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Reproduction: breeding assessment examination – stallion

ISSN 2398-2977


Synonym(s): Breeding fitness, Breeding soundness, Fertility assessment

Introduction

  • To determine if a stallion can reasonably be capable of rendering at least 75% of 40 or more mares pregnant when bred naturally, or 120 mares when bred artificially, in one breeding season with reasonable mare management and fertility.
  • Determining the probability of a male (stallion) not likely to be infertile. Importantly the breeding assessment of the stallion (male) is not designed to predict fertility, but rather decrease the likelihood of infertility.
  • A more rational definition would be, to determine the likelihood of a stallion to obtain at least a 60% per cycle pregnancy rate at first pregnancy diagnosis scan, assuming normal mare fertility and good management.

Uses

Advantages

  • Pre-season breeding assessment.
  • To assess the likelihood of a young or first season stallion not being infertile.
  • To assess why a stallion might be infertile or sub-fertile.
  • Assessing abnormal reproductive behavior.
  • To determine the presence of a pathogenic organism in the reproductive tract.
  • As part of a pre-purchase and or examination for insurance purposes.
Breeding soundness exams do not form part of a routine five stage pre-purchase examination.
  • To determine the likely size of his seasonal breeding ‘book’.

Disadvantages

  • The reported correlation between many in vitro fertility parameters and pregnancy outcomes (fertility) are typically considered modest.

Technical problems

  • Examinations associated with the breeding assessment examination can include relatively simple tests, such as measuring scrotal width; or could require sophisticated laboratory support such as flow cytometry for sperm chromatic structure analysis (SCSA).
  • A safe environment to examine the stallion, and to be able to collect a representative ejaculate from that stallion. Appropriate equipment and facilities to accurately assess the volume, color, spermatozoa concentration, motility, morphology and total number of ejaculated spermatozoa.

Safety

  • Appropriate personal protective equipment should be worn by all staff.
  • This is not limited to, but includes:
    • Helmets.
    • Body protection.
    • Suitable clothing such as coveralls/overalls.
    • Sturdy footwear: possibly boots with a toe cap.
    • Gloves.
    • Biosecurity procedures for all processes as appropriate.

Alternative techniques

  • Ultimately, fertility is determined by the ability of the stallion to successfully impregnate mares; this can be carried out by collecting and evaluating the appropriate data.

Time required

Preparation

  • Variable, depending on whether the examination takes place ‘in the field’, or at a designated fertility facility.
  • 15 min.

Procedure

  • Variable, depending on the stallion’s demeanor, the facilities, the ancillary examinations carried out, and the ease of semen collection for assessment.
  • Approximately 60 min for stallion identification, examination, and semen collection.
  • Approximately 30 min for the initial semen assessment and collection of samples.

Decision taking

Criteria for choosing test

  • The overarching assessment is that of determining the likelihood of the stallion not being infertile.
  • There are many procedures and tests associated with this evaluation, which can be included depending on the reason for the assessment, and the results/outcomes of the previous tests in the procedure.

Risk assessment

  • Safety of personnel, the animals (stallion and if necessary, a ‘mount mare’), facilities and equipment.
  • Assessing the breeding soundness of stallions is (potentially) dangerous. All personnel should be trained in procedures to protect the safety of humans and horses. All personnel should wear protective clothing, eg hard hat, gloves, body protector and robust footwear. An experienced stallion handler is important.

Requirements

Personnel

Veterinarian expertise

  • Appropriately trained and competent in dealing with the animals (mares and stallions) as necessary, to collect and appropriately interpret findings, and to handle and assess the ejaculate.

Anesthetist expertise

  • Generally not required.
  • Depends on age and demeanor of the stallion.
  • Sedation might be necessary to palpate the genitalia, for swab collection, or for ancillary diagnostic tests, eg endoscopy/radiography as necessary.

Nursing expertise

  • Technical staff include:
    • A competent stallion handler, familiar with the breeding process and procedure.
    • A competent person to handle the mount or teaser mare as necessary.
    • A competent person to collect the semen (technician or veterinarian).
    • A competent person to ensure the safety and welfare of the semen collector, other staff present, and the animals.

