A meta-analysis of the role of subpopulation analysis in the qualification and quantification of sperm sample quality, and the relationship with male fertility

Thesis (MScAgric)--Stellenbosch University, 2021.

ENGLISH ABSTRACT: Given the predicted increase in the world population by 2050, the expected demand for animal products places increasing pressure on livestock farmers to farm as sustainable and cost-efficient as possible, taking environmental challenges such as global warming into account. The identification and selection of animals that have a superior ability to cope with and produce under conditions that are challenging, will assist livestock producers to contribute directly to food security. The development and application of assisted reproductive technologies (ART’s) such as artificial insemination (AI) and in vitro embryo production (IVEP) and transfer, offers livestock producers the opportunity to overcome limitations such as the seasonal nature of reproduction in certain species, and long generation intervals. The successful application of ART’s, however, requires the identification of quality spermatozoa that will be able to participate in fertilization. One of the limitations of current sperm sample evaluation protocols, is the inherent subjective nature of the evaluation protocols, which is largely due to the use of trained technicians, which in turn dictates that a certain level of experience is also required. This lead to the development of computer-assisted sperm analysis (CASA) systems, with the aim to overcome the limitation of the subjective nature of sperm sample evaluation protocols, and thus biased values obtained. Sperm samples, whether ejaculate or epididymal in origin, naturally are characterized by a degree of heterogeneity, which contributed to the development of a sperm subpopulation approach to quantify sperm sample quality, and the fertilizing ability of spermatozoa in said samples. Knowledge of sperm fertilizing ability is important in determining male fertility under natural mating conditions (field fertility) or for use in ART’s such as IVEP. A meta-analysis was conducted to establish and quantify the use of sperm subpopulations to quantify male fertility by doing an in-depth analysis of existing peer-reviewed publications using specific criteria. All available web-based databases were used for this search, and publications that met all the selection criteria were thoroughly explored. Datasets for CASA-generated motility- and sperm head morphometry parameters were generated, and subjected to statistical analyses using a mode approach, where species, sperm subpopulation, type of CASA system used, and type of medium used, were considered as fixed effects. Findings from this study highlighted the inconsistency between studies, as well as lack of an identified link between sperm subpopulations and male fertility, whether under field conditions or for use of spermatozoa in ART’s. A lack of consensus amongst authors regarding subpopulation classification, especially in terms of the pre-determined cut-off values and thus the descriptive categories that are used to classify and describe sperm clusters, whether it be for sperm motility (i.e. fast, medium, and slow) or sperm head morphometry (i.e. large, short, round, elliptical, etc.), was reported. Given the multi-factorial nature of fertilization, standardization between research group and laboratories in terms of cut-off values for sperm subpopulations is warranted to allow for the establishment of whether a sperm subpopulation approach in quantifying sperm fertilising ability is firstly feasible, and secondly whether CASA can be incorporated in livestock and wildlife management programs as a management tool. It is recommended that future studies include both sperm motility and sperm head morphometry in the study designs, as this will provide a more accurate indication of sperm fertilising potential, and thus a male´s fertility. Additionally, studies should report more detail on sample preparation, processing, and imaging, which will allow improved quantification of results and standardization between laboratories. Furthermore, there is under-representation for various species, and more studies are warranted to allow for the development of standardized species-specific protocols, which in turn can lead to the establishment of reproductive indices for animals, under field conditions and/or for use of their gametes in ART’s, which in turn will assist livestock and wildlife managers to select species with an ability to cope under certain production conditions. Selection of wildlife species that can cope with challenging environmental conditions, will assist in maintaining of ecosystem stability, and also allow for the use of spermatozoa obtained from such animals, to be used in ART’s in the establishment of genome resource banks for said species.

AFRIKAANSE OPSOMMING: Gegewe die voorspelde toename in die wêreldbevolking teen 2050, plaas die verwagte vraag na diereprodukte toenemende druk op veeboere om so volhoubaar en kostedoeltreffend as moontlik te boer, met inagneming van omgewingsuitdagings soos aardverwarming. Die identifisering en seleksie van diere wat die beste vermoë het om onder moeilike omstandighede te produseer, sal veeprodusente help om direk tot voedselsekerheid by te dra. Die ontwikkeling en toepassing van ondersteunende reproduksietegnieke (ORT’s), soos kunsmatige inseminasie (KI) en in vitro embrioproduksie (IVEP) en oordrag, bied veeprodusente die geleentheid om beperkings soos die seisoenale aard van voortplanting by sekere spesies en lang generasie-intervalle te oorkom. Die suksesvolle toepassing van ORT’s vereis die identifisering van spermatozoë van gehalte wat aan vrugbaarheid kan deelneem. Een van die beperkings van die huidige spermmonster-evalueringsprotokolle is die inherente subjektiewe aard van die evalueringsprotokolle, wat grotendeels te wyte is aan die gebruik van opgeleide tegnici, wat weer bepaal dat 'n sekere vlak van ervaring ook nodig is. Dit het gelei na die ontwikkeling van rekenaargesteunde spermanalise (RGSA) stelsels, met die doel om die beperking van die subjektiewe aard van spermmonster-evalueringsprotokolle, en sodoende bevooroordeelde waardes, te oorkom. Spermmonsters, hetsy ejakulaat of epididimaal van oorsprong, word natuurlik gekenmerk deur 'n mate van heterogeniteit, wat bygedra het tot die ontwikkeling van 'n spermsubpopulasie-benadering om die kwaliteit van die spermmonsters te kwantifiseer, en die bevrugtingsvermoë van spermatozoë in genoemde monsters te kwantifiseer. Kennis van spermvrugbaarheidvermoë is belangrik om manlike vrugbaarheid te bepaal onder natuurlike paringstoestande (veldvrugbaarheid) of vir gebruik in ORT’s soos IVEP. 'n Meta-analise is uitgevoer om die gebruik van spermsubpopulasies om die vrugbaarheid van manlike diere te bepaal, vas te stel en te kwantifiseer deur 'n diepgaande analise te doen van bestaande portuurbeoordeelde publikasies volgens spesifieke kriteria. Alle beskikbare webgebaseerde databasisse is vir hierdie soektog gebruik en publikasies wat aan al die seleksiekriteria voldoen, is deeglik ondersoek. Datastelle vir RGSA-gegenereerde motiliteits- en spermkop morfometrie-parameters is gegenereer en onderwerp aan statistiese ontledings met behulp van 'n modusbenadering, waar spesies, subpopulasie van die sperma, tipe RGSA-stelsel en die gebruikte medium, as vaste effekte beskou word. Bevindinge uit hierdie studie het die teenstrydigheid tussen studies beklemtoon, sowel as die gebrek aan 'n geïdentifiseerde verband tussen spermsubpopulasies en manlike vrugbaarheid, hetsy onder veldtoestande of vir die gebruik van spermatozoë in ORT’s. 'n Gebrek aan konsensus tussen outeurs rakende subpopulasie-klassifikasie, veral in terme van die voorafbepaalde afsnywaardes en dus die beskrywende kategorieë wat gebruik word om spermklusters te klassifiseer en te beskryf, hetsy vir spermmotiliteit (d.w.s. vinnig, medium en stadige) of spermkopmorfometrie (d.w.s. groot, kort, rond, ellipties, ens.), is gerapporteer. Gegewe die multifaktoriese aard van vrugbaarheid, is standaardisering tussen navorsingsgroep en laboratoriums in terme van afsnywaardes vir spermsubpopulasies, geregverdig om vas te stel of 'n spermsubpopulasiebenadering in die kwantifisering van die bevrugtingsvermoë van die sperm eerstens haalbaar is, en tweedens of RGSA in vee- en wildbestuur opgeneem kan word programme as 'n bestuursinstrument. Dit word aanbeveel dat toekomstige studies sowel spermmotiliteit as spermkopmorfometrie in die studieontwerpe insluit, aangesien dit 'n akkurater aanduiding van die bevrugtingspotensiaal vir sperms en dus die vrugbaarheid van 'n manlike dier sal gee. Daarbenewens moet studies meer besonderhede oor voorbereiding, verwerking en beelding van monsters rapporteer, wat verbeterde kwantifisering van resultate en standaardisering tussen laboratoriums moontlik maak. Verder is daar onderverteenwoordiging vir verskillende spesies, en meer studies is geregverdig om die ontwikkeling van gestandaardiseerde spesiespesifieke protokolle moontlik te maak, wat weer kan lei tot die daarstelling van reproduksie-indekse vir diere, onder veldtoestande en/of vir die gebruik van hul gamete in ORTs, wat weer vee- en wildbestuurders sal help om spesies te selekteer wat die vermoë het om sekere produksietoestande die hoof te kan bied. Die seleksie van wildspesies wat die uitdagende omgewingstoestande die hoof kan bied, sal help om die stabiliteit van die ekosisteem te handhaaf, en ook die gebruik van spermatozoë wat van sulke diere verkry word, te gebruik in ORT’s vir die instelling van genoombronne vir genoemde spesies.

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