Typescript (photocopy). / Digitized by Kansas Correctional Industries

This study was conducted to determine the relationships among the linear type traits of rump angle, rump width, rump length, rear legs side view, rear legs position, rear legs rear view, tailhead, vulva angle, mobility, pasterns, foot angle, and toes with reproductive performance (days open and times bred) in Holstein cows and to develop indices to predict reproductive performance from mathematical functions of the anatomical traits. Two trials were conducted. The first trial involved 7630 registered Holstein cows from Oregon and California. The regression analysis (R²) showed only 1.1% of the variability of times bred and 1.3% of the variability of days open was accounted for by the rump and rear leg type traits. In the second trial, 8155 Holstein cows, both registered and grade, were analyzed using the linear type traits of rump angle, rump width, rear legs side view, rear legs position, rear legs rear view and foot angle.. Grade and registered cows were analyzed separately to determine if differences in management between them would be reflected in the statistical analysis. Evaluator, lactation number (parity), season, geographic location, and the interaction of evaluator and lactation number had a significant effect on most of the type traits and the scorecard category (General Appearance, Mammary System, Dairy Character and Body Capacity) scores examined. The effects of these variables were statistically removed and the residuals of the type traits were used in the final regression analysis. Using stepwise regression analysis, several non-significant traits were omitted from the final model. The analysis used days open and times bred as dependent variables. Lactation number, mature equivalent milk, foot angle, rump width and their respective quadratics were independent variables, as were season calved and geographic location. The regression analysis (R²) indicated that 5.3% of the variability in days open and 4.7% of the variability in times bred in registered cows was accounted for by the type traits, foot angle and rump width, respectively, when the effects of season calved, geographic location, lactation number and mature equivalent milk were included in the model. For the grade cows the regression analysis (R²) indicated that 3.5% of the variability in days open was accounted for by foot angle. None of the type traits examined had a significant effect on times bred. This study detected no significant influence of rump angle or rear leg position, as described by the HFA linear classification program, on reproductive performance. However, our analysis indicated that fertility decreased as rear foot angles became more steep in grade and registered cows and as rump width increased in registered cows. / Graduation date: 1991

The aging phenomena is a process to which all organisms eventually succumb. The universality of this phenomena suggests that there may be one overwhelming factor involved. The exact biochemical basis of aging is still unclear. Free radicals such as the superoxide radical (O₂⁻) and the hydroxyl radical (OH⁻), formed in biological oxidation reactions may be responsible for cellular aging. Because of the high reactivity of the O₂⁻ and the OH⁻ they can produce extensive damage to lipids, proteins, and nucleic acids. In this study we have developed an <i>in vitro</i> quiescent model using density dependent bovine embryonic lung fibroblast (BELF). The effect of this process on the antioxidant defense enzymes such as, the superoxide dismutases, catalase, glutathione reductase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase, was studied. We have also extensively monitored the levels of free radical in the cell by both direct and indirect methods. The results indicate that no significant (p<0.05) changes in the activity of any of the major antioxidant enzymes in our quiescent model. Significant increases were observed in the intracellular levels of lipofuscin (age pigment) with time, but no changes in the generation of free radicals were observed using electron spin resonance spectrometry, cytochrome <i>c</i> reduction or spectrofluorometric techniques (caution should be placed in statistical interpretation of the data because of the small sample size in some experiments). The transcriptional and translational controls of the one of the major antioxidant defense enzymes (manganese superoxide dismutase) in bovine embryonic lung fibroblasts, human pulmonary artery endothelial cells (HPAE) and bovine PAE cell lines were also studied. Our preliminary data suggest that inhibitors of protein and RNA synthesis both cause a significant decrease in the induction of the manganese SOD in bovine pulmonary endothelial cells. / Master of Science

