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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
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What is theriogenology?
veterinary specialist for reproduction
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How long is a dog's estrous cycle? When is it considered abnormal?
- normal: averages 6 months
- abnormal: < 4 or > 12 months
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What are the different stages of the estrous cycle?
- proestrus
- estrus
- diestrus
- anestrus
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How long does a dog's proestrus last?
3 - 17 days
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What are the signs of proestrus for a dog?
- vulva swollen
- vaginal bleeding
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Are male dogs attracted to a female dog during proestrus?
yes
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How long does a dog's estrus stage last?
3 - 21 days
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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
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When does a dog ovulate?
ovulates usually in 48 - 72 hours as they come into estrus
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Which stage is standing heat for a dog?
estrus
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Why is it hard to spay a dog while they are in heat?
blood vessels rupture easily and can cause hemmorrhaging
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How long does the diestrus stage last in dogs?
lasts 2 months
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What are the different signs of diestrus in a dog?
- discharge ceases
- vulva returns to normal
- pregnancy or pseudocyesis
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What is pseudocyesis?
false pregnancy
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What is another name for diestrus?
metestrus
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How long does the anestrus period last in a dog?
lasts 4 - 10 months
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What is anestrus in a dog?
period of sexual inactivity
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When a dog is spayed, what stage are they permanently in?
anestrus
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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
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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
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Which test can we use to test for brucellosis?
- D-Tec CB slide test
- blood culture or AGID test (definitive tests)
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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
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Are false positives common with D-Tec CB tests?
- yes
- a negative is a negative, but a positive does not necessarily mean its positive
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What is the most common cause of infertility in dogs?
breeding at the wrong time
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What tests can we use to help breed dogs at the correct time?
ovulation timing tests
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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
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What are some examples of ovulation timing tests for dogs?
- Target test
- ICG Status-Pro
- ELISA test
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What do all ovulation timing tests measure in dogs?
progesterone levels
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When should we do the ovulation timing test for dogs?
- start in proestrus for a baseline
- then repeat every 2 - 3 days
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How long does fresh dog semen last? Chilled? Frozen?
- fresh: about 5 - 7 days
- chilled: about 48 hours
- frozen: about 24 hours
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How does the ICG Status Pro ovulation timing test work in dogs?
as hormone increases, color fades on test
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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
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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
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Why do we do vaginal cytology for a dog?
- to identify cellular reactions
- to help identify stage of bitch's estrous cycle
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When doing a vaginal cytology should we check the entire slide?
yes
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Which magnification do we use for vaginal cytology?
400x
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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
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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
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Describe parabasal cells.
- smallest cell
- high N:C ratio
- round nuclei
- noncornified/nonkeratinized
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When do we normally see parabasal and intermediate cells?
- seen in early proestrus, diestrus, and in anestrus
- don't usually see them in estrus
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Describe intermediate cells.
- larger than parabasal cells
- round nucleus
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Descri superficial intermediate cells.
- angulated, folded borders
- same squamous cells we see in urine
- cornified/keratinized
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Describe superficial cells.
- largest cell
- nuclei faded or pyknotic
- angulated, folded borders
- cornified/keratinized
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What stages do we see superficial cells?
proestrus and estrus
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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
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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)
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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
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On a cytology, which are the two stages that are hard to tell the difference between?
proestrus and estrus
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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
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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
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What are metestrum cells?
epithelial cells with segs in it
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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)
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What do the cells look like on a vaginal cytology during anestrus?
- non-cornified epithelial cells (parabasal and intermediate cells)
- occasional segs
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What do we use to collect vaginal cultures?
guarded swabs
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How do we use guarded swabs for vaginal cultures?
- insert into vagina
- push swab out
- pull swab back in
- remove and cap
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How can we diagnose pregnancy in dogs?
- palpation
- ultrasound (18 - 20 days)
- rads (after 42 days)
- relaxin test (ELISA test)
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How long is gestation in dogs?
about 63 days
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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
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What kind of breeders are cats?
seasonal long day breeders
-
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What kind of ovulators are cats?
induced ovulators (ovulates 24 hours after being bred)
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If there is a fertility problem in cats is it usually due to mistiming?
no
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What is the problem if there is infertility in cats?
systemic/endocrine problems
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Is a vaginal cytology useful in cats?
no
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What kind of breeder is cows?
nonseasonal
-
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How long is the estrous cycle in cows?
20 - 21 days
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How long does estrus last in cows?
12 - 18 hours
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When is AI used in cows?
mainly for dairy cows
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What is the estrus behavior in cows?
bellow, aggression
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What percent of estrums are missed in cows?
30 - 40%
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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
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What can progesterone tests be used for in cows?
- diagnose pregnancy
- heat detection
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What does progesterone tests use to test for cows?
-
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
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What are examples of milk progesterone tests?
- Ovusure
- Progestassay
- Enzygnost Milk Progesterone VET
- EstruCHEK
- Calfcheck
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Can we do embryo transfer in cows?
yes
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What kind of breeder are horses?
seasonal long day breeders
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What are the estrual behavioral signs in horses?
- vulval winking
- standing
- squating to urinate
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How long is a horse's estrous cycle?
21 days
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How long does estrus last in a horse?
4 - 9 days
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How often do we breed a horse during estrus?
twice (early and later)
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How long is diestrus in a horse?
14 - 15 days
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If a horse has a short estrus what days should we breed them?
days 1 and 3
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If a horse has a long estrus what days should we breed them?
days 3 and 6
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What is often required for uterine cultures in horses?
prebreeding
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When do we do uterine cultures in horses?
if infertility is seen
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How do we do a uterine culture in horses?
VMD rectally guides guarded swab into uterus
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What kind of sample do we need for progesterone tests in horses?
serum or plasma
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What do we use progesterone tests in horses?
to diagnose pregnancy
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When do we do progesterone tests in horses?
- test at day 16 after estrus signs seen
- or day 18 after ovulation
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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
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Why do we monitor progesteron during pregnancy?
if progesterone levels drop then they will lose the foal
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What does Predit-A-Foal do?
test to predict when the mare will give birth
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When do we do the Predit-A-Foal test?
- test 10 days before due date
- or when milk is first seen (test milk)
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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
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How do we do a equine endometrial cytology?
- flush 50 ml of 0.9% saline in
- aspirate back
- centrifuge sample and make smears
- stain
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What are the different patterns we see on an equine endometrial cytology?
- inactive pattern
- transitional patterns
- active patterns
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When do we see inactive pattern on an equine endometrial cytology?
anestrus in winter
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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)
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What does a transitional pattern on an equine endometrial cytology look like?
- ciliated columnar cells
- some cuboidal epithelial cells
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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
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When do we see an active pattern on an equine endometrial cytology?
during the period of coming into heat
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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
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What is urine pooling?
urine flows backwards into the uterus
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Why can urine pooling be a problem for mares?
can interfer with a mare getting pregnant
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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
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This is a dog. What phase of the estrous cycle is this?
anestrus
- non-cornified epithelial cells (parabasal and intermediate cells)
- occasional segs
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What is this?
a cow embryo
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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
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This is a sample from a dog in diestrus. What is the cell in the top right corner?
metestrum cell
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This is a dogs estrous cycle. What do the different lines represent?
- blue: estrogen
- green: luteinizing hormone
- red: progesterone
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What kind of test is this?
D-Tec CB for dogs
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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
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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
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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
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What is this?
guarded swabs
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What test is this?
