Thesis (MSc)--Stellenbosch University, 2004. / Full text to be digitised and attached to bibliographic record. / ENGLISH ABSTRACT: Different synchronization and superovulation treatments were evaluated in the South African Boer goat (n = 367). Two progestagen implants, Synchro-Mate-B (SMB)/Crestar™ and Controlled Internal Drug Releases (CIDR), containing 3mg norgestomet and 0.33gm of natural progesterone, respectively, were used in the synchronization treatments. A luteolytic agent, Estrumate (Cloprostenol) 125)lg, was administered 12h before progestagen withdrawal. Synchronization treatment groups were: 1) 5MB x 1 (n = 123), one dose of 5MB for 13 to 17 days; 2) 5MB x 2 (n = 32), two doses of 5MB implanted for 10then 17 days; 3) CIDR x 1 (n = 187), one dose of CIDR; 4) CIDR x 2 (n = 25), two doses of CIDR, inserted for 9 to 17 days. On day 1 of the treatment, 0.5mg of estradiol cypionate (ECP) was administered to a group of randomly chosen goats (n = 112). Superovulation treatments consisted of Ovagen ™ or Embryo-STM. An additional single dose (300 UI) of Pregnant Mare Serum Gonadotropin (PMSG) was administered to a group of randomly chosen does. Superovulation treatment groups were: 1) OV alone (n = 147), Ovagen 9 mg every 12h, 8 times starting 72h prior to progestagen removal; 2) OV + PMSG (n = 164), same treatment as 1 plus 300 ru ofPMSG once 48h prior to progestagen removal; 3) E-S alone (n=16), Embryo-S 25 units twice a day, 8 times starting 72h before progestagen removal; 4) E-S + PMSG (n=40), same treatment as 3 plus 300 ru of PMSG once 48h prior to progestagen removal. Most does were naturally bred to bucks. Embryos were collected using the surgicallaparascopic procedure on day 6 and corpora lutea counted. Data were not normally distributed and therefore analyzed using a nonparametrie test (Wilcoxon, 1945 and Kruskal- Wallis, 1952) with outcome variable using the Mixed Procedure of SAS and the Tukey test. Differences were considered significant at p<0.05. Slightly more CL were on the left (52%) than on the right (48%) ovary. Superovulation treatment was significantly associated (p<O.OOl) while synchronization treatment was only marginally associated (p=0.06) with ovulation rate. Ovagen alone and Ovagen + PMSG were significantly more effective (p<0.05) than Embryo-S alone or Embryo-S + PMSG in influencing ovulation. Only synchronization treatment with 2 doses of CIDR was significantly more (p=0.04) effective in producing a high ovulation rate. Superovulation treatment was significantly (p=0.02) associated with the number of transferable embryos while synchronization treatment was not. Ovagen + PMSG was significantly (p=0.02) effective in producing more transferable embryos than Embryo-S + PMSG. Both superovulation and synchronization treatments were significantly (p<0.05) associated with producing unfertilized oocytes. Effectiveness of addition of ECP was shown in its association (p=0.05) with better quality embryos in univaraete analysis, though it did not have significant effect in the multivariate model. Though there was apparent advantage of CIDR over 5MB, no significant difference in ovulation rate or embryo quality was associated with synchronization treatments. Effectiveness of Ovagen over Embryo-S was demonstrated and addition ofPMSG improved embryo quality. / AFRIKAANSE OPSOMMING: Verskillende sinkronisasie en multi-ovulasie behandelings is ge-evalueer in die Suid-Afrikaans Boerbok (n= 367). Twee progestagene, Synchro-Mate-B (SMB)/Crestar™ en Controlled Internal Drug Releases (CIDR), bevattende 3mg norgestomet en 0.33gm natuurlike progesteroon, respektiewelik, is gebruik tydens die sinkronisasiebehandelings. 'n Luteolitiese middel, Estrumate (Cloprostenol) 125J.lg, is toegedien 12 h voor progestageen verwydering. Sinkronisasie behandelings groepe was: 1) 5MB x 1 (n = 123), een dosis 5MB vir 13 tot 17 dae; 2) 5MB x 2 (n = 32), twee dosisse 5MB implante vir 10 tot 17 dae; 3) CIDR x 1 (n = 187), een CIDR vir die hele periode; 4) CIDR x 2 (n = 25), twee CIDRs, vir 9 tot 17 dae. Op dag 1 van die behandeling is 0.5mg estradiol cypionate (ECP) aan 'n willekeurige groep bokooie toegedien (n = 112). Multi-ovulasie behandelings het bestaan uit Ovagen™ of Embryo-S™. 'n Bykomstige dosis (300 UI) Dragtige Merrie Serum Gonadotrofien (PMSG) is toegedien aan 'n willekeurige groep ooie. Multi-ovulasie behandelingsgroepe was: 1) OV alleen (n = 147), Ovagen 9 mg elke 12h, 8 keer beginende 72 h voor progestageen verwydering; 2) OV + PMSG (n = 164), selfde behandeling as in (1) plus 300 IV PMSG eenmalig 48h voor progestageen verwydering; 3) E-S alleen (n=16), Embryo-S 25 eenhede tweemaal per dag, ag inspuitings beginende 72h voor progestageen verwydering; 4) E-S + PMSG (n=40), selfde behandeling as in (3) plus 300 IV PMSG eenmalig 48h voor progestageen verwydering. Die meerderheid ooie is natuurlik deur ramme gedek. Embrio's is gekollekteer deur gebruik te maak van die chirurgieslaparoskopiese metode op dag 6 en die aantal corpora lutea getel en aangeteken. Aangesien die data nie 'n eweredige verspreiding gehad het nie, is dit geanaliseer deur gebruik te maak van 'n nie-parametriese toets (Wilcoxon, 1945 en Kruskal-Wallis, 1952) met variërende uitkomste deur die Gemengde Prosedure Toets van SAS en die Tukey toets. Verskille is as beduidend aanvaar met 'n P-waarde van <0.05. Onbeduidend meer CLs is op die linker (52%) as op die regter (48%) ovarium opgemerk. Multi-ovuasie behandelings was beduidend geassosieer (p<0.001) met ovulasietempo, terwyl sinkronisasie behandelings net marginaal geassosieer was (p=0.06) met ovulasietempo. Ovagen alleen en Ovagen + PMSG was beduidend meer effektief (p<0.05) as Embryo-S alleen of Embryo-S + PMSG om ovulasie te beïnvloed. Slegs die sinkronisasie behandeling met 2 dosisse CIDR was beduidend meer (p=0.04) effektief om 'n hoër ovulasietempo te veroorsaak. Multi-ovulasie behandeling was beduidend geassosieer met die aantal oordraagbare embrio's, terwyl sinkronisasie nie dieselfde tendens gewys het nie. Ovagen + PMSG het beduidend meer (p=0.02) oordraagbare embrio's opgelewer as Embryo-S + PMSG. Beide multi-ovulasie en sinkronisasie behandelings was beduidend geassosieer (p<0.05) met onbevrugte oosiete. Die rol van die byvoeging van ECP is getoon in die assosiasie daarvan (p=0.05) met beter kwaliteit embrio's in 'n eenvariante analiese, alhoewel dit nie 'n beduidende effek op die multi-variante model gehad het nie. Alhoewel dit blyk dat CIDR 'n beter reaksie as 5MB gee, kon geen beduidende verskil in die ovulasietempo of embriokwaliteit opgewys word nie. Die groter effektiwiteit van Ovagen oor Embryo-S is gedemonstreer, terwyl die byvoeging van PMSG embriokwaliteit verbeter het.