Other involvement

  • Laboratory staff as necessary.

Materials required

Minimum equipment

  • Identification:
    • As appropriate:
      • Age, color, breed.
      • Identification forms (passport number).
      • Photography.
      • Brand identification.
      • Microchip reader.
  • General examination:
    • Appropriate restraint: bridle and lead rope. Many prefer to use a Chiffney bit when the stallion is being used for breeding, for added control and to help the stallion to distinguish between his breeding and ridden roles.
    • Thermometer, stethoscope, gloves, torch/ophthalmoscope, hoof testers.
  • Reproductive examination:
    • Device to measure scrotal width, trans rectal gloves and lubricant, ultrasound machine.
    • Swabs for sample collection (typically three/four) and appropriate transport medium (charcoal if assessing for Taylorella equigentalis).
  • Semen collection:
    • Facility to enable penile examination when erect, and to safely take appropriate swabs as necessary.
    • Basic equipment washing facilities.
    • Some sort of device to mount: ‘mount mare’ or ‘phantom’ device.
    • Appropriate collection device: artificial vagina (AV) and collection bottle.
  • Semen assessment:
    • Accurate ejaculate measuring device: typically, 10-150 mL volume range.
    • Means of assessing the sperm concentration, eg ‘Spermacue’ or equivalent automated device; hemocytometer with appropriate dilution.
    • Extender solution to assist in motility assessment.
    • Appropriate microscope with heated stage to assess motility, and if desired to determine morphology. For morphological assessment, a good quality microscope with differential interference contrast (DIC) or phase contrast and sufficient resolution to observe individual spermatozoa at ≥x1000 magnification under oil immersion is required.
    • Stains and microscope slides as appropriate for assessment of spermatozoa morphology, (eg Eosin/Nigrosin, Indian Ink), or cells other than spermatozoa (COTS) such as ‘Diffquick’. Ten percent buffered formol saline (10% BFS) solution for collection of samples for wet mount morphological assessment via DIC.
  • Semen safe syringes, needles and containers to aspirate and collect samples from the ejaculate.
  • Access to a laboratory for reliable assessment of collected samples.
  • Ancillary examinations: this is not part of the routine breeding evaluation, but endoscopy of the urethra or bladder, and radiography or contrast radiography might be considered necessary depending on reasons for, and findings in the fertility examination.
Air embolisms have been reported to occur during urethral endoscopic examination; it is a rare but reported complication.The use of ‘soundness’ to describe the evaluation is commonly used. The potential implication of soundness is the animal is either not sound, or sound, which is not feasible with prospective fertility examinations that are not directly assessing pregnancy outcomes.


Ideal equipment

  • In addition to equipment above:
    • A safe enclosed and roofed facility with a firm and reliable surface to enable mounting and thrusting by the stallion.
    • A ‘phantom’ mount in association with a ‘teaser’ mare.
    • Stocks to safely examine the stallion, including trans rectal palpation of the reproductive tract as deemed necessary.
    • Ultrasound machine with a 5MHz (or higher) linear transducer with a good quality image, and the ability to capture those images as necessary.
    • An in-house processing laboratory to enable clean use of the AV at the appropriate (c. 45°C/113°F) temperature and pressure, and to prevent the possibility of cold shock when processing the collected ejaculate.
    • Filters to efficiently remove any gel fraction from the ejaculate.
    • A Phase Contrast, or Differential Interference Contrast (DIC) microscope.
    • An appropriately sized (6 mm x 1.2 m) endoscope for urethral examination as necessary. The possibility of an air embolism has been reported during this procedure.
    • Radiography equipment as might be deemed necessary for orthopedic or other investigations.
    • Computer Assisted Spermatozoa Analysis (CASA) device.

