The influence of genotype on sperm motility and sperm head morphometry of Merino (Ovis aries) sheep

Boshoff, Ninja Hettie (2014-04)

Thesis (MScAgric)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: The application of assisted reproductive biotechnologies in sheep flocks is hampered by the susceptability of ovine sperm to cryodamage. There is still considerable scope in the improvement of cryopreservation protocols for ovine sperm to minimize the degree of damage to sperm during the cryopreservation process. Pre-cryopreservation processing has a definite effect on the survivability, motility, and fertilizing ability of sperm. Little information is however available on the potential contribution of the genetic make-up of rams, divergently selected for fecundity, on the ability of sperm to offer resistance to the damage caused by cooling, cryopreservation and thawing. The study aimed to investigate the influence of genetic selection for prolificacy (i.e. High Merino Line and Low Merino Line in terms of fecundity) on the ability of ovine sperm to offer resistance to cryodamage. The study investigated the effect of pre-cryopreservation processing by comparing motility and morphometry traits recorded for fresh- and post-thaw Merino ejaculated and epididymal sperm samples obtained form the High and Low lines, respectively. The effect of different sperm concentrations, equilibration periods and the addition or omission of seminal plasma from cryopreserved samples on the viability and morphometrical traits were also investigated. Ejaculate samples were collected by means of the artificial vagina (AV) method from 8 High Line rams and 7 Low Line rams. Epididymal samples were collected from 6 rams of each of the High and Low lines respectively, by recovering the epididymal sperm via aspiration from the cauda epididymides post mortem. Ejaculate samples were subjected to macroscopic and microscopic evaluation, and epididymal samples only to microscopic evaluation, for which the Sperm Class Analyzer® program was used for the evaluation of motility and morphometric measurements. Sperm motility recordings were captured at 100 frames per second. From findings of the study, it was concluded that genotype had no positive influence on the conception rate of the ewes mated to the High or Low Line rams, even though the rams from the two lines differed significantly in terms of their serving capacity. When sperm morphometry was evaluated for fresh ejaculate samples, the two lines differed significantly in terms of the morphometric traits elongation and ellipticity. Epididymal and ejaculated sperm obtained from Low Line rams had broader and rounder heads, compared to sperm obtained from High Line rams. When morphometry was assessed for sperm samples between the two methods of sperm recovery (collected with an AV or recovery via aspiration from the cauda epididymides of sacrificed rams), no morphometrical differences were observed. Significant differences were reported for the majority of the sperm motility traits (i.e. percentage motile, rapid-, medium-, slow swimming, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), and amplitude of the lateral head displacement (ALH)) recorded for ejaculated and epididymal sperm. The motility traits ALH and beat-cross frequency (BCF) analysed for epididymal sperm differed significantly between the two lines. When epididymal sperm were evaluated post-thaw, it became evident that the sperm obtained from the High Line rams had a larger acrosome surface cover when compared to that of the Low Line ram sperm. The addition of seminal plasma to epididymal samples did not result in an improvement of the preservation of sperm motility. It is known from the literature that cryopreservation causes a decrease in sperm head size. Head width was unaffected by cryopreservation with the addition of seminal plasma in this study, indicating a potential benefit with the use of seminal plasma in the cryopreservation protocol of epididymal ram sperm. The study compared two pre-processing techniques, i.e. the more time consuming swim-up technique (SUT) with a more time-efficient ‘flush technique’ (FT) to optimize the pre-processing protocol for motility assessment of sperm samples before cryopreservation of ram sperm. Comparison of the SUT and FT indicated that almost all of the motility parameters measured using the FT compared favourably with those obtained using the SUT. The results indicated that the FT can be used a more time-efficient technique to use for determining the motility of a sperm sample prior to cryopreservation. In conclusion, line differences associated with reproduction were observed in terms of the serving capacity of the rams, with selection for fecundity influencing the morphometric traits elongation and ellipticity for sperm obtained from the two lines. Future studies should be aimed at investigating morphometric traits of ovine sperm, to correlate it with fertilizing ability of sperm post-thaw and ensure optimal cryopreservation processing.

