. Are male dogs attracted to a female dog during proestrus?
    yes
  8. How long does a dog's estrus stage last?
    3 - 21 days
  9. What are the signs for estrus in a dog?
    • vulva still swollen
    • still has bloody discharge
    • tail flags/usually will stand
    • may seek out males
  10. When does a dog ovulate?
    ovulates usually in 48 - 72 hours as they come into estrus
  11. Which stage is standing heat for a dog?
    estrus
  12. Why is it hard to spay a dog while they are in heat?
    blood vessels rupture easily and can cause hemmorrhaging
  13. How long does the diestrus stage last in dogs?
    lasts 2 months
  14. What are the different signs of diestrus in a dog?
    • discharge ceases
    • vulva returns to normal
    • pregnancy or pseudocyesis
  15. What is pseudocyesis?
    false pregnancy
  16. What is another name for diestrus?
    metestrus
  17. How long does the anestrus period last in a dog?
    lasts 4 - 10 months
  18. What is anestrus in a dog?
    period of sexual inactivity
  19. When a dog is spayed, what stage are they permanently in?
    anestrus
  20. Before we breed a dog, what do we need to ask and do first?
    • should the bitch be bred? (looks, diseases, abnormalities)
    • test for brucellosis
  21. Why is it important to test for brucellosis before breeding a dog?
    • causes infertility
    • is contagious (don't want to spread it to a perfectly good breeding dog and then ruin their career as a breeding dog)
    • zoonotic - don't want to spread it
  22. Which test can we use to test for brucellosis?
    • D-Tec CB slide test
    • blood culture or AGID test (definitive tests)
  23. How do we do the D-Tec CB slide test?
    • Do rapid slide agglutination test (RSAT) first (mix plasma with reagent and look for clumping)
    • If the rapid slide agglutination test comes up positive, then do 2 - ME RSAT
    • If the 2 - ME RSAT comes up positive then sdent it out to the lab
  24. Are false positives common with D-Tec CB tests?
    • yes
    • a negative is a negative, but a positive does not necessarily mean its positive
  25. What is the most common cause of infertility in dogs?
    breeding at the wrong time
  26. What tests can we use to help breed dogs at the correct time?
    ovulation timing tests
  27. Other than breeding a dog at the wrong time, what are other things that could cause infertility?
    • endocrine disorder
    • hypothyroidism
    • infection
    • anatomical defect
    • sterile male
    • poor semen quality
  28. What are some examples of ovulation timing tests for dogs?
    • Target test
    • ICG Status-Pro
    • ELISA test
  29. What do all ovulation timing tests measure in dogs?
    progesterone levels
  30. When should we do the ovulation timing test for dogs?
    • start in proestrus for a baseline
    • then repeat every 2 - 3 days
  31. How long does fresh dog semen last?  Chilled?  Frozen?
    • fresh:  about 5 - 7 days
    • chilled:  about 48 hours
    • frozen:  about 24 hours
  32. How does the ICG Status Pro ovulation timing test work in dogs?
    as hormone increases, color fades on test
  33. How does the Target test ovulation timing test work in dogs?
    • look for simple color change
    • from blue to white
    • breed 2 days after faint blue
  34. When should we inseminate a dog?
    • ovulates 2 days post LH peak
    • eggs must mature 2 - 3 days
    • fertile 4 - 7 days post LH peak
    • days 5 - 6 are the best to inseminate
  35. Why do we do vaginal cytology for a dog?
    • to identify cellular reactions
    • to help identify stage of bitch's estrous cycle
  36. When doing a vaginal cytology should we check the entire slide?
    yes
  37. Which magnification do we use for vaginal cytology?
    400x
  38. How do we collect a sample for a vaginal cytology?
    • gently roll on roof of vagina
    • avoid the vestibule at the bottom of the vagina because we will see weird squamous cells that are sloughing off
    • roll on a slide
    • air dry
    • stain
  39. What kind of cells do we see on a vaginal cytology (order from deepest cells to most superficial cells)
    • basal cells (not seen on a cytology)
    • parabasal cells
    • intermediate cells
    • superficial intermediate cells
    • superficial cells