Minimum consumables

  • Four swabs for microbial assessment (with charcoal transport medium).
  • Clean/lined bucket with warm water for washing the penis.
  • Cotton wool.
  • Paper towel.
  • Gloves: hand and transrectal.
  • Lubricant: for ultrasonography (or methylated spirit for trans cutaneous ultrasonography), and sterile/aseptic for AV lubrication.
  • Microscope slides and cover slip.
  • Isotonic diluent, eg phosphate buffered saline (for individual spermatozoa motility assessment).
  • Report delivery.

Ideal consumables

  • As for minimum consumables, plus:
    • Eppendorf tubes, or similar, with 1 mL 10% BFS for wet mount morphological spermatozoa assessment.
    • Transparent semen extender for motility assessment.

Other requirements

  • A clean and temperature stable environment.

Preparation

Pre-medication

  • Typically not required.
  • If needed for parts of the examination, eg palpation of the reproductive tract or swab collection, then a combination of an α2receptor agonist and an opioid at the dose considered to be appropriate may be used.
  • Historically, the use of the sedative acepromazine (ACP) in breeding stallions has been advised against due to risk of penile prolapse/priapism Penis: paralysis/priapism/paraphimosis Penis: paraphimosis.
Sedation with an α2receptor agonist and an opioid should not be used when the stallion is attempting to mount the mare or phantom, due to the risks to the horse and personnel associated with the horse being ataxic/unbalanced.
  • Diazepam Diazepam (approximately 25 mg/500 kg; 0.05 mg/kg) might be useful to alleviate apprehension in a given animal. However, whilst this treatment may be appropriate for animals in which lack of libido Male: lack of libido is due to psychological inhibition (for example a stallion which has been previously kicked by a mare), if the stallion is simply a novice or ‘slow breeder’, then patience and training are much more appropriate.

Site preparation

  • Scrotum/testes:
    • The testes are paired laterally flattened cylinders, relatively resilient and toned with a rubbery consistency within the scrotal sac.
    • The testes can be most easily palpated immediately after a semen collection, when they are descended, and the stallion is relaxed.
    • Both testes should be descended, palpable individually, and freely movable within the scrotal sac.
    • The head of the epididymis should be rostral, and the tail caudally in the scrotum. Occasionally a hemi-rotated testis is detected which should be noted on the examination report; it is not necessarily considered pathological in all cases.
    • There should be no other palpable swellings, nor fluid detected on or around the testes.
    • The total scrotal width should be measured, with the testes retained ventrally in the scrotum and the maximum distance measured (digitally, with calipers, or ultrasonographically).
  • The stallion will then need to be ‘teased’ by a mare to obtain an erection; initially whilst restrained in stocks, or by the handler.
  • Prepuce and penis:
    • If collecting swabs for potential bacterial venereal pathogens, it is best to collect them prior to washing the penis. Three or four swabs prior to semen collection:
      • Shaft of the penis/prepuce.
      • Urethral fossa.
      • Distal urethra.
      • Pre-ejaculatory fluid: if difficult to collect/swab pre-ejaculatory fluid the semen can be swabbed after collection.
    • The preputial orifice can be visualized and palpated at this time for any gross abnormalities such as skin lesions.
    • Washing the stallion’s penis:
      • Clean warm water only, no disinfectant nor surfactant/soap should be used to wash the penis and prepuce.
      • Wear gloves.
      • Use a cup or cotton wool to remove the water from the lined bucket to clean the penis; ensuring that the water in the bucket is not contaminated by previous washing procedures.
      • The penis should be divided into three zones: with the aim of having the glans penis (including the urethral fossa which occasionally has a smegma ‘bean’ that should be removed) as the cleanest part after washing, followed by the distal shaft of the penis, and finally the base of the prepuce really only needs gross contaminants removed.
      • Dry the penis using paper towel or ‘squeeze dried cotton wool’, to prevent leaving strands of material on the penis.

