Basic canine andrology

Question 1

For spermatogenesis there is an endocrine requirement: (3)

Answer 1

o GnRH production from the hypothalamus

o FSH/LH from the anterior lobe of the pituitary

o Secretion of gonadal steroids (testosterone/estrogen)

Question 2

Who has bulbourethral glands?

Answer 2

All domestic animals except for dogs have bulbourethral glands.

Question 3

The gonadal steroids are produced by

Answer 3

the Leydig Cells in the interstitium of the testicles.

They contain LH receptors so they can respond to LH and produce progesterone which is converted to testosterone and estrogen. (some of this testosterone goes to the sertoli cells)

They are the Male equivalent to the female theca internal cells.

Question 4

Sertoli cells are

Answer 4

❖ Somatic cells anchored at the basal membrane in the seminiferous tubuli.

❖ Male equivalent to the follicular granulosa cells.

❖ Produce a variety of substances: androgen binding protein, sulfated
glycoprotein, transferrin, inhibin.

❖ Important for physical support, nutrition and phagosytosis, blood/testis barrier.

❖ Convert testosterone (received from leydig cells) to dihydrotestosterone (DHT) and estrogen→ negative feedback on GNRH neurons.

Question 5

What test do you do to check a male dogs testosterone?

Answer 5

testosterone stimulation test

Question 6

Tight junctions between sertoli cells divide
seminiferous epithelium into (2)

Answer 6

 basal compartment (houses spermatogonia and early primary spermatocytes)

 adluminal compartment (houses all the other germ cells)

 They play an important role in the
blood/ testis barrier

Question 7

Spermatogenesis in the dog takes?
in the cat?

Answer 7

Spermatogenesis in the dog takes 63-65 days and for the cat 47 days.

Question 8

In case of bad semen quality upon collection, what should you do?

Answer 8

wait at least 8 weeks (because that’s how long spermatogenesis takes →

do a new collection, to see if there is permanent damages or the semen quality is normal again →

we normally collect 2-3 times before deeming the dog unusable if his semen quality doesn’t improve.

Question 9

Puberty in dogs, normally occurs at

Answer 9

9 month of age on average (5-12 still normal) its very breed dependent→

small breeds often earlier in puberty→

caused by increasing production of testosterone.

Signs:
❖ The male is lifting it’s leg against something when urinating.

❖ The testicles and preputium is growing.

❖ Increased interest in females in estrus ( and in general).

❖ The whole body end expression becomes more masculine.

Question 10

Semen quality.
Male dogs can be fertile up till how old?

Answer 10

from 6 month- 10 years of age….. depending on the breed .

 The quality of the semen decreases for some dogs after 6 years of age.

 Big difference depending on the breed.

 Fertility is inherited.

 Semen quality can vary during lifetime of the dog and during the time of the year.

Question 11

Reasons to perform semen evaluation. (5)

Answer 11

1. Dogs that have been in sexual rest for a long time (> 6 months)
2. If there is a known problem with bad fertility in the family
3. If several bitches don´t get pregnant after successful matings
4. Before preparation of frozen- or chilled semen
5. Before a young male is taken into breeding

Question 12

Methods for semen collection. (2)

Answer 12

Manual masturbation:
❖ The result is ALWAYS best with a teaser bitch in heat.

❖ Otherwise use a swab from vulva of a bitch in heat (can be frozen→ unfreeze).

Electroejaculation/urethral catheterization:
❖ The animals are sedated

❖ Used in cats and wild canids/felids

Question 13

Manual masturbation Semen collection process.

Answer 13

Do the collection where the male dog feels comfortable (outside, in the room for estrus control). Don’t have too many persons in the room -> sometimes send the owner out (hunting dogs).

 Have a teaser bitch → let the dogs say hello to each other and let him sniff her vulva before touching him.

❖ Clean the genital area if necessary with a dry towel.

❖ Use collection tubes of glass, plastic with cones or condoms. Use gloves if you want (but some dogs don’t like that).

Massage the bulbus glandis in the prepuce until the erection starts.

