Effects of cryopreservation of bovine gametes on fertilizing potential and in vitro embryo development

Dissertation (PhD) -- University of Stellenbosch, 2007.

ENGLISH ABSTRACT: Cryopreservation of gametes has significant importance in the advancement of assisted reproductive technologies (ART) in the management of livestock and laboratory animal species, conservation of biodiversity, and treatment of human infertility. Cryopreservation also reduces the cost, genetic drift and diseases associated with maintaining live animals and cell lines. The increasing use of mammalian gametes obtained from the testis, and excurrent ducts, and the ovaries in ART has, therefore, been enhanced by the cryopreservation. There is need to maximize survival of cryopreserved gametes and the ability of cryopreserved gametes to produce embryos. Gametes from bovine ovaries and testes obtained from abattoirs, and culled buffalo testes were used to examine the effects of cryopreservation of gametes on embryo development, and the ability of frozen-thawed African buffalo ( Syncerus caffer caffer) epididymal sperm in in vitro production of cattle x Buffalo hybrid embryos. Effects of a commercial tris-egg yolk-based extender, Biladyl® (BIL) and modified Tyrode's lactate (MTL) on sperm fertilizing potential were compared by evaluating sperm motility, viability, and membrane and acrosome integrity. Tyrode's lactate medium was supplemented with 20 % foetal bovine serum (FBS) and 0.95 M glycerol (GL Y), ethylene glycol (EG) or dimethyl sulfoxide (DMSO). Pre-freezing effects of the extenders and cryoprotectants were minimal in all treatments. Post-thaw parameters were lower (P < 0.05) than the prefreezing parameters and the highest difference (P < 0.0001) was observed after 0 h of equilibration. Post-thaw motility and viability of treatments equilibrated for 2 h and 4 h in MTL, and frozen with GL Y or EG were not different from the control (P > 0.05). Significant differences in post-thaw membrane and acrosomal status occurred between the control and treatments equilibrated for 2 h and 4 h. Using MTL with GL Y or EG, and 2 h or 4 h of equilibration produced results comparable to the control. However, freezing bull epididymal spermatozoa without equilibration is not recommended, and DMSO should only be used in MTL in the absence of GL Y and EG. Bovine epididymal spermatozoa cryopreserved in BIL and MTL after equilibration for 2 h and 4 h was used to inseminate in vitro matured bovine oocytes. In vitro embryo development was assessed to compare the efficacy of BIL and MTL in cryopreserving the fertilizing potential of bovine epididymal spermatozoa. Cleavage rates varied between the treatments, and were lower (P < 0.05) for the treatments than that of the control (BIL). As embryo development progressed, differences between treatments decreased except for sperm cryopreserved in DMSO that maintained a lower (P < 0.0001) development rate than other treatments and the control. Embryo development did not differ (P > 0.05) when sperm equilibrated for 2 h or 4 h was cryopreserved in BIL. However, embryo development was comparable when sperm equilibrated for 2 h or 4 h in MTL was cryopreserved with GL Y or EG. There was no difference in blastomere numbers of embryos of all treatments equilibrated for 4 h. Spermatozoa cryopreserved in MTL containing 0.95 M GL Y or EG, but not DMSO, produced embryos at rates comparable to BIL. Therefore, MTL supplemented with 20% FBS and 0.95 molar concentration of GL Y or EG can be used as a substitute for commercial extender such as BIL for