  40. Describe parabasal cells.
    • smallest cell
    • high N:C ratio
    • round nuclei
    • noncornified/nonkeratinized
  41. When do we normally see parabasal and intermediate cells?
    • seen in early proestrus, diestrus, and in anestrus
    • don't usually see them in estrus
  42. Describe intermediate cells.
    • larger than parabasal cells
    • round nucleus
  43. Descri superficial intermediate cells.
    • angulated, folded borders
    • same squamous cells we see in urine
    • cornified/keratinized
  44. Describe superficial cells.
    • largest cell
    • nuclei faded or pyknotic
    • angulated, folded borders
    • cornified/keratinized
  45. What stages do we see superficial cells?
    proestrus and estrus
  46. What besides squamous cells, what other cells do we see in a vaginal cytology?
    • neutrophils
    • erythrocytes during cycle
    • bacteria because the vagina is not sterile
  47. What kind of cells do we see during proestrus on a vaginal cytology?
    • noncornified epithelial cells (parabasal and intermediate cells)
    • erythrocytes
    • as progress see an increase in epithelial cell cornification (become keratinized, karyolysis, angular cells)
  48. Why could we potentially see transitional cell tumors in a vaginal cytology?
    urine can drip into the vagina and when we obtain our sample we might get a little urine in it
  49. On a cytology, which are the two stages that are hard to tell the difference between?
    proestrus and estrus
  50. What kind of cells do we see during estrus on a vaginal cytology?
    • cornified epithelial cells
    • nucleus fading/missing
    • may see rbcs
    • segs are rare
  51. What kind of cells do we see during diestrus on a vaginal cytology?
    • abrupt retrun to non-cornified epithelial cells
    • neutrophils
    • may see metestrum cells
  52. What are metestrum cells?
    epithelial cells with segs in it
  53. Why is diestrus important to recognize on a vaginal cytology?
    • if they are in diestrus then they are unable to be bred anymore
    • if you know the first day of diestrus then you can estimate whelping (56 - 57 days from day 1 of diestrus)
  54. What do the cells look like on a vaginal cytology during anestrus?
    • non-cornified epithelial cells (parabasal and intermediate cells)
    • occasional segs
  55. What do we use to collect vaginal cultures?
    guarded swabs
  56. How do we use guarded swabs for vaginal cultures?
    • insert into vagina
    • push swab out
    • pull swab back in
    • remove and cap
  57. How can we diagnose pregnancy in dogs?
    • palpation
    • ultrasound (18 - 20 days)
    • rads (after 42 days)
    • relaxin test (ELISA test)
  58. How long is gestation in dogs?
    about 63 days
  59. What type of vaginal flora will we see on a culture of a dog's vagina?
    • Pasteurella multocida
    • beta hemolytic strep
    • E. coli
    • Mycoplasma
    • Strep
    • Staph
    • Pseudomonas
  60. What kind of breeders are cats?
    seasonal long day breeders
  61. Are cats polyestrus?
    yes
  62. What kind of ovulators are cats?
    induced ovulators (ovulates 24 hours after being bred)
  63. If there is a fertility problem in cats is it usually due to mistiming?
    no
  64. What is the problem if there is infertility in cats?
    systemic/endocrine problems
  65. Is a vaginal cytology useful in cats?
    no
  66. What kind of breeder is cows?
    nonseasonal
  67. Are cows polyestrus?
    yes
  68. How long is the estrous cycle in cows?
    20 - 21 days
  69. How long does estrus last in cows?
    12 - 18 hours
  70. When is AI used in cows?
    mainly for dairy cows
  71. What is the estrus behavior in cows?
    bellow, aggression
  72. What percent of estrums are missed in cows?
    30 - 40%
  73. How can we determine when the cows are ready to breed?
    • watch cows 20 - 30 minutes in the AM an dPM
    • if a cow mounts another cow in the AM and stands to be mounted in the PM then breed the cow the next AM
    • Kmar heat detector - strap on the back, has a pack with dye in it and when the cow is mounted it breaks the pack and the dye gets on the cows back
  74. What can progesterone tests be used for in cows?
    • diagnose pregnancy
    • heat detection
