Thesis (MScAgric)--Stellenbosch University, 2014. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/86301 |
| Date | 04 1900 |
| Creators | Boshoff, Ninja Hettie |
| Contributors | Lambrechts, Helet, Cloete, Schalk, Stellenbosch University. Faculty of AgriSciences. Dept. of Animal Sciences. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xviii, 169 p. : ill. |
| Rights | Stellenbosch University |
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