When assessing bull fertility, careful consideration must be given to the entire bull breeding soundness examination (BBSE) process. The purpose of this article is to share some advice on semen handling and evaluation techniques, to reduce the chance of artefacts occurring and increase trust in interpretation of results.
All measures should be taken to ensure that the sample we see down the microscope is a representative ‘biopsy’ of what would be ejaculated during natural service. However, with each handling process and exposure to the environment the semen cells can become damaged and further removed from this.
Cold shock is challenging to overcome but preparation and suitable equipment can help avoid premature death and artificially low motility of the cells. A small incubator, a slide warming bench and a heated microscope stage are worth the investment. The laboratory equipment should be set up before the bull examination to allow the kit to reach the optimal temperature of approximately 37°C (as per the manufacturer's instructions) (Appell et al, 1977). Slides, coverslips, pipette tips, collection tubes, collection sheaths, phosphate buffer solution (PBS) and nigrosin-eosin (NE) stain can be heated quickly on the warming bench and should be maintained at temperature in the incubator. Access to electricity should be checked (before arrival) to ensure the set-up is as close to the crush as possible, to reduce the time between collection and semen examination. The collection tube should remain close to body temperature during and after the collection too. Keep the pre-warmed tube in a pocket while carrying out the clinical examination, in hand during collection and in the incubator during evaluation. On really cold days insulating the tube with cotton wool can help.
Steps to avoid contamination of the sample should be taken. Trim the hair around the bull's prepuce to remove debris. This is best done at the start of the electroejaculation (EEJ) cycle, taking care not to get injured. Collection tubes with screw top lids can be useful post-collection, on rainy days, to prevent water contamination. Exposure to water or hypotonic solutions distorts the cells leading to morphological changes such as bent tails, coiled tails and pseudodroplets (Revell, 2010).
Progressive motility is a key area of the British Cattle Veterinary Association (BCVA) bull pre-breeding examination certificate. The percentage value indicates the cell's ability to move through the reproductive tract to fertilise the oocyte, and this assessment is made by diluting the semen to better visualise individual sperm cell motion and direction. This is best done by mixing one drop of semen using an adjustable micro pipette with 0.5 ml of pre-warmed PBS in a centrifuge vial (Mueller, 2018). Saline may be used, but motility may be adversely affected where the pH is lower. There are no minimum standards when considering both the volume and appearance of the semen sample since EEJ collection will not be comparable to that of a natural collection. However, when preparing the sample, it is still useful to grade the concentration on appearance according to Revell's (2010) system (see Box 1) to help ascertain the size of the drop required. Only 25 µl is needed where samples are dense, like cream, as opposed to 100 µls from a watery, dilute sample. Change the pipette tip before taking up the diluted sample and place a drop on to a slide with a coverslip over the top.
Box 1.Guide to semen examinationGross motility:
- One undiluted drop of semen
- Slide only
- Score out of 5
Progressive motility:
- Mix 0.5 ml PBS & one drop of semen in vial (see below)
- Slide and coverslip
- Satisfactory ≥60%
| Appearance | Number sperm (x106/ml) | Drop size (µl) |
|---|---|---|
| Water | <300 | 100 |
| ‘Semi-skimmed’ milk | 300–500 | 75 |
| ‘Whole’ milk | 500–1000 | 50 |
| Cream | >1000 | 25 |
Morphology:
- Mix 270 µl NE and 30 µl semen in vial (or any 9:1 ratio)
- Make smear on slide
- Use oil immersion lens
- Satisfactory ≥70%
Sense-check:
- Mix 1 ml formal saline and 0.02 ml semen
- Slide and coverslip
Mueller (2018) and Revell (2010). Scores are according to the BCVA bull pre-breeding examination certificate
Consider low progressive motility (<60%) alongside a thorough history. A high dead count, low progressive motility, and high numbers of cells with detached heads and swollen acrosomes (McAuliffe, 2010) may not reflect the bull's true fertility status and instead can be attributed to sperm accumulation where senescence has occurred. This happens if a bull has not been working recently and these bulls should be retested.
Morphological examination is the assessment of the physical structure of the cells which affects their ability to function and fertilise oocytes. Good morphological assessment preparation helps avoid producing overcrowded smears. A NE stain to semen ratio of 9:1 (Mueller, 2018) works well for examining cell morphology (see Box 1). Instead of messily mixing it on the slide, pre-warm 9 drops of NE in a centrifuge vial, then add one drop of semen to it. Leave it to stand for 5 minutes to allow any dead cells to take up the stain. However it is important to avoid leaving it for any longer since an artificial number of bent tails may occur.
If you do suspect morphological changes due to stain mishandling, a ‘sense-check’ can be carried out by examining semen that has been fixed in formal saline. It is possible to assess the cell integrity long after the visit due to this preservation. Mix 0.02 ml of semen with 1 ml of pre-drawn-up and warmed formal saline in a centrifuge vial. Examine a drop under a coverslip using oil immersion. The dead cells appear to move in the clear media making it easier to assess for cytoplasmic droplets. They will float by if not attached; unlike in stained, dry smears a cytoplasmic droplet may appear to be attached but is in fact just overlying the tail. It is also easier to assess the cell for nuclear defects, including vacuoles with this method.
Of course, samples can be sent to laboratories for computer assisted semen analysis (CASA) and flow cytometry to obtain objective results for motility and concentration. Technology also continues to develop with portable systems becoming more widely used on farm to assess motility and survivability. These tools certainly have a place in practice but still rely on accurate collection techniques.