Thesis (M. Sc. (Animal Production)) -- University of Limpopo, 2015 / The objectives of this study were: 1) To determine the effect of oocyte retrieval techniques (slicing and aspiration) on the quality and quantity of cattle oocytes, 2) To evaluate the effect of different concentrations of hormones on the maturational rate of cattle oocytes selected by brilliant cresyl blue staining, 3) To evaluate fertilization rate and cleavage/embryonic development of oocytes with or without cumulus cells, and 4) To compare the effect of fresh and frozen thawed semen on the fertilization rate of cattle oocytes. In Experiment 1: oocytes were recovered from abattoir derived ovaries using slicing and aspiration. The recovered oocytes were exposed for 90 minutes to 26μM of brilliant cresyl blue (BCB) stain and classified according to the colour of their cytoplasm: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). There was no difference (P>0.05) in the quality of oocytes recovered using slicing (60.7 %) or aspiration (53.7 %) techniques. In experiment 2: The BCB selected and the non-selected immature oocytes were randomly allocated into medium 199 + 10 % fetal bovine serum (FBS) maturation media. The media was supplemented with three different concentrations of hormones as treatments (T). The T1 (0.5 μg/ml of follicle stimulating hormone (FSH), 5mg/ml of luteinizing hormone (LH) and 2 μg/ml of estradiol (E2) as the control group. Then, T2 (1 μg/ml of FSH, 6 mg/ml of LH and 2.5 μg/ml of E2) and T3 (1.5 μg/ml of FSH, 7 mg/ml of LH and 4.5 μg/ml of E2). Maturation rate of oocytes was determined by the protrusion of the first polar bodies 24 hours following in vitro maturation. Treatment 2 yielded higher (P<0.05) maturation rate for both BCB+ (65.6 %) and without BCB (60.3 %) oocytes with T1 giving lower (P<0.05) maturation rate for BCB+ (22 %) and without BCB (16 %) oocytes. However, BCB- oocytes had lower (P<0.05) polar body extrusion (3.03 %, 8.1 % and 2.2 %) for T1, T2 and T3, respectively. In Experiment 3: one group of the presumptive zygotes was denuded of cumulus cells and the other group was cultured with cumulus cells. The presumptive zygotes were in vitro cultured in SOF-BSA and changed to SOF-FBS after 48 hours. High fertilization/cleavage rate was observed in oocytes cultured with cumulus cells (29.0 %) compared to the denuded oocytes (20.0 %) for 2-4 cells stage. Day 7 blastocysts were more (P<0.05) on oocytes cultured with cumulus cells (32 %) compared to denuded oocytes (13 %). In experiment 4: The matured oocytes were fertilized using fresh and frozen thawed semen. The oocytes fertilized with frozen thawed semen obtained a better number of 2-4 cell cleavage (23 %) when compared to fresh semen (19 %). Oocytes that were fertilized with frozen thawed semen also obtained higher morula (13 %) and blastocyst (8 %) compared to fresh semen with morula (3.4 %) and blastocyst (2 %). In conclusion, immature oocytes that were exposed to BCB+ and cultured in M199 supplemented with 10 % FBS, 0.5 μg/ml of FSH, 5 mg/ml of LH and 2 μg/ml of E2 had a higher (P<0.05) number of matured oocytes (extrusion of first polar body) compared to those that were not exposed to BCB (no BCB). Oocytes that were cultured with cumulus cells yielded a higher (P<0.05) number of cleaved embryos compared to the denuded oocytes. Slicing yielded a higher (P<0.05) number of oocytes, however the quality of oocytes recovered was similar compared to those recovered by the aspiration technique (P>0.05). Oocytes fertilized with frozen thawed semen yielded higher (P<0.05) number of 2-4 cell, morula and blastocyst when compared with oocytes that were fertilized using fresh semen. Keywords: ovaries, oocytes, slicing, aspiration, COCs, BCB, polar body and cattle

The uterine protein milieu of cyclic and ovariectomized (ovx) cows treated with estradiol-17S (E-2) and progesterone (P) alone or in combination was studied by various biochemical methods. Uterine lumenal fluids (UF) were collected nonsurgically via a foley catheter. Sephacryl S-200 chromatography revealed protein peaks specific to UF from cyclic cows had distribution coefficients at ambient temperature of .27 ± .02, .32 ± .02, .65 ± .03, .73 ± .06, and .91 ± .04; peaks at 4C were .35, .41 ± .04, .82 ± .05, 99 ± .04, and 1.20 ± .09. UF analyzed by HPLC had uterine specific protein peaks eluting at 7.27 ± .32, 15.42 ± .39, 17.71 ± .14, 18.31 ± .13, and 20.91 ± .38 ml. Native polyacrylamide disc gel electrophoresis at pH 8.3 revealed differences in the frequency of appearance between plasma and UF for proteins with relative mobilities (R_A) of .033, .415, .517, .709, .780, and .894. Mean relative percent collectively of the .894 band and albumin was higher (P < .01) in UF than in plasma. UF were obtained from ovx cows administered E-2 and Palone or in combination by Silastic® implant (I) after each of three consecutive 18 day treatment periods. During the third treatment period, supplemental daily steroid injections in the same ratios as found in the I were given to augment I hormone contributions. Proteins unique to UF after the first treatment period had R_A of .648, 1.13, and 1.66. One cow (8 g P, 2 mg E-2) had a basic protein migrating 1.35 cm toward the cathode. Proteins unique to UF after the second treatment period had mobilities of .645, 1.15, 1.22, 1.40,1.60, and 1.68. Three cows receiving treatments including four 2 g P I had a basic protein migrating 1.4 ± .3 cm (X ± S.D.) toward the cathode. Two cows in these same treatments had an additional basic protein migrating .2 to .3 cm into the gel. Acidic proteins unique to UF after the third treatment period had R_A of .638, 1.15, 1.24, 1.40, 1.58, and 1.67. The appearance of proteins with R_A greater than 1.15 was associated with treatments including P except one cow receiving E-2 only, having bands at 1.15 and 1.20. A basic protein migrating 1.34 ± .19 cm (X ± S.D.) towards the cathode was observed in 11 of 18 cattle receiving treatments including P compared with none from cattle not receiving P. These data indicate that UF from cyclic cows contains small molecular weight uterine specific proteins and at least one large molecular weight protein. At least four acidic proteins and two basic proteins appear to be induced by treatments including P. / Ph. D.

