1 CONTACT Peter Chenoweth pchenoweth@hotmail.com © 2024 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Citation: Clinical Theriogenology 2024, 16, 10600, http://dx.doi.org/10.58292/CT.v16.10600 Review Report Andrology laboratory review: evaluation of sperm morphology Peter Chenoweth,a Leo Brito,b Augustine Peter,c Dagmar Waberski,d Gary Althouse,b Christine Aurich,e Gaia Luvoni,f Regina Turner,b Natalie Fraser,g Cheryl Lopateh aSchool of Veterinary Sciences, College of Public Health Medical & Veterinary Sciences, James Cook University, Townsville, Queensland, Australia bDepartment of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA, USA cDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA dUnit for Reproductive Medicine of Clinics/Clinic for Pigs and Small Ruminants, University of Veterinary Medicine, Hannover, Germany eCentre for Artificial Insemination and Embryo Transfer, University of Veterinary Sciences, Vienna, Austria fDepartment of Veterinary Medicine and Animal Science, Università degli Studi di Milano, Milan, Italy gSchool of Veterinary Medicine, University of Queensland, Gatton, Australia hReproductive Revolutions, Case Road NE, Aurora, OR, USA Abstract Sperm morphology assessment has an important role in male fertility diagnosis and prognosis, both for humans and animals. Thus, it is important that relevant results are comparable and consistent. To achieve these aims, the following procedures are recommended: a. semen sample is suitably ‘fixed’ (e.g. in isotonic buffered formal-saline); b. sperm are examined at 1,000 x (phase or DIC microscopy); c.at least 200 sperm are counted; d. each sperm is placed into 1 category, only (e.g. normal, head, midpiece etc), and e. 70% ‘normal’ sperm is the threshold for a satisfactory sample. In addition, morphologists should be provided with relevant continuing education, upskilling, and monitoring programs. This review provides guidelines for the best performance of this assessment, as well as for avoiding pitfalls. Keywords: Sperm, morphology, fertility, animal Introduction A taskforce, representing the Association of Applied Animal  Andrology, American College of Theriogenologists, European College of Animal Reproduction, and Society for Theriogenology, was given the task of providing recommenda- tions on best methods of evaluating domestic animal semen quality. This is the third publication in a series, being preceded by reviews on sperm concentration1 and motility.2 Initial evalu- ation of semen is generally performed macroscopically and usually includes volume, color, and consistency. Semen quality is a term that usually includes sperm motility, viability, mor- phology, concentration, and seminal fluid composition,2 some of which were discussed.1,2 Of these attributes, sperm morphol- ogy is widely recognized as the semen characteristic most directly associated with fertility,3 despite the latter often being ill defined.4 Indeed, “the assessment of sperm morphology is probably the most useful and important aspect of the semen examination.”5 The microscopic assessment of sperm morphology is based on the premise that sperm shape is linked with sperm function,6 which is reinforced by evidence that abnormal sperm head shape is due to damaged DNA/chroma- tin.7 and thus represents a ‘useful tool’ for assessing potential male fertility.8 The procedure itself is simple to perform, and the results are considered to reflect sperm fertility, at least to a degree, and particularly where a large proportion of sperm are abnormal.9 Despite such considerations, wider acceptance of this procedure is constrained by variations in results, leading to a lack of confidence in their accuracy and relevance and reduced ability to directly compare data. For accuracy to be consistently achieved, sample handling, fixation and/or staining must all be optimal10 and standardized, as sperm morphology can be influ- enced by semen handling and observer variations,11,12 thus chal- lenging consistency and objectivity. Thus, this review aims to provide current guidelines for this procedure in order to help achieve greater consistency in its application, leading to improved acceptance of this important component of male fer- tility assessment. mailto:pchenoweth@hotmail.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/CT.v16.10600 2 Citation: Clinical Theriogenology 2024, 16, 10600, http://dx.doi.org/10.58292/CT.v16.10600 Equipment To record sperm morphological defects, they must first be observed and recognized. The first consideration requires appropriate magnification. A variety of methods are available, with the decision to use a particular procedure often being influenced by its relative complexity, cost effectiveness, expe- dience and the degree of fine detail required. Equipment list for a basic andrology laboratory is provided.13 Here, it is incontestable that optimal results depend upon using the best available equipment available in concert with appropriate semen handling and sample preparation. In turn, recognition of particular sperm defects, and an understanding of their rel- ative significance, benefits from a good understanding of cur- rent knowledge and developments in physiology and cell biology. Equipment used for the microscopic examination of sperm includes brightfield microscopy, ordinary phase-contrast microscopy, differential interference contrast phase micros- copy (DIC), computer-assisted sperm analysis (CASA) config- ured for morphology, and electron microscopy. Brightfield microscopy has traditionally been used for routine andrologi- cal work as it has the advantages of relatively low cost and ease of use. It is, however, best suited for use with fixed, stained specimens, whereas phase microscopy and DIC are usually pre- ferred for unstained specimens.14 An earlier review15 provided commonly used semen stains and a recommended equipment list for a basic andrology laboratory was suggested.13 Applications using CASA systems for sperm morphology assessment (i.e. automated sperm morphology analyses) are rapidly gaining traction.11,16,17 These have advantages of high- speed, evaluation of large numbers of sperm, providing con- sistent, easily quantifiable results and thus reducing the significant variation that can occur between technicians and laboratories. However, the current lack of standardization methods makes comparisons difficult.18 In addition, CASA employs negative phase-contrast microscopy that is less than ideal for the recognition of nuclear diadems and vacuoles. Electron microscopy, although complex and expensive, is a most useful tool for depicting sperm ultrastructure, although this technique does not lend itself to quantitative applica- tions.19,20 Both transmission and scanning electron micros- copy are now widely used in andrology.6 The generally recommended magnification of 1,000 x for the microscopical assessment of sperm morphology can be achieved using bright-field microscopy with an oil immersion objective and stained slides (e.g. using an eosin-nigrosin stain). However, it is considered preferable to use either ordinary phase or DIC microscopy in conjunction with a ‘fixed’ (i.e. ‘wet’) semen sample. Buffered formal saline,21 is widely used as a fixative for this purpose. In routine clinical applications, it is often expedient to ‘count’ 100 sperm per sample, although this number is at the lower end of recommended estimates of sam- ple size, especially when confidence limits are taken into account.12,22,23 For example, World health organization (WHO) recommends counting at least 200 sperm for human semen assessment.24 However, counting more sperm did not lead to a change in bull semen morphology classification, even when as many as 400 sperm were counted.12 Thus, this review supports the conventional approach of characterizing 100 to 200 sperm for routine bull sperm morphology assessment. Sperm morphology categorization Following systems categorize animal sperm morphology:25 1. Origin of the defect (e.g. primary and secondary abnormalities26) 2. Potential impact on fertility (e.g. major and minor abnormalities27) 3. Localization of defect on sperm (e.g. head, midpiece, and tail defects28,29) 4. Compensable and uncompensable sperm defects30 5. Systematic sperm defects31,32 6. Genetic sperm defects33 The most widely used system, at least in animal andrology laboratories, is considered to be number 3, above,24,28,29 which is also the simplest and least ambiguous system of those above. Interpreting and reporting sperm morphology Sperm morphology reports should include such details as: a. the criteria used to categorize different defects;25 b. the materi- als and methods used to prepare the samples for examination (e.g. fresh or frozen-thawed semen, fixatives, dilutions, and staining); c. relevant microscopic and/or imaging details; and d. the reference values used for final summation and conclusions.34 Standardization of sperm morphology assessment It would be very useful to achieve consensus on sperm mor- phology techniques and interpretations to facilitate research and to reduce misunderstandings and differences that can result in economic loss, conflict, and personal distress. Adoption of standardized procedures for semen analysis, including sperm morphology, would allow objective compari- son of results, in turn improving confidence in the process.35 Despite this, various attempts to standardize sperm morphol- ogy have not been widely adopted. This is probably due to dif- ficulties in harmonizing differences in semen preparation and staining, microscope systems and optics and differences among technicians in their training, competence, and interpretation of results. This was illustrated that morphology evaluations of stallion sperm varied with both technician and methodology.36 Here, wet-mount preparations examined by phase-microscopy produced better results than stained smears examined with bright-field microscopy. Two techniques (‘wet’ preparation using DIC phase-contrast microscopy and eosin