ICG Status-Pro
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This is an equine endometrial cytology. What pattern is this?
inactive pattern
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This is an equine endometrial cytology. What pattern is this?
inactive pattern
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This is a horse. What is going on?
inflammatory reaction
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This is a dog. What phase of the estrous cycle is this?
proestrus
- noncornified epithelial cells (parabasal and intermediate cells)
- rbcs
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This is a dog. What phase of the estrous cycle is this?
proestrus
- noncornified epithelial cells (parabasal and intermediate cells)
- rbcs
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This is a dog. What types of cells are these?
superficial cells
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This is a dog. What type of cell is this?
superficial intermediate cells
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This is a dog. What is this?
transitional cell tumor
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This is a dog. What is this?
transmissible venereal tumor
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This is a dog. What is the cell on the top? Which one is on the bottom?
- parabasal cell
- intermediate cell
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What type of cytology is this?
vaginal cytology
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This is a dog. What's wrong?
vaginitis
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This is a dog. What's wrong?
vaginitis
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This is a dog. What's wrong?
vaginitis
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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
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What are the different types if semen collection techniques?
- artifical vagina
- electroejaculation
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Should we keep semen warm after collection?
Yes
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When should we analyze semen after collecting it?
Immediately
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What are the 4 different aspects about semen do we analyze?
- gross characteristics
- cell (sperm) counts
- motility
- morphology
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What about the gross characteristics of semen do we make note of?
- color/transparency
- volume collected
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What is the normal color of semen?
grey to white
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The whiter the semen, the _____.
more concentrated
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What colors of semen are due to contamination?
- red: blood
- brown: poop
- yellow: urine
- green: puss
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What is the normal pH of a dogs semen?
6.2 - 6.9
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As the semens pH increases, what else increases?
Prostatic fluid
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What could a high semen pH be due to?
Inflammation
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What could change the characteristic of semen?
- day length
- temperature
- disease status
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What are the three fractions of semen?
- presperm
- sperm rich
- clear prostatic fluid
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What does presperm look like? About how many cc?
-
What does sperm rich look like? About how many cc?
- Cloudy-white, thick
- 0.5 - 3cc
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About how many cc do we get of clear prostatic fluid?
3-20cc
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Which part of the semen fraction do we want for artificial insemenation?
the sperm rich fraction
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When do we check sperm motility?
Immediately after collection and under a microscope
-
What kind of movement do we want from sperm?
Vigorous forward movement by large numbers
-
We rank sperm motility from 0-5. What is considered normal?
4.5 is normal motility
-
What can adversely affect sperm motility?
-
When is motility considered abnormal?
If < 50% are not moving in a forward direction
-
Define necrospermia.
dead sperm
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What is live/dead stain?
- stain to determine if sperm is dead or alive
- if it is dead it will stain red
-
What do we use to count sperm?
- Automated counter
- hemocytometer
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Total number of sperm is _____ than concentration of sperm.
more important
-
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
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How do we get the total number of sperm?
multiply # of sperm/ml by the total volume of ejaculate
-
What is the normal sperm count for dogs? Toy dog breeds?
- dogs: 300 - 500,000,000
- toy: 100 - 200,000,000
-
-
What are we more concerned with, sperm counts or mobility/morphology?
motility and morphology
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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
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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
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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
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Abnormal sperm is divided into what 2 categories?
primary and secondary
-
What is primary abnormal sperm?
- occurs during spermatogenesis
- malformed heads
-
What is secondary abnormal sperm?
- after sperm formed, in transit
- kinked tails, broken heads
-
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
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What is wrong with these sperm?
- coiled midpiece
- proximal droplet
-
What's wrong with this sperm?
coiled tail
-
What's wrong with this sperm?
detached head
-
What's wrong with this sperm?
distal droplet
-
What's wrong with this sperm?
double head
-
What's wrong with this sperm?
elongated head
-
What's wrong with this sperm?
elongated head
-
What is this?
how we can estimate a cell count using PCV
-
What kind of stain was used?
live/dead stain - the red means their dead
-
What wrong with this sperm?
misshapen head
-
What's wrong with this sperm?
misshapen head and proximal droplet
-
What's wrong with this sperm?
proximal droplet
-
What is this used for?
sperm count
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