4. MATERIALS AND METHODS
4.2 Sperm evaluation method
Viability staining, fixing and acrosome staining
Dilution of the semen samples: Stallion spermatozoa are sensitive to pH, osmolality and temperature changes. Therefore, phosphate-buffered saline (PBS) containing 0.06% K2HPO4 anhydrate and 0.825% NaCl (pH: ~ 6.8) was used for semen dilution at the same temperature as the sperm specimen was. Dilution rate depends on the status of the semen samples, spermatozoa concentration and extender type. For example
sperm concentration in the pellet after separation (Experiment 3) was fairly low (in the range of 1-62 million/ml at the various treatment and stallions), therefore to count enough sperm for the correct evaluation after further dilution with PBS was almost impossible in many cases. However proteins of seminal plasma and extender or glycerol did not disturb the staining with their binding affinity to the viability dye, the solutions were clean enough to use them without additional dilution for making smear.
Fresh sperm and cooled semen - diluted in 1:1 or 1:2 rate with NFDSM-Glucose or egg-yolk-skim-milk-based (EY-SM) extender-, was diluted 1:4 with PBS before viability staining. Both the centrifuged and frozen/thawed semen processed at a final concentration of 100-200 million cells/ml in freezing extender containing egg-yolk and glycerol, diluted 1:9 with PBS just before making smear.
The viability test stain contained 0.16% Chicago sky blue 6B (Sigma-Aldrich St.
Louis, MO, USA, C-8679). The working solution was made from a 2.6% stock solution in distilled water -which is isotonic-, diluted 1:15 with PBS or 0.27% trypan blue working solution was prepared from 0.4% stock solution (Sigma T-8154) diluted 2:1 with PBS. Both staining solutions are isotonic, have neutral pH, and are stable for a year in eye-drop bottles at room temperature. In the Experiment 1 the two viability stains were compared. Semen samples in the Experiment 2 and 4 were collected partly during that period when the works on improving and validation of the modified complex staining method were being performed. Therefore in some cases only smears stained by TB were available and from the most sperm samples, smears stained with both viability dyes were made because these specimens were also used for the densitometry and methodology comparative study. In these cases I used the CSB-stained samples for the two experiments mentioned above. In the Experiment 3 semen samples stained by CSB were analysed.
The fixative was composed of 86 ml 1 N HCl plus 14 ml of 37% formaldehyde solution and 0.2 g neutral red (Sigma N2880); it is stable for a year at room temperature and may be used repeatedly. The acrosome stain was 7.5% Giemsa stock solution (Sigma GS-500) in distilled water prepared freshly before use.
Staining procedure
Equal drops (20 µl) of viability stain and diluted semen were mixed gently on a slide flatly with the corner of another slide without scratching and touching the glass surface. Liquid layer is formed between the two slides and the droplets get mixed up with a slightly movement of the slides. The slides were attached parallel to each other and pulled to make two smears. The smears were air dried nearly vertically at room temperature. After drying, slides were fixed for 4 min. Both sides of the slides were
rinsed with tap water and distilled water, then stained in Giemsa solution in an uncovered staining jar (not more than 14 slides per jar with 16 spaces) at 25-40° C for 2-4 hrs. Slides were rinsed with tap water, then differentiated in distilled water for 2 min, air dried in a nearly vertical position, and cover slipped with Entellan (Merck 1.07960, Darmstadt, Germany). Slides were evaluated at 1000 X magnification using oil-immersion objective and a yellow filter for better live/dead differentiation.
Viability evaluation
- Three hundred cells were counted on each slide and classified into five categories (Fig. 5) in Experiment 1 and Experiment 3.
IHITDA
IHDT DHIT DHDTDA
Intact= intact head, tail, acrosome
IHITDA = intact head, tail, damaged (or lost) acrosome
IHDT = intact head, damaged tail
DHIT = damaged head, intact tail
DHDTDA = damaged head, tail, acrosome
Intact
SPERM CATEGORIES
Figure 5. Different sperm categories classified for the viability evaluation in Experiment 1 and 3
- Eight sperm categories were classified based on membrane integrity combined morphology (Figures 6-7) for the light microscopy examination in Experiment 2 and Experiment 4. Two-three hundred cells were evaluated in each sample.