freezing bovine epididymal sperm, and possibly also of other species, for use in ARTs. Frozen-thawed bovine cauda epididymal sperm were subjected to a second freeze-thaw cycle in BIL and MTL, and used for in vitro fertilization (IVF) of in vitro matured bovine oocytes. Cleavage, morula and blastocyst rates were lower (P < 0.05) for oocytes inseminated with sperm that underwent one freeze-thaw cycle in MTL and two freeze-thaw cycles in BIL and MTL (treatments), than the control (one freeze-thaw cycle in BIL). Embryo expansion and/or hatching on days 7-15, were not different (P > 0.05) among the treatments, but embryo expansion and/or hatching on the same days were lower (P < 0.05) when oocytes were inseminated with sperm refrozen in BIL. The blastomere numbers of embryos from sperm of freeze-thaw cycle one in BIL (119 ± 52.8) and MTL (130 ± 43) were not different (P > 0.05) from each other, but were higher (P < 0.05) than that of embryos from sperm refrozen in BIL (58 ± 29). It was concluded that bovine epididymal sperm can be refrozen in BIL, and MTL containing 20 % FBS, EG and used in ARTs. Hybridization between cattle and its closest African wild relative, the African buffalo (Syncerus caffer caffer) was investigated. In an attempt to produce pre-implantation cattle x buffalo hybrid embryos in vitro, matured bovine oocytes were subjected to standard IVF procedure with either homologous bovine (n = 1166 oocytes) or heterologous buffalo (n = 1202 oocytes) frozen-thawed epididymal sperm. After IVF, 67.2% of the oocytes inseminated with homologous sperm cleaved. In contrast, insemination with buffalo sperm resulted in a 4.6% cleavage rate (P < 0.0001). Cleavage was also slower in hybrids than in cattle embryos. Up to 52.2% of the cleaved homologous embryos progressed to the morula stage compared with 12. 7% for the hybrids (P < 0.0001 ). No hybrid embryos developed beyond the 16-cell stage, while 40.1 % of cleaved bovine embryos developed to the blastocyst stage. Developmental anomalies such as polyspermy, uneven cleavage, vacuolization and absence of nuclei in some blastomeres were common in the hybrid embryos. It was concluded that interspecies fertilization of cattle oocytes with African buffalo epididymal sperm occurs in vitro, and that the barrier to hybridization occurs in the early stages of embryonic development. Chromosomal disparity is likely the cause of the fertilization abnormalities, abnormal development and subsequent arrest impairing formation of pre-implantation hybrid embryos. Investigation into the developmental abnormalities including reciprocal hybridization and genetic studies of the hybrid embryos are recommended. The effects of supplementing oocyte maturation medium with 100 μM cysteamine, and the use of a copper-wire cryoloop for vitrification of the in vitro matured bovine oocytes in the production bovine embryos in vitro was examined. Cysteamine did not improved the cleavage rate (P > 0.05), but improved morula and the blastocyst rates (P < 0.05) in one trial. In a second trial, cysteamine did not improve embryo development in the fresh oocytes and the cleavage rate of vitrified oocytes (P > 0.05), but improved the morula and the blastocyst rates (P < 0.05) of vitrified oocytes. Bovine oocytes can be successfully vitrified using a copper-wire cryoloop. Addition of cysteamine in maturation medium improves embryo development of vitrified-thawed mature oocytes. Cysteamine also improves embryo development in non-vitrified oocytes but this effect appears to be influenced by the pre-culture and culture conditions.