published_or_final_version / abstract / Obstetrics and Gynaecology / Master / Master of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

No description available.

Cryopreservation of fish embryos has proven to be a difficult problem in cryobiology. Three main difficulties have been identified or suspected: 1) embryo membrane permeability barriers to cryoprotectants and water; 2) high chilling sensitivity of the embryos; and 3) the twocompartment nature of the embryos with a large yolk. Using the zebrafish embryo as a model system, these limiting factors and possible approaches to overcoming them were investigated with a view to developing an effective procedure for fish embryo cryopreservation. Compared to previous studies, vitrification of zebrafish embryos on gold electron microscope grids using methanol as the cryoprotectant resulted in improved morphological survival, being -50% for early stage (I-cell and 64-cell) and ~ 80% for late stage (50%epiboly, 6-somite and prim-6) embryos, but no embryo showed viability. Poor cryoprotectant permeation and embryo dehydration, and consequently intraembryonic ice formation, remained as the main problem for vitrification. Embryo chilling sensitivity studies suggested that later stage zebrafish embryos were sensitive to cold shock injury arising from rapid cooling followed by being held for an extended exposure period (1 h) at 0 or -5°C. Studies on embryo developmental arrest by anoxia showed that chilling injury in zebrafish embryos was probably not associated with their high development rate. However, the chilling sensitivity of zebrafish embryos was found to be related to the amount of yolk present. Yolk-reduced embryos at prim-6 and high-pec stages became less sensitive to chilling at O°C. Differential scanning calorimetry studies on the depression of intraembyonic nucleation temperatures by cryoprotectants revealed that multi-punctured embryos at 6-somite and prim-6 stages became significantly more permeable to methanol and propylene glycol when compared with their nonpunctured controls. Puncturing of the yolk-sac of fish embryos to reduce yolk content, and the increased permeability to cryoprotectants that this promotes, may offer a new approach to surmounting the difficulties confronting the cryopreservation of fish embryos.

