Semen Analysis

1. Semen Analysis Clinical Pathology

2. Semen Collection • Semen is often collected into an artificial vagina, usually while a teaser bitch is present. • An artificial vagina may be made of latex or disposable plastic.

3. May use electoejaculation in which a probe is attached to the pelvic nerves to stimulate ejaculation.

5. Semen (3 fractions) • 1st- mainly prostatic fluid, few sperm • Released during the period of vigorous thrusting • 2nd- Sperm rich portion of ejaculate • 3rd- Mainly prostatic fluid, few sperm, majority of total volume of ejaculate. • Buck, bulls, tom, ram you should collect all three • Stallions, dogs, boars- collect 2nd and 3rd portions seperately

6. Semen Handling Techniques • Avoid marked temperature changes • Avoid exposure to water, disinfectants • Use clean, dry, warm equipment (37 C or 98 F) • Slides, coverslips, pipettes, stains. • Process soon after collection

7. Semen Evaluation • Color and consistency (normal is milky and moderately viscous) • Volume • Wave motion/sperm motility • Spermatozoa concentration • Morphology • Ratio live:dead • Presence of foreign cells/material

8. Volume of Ejaculate • Measured in volumetric flask • Method of collection affects volume • Electroejaculation- volume is larger • Teasing with a female-volume is larger • Species variation • Dogs: 10-40 ml • Stallion: 65 ml • Tom: 0.5 ml • Volume does not necessarily correlate with fertility

9. Sperm Motility • Motility correlates with fertility • Improper handling can affect motility • Evaluate immediately after collection • Place a drop of semen on a warm slide, immediately cover with coverslip • Dilute with warm saline if high concentration of sperm

10. Classes of Sperm Motility • Can be classified as good, very good, fair, or poor. • Normal sperm should have greater than 70% motility • Examine under 100 x, may need to dilute concentrated samples • Poor is when there is less than 40% motility

11. Wave Motion • Under low power 40x, look for swirling • Progressive motility- sperm are moving around all over slide • Non-progressive motility- sperm are only swimming in a similar pattern

13. Sperm Concentration • Most important characteristic • Dilute a portion 1:100 with saline or red cell Unopette. • Using a hematocytometer, count total of sperm in the central grid • Multiply the number by 2 million • Boars/Stallions: 150 M/ml • Bulls: 1200 M/ml • Dogs: 300 M/ml • Cats: 1700 M/ml

15. Live:Dead Sperm Ratio • Place 1 drop eosin/nigrosin stain (make sure it is warm) and mix gently with a drop of semen on a warm slide. • After several seconds, smear like blood smear. • Live sperm resist staining-appear white against a blue-black background • Dead sperm take up the eosin and stain pinkish red • Examine and observe 200 cells

17. Sperm Morphology • Can examine on eosin/nigrosin stained smear • Other stains: india ink, H&E, Wrights • Observe 100-500 cells • Record % of abnormal cells • Divide problems into head, neck, midpiece, and tail problems. • Primary abnormalities occur during sperm production. • Secondary occur from storage in the epididymis until the smear is made

19. Double headed Sperm

20. Misshapen Head

21. Elongated Head

22. Pear shaped head and bent midpiece

23. Proximal Droplet

24. Distal Droplet

25. Detached Head

26. Bent Tail or Midpiece

27. Coiled Tail

29. http://www.vivo.colostate.edu/hbooks/pathphys/reprod/semeneval/conc.htmlhttp://www.vivo.colostate.edu/hbooks/pathphys/reprod/semeneval/conc.html