Microscope Requirements

Transcription

1 SEMEN EVALUATION

2 EQUIPMENT

3 Microscope Requirements Good quality lenses Phase-contrast preferred for % progressive motility evaluations Objectives 10X, 20X*, 40X*, 100X, minimum Heated stage preferred *Preferably phase-contrast

4 EVALUATION OF MOTILITY Semen must be kept warm ( o C) Evaluate immediately after collection Motility declines rapidly on an unheated stage On an unheated stage, the first slide is usually for focusing so subsequent slides can be evaluated immediately If no phase-contrast, use low light

5 Evaluation of Wave Motion Only useful for concentrated samples (usually ruminant semen) Place a drop of semen on a warm slide (no cover slip) and evaluate under <125X magnification (Gross Motility)

6 Wave Motion Depends on: High concentration Percentage of progressively motile cells Speed of progression

7 Gross Motility Rating: VG rapid dark swirls G slower swirls and eddies F no swirls, but prominent individual cell motion P little or no individual cell motion If gross motility is >good, further motility evaluation is usually unnecessary

8 Evaluation of Percentage Motility 5 µl semen on a warm slide Gently press down warm cover slip

9 Evaluation of Percentage Motility Evaluate at X Avoid examining near cover slip edge and near air bubbles Evaluate several fields and >2 slides

10 Standards Progressive Motility Minimum >60% VG % G 60-79% F 40-59% P - <40%

11 SPERM CELL CONCENTRATION Only estimated with routine evaluation on ruminants: VG grainy appearance with billion sperm/ml ml G opaque milk-like like with billion sperm/ml ml F skim milk-like like with billion sperm/ml ml P translucent or watery with <0.25 billion sperm/ml ml Semen obtained by electroejaculation may not reflect true volume and concentration

12 VOLUME & CONCENTRATION Precise estimates required when Evaluating semen when the entire ejaculate is obtained: concentration x volume = total sperm in ejaculate Calculating dose volume: concentration x %motile (+/- x %normal) = %(normal) motile/ml ml. E.g., If this is 50x10 6 and 500x10 6 are required, the dose is 10 ml.

13 SEMEN VOLUME

14 SPERM CELL CONCENTRATION Focus on the grid in hemacytometer chamber first This avoids broken cover slips

15 SPERM CELL CONCENTRATION Usually dilute to 1:100 with water or with the Unopette system Platelet/leukocyte system dilutes 1:100 Sometimes get clumping of cells with straight leukocyte system

16 The Unopette System

17 Loading the Chamber Flush capillary tube Mix solution well Fill both chambers Let sperm settle a few minutes before counting

18 Pipette Method Dilute 1:100 E.g., 20 E.g., 20 µl semen into into 1.98 ml water

19 Pipette Method Mix well before withdrawing diluted sample Pipetter used to fill both chambers Let sperm settle before counting

20 Assuming a 1:100 Dilution Count at ~200X, phase- contrast or low light Count all cells in the 4 corner squares (each contains 16 smaller squares) Divide by 4 and multiply by 10 6 to get concentration of cells/ml

21 Automated Methods to Determine Sperm Cell Concentration

22 SPERMATOGENESIS Sperm develop in the seminiferous tubules This takes ~40-70 days, depending on the species

23 SPERMATOGENESIS Sperm pass from the seminiferous tubules (ST) into the rete testes (RT) in the mediastinum Efferent ducts (ED) then transport sperm to the epididymis

24 EPIDIDYMAL TRANSPORT This requires days, depending on the species The cytoplasmic droplet passes down the tail and is usually shed in the ejaculate

25 EVALUATION OF MORPHOLOGY Sperm in the ejaculate reflect events that occurred <2 2 months ago Abnormal sperm were formed weeks before appearing in the ejaculate

26 Sperm Cell Morphology Use eosin-nigrosin nigrosin stain Nigrosin provides a dark background Sperm cells are white unless eosin penetrates a damaged membrane

27 Sampling Use a non-heparinized capillary tube or wooden applicator stick The higher the concentration, the smaller the amount to get correct concentration on slide

28 Preparing the Slide 1

29 Preparing the Slide 2

30 Examination of the Slide Dry slide immediately Check slide at 200X Evaluate morphology under oil immersion at >1000X Evaluate at least 100 cells

31 Normal Morphology From Garner DL, Hafez ESE. Spermatozoa and seminal plasma. In: Hafez B, Hafez ESE, eds. Reproduction in Farm Animals. 7th ed. Philadelphia: Lippincott L Williams & Wilkins, 2000:98.

32 Comparative Morphology From Garner DL, Hafez ESE. Spermatozoa and seminal plasma. In: Hafez B, Hafez ESE, eds. Reproduction in Farm Animals. 7th ed. Philadelphia: hia: Lippincott Williams & Wilkins, 2000:98.

33 Head Defects Pinched base or moderately pyriform heads probably do not cause infertility if nearly all cells have these shapes (photos courtesy of AD Barth)

34 Head Defects That Affect Fertility Severe pyriform shape Equatorial vacuoles Large vacuoles The significance of small, single apical vacuoles is unknown (photos courtesy of AD Barth)

35 Examples of Other Head Defects That Affect Fertility Large or small heads Usually not seen in high numbers Abnormal DNA condensation Not detected with ordinary stains; requires Feulgen stain

36 Knobbed Acrosomes Unable to fertilize (Photos courtesy of AD Barth)

37 Acrosome Defects usually only seen on frozen-thawed semen

38 Defects That Apparently Do Not Abaxial tail attachment, if no accessory or double tail is present (photo courtesy of AD Barth) Distal cytoplasmic droplets Affect Fertility

39 Defects That Affect Fertility (photo courtesy of AD Barth)

40 Possible Artifacts Verify by Checking Wet Mount Distal midpiece reflexes and bent principal pieces with no droplet enclosed Bowed midpieces Loose normal heads (photo courtesy of AD Barth)

41 Minimum Standards - Morphology A minimum of 70% normal cells Compensable defects such as midpiece defects may be of less concern as long as there are sufficient normal, motile cells Certain head defects are non-compensable and therefore of more concern

42 CELLS OTHER THAN SPERM (COTS)

43 CELLS OTHER THAN SPERM White blood cells (and other COTS) are best detected by staining a dried semen smear with Wright-Giemsa or Diff Quik stains WBCs indicate infection in the tract

44 CELLS OTHER THAN SPERM Cells shed from the seminiferous epithelium indicate testicular degeneration This may condition may improve or worsen with time

45 ASSIGNMENT Classify >100 cells Work alone or in small groups Hand in results Include species evaluated List name(s) ) of evaluator(s)

46 Categorize Cells By Percentage Head defects Midpiece defects Principal piece defects Normal detached heads Proximal droplets Acrosome defects Minor defects (e.g., distal droplet, simple bent principal piece) Normal