IBT
Sperm with intact membranes
„Intact”
IHITIA IPD IDD
IHITIA = intact head, tail, acrosome, normal morphology
IPD = intact head, tail, acrosome;
proximal cytoplasmic droplet IDD = intact head, tail, acrosome;
distal cytoplasmic droplet IBT = intact cell with bent, curved,
broken midpiece or tail Figure 6. Spermatozoa categories with intact membranes
IHITDA IHDT DHIT DHDTDA
IHITDA = intact head, tail, damaged (or lost) acrosome
IHDT = intact head, damaged tail
DHIT = damaged head, intact tail
DHDTDA = damaged head, tail, acrosome
Figure 7. Sperm types with damaged membrane of any part of the cell
- In the statistical analysis of Experiment 2 additional and combined categories were also evaluated:
1. Damaged spermatozoa with CD [DCD]
2. Damaged) sperm with bent, curved, broken midpiece or tail [DBT]
3. All cells with intact membranes [IHITA + IPD + IDD + IBT] Intact
4. Intact sperm with CD + Intact sperm with bent tail [IPD + IDD + IBT] ICDBT 5. All (intact and damaged) spermatozoa with CD [IPD + IDD + DCD] IDCD
6. All (intact and damaged) sperm with bent, curved, broken midpiece or tail [IBT + DBT] IDBT
7. All (intact and damaged) spermatozoa with CD or BT [IDCD + IDBT] IDCDBT
DCD
DCD= damaged spermatozoa with cytoplasmic droplet
Figure 8. Damaged spermatozoa with cytoplasmic droplet
DBT
DBT = damaged spermatozoa,
with bent, curved, broken midpiece or tail
Figure 9. Damaged spermatozoa, with bent, curved, broken midpiece or tail Different sperm types are shown in Fig. 10:
Figure 10. Microscopic picture of different sperm types (CSB/Giemsa staining)
Morphological evaluation
- Three hundred cells were classified in 5 simple morphological categories in Experiment 3:
1. normal
2. proximal cytoplasmic droplets (PD) 3. distal cytoplasmic droplets (DD) 4. midpiece and tail defect (midp+tail) 5. abnormal head (head)
- Cells were classified in 9 morphologic categories in Experiment 4:
1. Normal morphology (normal)
2. Head abnormalities (head) (microcephal, macrocephal, tapered, pyriform, nuclear vacuoles, acrosome defects)
3. Midpiece defect (midp) (swollen, bent, DMR, mitochondrial sheath defect, corkscrew, bowed)
4. Tail abnormalities (tail) (broken, bent, hairpin-curved, distal coiled tail) 5. Coiled tail defect (coiled) (tightly coiled tail, dag-like defect)
6. Detached head (detached)
7. Proximal cytoplasmic droplet (PD) 8. Distal cytoplasmic droplet (DD)
9. Multiple forms (multiple) (e.g. double midpiece, head, tail)
The standard counting and classifying system was used where a sperm cell was placed in one class of abnormality only (the most primary one) and identified the percentage of each of nine sperm categories. However increased proportion of sperm with multiple defects is also noted during the evaluation. Both live and dead spermatozoa were assessed for morphology during the count. Three hundred cells were evaluated in each sample to determine the percentage of different cell types. Classification of sperm abnormalities was based on previous publications and guidelines (Barth and Oko 1989, Kenney et al. 1990, Card 2005, Brito 2007, Varner 2008). Acrosome defects (knobbed, vacuoles, detached) were not enumerated separately from head defect here, however counted into distinct category among live cells (IHITDA) during viability evaluation. Distal midpiece reflex (DMR) defect was classified in midpiece defect according to Card (2005) and Brito (2007). Coiled tail defect (tightly coiled tail, dag-like defect) was enumerated in separated category from other tail defects because this can be caused by disturbance of spermatogenesis. Different sperm types are shown in the microscopic pictures in the Appendix.