nigrosin stained smears) were compared using microscopy and it was concluded that, although the results had some qualitative dif- ferences, the final breeding soundness examination classifica- tion of bulls did not differ.37 It is a reassuring fact that veterinary practitioners were 92% in accordance when categorizing bull semen morphology38 and there was little difference in the types of sperm defects observed in tropical Bos indicus bulls compared to temperate Bos indicus bulls.39 Despite this, there remains a relative lack of confidence in sperm morphology results from both animal and human andrology laboratories.18,40,41 Sperm abnormality thresholds An early observation was that there was a ‘threshold’ of observable sperm morphological abnormalities above which http://dx.doi.org/10.58292/CT.v16.10600 Citation: Clinical Theriogenology 2024, 16, 10600, http://dx.doi.org/10.58292/CT.v16.10600 3 fertility became compromised. This threshold, ~ 30%, has remained remarkably consistent. In bulls, this was illustrated in natural mating trials in Texas,42 where bulls preselected for good sperm morphology (> 70% normal sperm) achieved significantly more pregnancies than those that were unse- lected for sperm morphology. Fertility is compromised by morphological anomalies by themselves or due to the cor- relation of sperm morphology to other variables (e.g. DNA integrity in boars43). In felids, a threshold should be set lower than in other species, because domestic and wild are gener- ally affected by teratozoospermia (< 40% morphologically normal sperm).44 Since then, similar conclusions have been reached from trials in areas as diverse as in vitro fertilization and intrauterine insemination and studies on sperm DNA damage. The pathogenesis of this relationship has not yet been well elucidated. In pigs, most breeding organizations have defined thresholds between 15 and 30% for abnormal sperm, with or without specification of thresholds for sperm with cytoplasmic droplets (15-30%).45 It is important to note that such thresholds only apply when the observed spermio- gram is representative of normal, generalized spermatogenic stress. In some situations, such as immaturity or gossypol tox- icity, the particular abnormalities encountered may be more indicative. Stains and preparations Various stains and preparations have been used in evaluating human and animal sperm microscopically and a representa- tive list is provided (Table 1 in Appendix). There are useful references.15,46 Supra-vital stains (e.g. eosin nigrosin) are commonly used for semen staining in the field as they are simple to use, and they can depict sperm morphology rea- sonably well in addition to providing an insight into sperm vitality.47,48 However, differential-interference phase contrast microscopy of ‘fixed’ (i.e. unstained) samples at 1,000 x is regarded as the ‘gold standard’ for depicting certain types of sperm abnormalities, particularly acrosomal,49,50 as well more subtle sperm head or midpiece defects. However, either stained or unstained methods produced similar results in terms of bull classification.51 Here, it is considered that some approaches recommended for human semen assessment, such as using the Papanicolaou stain and strict criteria for morphology categorization,3 are not easily adopted for ani- mal semen assessment, due to problems of logistics and complexity. Despite this, morphological indices for canine sperm have been developed52 based upon those described in the WHO laboratory manual for the examination and pro- cessing of human semen.24 It is important to note that such thresholds only apply when the observed spermiogram is representative of normal, generalized spermatogenic stress. In some situations, such as immaturity or gossypol toxicity, the particular abnormalities encountered may be more indicative. Semen handling and preparation Sperm morphological defects may occur both pre- and poste- jaculation,53 with the latter including collection, handling and cryopreservation procedures,54 as well as the staining method employed.10 Semen collection methods as well as collection frequency can influence sperm morphology, and this can vary with species.55 In addition, sperm morphology can be influ- enced by environmental factors such as pH,56 bacteria, and inflammatory products,57–59 and age of the donor; sperm mor- phology declined in stallions after 11-14 years of age60 and in dogs after 7 years.61,62 If a representative semen sample has been obtained, then care should be taken to avoid subsequent sperm damage by pro- tecting sperm viability and/or integrity during handling and processing. Factors that can affect results include sample prepa- ration, species, extender or medium, objective magnification and ‘quality’ as well as operator knowledge and experience.12 Defects that can be attributed to poor semen handling and those linked with poor preparation of semen smears are listed (Tables 2 and 3, respectively, in Appendix). A checklist of fac- tors that could