Anatomical features of bovine cervical epithelium were investigated with respect to mucus secretion and sperm transport. Techniques included: 1. Surface staining of fixed tissue blocks in Harris' hematoxylin followed by steriomicroscopic examination, 2. Model construction from serial sections using both computer-aided and plexiglas assembly of epithelial tracings, 3. Histochemical investigation using five follicular phase animals (four bred naturally 8 to 12-h before slaughter) and three luteal phase animals. Cross-sections of two samples from each quarter cervix were stained with (1) Alcian blue (AB) at pH 1.0 (sulfomucins) and at pH 2.5 (sialomucins and sulfomucins); (2) periodic acid Schiff (PAS) (neutral mucins) and AB (pH 2.5); (4) high iron diamine (HID) (neutral and sulfomucins) with AB (pH 2.5). Additional samples were processed for ultrastructural examination. The mucosa was characterized by longitudinal primary and secondary folds which maintained continuity throughout the cervix, with numerous tertiary shallow ’grooves’ apparent on all epithelial surfaces. No evidence was found suggesting blind-ending glands or crypts. Staining results in follicular animals indicated a predominance of neutral and sulfated mucins in apical areas with secreted mucins extending as sheets from these areas toward the central canal. In basal areas (within grooves) sialomucin production was predominant with secreted mucins evident within grooves and between neutral mucin layers. In luteal phase animals, sulfated and neutral mucins were abundant in both basal and apical areas, while sialomucin production was decreased. Using light and transmission electron microscopy, spermatozoa observed within the cervix appeared unidirectionally opposed to ciliary beat. Suggested privileged paths for the transport of viable spermatozoa are within grooves, where sialomucins were most predominant. / Master of Science / incomplete_metadata

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: In this study, five trials were conducted to examine in vitro and in vivo degradation, excretion and absorption parameters of melamine (MEL) in dairy cows that have not been studied before or where limited information is available. The first two trials were in vitro studies conducted to determine the extent of MEL degradation in rumen liquor and the effects of MEL on ruminal ammonia (NH3) and volatile fatty acid (VFA) concentrations. For both trials, rumen liquor was collected from ruminally cannulated lactating Holstein cows. For the first and second trial, rumen liquor was collected from three and two cows, respectively. For both trials, Erlenmeyer flasks contained 1 g substrate and 100 mL incubation medium consisting of 20 mL rumen liquor and 80 mL reduced buffer solution. In the first trial, each flask contained 100 mg of MEL, resulting in an initial MEL concentration of 1000 mg/L. The flasks were incubated at 39° C for 0 (Control), 6, 24 or 48 hours under strictly anaerobic conditions. In all the trials, MEL concentrations were determined by LC/MSMS. MEL degradation was low after 6 and 24 h of incubation (3.2 and 5.5%, respectively) and increased to 13.6% after 48 h of incubation. In the second trial where VFA and NH3 concentrations were determined, the flasks contained either 0 (Control), 0.2 (T1) or 0.4 mg (T2) of MEL. The flasks were incubated for 6, 24 or 48 h. Treatment had no effect on individual or total VFA concentrations or NH3 concentrations at 6 and 48 h. At 24 h, T2 resulted in an inexplicable higher NH3 concentration. This study showed that the addition of melamine would not result increased rumen NH3 concentrations in vitro. Melamine would also not affect the production of different VFA’s. Therefore, it was concluded that the rumen micro-organisms present in rumen liquor would be unable to utilize MEL as a source of nitrogen and that the microbial production of VFA’s remains unaffected by the presence of MEL. In the third trial, MEL excretion in lactating cows was determined. Five cows were randomly allocated to treatments according to a 5 x 5 Latin square design. Cows received the treatment diets for 7 d followed by 8 d of MEL withdrawal during each of the five periods. The experimental treatments were formulated to provide a daily MEL intake of 0 (M0), 500 (M1), 1000 (M2), 5000 (M3) or 10000 mg (M4) via 15 kg of dairy concentrate pellets. Calculations based on the work of Newton & Utley (1978) suggested that a melamine intake of 0.16 g/kg of live weight would not result in detrimental health effects of ruminant animals. Therefore, a 600 kg lactating dairy cow should not be at risk when consuming 100 g of melamine. In this trial, the highest melamine treatment (M4 = 10 