Other preparation

  • Ensure that the artificial vagina (AV) is appropriately prepared; this should occur immediately prior to the stallion penis washing:
    • The size of the vagina is appropriate for the stallion .
    • The temperature (typically more than 40°C/104°F but <50°C/122°F) of the water jacket is correct as is the pressure (ability to insert an arm into the AV that is about the size of the stallion’s penis) within the water jacket.
    • Apply sufficient, but not excessive, sterile/aseptic non-spermatoxic (no preservative) lubricant gel (carboxymethyl cellulose) to the inside of the AV:
      • The inserted gloved arm in the AV should move relatively freely within the jacket of the AV.
      • The inside of the AV can be kept moist by leaving the glove within the AV and removing it just prior to semen collection.
  • If using a phantom mount, ensure that it is secure and the correct height for the particular stallion.

Restraint

  • Ensure that the ‘teaser’ mare or ‘mount mare’ is in estrus.
  • Ensure appropriate restraint of the mare, such as with a nose twitch (when necessary), and with padded boots on her hind limb hooves.
  • The stallion should be appropriately controlled with a bridle and long lead rope (typically a Chiffney bit). The rope must not be so long that it presents a risk of tangling but must be long enough for when the stallion rears up onto the dummy/mare.
For safety purposes, all stallions should be considered potentially dangerous animals and treated accordingly.

Technique

Approach

Step 1 - Correct placement and control of the stallion in the serving/collection shed/barn

  • Ensure there are sufficient suitably trained personnel to undertake the procedure.
  • Typically, minimum staff:
    • Stallion handler.
    • Teaser mare handler if a teaser mare is needed.
    • Semen collector/AV handler.

Step 2 - Teasing the stallion

  • The stallion is moved from the penis cleaning area, to the designated area in the collection shed/barn where he is controlled by the stallion handler.
  • The tease mare is moved safely towards the stallion in order to induce an erection:
    • Some stallions will not require teasing.
    • Those that do require teasing, it is important that the correct behavioral cues are allowed to be conveyed to the stallion by the tease mare in estrus.
    • In summary, the mare should be taken to the stallion ‘face to face’ in the first instance.
      • When interest is shown in the mare by the stallion (sniffing her, and initiation of an erection), she should be turned about so that the stallion is able to visualize the ‘winking’ perineum of the mare.
      • The mare typically decreases weight on a fore limb, turns her head backwards, and moves in a ‘jerky’ manner to illustrate her readiness to be mounted. Do not allow the mare to continually walk around the stallion, standing to be mounted is an important cue for sexual behavior.
    • The process is continued until the stallion obtains an erection.
  • The semen collector with the correctly prepared AV takes their position on the left side of the stallion, at a distance that does not adversely impact his arousal behavior and in a position which reduces the risk of being struck by hind or forelegs.
Ensure that an insulated collection bottle is attached to the AV.


Step 3 - Preparation for mounting

  • When the stallion has an erect penis, he is encouraged to mount the mare or the phantom by moving the teaser mare into an appropriate position, with the mare either standing to be mounted, or towards the phantom for a phantom collection.
  • As the stallion handler prepares to encourage, or allow the stallion to mount, the person with the AV approaches the left flank of the stallion.
  • Typically, the approach is from the left side of the stallion, with the AV held in the left hand of the semen collector.
  • As the stallion mounts, the collector deviates, without grasping, the penis towards the collector (left side of the horse) with the right hand, and introduces the penis into the AV.
  • The AV is held adjacent to the phantom or mare, as the stallion has about 10 thrusts.
  • The stallion collector needs to maintain stability and balance, which can be facilitated by resting their head on the flank of the stallion:
    • Ensure that:
      • The forelimbs of the stallion do not impact on the collector.
      • The stallion does not fall onto the collector.
      • The hindlimbs of the stallion do not crush the feet of the collector.
      • The AV opening advances to the base of the penis: if the stallion does not ejaculate, applying a hot (60°C/140°F) wet plastic wrapped towel to the base of the penis can assist with inducing ejaculation.

