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system has the toughest standard, requiring individual motility to be greater than 70%. The ACV, BCVA, and WCABP systems don't consider that the bull meets the requirements for this standard unless it is greater than 60% motile. But the ACV system also accepts that although semen quality may not be suitable for freezing, bulls used solely for natural mating can have commercially acceptable fertility if the motility is greater than 30%. The ACV system therefore allows a “qualified” rating for this standard for individual motility scores between 30 and 60%. The SFT lower threshold for individual motility has been 30% since the first iteration of its BBSE system in the 1970s. This figure recognizes the need to confirm there are motile spermatozoa, but also recognizes that assessments are made in varied field conditions that can adversely affect motility.
Laboratory Assessment of Semen
Semen Morphology
The standards from all regions recommend collection of semen and the assessment of sperm morphology when performing a BBSE. The South African, BCVA, and SFT standards explicitly state that bulls must exceed the morphology threshold of 70% normal spermatozoa to pass the examination and receive the respective certification. The ACV risk‐assessment model explicitly provides for the option of no morphological evaluation but mandates the compulsory reporting on their certificates of whether morphology is included in the assessment. In this latter standard, options are provided for separating the semen evaluation into “bull‐side” assessment (see section “General Concepts”) and morphological assessment. If morphological assessment is performed in the ACV system, a count of 70% normal is required to meet the category requirements, but a “Qualified” assessment can be assigned to this category for bulls with normal sperm percentages between 50 and 69%, which is justified based on industry‐specific research [25].
All regions other than the ACV recommended morphology be assessed with either a nigrosin‐eosin stained slide or with a preserved, fixed sample under phase‐contrast or differential interference contrast (DIC) microscopy. The ACV does not provide the option of nigrosin‐eosin stained slides for morphology, instead recommending that bull‐side samples be preserved in warmed fixative, such as formal buffered saline, or phosphate‐buffered saline (PBS) glutaraldehyde for subsequent assessment.
There are definitive recommendations for the morphological classification of sperm in the SFT, ACV, and BCVA systems. In these systems sperm abnormalities are recorded as seen. There is no clear recommendation regarding morphology classification in the South African system, but the inference is a focus on assessing the number of normal spermatozoa, with abnormalities individually recorded. The conclusion is that morphology assessment for all regions has moved away from previous classification systems such as primary/secondary or major/minor abnormalities. There is still reference to the compensable/uncompensable classification method in the SFT and ACV manuals, with suggestion it may be suited to assessing sperm morphology in samples that will be inseminated, but not relevant to the BBSE process [10, 23].
One specific morphological defect that is the subject of varied opinion is that of distal cytoplasmic droplets (DDs). The SFT no longer considers DDs as having an adverse effect on fertility and counts them as normal (technically a variation of normal) in the spermiogram. The ACV had previously recommended counting DDs as normal sperm, but currently they are recorded as abnormal sperm. The ACV recommends that if the percentage of DDs is great enough to influence whether a bull meets the morphology standard, then the percentage of DDs, accompanied by an interpretation, should be clearly noted in the comments section for this category so that vendors and purchasers can make an informed decision.
The range of regulatory control over morphology assessment processes and efforts to standardize the procedure varies considerably between regions. The South African and BCVA systems simply state that morphology be performed as part of the evaluation, relying on veterinary undergraduate training and personal continuing professional development (CPD) to maintain integrity of the process and interpretation. The SFT provides a detailed manual on sperm morphology assessment and details current knowledge of the influence of morphology on bull fertility to assist practitioners with their interpretations. The ACV system also provides a manual with detailed morphological assessment and interpretative information. But uniquely, while any veterinarian may perform their own morphology assessments, the ACV strongly recommends samples be sent to an ACV endorsed andrology/morphology laboratory to ensure all bulls are assessed by a standard procedure under high‐quality optics. The inference from the ACV recommendation is that current Australian undergraduate veterinary training and the quality of microscope optics in the clinical setting may not adequately equip veterinarians for performing repeatable and reproducible morphological evaluations that will stand up to commercial scrutiny. While this position is not necessarily relatable to other regions, it does raise the question of whether the assessment, recording, and reporting of sperm morphology should be subjected to quality assurance practices that are followed for many other laboratory processes. The commercial implication of aberrations in morphology assessment or interpretation is that the sale price of any bull failing to meet minimum standards for sperm morphology will be reduced or the real‐world fertility may not reconcile with the anticipated fertility. It therefore seems reasonable for a producer to expect that standards are being applied in an equitable way regardless of the personnel performing the evaluation.
The Use of Vital Stains
In all systems except that of the ACV, the use of nigrosin‐eosin staining is one of the recommendations for preparing samples for morphological examination. None of the regions recommends incorporating the Live/Dead aspect of vital staining into the BBSE assessment. The ACV does not recommend the use of vital stains for morphology assessment due to the recommendation of utilizing an endorsed morphologist for the assessment process. In this scenario, the morphologists unanimously prefer samples in fixative such as formal buffered saline (FBS) solution as this enhances the detection of defects under phase‐contrast or DIC microscopy. There is also concern that the osmolality of the vital stain may vary widely if not stored carefully, adversely affecting sperm morphology.
White Blood Cells in Semen
The ACV and SFT manuals both describe the detrimental effects of white blood cells (WBCs) in the semen. The ACV suggests that if more than several WBCs are identified per high‐power field or if purulent material is observed grossly in the ejaculate, the veterinarian should use clinical judgment regarding treatment and prognosis, with a Q or X applied to the category.
Discussion
While exploring the evolution of the BBSE throughout the world, it is apparent from all regions that variation in the early processes of assessment, interpretation, and reporting of results led to uncertainty in the outcomes of the testing. There was a perception from seed‐stock producers and their veterinarians that bull sale opportunities may not be equitable if some bulls were placed under more or varied scrutiny compared to contemporaries. In most regions this variation was arguably the biggest driver toward a standardized BBSE procedure. Regardless of other reasons for performing a BBSE, the requirement for a process that provides equitable comparison of all sale bulls is critical to the integrity of the BBSE. The understanding is that the primary driver to standardizing the approach to BBSE may not necessarily be the science behind the ideal of improving bull fertility, but more the necessity to make the collection and interpretation of data on which bulls are compared when presented for sale as equitable as possible. If this goal is not achieved, then the credibility of BBSE will be reduced to the point of it no longer being considered by producer bodies to be relevant.
When reviewing BBSE standards from different regions, most provide clear guidelines on what aspects are compulsory and what aspects are suggested recommendations. Ideally these compulsory and suggested processes can be tailored to different bull management scenarios. The South African and BCVA systems have addressed this requirement by stipulating “Use Classes” (Figure 7.5). On similar lines, the ACV system works on a risk assessment format where non‐compulsory aspects of the testing can be included or removed depending on the management