Equis ISSN 2398-2977

Semen: evaluation

Contributor(s): Madeleine L H Campbell, John Dascanio, Jean Pierre Held, Rob Lofstedt, Graham Munroe, Vetstream Ltd

Overview

  • Semen evaluation is part of any fertility investigation of a stallion and provides an overall indicator of semen quaility.
  • The number of progressively motile sperm (those swimming forward in straight lines) best indicates semen quality and fertilizing capacity.
  • Poor semen quality could be caused by infection, disease, congenital factors or processing
  • Evaluation may also be used in stud management regimes to avoid overuse of a stallion or assess the suitability of a stallion for artificial insemination programs.

Sampling

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Tests

Methodologies

  • Filter semen from gel   Semen: gel fraction  if a filter was not incorporated into the artificial vagina. Commercially available filters are used.
  • The semen is then evaluated as follows.

Appearance

  • Milky white and thinner consistency than bull semen.
  • Opaque with a neutral smell.

Volume

  • Gel included volumes vary with stallion and degree of sexual stimulation.
  • Normal values for total volume 15-100 ml. 

Motility

Accurate assessment requires experience and quality phase-contrast microscope.

Should be assessed within 5 min of collection.

  • A drop of semen is placed onto a pre-warmed microscope slide (37-38°C) and covered with a warmed coverslip.
  • Estimate the percentage of sperm that are motile for total motility, ie moving at all (60-80%).
  • Estimate the number of sperm swimming rapidly and progressively, ie in a relatively straight direction, to give the percentage of progressively motile sperm (40-70%).
  • Total number of progressively motile sperm = volume x concentration x % progressively motile.
  • This is usually a subjective "by eye" observation and will vary from observer to observer.

Always err on the side of being cautious, ie of underestimating motility, as that way you will provide a generous rather than an insufficient insemination dose per mare. 

Computerized motility analysis equipment can be used but is expensive.

Diluting semen in warmed semen extender may assist motility assessment in very concentrated samples.

pH

Should be assessed within 10 min of collection.

  • 7.2-7.6.
  • The pH of second samples is usually greater than that of the first.
  • Abnormal pH might be consistent with urospermia   Semen: urosemen  .

Alkaline phosphatase

Concentration

Use a hemocytometer or calibrated spectrophotometer.

  • Dilute semen in buffered formol saline or acetic acid 1:100 (or 1:10 if sample very dilute).
  • Fill hemocytometer chamber with dilution.

Count both sides of hemocytometer if there is >10% difference between both sides then repeat test.

  • Count heads only in five small squares located in the large central square of the hemocytometer. Do not include those heads on the right and bottom lines.
  • The final concentration if the dilution used was 1:100 = the number of heads counted multiplied by 5 x 10(*9).
  • Normals depend on collection circumstances but usually vary from 50-900 x 10(*6)/ml.
  • Concentration x volume = total number of sperm per ejaculate; this may range from 4-12 billion.

Sperm count should halve approximately between each successive collectionon the same day.

  • After 5-7 daily collections, the number of sperms collected represent the daily sperm output of the stallion.

Spectrophotometers, although quick to use, are inaccurate at identifying sperm cells, eg they may interpret white blood cells as being sperm cells, and give a falsely elevated sperm count. Assessment using a hemocytometer is the gold standard for calculating semen concentration.

Morphology

Accurate assessment requires experience and quality phase-contrast microscope.

Morphology can be enhanced using a wet mount.

  • Add a drop of semen to a drop of nigrosin eosin stain on a microscope slide and smear with a second slide as for preparing blood films.
  • The stain will be taken up across damaged cell membranes but not across intact cell membranes: thus live cells look white and dead cells look pink.
  • Assess under (x40) or (x100) oil immersion.
  • 200 sperm are counted for percentage normal live, normal dead, abnormal live and abnormal dead.
  • Abnormalities include acrosome abnormalities, tail-less heads, proximal droplets, distal droplets, bent or misshapen midpieces, and bent or coiled tails.

It is important to distinguish genuine abnormalities which relate to spermatogenesis, or sperm maturation from abnormalities caused by the handling of the semen.

Longevity of motility

  • Examine the raw and extended semen (extended to 50 million sperm/ml by dilution with an extender) kept at 4 and 22°C every hour for 6 h then at 24 h and 48 h for progressive motility.
  • Extension is necessary because sperm cells, being highly specialized, have a high metabolic rate and produce waste products, which will kill off the cells if they are not diluted in extender.
  • There is a wide range of commercial extenders available, which typically contain nutrients (most commonly skimmed milk) and antibiotics, and is buffered for pH and for osmolarity.
  • There is inter-stallion variation in semen compatibility with different semen extenders. When a stallion's semen is first being assessed it should be tested with a range of different extenders to identify the one which suits that stallion best.

 In vitrolongevity however poorly correlates with in vivolongevity in some stallions.

Should have at least 10% progressive motility at the end point.