  75. What does progesterone tests use to test for cows?
    • milk
    • does not use blood
  76. How do we use milke progesterone tests to diagnose pregnancy in cows?  What percent accuracy is this?
    • test day 20, if positive then they cow is pregnant
    • if not, then rebreed
    • retest on day 24 to make sure it was not a false positive
    • if still positive on day 24, then it is 85% accurate
  77. What are examples of milk progesterone tests?
    • Ovusure
    • Progestassay
    • Enzygnost Milk Progesterone VET
    • EstruCHEK
    • Calfcheck
  78. Can we do embryo transfer in cows?
    yes
  79. What kind of breeder are horses?
    seasonal long day breeders
  80. What are the estrual behavioral signs in horses?
    • vulval winking
    • standing
    • squating to urinate
  81. How long is a horse's estrous cycle?
    21 days
  82. How long does estrus last in a horse?
    4 - 9 days
  83. How often do we breed a horse during estrus?
    twice (early and later)
  84. How long is diestrus in a horse?
    14 - 15 days
  85. If a horse has a short estrus what days should we breed them?
    days 1 and 3
  86. If a horse has a long estrus what days should we breed them?
    days 3 and 6
  87. What is often required for uterine cultures in horses?
    prebreeding
  88. When do we do uterine cultures in horses?
    if infertility is seen
  89. How do we do a uterine culture in horses?
    VMD rectally guides guarded swab into uterus
  90. What kind of sample do we need for progesterone tests in horses?
    serum or plasma
  91. What do we use progesterone tests in horses?
    to diagnose pregnancy
  92. When do we do progesterone tests in horses?
    • test at day 16 after estrus signs seen 
    • or day 18 after ovulation
  93. What are other reasons to do progesterone testing?
    • monitor progesterone during pregnancy
    • to confirm ovulation
    • to confirm estrus if no signs are seen
    • determine if corpus lutem is present if acyclic or abnormal cycles
  94. Why do we monitor progesteron during pregnancy?
    if progesterone levels drop then they will lose the foal
  95. What does Predit-A-Foal do?
    test to predict when the mare will give birth
  96. When do we do the Predit-A-Foal test?
    • test 10 days before due date
    • or when milk is first seen (test milk)
  97. How do we do the Predict-A-Foal test?
    • mix milk and test solution
    • dip test strip into tube
    • if mauve color in 4 - 5 squares shows up then theres an 80% chance the mare will foal in 12 hours
  98. How do we do a equine endometrial cytology?
    • flush 50 ml of 0.9% saline in
    • aspirate back
    • centrifuge sample and make smears
    • stain
  99. What are the different patterns we see on an equine endometrial cytology?
    • inactive pattern
    • transitional patterns
    • active patterns
  100. When do we see inactive pattern on an equine endometrial cytology?
    anestrus in winter
  101. What does an inactive pattern on an equine endometrial cytology look like?
    • ciliated and non cuboidal to low columnar cells
    • cells often arranged in groups (looks like honeycomb or fence)
  102. What does a transitional pattern on an equine endometrial cytology look like?
    • ciliated columnar cells
    • some cuboidal epithelial cells
  103. When do we see a transitional pattern on an equine endometrial cytology?
    • often seen in the fall/spring
    • then changes to inactive pattern as winter approaches
  104. When do we see an active pattern on an equine endometrial cytology?
    during the period of coming into heat
  105. What does an active pattern on an equine endometrial cytology look like?
    • mostly non-ciliated columnar cells (foamy cytoplasm, oval nuclei with stippled chromatin and single nucleolus)
    • few lymphs and some mucus
  106. What is urine pooling?
    urine flows backwards into the uterus
  107. Why can urine pooling be a problem for mares?
    can interfer with a mare getting pregnant
  108. What does a urine pooling pattern look like on an equine endometrial cytology?
    • moderate number of neutrophils
    • abundant of tall columnar cells (cytoplasm foamy or granular)
    • groups of epithelial cells with large nuclei and prominent nucleoli
    • urine crystals

