A protocol for liquid storage and cryopreservation of ostrich (Struthio camelus) semen

Smith, Algina Maria Johanna (2016-03)

Thesis (PhD)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: The aim of this study was to develop a species-specific protocol for short (liquid) and long (cryopreserved) term storage of ostrich semen. Effective storage followed by artificial insemination (AI) could assist to overcome the current industry limitations, such as poor egg fertility as well as low embryo and chick survival. Initially, factors influencing sperm storage in other avian species were considered to identify the steps needed to develop an ostrich-specific protocol. Secondly, it was necessary to adapt a set of in vitro functions to quantify and classify sperm of the ostrich, even before storage processing could commence. This approach allowed the evaluation of ejaculates and detected differences in sperm function caused by semen processing. The classification of ostrich sperm characteristics through in vitro sperm functions of sperm motility, viability and membrane integrity allowed for ostrich semen to be assessed for quality. Males differed from one another in terms of motility, progressive motility, sperm swimming speed and the linearity of swimming. Males may be screened using these traits to determine storage suitability and inclusion in an AI programme to optimize fertility. Semen dilution immediately upon collection is a prerequisite for the further assessment of sperm. An ostrich specific diluent (OS1) based on macro mineral composition and concentration of ostrich seminal plasma maintained sperm function during short-term storage for 24 hours at 5 °C without a significant loss in sperm quality. The maintenance of sperm function and specifically swim quality over extended storage periods of 48 hours depended upon a dilution medium (OS2) balanced for macro and micro minerals in ostrich seminal plasma. However the rate at which the semen is diluted as well as the dilution temperature is vital for optimum sperm function. An interpolated rate of 1:6 (semen: diluent) together at a dilution temperature of ≥ 21 °C gave the best results. Slow cooling (1 °C/minute) after dilution to 5 °C reduce metabolic activity and maintained sperm function after 24 hour short term storage for AI. Fertilized eggs were obtained after AI with semen in liquid storage proving that short term storage of ostrich semen is feasible. The semen of certain ostrich males were more resistant to processing stressors associated with short-term storage than others. A suitable cryoprotectant (CP) at the best inclusion level was needed for the indefinite cryopreservation storage of ostrich semen. Dimethylacetamide (DMA) was the CP of choice. Ostrich semen could be equilibrated for storage in liquid nitrogen (LN) without a significant reduction in normal sperm morphology at an inclusion level of 16 % DMA. Fast freezing in a programmable freezer at 10 °C/minute to -30 °C or in a LN Styrofoam box with a plate set at 3.5 cm LN sustained sperm motility best after thawing. The utility of the latter cryopreservation protocol was supported by an in vivo fertility assessment of the cryopreserved semen after AI. Further research on female factors as well as the refinement of the AI regime is needed for optimizing both the in vitro and in vivo protocols for the ostrich.

AFRIKAANSE OPSOMMING: Hierdie studie se oogmerk was om ‘n spesie-spesifieke werkswyse vir die lang- en korttermynberging van volstruissemen te ontwikkel. Doeltreffende berging, gevolg deur kunsmatige inseminasie (KI) kan daartoe bydra dat bedryfsbeperkings soos swak vrugbaarheid van eiers sowel as swak embrio- en kuikenoorlewing die hoof gebied kan word. Faktore betrokke by die stoor van semen in ander spesies is aanvanklik oorweeg ten einde die stappe benodig vir die ontwikkeling van ‘n spesie-spesifieke werkswyse vir die lang- en korttermyn berging van volstruissemen te identifiseer. Verder is die in vitro-funksies vir die kwantifisering en klassifisering van semen aangepas vir volstruise voordat berging in aanvang geneem het. Hierdie benadering het dit moontlik gemaak om ejakulate te evalueer en verskille in spermfunksie as gevolg van die proses te beskryf. Dit was moontlik om in die in vitro-funksies beweeglikheid, lewenskragtigheid en membraan-integriteit van volstruissperme vir gehalte aan te slaan. Die semen van volstruismannetjies het verskil ten opsigte van sperm beweeglikheid, doelgerigte beweeglikheid, swemspoed en reglynige beweging. Mannetjies kan op grond van hierdie eienskappe tot ‘n KI-program vir verbeterde vrugbaarheid toegelaat word. Die verdunning van semen direk na kolleksieis ‘n voorvereiste vir die verdere aanslag van semen. Inligting oor die makro-mineraalsamestelling van seminale plasma is bekom en gebruik om ‘n verdunningsmengsel spesifiek vir volstruise saam te stel (OS1) om sperme vir solank as 24 uur by 5 °C te berg, sonder om sperm kwaliteit prys te gee. Spermfunksie en swemgehalte is behou vir solank as 48 uur deur verdere ontwikkeling van hierdie verdunningsmengsel deur mikro-minerale ook in ag te neem (OS2). Die vlak van verdunning sowel as die temperatuur van die verdunningsoplossing is egter belangrik om spermfunksie te behou. ‘n Ge-interpoleerde verdunningstempo van 1:6 (semen:verdunningsmengsel) by ‘n verdunningstemperatuur van ≥ 21 °C het die beste gevaar in terme van semen kwaliteit. Stadige afkoeling (1 °C/minuut) na verdunning tot by 5 °C het die metaboliese aktiwiteit van sperme laat afneem en dit moontlik gemaak om semen vir tot 24 uur voor KI te berg. Daar is bevrugte eiers gekry na KI met semen in vloeibare berging, wat daarop dui dat die korttermynberging van volstruissemen uitvoerbaar is. Sommige volstruis mannetjies se sperme was meer weerstandbiedend teen proseseringstremmings, met korttermynberging, as andere. ‘n Aanvaarbare vriesbeskermingsmiddel (CP) teen die beste toedieningsvlak is ondersoek vir die onbeperkte berging van volstruissemen. Dimetielasetamied (DMA) was die verkose CP. Volstruissemen kon vir berging in vloeibare stikstof (LN) ge-egaliseer word sonder ‘n betekenisvolle verlaging in normale spermmorfologie by ‘n insluitingspeil van 16 % DMA. Versnelde bevriesing in ‘n programeerbare bevriesingsapparaat teen 10 °C/minuut tot by -30 °C of in ‘n polistireen houer met LN op die bodem met ‘n bord op ‘n hoogte van 3.5 cm bokant die LN het sperm-beweeglikheid die beste na ontdooiing behou. Die toepaslikheid van laasgenoemde bevriesingswerkswyse is bevestig deur ‘n in vivo vrugbaarheidsaanslag na die KI van wyfies met bevrore semen. Verdere navorsing op wyfie-faktore sowel as die verfyning van die KI-protokol word benodig om beide in vitro- en in vivo-werkswyses te optimiseer.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98729
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