Crocodilians and birds comprise sister taxa of archosaurs, the development of these vertebrates occurs within an egg case that leaves developing embryos susceptible to fluctuations in the nesting environment. Studies suggest that sub-optimal conditions alter morphological growth and cardiovascular physiology. Regulation of the cardiovascular system is immature in the subjects studied, and embryos may rely on humoral rather than neural control of the cardiovascular system. The primary focus of this dissertation was to assess regulatory mechanisms responsible for maintenance of arterial pressure and heart rate. Dehydration stress had marked effects on embryo growth, and altered baseline cardiovascular parameters, while leaving the response to humoral regulator, angiotensin II (Ang II), unaffected. However, dehydrated alligator embryos developed cholinergic tone on heart rate. Hypoxic incubated chicken embryos were reduced in embryo mass, and altered response to humoral regulatory components Ang I and adenosine in addition identifying a novel regulatory component of the cardiovascular response to acute hypoxia. Collectively, these studies add to the existing knowledge of cardiovascular physiology in embryonic archosaurs and suggest that some components of cardiovascular regulation are plastic following developmental stress.

Cryopreservation of fish embryos was studied using zebrafish embryo as a model system. Cryoprotectant toxicity, chilling sensitivity and embryo membrane permeability were investigated with a view to developing optimum protocols for cryopreservation of the embryos using controlled slow cooling, non-freezing storage and vitrification. Methanol was found to be the most effective cryoprotectant for controlled slow cooling and non-freezing storage of zebrafish embryos with 11 %heart beat stage embryos surviving after controlled slow cooling to -25°C. Zebrafish embryos were found to be very chilling sensitive with early developmental stages being the most sensitive to chilling injury. Embryo developmental stages after closure of the blastopore (>12-h), especially post heart beat stages were much more resistant to cryoprotectant toxicity and chilling injury. Heart beat stage (27-h) embryos proved to be the best embryo developmental stage for controlled slow cooling and non-freezing storage. Dechorionated embryos are more sensitive to cryoprotectant toxicity and chilling injury. The sensitivity to chilling injury of zebrafish embryos limited the use of controlled slow cooling and non-freezing storage for long term cryopreservation. The attempts at cryopreservation of zebrafish embryos using vitrification produced no embryo survival, although up to 32 % embryos remained morphologically intact immediately after vitrification. Poor cryoprotectant permeation, dehydration and consequently ice formation within the egg are probably the main factors on effecting embryo survival. The results of zebrafish embryo permeability studies demonstrated that the chorion of the embryos was permeable to water and cryoprotectants, whilst the vitelline (plasma) membrane was an effective permeability barrier. The inability to achieve sufficient penetration of the vitelline membrane by cryoprotectants poses severe problems for long term cryopreservation, which need to be overcome, possibly by permeabilisation of the vitelline membrane, before successful cryopreservation can be achieved.