  109. This is a dog.  What phase of the estrous cycle is this?
    anestrus

    • non-cornified epithelial cells (parabasal and intermediate cells)
    • occasional segs

  110. What is this?
    a cow embryo

  111. This is a dog.  What phase of the estrous cycle is this?
    diestrus

    • see neutrophils
    • may see metestrum cell (epithelial cells with seg in it)
    • abrupt return to non-cornified epithelial cells

  112. This is a sample from a dog in diestrus.  What is the cell in the top right corner?
    metestrum cell

  113. This is a dogs estrous cycle.  What do the different lines represent?
    • blue:  estrogen
    • green:  luteinizing hormone
    • red:  progesterone

  114. What kind of test is this?
    D-Tec CB for dogs

  115. This is a dog.  What phase of the estrous cycle is this?
    estrus

    • cornified epithelial cells (50% superficial cells, nucleus fading/missing)
    • may see rbcs
    • segs are rare

  116. This is a dog.  What phase of the estrous cycle is this?
    estrus

    • cornified epithelial cells (50% superficial cells, nucleus fading/missing)
    • may see rbcs
    • segs are rare

  117. This is a dog.  What phase of the estrous cycle is this?
    estrus

    • cornified epithelial cells (50% superficial cells, nucleus fading/missing)
    • may see rbcs
    • segs are rare

  118. What is this?
    guarded swabs

  119. What test is this?
    ICG Status-Pro

  120. This is an equine endometrial cytology.  What pattern is this?
    inactive pattern

  121. This is an equine endometrial cytology.  What pattern is this?
    inactive pattern

  122. This is a horse.  What is going on?
    inflammatory reaction

  123. This is a dog.  What phase of the estrous cycle is this?
    proestrus

    • noncornified epithelial cells (parabasal and intermediate cells)
    • rbcs

  124. This is a dog.  What phase of the estrous cycle is this?
    proestrus

    • noncornified epithelial cells (parabasal and intermediate cells)
    • rbcs

  125. This is a dog.  What types of cells are these?
    superficial cells

  126. This is a dog.  What type of cell is this?
    superficial intermediate cells

  127. This is a dog.  What is this?
    transitional cell tumor

  128. This is a dog.  What is this?
    transmissible venereal tumor

  129. This is a dog.  What is the cell on the top?  Which one is on the bottom?
    • parabasal cell
    • intermediate cell