Step 4 - Semen collection

  • Usually, within about 20 seconds within the AV, the stallion starts ejaculating Semen: collection.
  • Ejaculation can be detected by:
    • The collector palpating the pulsations in the ventral penis- peristalsis of the urethra.
    • ‘Flagging’ of the stallion’s tail.
  • Once ejaculation begins, the distal part of the AV should be lowered slightly to facilitate the flow of the ejaculate into the collection device.
  • If necessary, a third/fourth swab should be collected from the distal urethra, in case there are organisms within the ejaculate that would not necessarily be detected with pre-ejaculatory swabs.

Step 5 - Semen processing

  • Once the AV is removed from the stallion, with the collection device ventrally, the water is removed from the AV to enhance semen drainage into the collection device.
  • The ejaculate is expeditiously assessed, preferably in an adjoining laboratory.

Step 6 - Semen assessment

  • The semen container is quickly moved into a climate-controlled environment: minimal dust, temperature variation, incident sunlight.
  • Processes Semen: evaluation:
    • Where gel is present it needs to be removed from the ejaculate as soon as possible using a gel filter, or suction via a syringe.
    • The semen should not drop below the temperature that can induce ‘cold shock’ changes (20°C/68°F), nor increase above 37°C/98°F.
    • Assessment:
      • Color.
      • Volume (and pH).
      • Sperm concentration: automated device or manual counting.
      • Collection of an aliquot (1-2 drops of raw semen in 1 mL 10% BFS) of semen for DIC/phase contrast microscopy.
      • Sperm motility: via CASA, manually via microscopy, or a combination. Typically using an aliquot of 1:1 extended sample with appropriate extender at 37°C/98.6°F.
      • Staining of a portion of the ejaculate/semen with an appropriate stain, eg eosin and nigrosine stain, for morphological microscopic assessment on a microscope slide
      • Collection of other samples that might be necessary, such as snap freezing an aliquot for sperm chromatin structure assay (SCSA), or for culture for potential pathogens, or alkaline phosphatase if azoospermic Semen: azoospermia/oligospermia.
  • Transrectal palpation and ultrasonography of the reproductive tract might be necessary ancillary procedures, depending on the reason for the assessment, and the findings during the examination process.
  • Testicular biopsy if indicated by initial test results Testis: biopsy.

Core procedure

Step 1 - Interpretation of results - general examination

  • The stallion should be ‘fit for purpose’, which will depend on the number of mares and method of breeding for that particular animal.
  • The stallion is typically required to be able to safely visualize, move to, and mount either the mare or phantom, with an erection, resulting in an ejaculate that results in a pregnancy.
  • The stallion must:
    • Visualize.
    • Interact with.
    • Mount a mare or phantom.
    • Ejaculate: spermatozoa that are likely to render the mare pregnant.
  • Any reason for him being unable to carry out these procedures will render him unfit for breeding purposes, eg severe orthopedic disease.

Step 2 - Interpretation of results

Reproductive examination - external genitalia
Penis and prepuce
  • The penis should normally be withdrawn into the preputial orifice/opening.
  • And should protrude from the orifice when erect.
  • Bacterial venereal pathogen (Taylorella equigenitalis Taylorella equigenitalis and Klebsiella pneumoniae Klebsiella pneumoniae capsule types 1,2,5 (and 14), and reportedly Pseudomonas aeruginosa Pseudomonas aeruginosa with limited evidence) isolation will require appropriate notification and treatment depending on jurisdiction.
  • The stallion must be able to ejaculate semen containing spermatozoa.
Scrotum and testes
  • The glabrous skin of the scrotum should be lesion free with no resentment to palpation.
  • Only testes and epididymes should be palpable within the scrotum, eg no free fluid nor abdominal contents detectable.
  • Both of the testes should be freely movable within the scrotum, with appropriate resilience and tone and no palpable swellings, excluding the epididymis.
  • Testes should be similar sizes, with the same resilience and tone.
  • The head of the epididymis should be rostrally, and the tail caudally, orientated.
  • The total scrotal width should meet the breed standard, typically be greater ≥80 mm.
  • Scrotal volume could be calculated if the three dimensions of the testis are measured:
    • Vol (mL/cm3) = 4/3π x width/2 x height/2 x length/2in cm; for each testis and combine for a total testicular volume (TTV).
  • Daily Sperm Output (DSO) can be predicted relatively accurately:
    • DSO (x109) = 0.024 (TTV) – 1.26 (better correlation than with TSW).
  • Assessment is important to exclude obvious pathology such as castration, cryptorchidism, herniation, fluid accumulation (eg varicoceles), rotation/torsion, testicular degeneration/hypoplasia.