As bacteria are becoming resistant to conventional antibiotics, researchers are looking for new ways to combat microbial infection. We have begun to adopt genetic and functional genomic approaches to define the molecular determinants of pathogen resistance in the dung beetle, Euoniticellus intermedius. This dung beetle survives microbe-rich environments such as dung. This ability makes it a potential model for the study of infectious agents and ecological damage. To date, E. intermedius has not been studied at the molecular level. In this study, a range of complimentary analytical techniques were used to characterize the E. intermedius embryonic cell line established in our laboratory. These techniques characterize morphology, growth characteristics, karyotype, isoenzyme patterns and embryonic development. Complete characterization of the E. intermedius cell line is essential for the cell banks and for the regulatory requirements in biopharmaceutical production. This study followed gene sequences and their comparisons for both adult and cell line to confirm that the E. intermedius (EISA08) cell line is originated from the embryonic E. intermedius dung beetle. cDNA was synthesized from mRNA isolated from E. intermedius adult beetles and cell line (EISA08) was sequenced using GS (FLX) technology by a commercial facility, Inqaba Biotechnical Industries (Pty) Ltd, South Africa. In addition to characterization of the cell line, two genes, namely hopscotch and ribosomal protein S9 (RpS9), were selected from the Flylab genome data base. The E. intermedius database is a web-based system for the genome and transcriptome of the dung beetle to evaluate the immune system of the dung beetle (http://Flylab.wits.ac.za/). hopscotch was selected because it is believed to be involved in the JAK-STAT signalling pathway for anti-viral response, embryonic development and cell growth. Rsp9 was chosen as a loading control because it is expected to be a housekeeping gene. The conserved molecular signalling pathway JAK-STAT is used by E. intermedius (as in other insects and humans) for immune defence and early embryonic development. The project followed hopscotch and Rsp9 gene expression in all the E. intermedius life cycle developing stages; adult, pupae, larvae, embryo, and cell line cell growth, life cycle developing stages and embryonic development has was monitored. E. intermedius embryonic development is described as short germ-band. E. intermedius embryogenesis is regarded as basal and is observed in most arthropods. The study revealed that E. intermedius hopscotch is over expressed in the early developing stages, embryo, larvae, and pupae and in the newly established cell line EISA08. The results from this study lead to the suggestion that E. intermedius JAK-STAT pathway is activated early and has an important role in embryonic development, cell proliferation and immune defence. Studies of E. intermedius could provide more insight into the properties and evolution of innate immunity and embryonic development.

A Dissertation submitted to the Faculty of Science, University of Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. 2015. / Prdm1/Blimp1 is a transcription factor whose mechanism of action is mainly repression; however it has been identified as an activator in some cases. As a transcriptional repressor, it plays multiple roles during embryonic development, including neural crest specification. Prdm1 acts by repressing large sets of genes via sequence specific recruitment of co-repressors, many of which are epigenetic modifiers. Neural crest is a transient, migrating cell population that gives rise to a number of diverse cell lineages that form important structures in the vertebrate embryo. Examples of these include peripheral nervous system, melanocytes and cranial cartilage. Prdm1 is expressed during neural crest specification in Xenopus, zebrafish and lamprey. The expression of Prdm1 had not yet been investigated in the neural crest during chick embryonic development. The mechanism of Prdm1 action or the nature of possible binding partners that mediate its effects in the neural crest had not yet been addressed. Prdm1 binding partners are known to play important roles during embryonic development, yet in many cases no spatiotemporal expression analysis during early vertebrate development has been performed. Single and double in situ hybridization for Prdm1 and all the binding partners was performed to determine localization of mRNA during early stages of chick embryonic development. We report the expression patterns of Prdm1 and seven of its known or putative binding partners (Hdac1, Hdac2, Tle1, Tle3, G9a, Prmt5 and Lsd1) during early stages (HH4-HH10) of chicken embryogenesis. Prdm1 expression was observed in the neural plate border and pre-migratory neural crest during chick development. Six Prdm1 binding partners (except Tle1) are co- expressed with Prdm1 in the prospective neural plate border at HH4-HH6, and all seven show strong and specific expression in the neural plate border at HH7-HH8, suggesting all of them co-operate with Prdm1 during neural crest development in chick embryos. Future work will focus on protein interaction studies in order to directly demonstrate the association between Prdm1 and the binding partners it co-localizes with.

Thesis advisor: David Burgess / The contractile ring is pictured, analyzed, and even named under the basic assumption that the geometry of the structure begins and ends circularly and the ring is a single homogenous structure acting uniformly. However, under physiological conditions cell-to-cell adhesions force cells and therefore initial contractile rings into highly irregular and noncircular shapes. To investigate this basic assumption of contractile ring geometry, contractile ring shape of dividing sea urchin embryos was analyzed under three conditions: in seawater where cell-to-cell adhesion is strong, calcium free seawater where cell-to-cell adhesion is minimized, and in microfabricated chambers to artificially manipulate the initial contractile ring shape. We found that contractile ring geometry evolves over time to become circular even when it begins as an irregular shape due to cell-to-cell adhesions or artificial manipulation. By analyzing velocities of specific regions of the contractile ring, it became apparent that there is always a pattern of rounding regions of lowest circularity before overall ring contraction. This pattern suggests that the contractile ring is capable of producing varying forces in a coordinated manner. Therefore the contractile ring can not only be noncircular, but can also possess regions with different molecular and biophysical properties. / Thesis (MS) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.