adversely affect sperm morphology, as well as proposed solutions, is also available in the WHO laboratory manual for the examination and processing of human semen.24 Other cells in semen Semen consists of a fluid medium in which sperm are sus- pended. However, other cells and organisms can be present in semen, with many of these able to be observed microscopically. Occasional sightings of other cells do not necessarily indicate that a problem exists, although some are more clinically rele- vant than others.63 For example, increased numbers of leuko- cytes in semen can indicate infection, whilst being capable of directly causing sperm oxidative damage.64 Round spermatids, spermatocytes, and spermatogonia may indicate stress or dam- age to the spermatogenic epithelium. Bacteria are commonly observed in both fresh and stained semen,13,58 and are, in them- selves, capable of causing alterations to sperm DNA and mor- phology.59,65 Slide preparation, proper staining and appropriate microscopy are all important considerations for the recognition of nonsperm cells in semen.24 The recognition of major non- sperm inclusions (i.e. epithelial cells, macrophages, red blood cells, white blood cells, spermatogenic precursors, ‘round cells,’ epididymal cells, bacteria, contaminants, and debris), as well as appreciating their relative significance, should be an essential part of the sperm morphologist’s skillset. Conclusion Competent and accurate assessment of sperm morphology is an important component of male fertility diagnosis and prognosis. For sperm morphology assessment to be as useful as possible, it is important that results are comparable and consistent among and within veterinarians, technicians, and morphologists. This requires the combination of optimal technique with good equipment and its application within the context of relevant animal history, supported by appro- priate knowledge of male physiology and pathology. To encourage greater consistency, the following procedures are suggested as a basis for sample comparisons, within and among species: a. semen sample in a suitable fixative; b. use of phase/DIC microscopy at 1,000 x; and c. count of 200 sperm, and d; a 30% threshold for ‘abnormal’ sperm. It is also important that animal sperm morphologists have ongo- ing access to relevant continuing education, upskilling, and monitoring programs. Conflict of interest None to report. References 1. Brito LFC, Althouse GA, Aurich C, et al: Andrology laboratory review: evaluation of sperm concentration. 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Pozor MA, Zambrano GL, Runcin E, et al: Usefulness of dip quick stain in evaluating sperm morphology in stallions. Proc Am Assoc Equine Pract 2012;58:506-510. 74. Barth AD, Oko RJ: Abnormal Morphology of Bovine Spermatozoa. 1st edition, Ames, IA; Iowa State University Press: 1989. p. 302. 75. Erenpreiss J, Jepson K, Giwercman A, et al: Toluidine blue cytom- etry test for sperm DNA conformation: comparison with the flow cytometric sperm chromatin structure and TUNEL assays. Hum Reprod 2004;19:2277-2282. doi: 10.1093/humrep/deh417 76. Tsarev I, Bungum M, Giwercman A, et al: Evaluation of male fer- tility potential by Toludine Blue test for sperm chromatin struc- tural assessment. Hum Reprod 2009;24:1569-1574. doi: 10.1093/ humrep/dep068 77. Kovacs A, Foote RH: Viability and acrosome staining of bull, boar and rabbit spermatozoa. Biotech Histochem 1992;67:119- 124. doi: 10.3109/10520299209110020 78. Casarett GW: A one-solution stain for spermatozoa. Stain  Technol  1953;28:125-127. doi: 10.3109/10520295 309105113 http://dx.doi.org/10.58292/CT.v16.10600 https://doi.org/10.1093/humrep/deh417 https://doi.org/10.1093/humrep/dep068 https://doi.org/10.1093/humrep/dep068 https://doi.org/10.3109/10520299209110020 https://doi.org/10.3109/10520295 309105113 https://doi.org/10.3109/10520295 309105113 Citation: Clinical Theriogenology 2024, 16, 10600, http://dx.doi.org/10.58292/CT.v16.10600 7 Table 1. Stains and preparations used for assessing sperm morphology in animals Stain Details and source Uses, advantages, and disadvantages Eosin nigrosin Sources Lane Manufacturing, USA Minitube International www.minitube.com References15,66 A one-step differential membrane- dependent ‘supra-vital’ stain, easy to keep and simple to use. Employed for ‘live/ dead’ estimation as well as sperm morphology, although finer sperm structures may not be obvious. Recommended by the Society for Theriogenology. Modified Giemsa Sources www.sigmaaldrich.com www.fischersci.com References15,67 Commonly used in hematology for cellular depiction. Also useful for sperm morphology, particularly for acrosome definition. Williams stain Reagents from www.sigmaaldrich.com. www.fischersci.com References68,69 Double-stain method (carbol-fuchsin eosin counterstained with methylene blue). Good sperm morphology stain, despite requiring several steps. SpermBlue® SpermBlue® prestained slides Sources Microptic SL, Barcelona, Spain.   