Next,
1. Quickly retract the prepuce behind the Bulbus glandis (! otherwise can hurt the dog a lot)
2. Fixate penis/preputium tight behind Bulbus glandis
3. Rhythmical massage of the the bulbus glandis
4. Collect in 3 fractions (into 3 separate containers)

Question 14

Equipment for semen collection:

Answer 14

 plastic or soft rubber cups (always minimum 3)

 Condoms as an artificial vagina (fractions can´t be divided)

Avoid damage to the penis

Don´t mix the semen with soap or disinfectants.

Question 15

1. Fraction of ejaculate. (3)

Answer 15

 a small amount; contains urine, dead
sperm cells, epithelial cells

 This fraction is used to lubricate the vulva
before introduction of penis into
vestibulum

 Is delivered fast and while he is thrusting

Question 16

2. Fraction of ejaculate. (3)

Answer 16

 sperm-rich fraction contains the sperm cells that comes out in drops

 Is delivered before or while he is turning
and jumps off the bitch

 A volume of 0,5-3 ml depending on the
breed.

Question 17

3. Fraction of ejaculate. (4)

Answer 17

 prostatic fluid comes from the prostate, when the dogs stand opposite each other and comes out in bigger volumes than the sperm portion (squirts?)

 The volume can be up to 30 ml.

 Contains proteins and fructose. Can contain small amounts of urine.

 Penis is pulled backwards

Question 18

The 3 semen fractions:

Answer 18

1. fraction, the “waste” fraction
2. fraction, the sperm-rich fraction
3. fraction, the prostatic fraction

Question 19

The 2.fraction (sperm-rich) is evaluated for (3)

Answer 19

Motility: > 70% motile spermatozoa desired

Concentration: normal 200 millions to 2 billions

Morphology:
- not more than 25% defects
- live/dead ratio

Question 20

Semen Motility should be:

Answer 20

> 70% progressively motile spermatozoa desired, this would an excellent sample.

(aggressive forward movement)

Use a phase-contrast microscope. Warm slides to 37’C or use heat pad to avoid cold shocking the spermatozoa.

Question 21

Normal Semen Concentration:

Answer 21

normal 200 millions to 2 billions per ejaculate

Question 22

Semen Morphology should be

Answer 22

• not more than 25% defects

Assess live/dead ratio.

warm your slides before use! otherwise the spermatozoa tails will curl and you can mistake them for defects (image lower right example).

Question 23

Macroscopic examination of the sperm-rich fraction (2.). (3)

Answer 23

Volume:
- 3 to 30 ml (2. fraction only 0,5-3 ml!)
- If you get a low volume:
➔ Be sure you have collected the 2. fraction.

Color of 2. fraction:
Normally a milky white liquid but can be clear, reddish, green/yellow/brown.

Smell: none

Question 24

To make sure that its the 2. fraction that has been collected:

Answer 24

Measure Alkaline Phosphatase(ALKP)!
- normally : 5000 to 40 000 U/L

If low, can indicate:
 incomplete ejaculation
 a block through the epididymis

Question 25

What is the CASA system?
pros and cons?

Answer 25

CASA= Computer Assisted Sperm
Analysis

Pros:
 Accurate
 No subjective variation between
the persons testing the semen

Cons:
 Very expensive!

Question 26

If the semen sample is very concentrated, you should…

Answer 26

Dilute with:
 Seminal plasma
 NaCl, isotonic
 Semen extender

Before evaluating the motility!

Question 27

Semen Concentration = ?

How to get the above info?

Answer 27

total number of spermatozoa/ ejaculate

Automatic counting systems: Spermacue, Accuread, Casa, Nucleocounter, androscope, CASA-system

Manual counting: hematocytometer (Thomas, Makler chamber)

Question 28

Semen morphology eval. requires staining: (2)

Answer 28

Eoisin/nigrosin stain:
→ Put one drop of semen + 1 drop of stain
and make a thin solution, let it dry, count
200 (100) cells→ dead and living spermatozoa.

Spermac: stains the sperm acrosome cap green and the head of the spermatozoa red.

Question 29

Spermatozoa defects. (4)

Answer 29

Classification:
 A: head defects
 B: mid-piece defects
 C: tail defects
 D: distal/proximal droplets

Often more than one kind of defect
on the same spermatozoa.

