Semen Analysis Clinical Pathology

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.

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

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

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

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

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

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

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

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

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

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

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

Double headed Sperm

Misshapen Head

Elongated Head

Pear shaped head and bent midpiece

Proximal Droplet

Distal Droplet

Detached Head

Bent Tail or Midpiece

Coiled Tail

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