AFRKAANSE OPSOMMING: Kriopreservering van gamete speel 'n belangrike rol in die bevordering van ondersteunende reproduksietegnieke (ORT) tydens die bestuur van plaasdier- en laboratorium species, die bewaring van die biodiversiteit, en die behandeling van menslike infertiliteit. Kriopreservering verminder ook die koste, genetiese verskuiwing en siektetoestande wat met die bewaring van lewendige diere en sellyne geassosieer word. Die gebruik van soogdiergamete vanaf die testis en sy buise, en die ovaria tydens ORT word verder bevorder deur kriopreservering. Dit is dus aangewese om die oorlewing van gekriopreserveerde gamete en hul vermoe om embrio's te vorm, te optimaliseer Gamete vanaf beesovaria en -testis afkomstige vanaf die slagplaas, en uitgedunde buffels se testis is gebruik om die effek van kriopreservering op die ontwikkelingspotensiaal van gamete tot embrio's te ondersoek, sowel as die bevrugtingsvermoe van bevrore-ontdooide Afrika buff el ( Syncerus caffer caffer) epididymale sperme tydens die in vitro produksie van bees met buffel hibried embrio's. Die effek van 'n kommersieel beskikbare tris-eiergeel gebasseerde verdunner, Biladyl® (BIL) teenoor gemodifiseerde Tyrode's laktaat (MTL) op sperme se bevrugtingsvermoee is vergelyk deur spermbeweging, -oorlewing, en spermmembraan- en -akrosoomintegriteit te evalueer. Tyrode's laktaat medium is verryk met 20% fetale bees serum (FBS) en 0.95 molaar gliserol (GL Y}, etileen glikol (EG) of demetielsulfoksied (DMSO). Die effek van die verdunners en kriopreserveermiddels in al die behandelings was minimaal. Na-ontdooiingswaardes was laer (P < 0.05) as die waardes voor kriopreservering. Die grootste verskille is na 0 h van ekwillibrasie waargeneem. Na-ontdooiingsbeweeglikheid en -oorlewing van behandelings met 2 h en 4 h ekwillibrasie in MTL, en gevries met GL Y of EG, was nie verskillend van die kontrole nie (P > 0.05). Wesentlike verskille in membraan- en akrosoomstatus tussen die kontrole monsters en die behandelings ge-ekwillibreer vir 2 h en 4 h, het na ontdooiing voorgekom. Wanneer MTL met GL Y of EG, en 2 h of 4 h ekwillibrasie gebruik is, is resultate gelykstaande aan die kontrolegroep verkry. Kriopreservering van bees epididymale sperme sender ekwillibrasie is egter nie aan te beveel nie. DMSO behoort slegs met MTL in die afwesigheid van GLY en EG gebruik te word. Bees epididymale spermatosoa bevries in BIL en MTL na ekwillibrasie vir 2 h en 4 h is gebruik om in vitro gematureerde bees oosiete te fertiliseer. In vitro embrionale ontwikkeling is daarna beoordeel om die doeltreffendheid van BIL en MTL as kriopreserveermiddels vir die bevrugtingsvermoee van epididymale sperme te bepaal. Die tempo van seldeling het verskil tussen behandelings, en was laer (P < 0.05) vir die behandelings as vir die kontrole (BIL). Soos embrionale ontwikkeling gevorder het, het verskille tussen die behandelingsgroepe afgeneem, behalwe vir sperme bevries in DMSO wat 'n stadiger ontwikkelingstempo (P < 0.0001) as die ander behandelings en kontrole getoon het. Embrionale ontwikkeling het nie verskil (P > 0.05) wanneer sperme wat vir 2 h en 4 h ge-ekwillibreer is, in BIL gekriopreserveer is nie. Wanneer sperme vir 2 h of 4 h in MTL ge-ekwillibreer is, en in GL Y of EG bevries is, is soortgelyke resultate verkry. Daar was geen verskil in blastomeer aantal van alle groepe embrio's by sperme wat vir 4 h ge-ekwillibreer is nie. Sperme bevries in MTL met 0.95 molaar GL Y of EG, maar nie DMSO nie, het embrio's geproduseer soortgelyk aan BIL. Daarom kan aanvaar word dat MTL met 20% FBS en 0.95 molaar GL Y of EG as vervanging kan dien vir kommersiele verdunners soos BIL vir die bevriesing