The cooling rate should be close to -0.3°C/min to 4°C. This is easy to obtain as it is the cooling rate for a properly packaged Equitainer®.

Microbiology

  • Swabs should be taken for bacterial culture and sensitivity testing from the semen and immediately post semen collection from the urethra.
  • All stallions having their semen assessed should have been assessed for venereal disease prior to collection   Male: bacterial venereal disease screening  .

Endocrinology

  • Endocrine assessment may determine if semen/behavioral problems are due to hypothalamic-pituitary-gonadal axis dysfunction.

Further diagnostic tests

  • Chemical analysis.
  • Electron microscopy.
  • Chromatin assay.
  • Membrane integrity.
  • Acrosome reaction.

Availability

  • Universal.

Validity

Predictive value

  • The total number of progressively motile sperm is considered to be the best indicator of potential fertility.

Technique (intrinsic) limitations

  • The evaluation cannot provide an unequivocal guarantee of fertility of a stallion as some stallions yield high numbers of progressively motile sperm yet are still infertile and some stallions have poor-looking semen but acceptable pregnancy rates.
  • Unreliable guide to stallion fertility.
  • Results of clinical examination and mare factors will be involved.
  • Semen characteristics are highly variable and affected by iatrogenic damage to sperm, season, age, state of health, previous covering load, type of management.

Technician (extrinsic) limitations

  • Individual variation will be dependent on speed of sample handling and care of same.
  • Microscopic evaluation requires average skill.
  • Identification of contaminants such as urine may require more skill.
  • A series of semen evaluations and a full reproductive evaluation and assessment of historical data are necessary to reach a full diagnosis.
  • Experienced technicians become very consistent in estimating motility, etc.

Result Data

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Further Reading

Publications

Refereed papers

  • Recent references from PubMed and VetMedResource.
  • Crabtree J (2010) Prebreeding examination of the stallion 2. Semen collection and evaluation. In Pract 32 (2), 58-63.
  • Morrell J M, Dalin A M & Rodriguez-Martinez H (2009) Comparison of density gradient and single layer centrifugation of stallion spermatozoa: yield, motility and survival. Equine Vet J 41 (1), 53-58 PubMed.
  • Pycock J F & Samper J C (2004) Semen analysis: interpretation of findings - prediction of fertility. UK Vet (8), 9-11.
  • Pycock J F & Samper J C (2004) Evaluation of semen. UK Vet (7), 11-15.
  • Parlevliet J M & Colenbrander B (1999) Prediction of first season stallion fertility of 3-year-old Dutch Warmbloods with prebreeding assessment of percentage of morphologically normal live sperm. Equine Vet Educ 31 (3), 248-251 PubMed.
  • Graham J K (1996) Analysis of stallion semen and its relation to fertility. Vet Clin North Am Equine Pract 12 (1), 119-130 PubMed.
  • Veeramachaneni D N & Sawyer H R (1996) Use of semen as biopsy material for assessment of health status of the stallion reproductive tract. Vet Clin North Am Equine Pract 12 (1), 101-110 PubMed.
  • Jasko D J, Little T V, Lein D H & Foote R H (1992)Comparison of spermatozoal movement and semen characteristics with fertility in stallions-64 cases (1987-1988). J Vet Med Assoc 1:200 (7), 979-985 PubMed.
  • Jasko D J (1992) Evaluation of stallion semen. Vet Clin North Am Equine Pract (1), 129-148 PubMed.
  • Hurtgen J P (1992) Evaluation of the stallion for breeding soundness. Vet clin North Am Equine Pract 8, (1) 149-165 PubMed.
  • Colenbrander B, Puyk H, Zandee A R & Parlevliet J (1992) Evaluation of the stallion for breeding. Acta Vet Scand Suppl 88 (1), 29-37 PubMed.
  • Proceedings of the first European Symposium on production evaluation and preservation of stallion semen. Uppsala, Sweden, October 1-2, 1992. Acta Vet Scand Suppl 88 (1), 1-167.

Other sources of information

  • Horserace Betting Levy Board (2016) Codes of Practice. 5th Floor, 21 Bloomsbury Street, London WC1B 3HF, UK. Tel: +44 (0)207 333 0043; Fax: +44 (0)207 333 0041; Email: enquiries@hblb.org.uk; Website: http://codes.hblb.org.uk.
  • Campbell M L H (2009) How to Collect, Process and Certify Equine Semen. In: Proc 48th BEVA Congress. Birmingham. pp 41-43.
  • Frazer G S (2004) Assessment of Stallion Fertility. In: Proc 43rd BEVA Congress. Equine Vet J Ltd, UK. pp 184.
  • McKinnon A O, Voss J L (1993) Equine Reproduction. Lea & Febiger ISBN 0 8121 1427 2 (The definitive text).
  • Varner D (1991) Diseases and Management of Breeding Stallions. American Veterinary Publications. ISBN 0-939674-33-5.


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