www.micropticsl.com Fertility Technology Resources. www.fertilitystuff.com YouNing Biotech Co. Ltd. www.youning.com Reference10 A one-step stain for human or animal sperm that can be used on fresh, frozen and extended semen, as well as for automated sperm morphology analyses. Spermac® Sources Spermac laboratories www.spermac.com FertiPro NV www.fertipro.com, Minitube International www.minitube.com References70,71 Versatile, rapid, dual stain allowing separate visualization of the nucleus and cytoplasm, as well as good acrosome definition. Diff-Quik, Dip Quick® Sources Microptic SL, Barcelona, Spain. www.micropticsl.com Proiser R&D SL info@proiser.com References72,73 A Romanowsky stain used widely in clinical cytology. Commonly available in clinical settings. Can be used for combined assessment of morphology (recommended by WHO), seminal cytology and sperm DNA/chromatin stability. Aniline blue Reagents from www.sigmaaldrich.com www.fischersci.com Reference74 Histology stain. Also differentiates histones and protamines, and is a simple procedure. (Continued) Appendix http://dx.doi.org/10.58292/CT.v16.10600 http://www.minitube.com http://www.sigmaaldrich.com http://www.fischersci.com http://www.sigmaaldrich.com http://www.fischersci.com http://www.micropticsl.com http://www.fertilitystuff.com http://www.youning.com http://www.spermac.com http://www.fertipro.com http://www.minitube.com http://www.micropticsl.com mailto:info@proiser.com http://www.sigmaaldrich.com http://www.fischersci.com 8 Citation: Clinical Theriogenology 2024, 16, 10600, http://dx.doi.org/10.58292/CT.v16.10600 Table 1. (Continued) Stain Details and source Uses, advantages, and disadvantages Toluidine blue Reagents from www.sigmaaldrich.com www.fischersci.com References75,76 Useful to detect sperm chromatin abnormalities, as well as to depict morphology. Trypan blue Reagents from www.sigmaaldrich.com www.fischersci.com Membrane dependent ‘vital’ stain, used for ‘live-dead’ estimation as well as sperm morphology. Can be used with ‘fixed’ semen. Also useful for hematology and cell cultures; one-step procedure. Farrelly stain Source Minitube International www.minitube.com A 2-step contrast stain, useful for sperm morphology in samples which do not contain glycerol. Kovacs stain Reagents (trypan blue, congo-red and Giemsa) from www.sigmaaldrich.com Reference77 Combined sperm viability and acrosome stain. Casaretts stain Reagents (aniline blue, eosin B, Phenol) from www.sigmaaldrich.com. Reference78 One step stain which is useful for depicting different structures in human and dog sperm. Cell-Vu® Morphology slides Source TekEvent Pty Ltd info@tekevent.com Prestained slides, simple procedure. Can be used with undiluted semen. It provides good depiction of head, acrosome and tail. Modified Papanicolaou stain Reagents www.sigmaaldrich.com www.fischersci.com Reference13 A versatile cytology stain with a modified version used for sperm morphology, particularly human, and which is also useful for seminal cytology, including round cells in semen. Commonly available in clinics. The procedure is relatively complex and time consuming. Sperm stain Ready to use Source Microptic SL, Barcelona, Spain. www.micropticsl.com A rapid, 2-step Romanowsky stain used in human andrology. It is also useful for differential blood cell staining. Table 2. Sperm morphology problems associated with semen handling Causes Outcomes Nonphysiologic temperatures Contamination Rough handling Inappropriate extender Nonisotonic media Reduced percent intact acrosomes (PIA) Increased numbers of bacteria, sperm clumping Detached sperm heads Reduced PIA, increased crystal formation Reduced PIA, increased ‘bent’ midpieces and tails http://dx.doi.org/10.58292/CT.v16.10600 http://www.sigmaaldrich.com http://www.fischersci.com http://www.sigmaaldrich.com http://www.fischersci.com http://www.minitube.com http://www.sigmaaldrich.com http://www.sigmaaldrich.com mailto:info@tekevent.com http://www.sigmaaldrich.com http://www.fischersci.com http://www.micropticsl.com Citation: Clinical Theriogenology 2024, 16, 10600, http://dx.doi.org/10.58292/CT.v16.10600 9 Table 3. Sperm morphology problems associated with the preparation of semen slides Contributing factor Causes Sperm are disrupted and/or have signs of mechanical damage Rough smearing technique (including mixing using the edge of a glass slide) or the coverslip was disturbed prematurely Sperm are too sparse on the slide Poor mixing of sample Over-dilution of sample Excess stain The dried smear shows the appearance of ‘cracking’ Over-thick smear Slide exposed to excess heat while drying Areas of excessive stain accumulation occur on the slide Aged and/or unmixed stain Poor technique in making the smear Sperm are stained too darkly Stain was too thick or strong Staining time was too long Sperm are stained too lightly Stain was too thin or weak Staining time was too short A clear area resembling a ‘halo’ is observed above sperm heads Sperm movement before stain dried http://dx.doi.org/10.58292/CT.v16.10600