van bees epididymale sperme, en moontlik ook ander species, tydens ondersteunende reproduksie tegnieke. Bevrore-ontdooide kouda epididymale sperme was blootgestel aan 'n tweede bevriesingssiklus in BIL en MTL, en daarna gebruik vir in vitro bevrugting van in vitro gematureerde bees oosiete. Kliewing, sowel as morula en blastosist ontwikkeling was laer (P < 0.05) vir oosiete wat ge-insemineer is met sperme wat eenmalig bevries is in MTL and tweemaal bevries is in BIL en MTL, as in die kontrole groep (eenmalig bevries in BIL). Embriovergroting en -ontkieming vanaf dag 7-15 was nie verskillend (P > 0.05) tussen die behandelingsgroepe nie, maar embriovergroting en -ontkieming op dieselfde dae was laer (P <0.5) wanneer oosiete ge-insemineer was met sperme herbevries in BIL. Die aantal blastomere van embrio's geproduseer met sperme van vriessiklus een in BIL (119 ± 52.8) en MTL (130 ± 43) was nie verskillend (P < 0.05) tussen behandelings nie, maar was hoer (P < 0.05) as in embrio's geproduseer deur sperme herbevries in BIL (58 ± 29). Hieruit kan afgelei word dat bees epididymale sperme hervries kan word in BIL en MTL wat 20% FBS, en EG bevat, en as sulks gebruik kan word in ORT. Hibridisasie tussen beeste en sy naaste wilde Afrika familiegenoot, die Afrika buffel (Syncerus caffer caffer) is ondersoek. In 'n poging om bees x buffel hibried embrio's in vitro te produseer, is gematureerde bees oosiete blootgestel aan 'n standard IVF prosedure met of homoloe bees (n = 1168 oosiete) of heteroloe buffel (n = 1202 oosiete) bevrore-ontdooide epididymale sperme. Na IVF het 67.2% van die oosiete, ge-insemineer met die homoloe sperme, verdeel. In teenstelling daarmee het inseminasie met buffelsperme slegs 4.8% kliewing opgelewer (P < 0.0001 ). Verdeling was ook stadiger in die hibried- as in die beesembrio's. Tot 52.2% van die ontwikkelende homoloe beesembrio's het die morulastadium bereik, in vergelyking met 12. 7% van die hibriedembrio's (P < 0.0001 ). Geen hibriedembrio het verder as die 16-sel stadium ontwikkel nie, terwyl 40.1 % van die suiwer beesembrio's die blastosiststadium bereik het. Ontwikkelingsdefekte soos polispermie, oneweredige verdeling, vakuolisasering en die afwesigheid van kerne in sekere blastomere het algemeen in die hibriedembrio's voorgekom. Daar is tot die gevolgtrekking gekom dat interspecie bevrugting van beesoosiete met Afrika buffel epididymale sperme wel in vitro plaasvind , en dat die buffer teen hibridisasie in die vroee stadium van embrionale ontwikkeling plaasvind. Chromosomale ongelykheid is waarskynlik die oorsaak van bevrugtingsabnormaliteite, abnormale ontwikkeling en daaropvolgende staking in die ontwikkeling van pre-implantasie hibried embrio's. Ondersoeke na ontwikkelingsafwykings wat omgekeerde hibridisasie en die genetiese studie van die hibriedembrio's insluit, word aanbeveel. Die effek van die byvoeging van 100 μM sisteamien by die maturasiemedium, en die gebruik van 'n koperdraad kriolus vir die vitrifikasie van in vitro gematureerde beesoosiete tydens die produksie van beesembrio's, is ondersoek. Sisteamien het nie kliewing verbeter nie (P > 0.05), maar het morula en blastosist getalle in een eksperiment met gevitrifiseerde oosiete verhoog (P < 0.05). Beesoosiete kan suksesvol gevitrifiseer word deur van 'n koperdraad kriolus gebruik te maak. Die byvoeging van sisteamien by die maturasiemedium verbeter embrionale ontwikkeling in gevitrifiseerde-ontdooide gematureerde beesoosiete. Sisteamien verbeter ook embrionale ontwikkeling in nie-gevitrifiseerde beesoosiete, maar hierdie aksie van sisteamien word klaarblyklik be"invloed deur die prekultuur- en kultuurtoestande.

Doctoral