  130. What type of cytology is this?
    vaginal cytology

  131. This is a dog.  What's wrong?
    vaginitis

  132. This is a dog.  What's wrong?
    vaginitis

  133. This is a dog.  What's wrong?
    vaginitis
  134. Why would we do semen analysis?
    • prior to breeding
    • after breeding to look for cause of infertility 
    • breeding soundness exam
    • to look for disease in tract 
  135. What are the different types if semen collection techniques?
    • artifical vagina
    • electroejaculation
  136. Should we keep semen warm after collection?
    Yes
  137. When should we analyze semen after collecting it?
    Immediately
  138. What are the 4 different aspects about semen do we analyze?
    • gross characteristics
    • cell (sperm) counts 
    • motility
    • morphology
  139. What about the gross characteristics of semen do we make note of?
    • color/transparency
    • volume collected
  140. What is the normal color of semen?
    grey to white
  141. The whiter the semen, the _____.
    more concentrated
  142. What colors of semen are due to contamination?
    • red:  blood
    • brown:  poop
    • yellow:  urine
    • green:  puss
  143. What is the normal pH of a dogs semen?
    6.2 - 6.9
  144. As the semens pH increases, what else increases?
    Prostatic fluid
  145. What could a high semen pH be due to?
    Inflammation
  146. What could change the characteristic of semen?
    • day length
    • temperature
    • disease status
  147. What are the three fractions of semen?
    • presperm
    • sperm rich
    • clear prostatic fluid
  148. What does presperm look like?  About how many cc?
    • Clear
    • 0.25 - 2 cc
  149. What does sperm rich look like?  About how many cc?
    • Cloudy-white, thick 
    • 0.5 - 3cc
  150. About how many cc do we get of clear prostatic fluid?
    3-20cc
  151. Which part of the semen fraction do we want for artificial insemenation?
    the sperm rich fraction
  152. When do we check sperm motility?
    Immediately after collection and under a microscope
  153. What kind of movement do we want from sperm?
    Vigorous forward movement by large numbers
  154. We rank sperm motility from 0-5.  What is considered normal?
    4.5 is normal motility
  155. What can adversely affect sperm motility?
    • dirt
    • lube
    • chemicals
    • disease
  156. When is motility considered abnormal?
    If < 50% are not moving in a forward direction
  157. Define necrospermia.
    dead sperm
  158. What is live/dead stain?
    • stain to determine if sperm is dead or alive
    • if it is dead it will stain red
  159. What do we use to count sperm?
    • Automated counter
    • hemocytometer
  160. Total number of sperm is _____ than concentration of sperm.
    more important
  161. How do we use a hemocytometer to count sperm?
    • load semen in 20 ul pipette
    • add to large leukocyte unopette
    • load hemocytometer 
    • count heads in primary squares
    • multiply by 1,000,000 sperm/ejaculate
  162. How do we get the total number of sperm?
    multiply # of sperm/ml by the total volume of ejaculate
  163. What is the normal sperm count for dogs?  Toy dog breeds?
    • dogs:  300 - 500,000,000
    • toy:  100 - 200,000,000
  164. What is oligospermia?
    • Few sperm
    • < 100,000,000
  165. What are we more concerned with, sperm counts or mobility/morphology?
    motility and morphology 
  166. How do estimate the sperm count using PCV?
    • Spin sperm in hermatocrit tubes for 10 mminutes 
    • calculate % the same way we do blood
    • each % = 220,000,000
  167. How do we look at the morphology of sperm?
    • make smear and stain using diff quik or live/dead stain count at least 100 cells
    • note sperm abnormalities 
  168. What are the normal cells and how many should we see for each cell in sperm?
    • leukocytes:  2-4/hpf
    • erythrocytes:  5-10/hpf
    • prostatic cells:  10-20/hpf
    • other epithelial cells:  1-2/hpf
  169. Abnormal sperm is divided into what 2 categories?
    primary and secondary
  170. What is primary abnormal sperm?
    • occurs during spermatogenesis
    • malformed heads
  171. What is secondary abnormal sperm?
    • after sperm formed, in transit
    • kinked tails, broken heads
  172. What are the different types of sperm abnormalities?
    • small heads
    • big heads
    • no heads
    • kinked tails
    • broken tails
    • no tails
    • swollen midpiece 
    • coiled tails
    • double tails

  173. What is wrong with these sperm?
    • coiled midpiece
    • proximal droplet

  174. What's wrong with this sperm?
    coiled tail

  175. What's wrong with this sperm?
    detached head

  176. What's wrong with this sperm?
    distal droplet

  177. What's wrong with this sperm?
    double head

  178. What's wrong with this sperm?
    elongated head

  179. What's wrong with this sperm?
    elongated head

  180. What is this?
    how we can estimate a cell count using PCV

  181. What kind of stain was used?
    live/dead stain - the red means their dead

  182. What wrong with this sperm?
    misshapen head

  183. What's wrong with this sperm?
    misshapen head and proximal droplet

  184. What's wrong with this sperm?
    proximal droplet

  185. What is this used for?
    sperm count

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