AFRIKAANSE OPSOMMING: Die toepassing van ondersteunende reproduksie tegnieke in skaaptroppe word bemoeilik deur die onvermoë van ram sperme om weerstand teen bevriesingskade te bied. Daar is nog baie ruimte vir die verbetering van die bevriesingsprotokolle vir skaap sperm om die omvang van bevriesingskade te verminder. Voor-bevriesing verwerking het dan 'n besliste uitwerking op die oorlewing, beweeglikheid en bevrugtingsvermoë, van skaap sperme. Min inligting is beskikbaar oor die potensiële bydrae van die genetiese samestelling van ramme wat uiteenlopend op grond van vrugbaarheid geselekteer is, op die vermoë van skaap sperme om weerstand te bied teen die skade wat deur verkoeling, diepbevriesing en ontdooiing, veroorsaak word. Die doelwit van die studie was om die invloed van genetiese seleksie vir fekunditeit (d.i. Hoë Merino Lyn en Lae Merino Lyn in terme van fekunditeit) op die vermoë van skaap sperme om weerstand teen bevriesingskade te bied, te ondersoek. Die studie het getoets wat die bevriesing proses se effek op epididimale sperme is, deur sperm motiliteit en -morfometrie te vergelyk tussen vars gekollekteerde sperme en sperm monsters na ontdooiing. Die effek van verskillende sperm konsentrasies, ekwilibrasie tydperke en die byvoeging of uitsluiting van seminale plasma op die lewensvatbaarheid en morfometriese eienskappe van Merino ramsperme is ondersoek in die studie. Geëjakuleerde monsters is versamel met behulp van 'n kunsmatige vagina (AV) van 8 Hoë Lyn en 7 Lae Lyn ramme. Epididimale monsters is verkry van 6 ramme van elk van die Hoë en Lae Lyne, deur middel van aspirasie van die sperme uit die cauda epididimii nadoods. Geëjakuleerde sperm monsters is met behulp van makroskopiese en mikroskopiese metodes geëvalueer, en epididimale sperm monsters slegs mikroskopies geëvalueer, met behulp van die Sperm Class Analyzer® program wat vir die evaluasie van beweeglikheid en morfometriese afmetings gebruik is. Sperm beweeglikheids opnames is opgeneem teen 100 raampies per sekonde. Die resultate van die studie het aangedui dat genotipe geen effek het op besetting van die ooie gepaar met die Hoë of Lae Lyn ramme gehad het nie, terwyl die dekvermoë aansienlik tussen ramme van die twee lyne verskil het. Wanneer die morfometriese eienskappe van vars geëjakuleerde sperme vergelyk was, het die lyne beduidend in terme van die morfometriese eienskappe van verlenging (elongation) en elliptisiteit verskil het. Die epididimale en geëjakuleerde sperme verkry vanaf die Lae Lyn ramme het ʼn breër en ronder kopvorm getoon as sperme wat verkry is van die Hoë Lyn ramme. Wanneer die morfometriese eienskappe van sperme versamel met die twee verskillende metodes (d.i. kunsmatige vagina of aspirasie vanuit die cauda epididimides) vergelyk was, is geen morfometriese verskille waargeneem nie. Die meeste sperm beweeglikheidseienskappe (d.i. persentasie beweeglike, vinnig-, medium- en stadig-swemmende sperme, VCL, VSL, VAP en ALH) van geëjakuleerde en epididimale sperme het verskil. Die beweeglikheidseienskappe amplitude van die laterale verplasing van die spermkop (ALH) en frekwensie waarmee sperm sy eie pad kruis (BCF), soos bepaal vir epididimale sperme, het beduidend tussen die twee lyne verskil. Met die evaluering van epididimale sperme na ontdooiing was dit duidelik dat sperme verkry van die Hoë Lyn ramme 'n groter mate van akrosoom-oppervlak gehad het, in vergelyking met sperme van die Lae Lyn ramme. Die byvoeging van seminale plasma by epididimale monsters het nie bygedra tot 'n verbetering van spermbeweeglikheid nie. Bestaande literatuur dui aan dat diepbevriesing 'n afname in die kopgrootte van sperme veroorsaak. In hierdie studie het die byvoeging van seminale plasma ʼn verandering in kopgrootte voorkom, wat dui op ʼn potensiële voordeel om seminale plasma in die bevriesingsprotokol van epididimale ramsperme in te sluit. Die studie het twee beweeglikheid bepalingstegnieke vergelyk om te bepaal of die tydrowende “opswem” tegniek (SUT) vervang kan word met 'n meer tyd-doeltreffende "spoel tegniek” (FT) in die voorbevriesing verwerking protokolle van ram sperme. Vergelyking van die twee tegnieke het aangedui dat die meeste van die kinematiese eienskappe van die FT gunstig met die waardes soos verkry met die SUT, vergelyk het. Resultate het getoon dat die FT parameters goed vergelyk met die beweeglikheid parameters van die SUT, dus kan dit aangeneem word dat die FT ʼn meer tyd-doeltreffende tegniek is wat vergelykbare sperm beweeglikheidsinligting oor skaap sperm monsters voor bevriesing sal verskaf. In samevatting is verskille in terme van die dekvermoë en op morfometriese vlak, meer spesifiek die eienskappe van verlenging (elongation) en elliptisiteit, tussen die twee lyne waargeneem. In toekomstige studies moet die morfometriese eienskappe van skaapsperme verder bestudeer word, asook die korrelasie daarvan met die bevrugtingsvermoë na ontdooiing bepaal om sodoende die diepbevriesing protokolle van skaapsperme te optimaliseer.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/86301
This item appears in the following collections: