The effect of processing on the short- and long-term viability of epididymal African buffalo (Syncerus caffer) spermatozoa

Thesis (MScAgric)--Stellenbosch University, 2020.ENGLISH ABSTRACT: The Africa buffalo (Syncerus caffer) is one of the Big 5 that features strongly in the ecotourism and trophy hunting industries, and more recently, this species is sought after in game ranching operations. Optimal management of African buffalo in production systems however is hampered by genetic selection for traits without really knowing what the impact on reproduction is, and the diseases African buffalo carry. African buffalo also differ considerably from cattle and even water buffalo when their reproduction is considered. Little information is available on the processing of African buffalo sperm to yield quality samples that can contribute to a genome resource bank of this species, which can then be used for production and conservation purposes (i.e. where entire populations need to be eradicated due to e.g. Foot and Mouth Disease). The study therefore investigated the influence of sperm harvesting method (i.e. processed directly after culling or after 24h of intact storage at 4°C) on the viability of epididymal African buffalo spermatozoa. Spermatozoa aspirated from African buffalo epididymides were evaluated directly after aspiration or subjected to prolonged (i.e. 24h) liquid storage (in Ham’s F10) at 5°C to determine the effect of extended liquid storage on the motility, viability, morphology and acrosome integrity of the spermatozoa. Samples that were subjected to 24h of liquid storage post-aspiration were characterized by significantly poorer viability, midpiece abnormalities and total abnormalities. The prolonged intact cold storage of testes had a negative effect on the occurrence of tail abnormalities. Aspirated samples were subjected to cryopreservation in either Triladyl®, or Triladyl® supplemented with trehalose to determine the potential of trehalose supplementation to minimise the deleterious changes caused by cryopreservation. The addition of trehalose had a positive effect on the motility and viability of sperm samples; however, tail morphology was negatively affected. Cryopreserved sperm samples were thawed using two different thawing rates to determine the optimum thawing method to yield samples viable for use in in vitro fertilisation procedures or artificial insemination. The thawing rates included a slow thawing rate (37°C for 35 seconds), and a fast thawing rate (80°C for 5 seconds). A fast thawing rate is not recommended due to a significant decrease in the sperm viability. Lastly, flow cytometry was used to determine the potential of this objective analysis method to determine the post-thaw viability of aspirated epididymal African buffalo spermatozoa. Future recommendations include investigation of the influence of prolonged intact storage of testes post-mortem (i.e. up to 72 hours) and at different storage temperatures on epididymal African buffalo sperm viability. Different trehalose supplementation levels and the potential thereof to minimise the negative effect of processing and cryopreservation on spermatozoa warrant further studies. The range of parameters analysed using flow cytometry should be extended to include parameters such as morphology and acrosome integrity. The influence of extended boma holding stress, as experienced during routine TB monitoring periods, and the influence thereof on sperm viability, and in particular in the presence of elevated lactic acid levels warrants investigation.AFRIKAANSE OPSOMMING: Die Afrika-buffel (Syncerus caffer) is een van die Groot 5 wat ʼn gesogte spesie in die ekotoerisme- en trofeejagbedryf is en wat meer onlangs as gesog in wildboerderybedrywighede beskou word. Die optimale bestuur van Afrika-buffels in produksiestelsels word egter belemmer deur die genetiese seleksie vir eienskappe sonder om regtig te weet wat die impak op voortplanting is, en die siektes wat Afrika-buffels dra. Afrika-buffels verskil ook aansienlik van beeste en selfs waterbuffels as hulle voortplanting oorweeg word. Daar is min inligting beskikbaar oor die verwerking van Afrika-buffelsperm om kwaliteitmonsters te lewer wat kan bydra tot 'n genoomhulpbronbank van hierdie spesie, wat dan vir produksie- en bewaringsdoeleindes gebruik kan word (d.w.s. waar die hele bevolking uitgeroei moet word as gevolg van Bek-en-klouseer). Die studie het gevolglik die invloed van die oesmetode (d.w.s. direk verwerk na uitdunnning of na 24 uur van intakte berging by 4°C) op die lewensvatbaarheid van die epididimale Afrika-buffelsperme ondersoek. Sperme wat deur middel van aspirasie uit Afrika-buffel epididimii versamel is, is direk na aspirasie geëvalueer of aan langdurige (d.w.s. 24 uur) berging in Ham's F10 by 5°C beoordeel om die effek van langdurige berging op die beweeglikheid, lewensvatbaarheid, morfologie en akrosoom integriteit van die sperme te bepaal. Monsters wat aan 24 uur van berging na aspirasie onderworpe was, is gekenmerk deur aansienlik swakker lewensvatbaarheid, middelstuk abnormaliteite en totale abnormaliteite. Die langdurige intakte berging van testes het 'n negatiewe uitwerking op die voorkoms van stert abnormaliteite gehad. Aspireerde monsters is in óf Triladyl®, óf Triladyl® aangevul met trehalose gevries om die potensiaal van trehalose-aanvulling te bepaal om die nadelige veranderinge wat veroorsaak word deur diepbevriesing, te minimaliseer. Die toevoeging van trehalose het 'n positiewe invloed op die beweeglikheid en lewensvatbaarheid van spermmonsters gehad; die stertmorfologie is egter negatief beïnvloed. Die bevrore spermmonsters is teen twee verskillende ontdooiingtempo’s ontdooi om die optimale ontdooiingsmetode te bepaal om monsters lewensvatbaar te maak vir gebruik in in vitro bevrugtingsprosedures of kunsmatige inseminasie. Die ontdooiingstempo’s het 'n stadige ontdooiingstempo (37°C vir 35 sekondes) en 'n vinnige ontdooiingstempo (80°C vir 5 sekondes) ingesluit. 'n Vinnige ontdooiingstempo word nie aanbeveel nie as gevolg van 'n beduidende afname in die lewensvatbaarheid van die sperme. Laastens is die potensiaal van vloeisitometrie om as metode gebruik te word om die lewensvatbaarheid van die bevrore-ontdooide epididimale Afrika buffelsperme te bepaal, ondersoek. Toekomstige aanbevelings sluit in die ondersoek na die invloed van langdurige intakte berging van testes na uitdunning (dit wil sê tot 72 uur) en by verskillende bergingstemperature op die lewensvatbaarheid van die epididimale Afrika buffelsperme. Verskillende trehalose aanvullingsvlakke en die potensiaal daarvan om die negatiewe effek van prosessering en diepbevriesing op sperme tot die minimum te beperk, is 'n verdere ondersoek. Die reeks parameters wat met behulp van vloeisitometrie ontleed word, moet uitgebrei word om parameters soos morfologie en akrosoom integriteit in te sluit. Die invloed van langdurige aanhou in bomas, soos ondervind tydens roetine TB-moniteringstydperke en die invloed daarvan op die lewensvatbaarheid van sperme, veral in die teenwoordigheid van 'n verhoogde melksuurvlak, moet ook ondersoek word.Master