Question 30

Indications for insemination. (4)

Answer 30

 When natural mating for some reason is not possible (inexperienced animals,
antipathy between dogs).

 Use of chilled or frozen semen

 Prevention of venereal diseases

 Different laws about insemination around the world.

Question 31

Behavioral reasons that would contribute to insemination use. (2)

Answer 31

bad libido

bitch refuses to be mounted

Question 32

Breed reasons that would contribute to insemination use. (2)

Answer 32

size differences

anatomical problems (poor penis conformation)

Question 33

Methods for artificial insemination (AI). (3)

Answer 33

 Vaginal insemination

 Transcervical insemination (TCI) into the uterus (is always used for frozen semen). Intra-uterine insemination improves
the results.

 Surgical insemination (e.g. small volume of valuable semen)

Question 34

Semen “types”. (3)

Answer 34

 Fresh semen; collected by the veterinarian to be used immediately.

 Chilled semen; that is prepared with extender and stored at 4°C for max 4 days.

 Frozen semen; frozen in liquid nitrogen at -196°C and can be stored forever.

Question 35

When using AI and independent of
method, the most important thing is…

Answer 35

To determine the optimal time of
breeding.

Question 36

Answer 36

Catheter placed as far in as possible, then inflate the bulb with air, inject semen.

The back of the bitch is lifted for 10 minutes after insemination.

With accurate estrus control, good insemination technique and semen quality is good:

70 - 80 % pregnancy rate = results as good as for natural mating.

Question 37

TCI

Answer 37

Transcervical insemination

Question 38

Insemination with fresh semen.

Answer 38

 Extender is normally not needed, only if the 3. fraction is discolored (bloody,
green/yellow).

 Use all of the 2. fraction (sperm-rich) and some amount of the 3. fraction to dilute to
the optimal volume (2-6 ml).

Get rid of most of the supernatant to get the optimal volume.

Question 39

Pros and cons for the use of chilled semen.

Answer 39

Question 40

Extenders for chilled and frozen semen.
Extenders are added to buffer…? (4)

Answer 40

• changes in pH (citrate)
• to provide energy source to the spermatozoa (glucose, dextrose, lactose)
• to prevent growth of bacteria (dihydrostreptomycin)
• to prevent injury during cooling and freezing using cooling protectants (egg yolk
  and glycerol)

Only use extenders intended for canine.

Question 41

How to prepare chilled semen. (4)

Answer 41

Collect the dog and centrifuge the 2. fraction at: 700 rpm for 8 min

Get rid of the seminal plasma. Slowly add drops of chill extender (3-5 x extender), chill at 4°C and control the quality after 2 hours.

Take a few drops and put them in the fridge before export (if something goes wrong during transport).

The rest is packed in a insulated box with freezer elements → remember to do
semen evaluation before prepping it and sending it! → Export to the receiver.

Question 42

How to use chilled semen. (5)

Answer 42

 As soon as the box arrives; take one drop of the semen, heat it on the heating pad for a few minutes and evaluate it microscopically.

 Put it in the fridge in a precooled glass of water until use.

 Inseminate as soon as possible and don’t wait for more than 4 days.

 Before use, heat it up in the incubator until 37°C (it takes about 20 min).

 Inseminate by TCI (vaginally).

Question 43

Describe Frozen semen. (7)

Answer 43

 The semen can be saved indefinitely at -196°C nitrogen.

 Old lines of animals can be used to decrease inbreeding.

 New lines can be used across borders → the semen can be shipped all over
the world.

 Be aware of different import rules and certificates → special blood test at the
time of preparation or weeks after.

 Expensive to make AND expensive to send.

 Pregnancy rate lower compared to chilled semen.

Timing of ovulation is very important → insemination 3-4 days post-ovulation.

Question 44

How much semen do we need for AI?

Answer 44

 Always best to have enough for 2 AI.

 Minimum: 200 million normal spermatozoa per insemination.

 Never send poor quality semen without the consent of the receiver.

Question 45

Total insemination volume for TCI is

Answer 45

2-6 ml depending of the size of the
bitch. ALWAYS use TCI.