Step 3 - Semen assessment

  • One study recommends that two ejaculates collected an hour apart are assessed for volume, total spermatozoa, pH, and motility to enhance representative evaluation Semen: evaluation:
    • Volume should be similar between ejaculates.
    • The number of spermatozoa in the second ejaculate should be half that of the first.
    • pH should be similar (7.2-7.6) or slightly higher in the second ejaculate.
    • Motility should be similar to, or slightly increased, in the second ejaculate.
    • If spermatozoa numbers are important to assess ‘book size’, then the stallion should be collected to determine DSO/DSP after more than five daily collections.
  • Ejaculate assessment should include:
    • Gross appearance: typically ‘off white, and opaque’.
    • Initial motility (within five minutes of ejaculation): total (all spermatozoa that are moving) and progressive (those moving in a relatively straight line) motility is determined. Ideally this should be done both via microscopic assessment, and by a computer assisted sperm analysis (CASA).
    • pH, within 10 minutes of ejaculation, should be 7.2-7.6.
    • Spermatozoa morphological assessment (spermiogram):
      • Approximate dimensions are: head 6 µm, midpiece 10 µm and tail 60 µm.
      • Typically, ≥70% morphological normal spermatozoa is considered a minimum value required for a ‘fertile stallion’.
    • Spermatozoa longevity can be assessed if necessary, including with different extenders and at different temperatures.
    • The ejaculate can also be assessed for cells other than spermatozoa (COTS), especially inflammatory cells. Assessing dismount samples for COTS can be misleading.
    • Microbiological assessment of the ejaculate could be considered, remembering that an ejaculate is not expected to be sterile.

Step 4 - Summary

  • A stallion should have the following attributes:
    • Good libido, move freely, obtain an erection, mount the mare, with intromission, and ejaculate only semen.
    • A ‘normal’ size and shaped penis.
    • Be free of designated venereal pathogens, eg viral (EVA Equine viral arteritis (EVA)), bacteria (Taylorella equigenitalis Taylorella equigenitalis), fungi (Aspergillus spp Aspergillus spp), parasites (Habronema spp Habronema spp.
    • Free from dourine Penis: balanoposthitis (Trypanosoma equiperdum Trypanosoma equiperdum), and equine infectious anemia virus Equine infectious anemia virus (Coggin’s test negative).
    • Two normally palpable testes within the scrotum.
    • The stallion should ejaculate AT LEAST One billion (1x109) morphologically normal, progressively motile spermatozoa (sperm concentration x semen volume x % morphologically normal spermatozoa x % progressively motile spermatozoa) in the second ejaculate of each month of the year.
    • Scrotal circumference should be ≥80 mm.

Step 5 - Report

  • Ideally a report should be submitted to the owner/agent outlining the findings from the examination.
  • Examinations that were not carried out should be mentioned, and additional examinations pertinent to that case should be suggested.

Aftercare

Immediate Aftercare

Monitoring

  • For the standard examination and assessment, there is no need for any extraordinary after care.

Special precautions

  • Safe work and handling practices.

Outcomes

Further Reading

Publications

Refereed Papers

  • Recent references from PubMed and VetMedResource.
  • Whitesell K, Stefanovski D, McDonnell S & Turner R (2020) Evaluation of the effect of laboratory methods on semen analysis and breeding soundness examination (BSE) classification in stallions. Theriogenology 142, 67-76 PubMed.
  • Driessen B, Zarucco L, Kalir B & Bertolotti L (2011) Contemporary use of acepromazine in the anaesthetic management of male horses and ponies: a retrospective study and opinion poll. Equine Vet J