g/d) included a 10-fold safety factor from the suggested safe amount from the work of Newton & Utley (1978) and should not pose a health risk to the cows. Treatments had no effect on DMI, milk yield or milk composition. MEL was detected in the milk 8 h after initial MEL ingestion, increased rapidly and peaked on d 3 and was undetectable after 8 d. Treatments had no effect on MEL excretion efficiencies which ranged from 1.5 to 2.1%. The mean apparent digestibility of MEL was 78%. Mean faecal and urinary MEL excretions were 22 and 54 % of ingested MEL, respectively. Higher milk, urine and faecal MEL concentrations were observed with higher levels of dietary MEL. It was concluded that MEL appeared in the milk soon after first ingestion and a withdrawal period of 8 d was required for all milk, faecal and urine samples to reach undetectable levels of MEL. Urine and faeces were the primary routes for MEL excretion. The fourth trial was conducted to determine MEL absorption by the mammary gland in lactating dairy cows through arterio-venous (A-V) difference. Five cows received 10 g of MEL/d for three consecutive days. Day 3 of the trial was selected for commencement of blood sampling as previous studies (Cruywagen et al., 2009; Shen et al., 2010; Sun et al., 2011) reported the milk melamine concentration to reach a peak on d 3 of continuous melamine consumption by dairy cows. Early on d 3, catheters were inserted into the caudal superficial epigastric vein (milk vein) and caudal auricular artery. The blood sampling period commenced after residual milk removal from the udder following oxytocin administration. Blood from both locations were collected hourly for 9 hours. Following the final blood collection, oxytocin was administered again, catheters were carefully removed and cows were milked immediately thereafter. All blood samples were centrifuged and the decanted plasma was analysed for MEL, as well as for amino acid contents to calculate mammary blood flow. The positive MEL flux (calculated from A-V difference) confirmed net absorption of MEL into the mammary gland with an efficiency of absorption of 0.29%. Melamine excretion into milk was 5.63 mg/h. The mean plasma and milk MEL concentrations were 5.2 and 3.9 mg/kg, respectively. Melamine excretion efficiency to milk, expressed as percentage of the ingested amount, was 1.47%. It was concluded that melamine ingested by cows will result in net MEL absorption by the mammary gland, but that the absorption efficiency is low. The final trial of the study aimed to determine the effects that fermentation processes during the manufacturing of cheese, yoghurt and kefir would have on their MEL content if these products were made from MEL contaminated milk. Another objective was to determine if MEL in cheese would be degraded during the curing process. Cheese, yoghurt and kefir were made from milk with a MEL content of 6.77 mg/kg. The cheese was then cured for 2 wk at 6° C. The MEL contents of the yoghurt and kefir were 6.76 and 6.78 mg/kg, respectively, indicating that the different fermentation processes used in yoghurt and kefir production had no effect on their MEL content and that MEL was not degraded during the short fermentation periods. The percentage of milk MEL partitioned to whey and cheese were 97.4 and 6.5 %, respectively. It was concluded that the different fermentation processes involved during the manufacturing of yoghurt and kefir from MEL tainted milk did not decrease the MEL concentration. The milk MEL was predominantly partitioned to whey, with little MEL transferred to cheese. It was also concluded that MEL was not degraded in cheese during a 2-wk curing period. It was finally concluded that dietary MEL is readily absorbed by dairy cows and mainly excreted via the urine. The mammary gland has a low affinity for MEL absorption and approximately 2% of ingested MEL is excreted in the milk. When cheese is made from MEL tainted milk, the majority of MEL will concentrate in the whey fraction and only 6.5% will be present in the cheese. / AFRIKAANSE OPSOMMING: Vyf proewe is gedoen om in vitro- en in vivo-degradering, uitskeiding en absorpsie parameters van melamien (MEL) na te gaan waaroor daar min of geen inligting bekend was nie. Die eerste twee proewe was in vitro-studies, uitgevoer om die mate van MEL degradeerbaarheid in rumenvloeistof na te gaan, asook die invloed van MEL op rumen-NH3 en vlugtige vetsuur (VVS)-konsentrasies. Vir beide proewe is rumenvloeistof van lakterende, rumengekannuleerde Holsteinkoeie verkry. Vir die eerste en tweede in vitro-studies, was rumenvloeistof verkry vanaf drie en twee koeie, onderskeidelik. In albei proewe is 1 g substraat in Erlen-meyerflessies afgeweeg en 100 mL inkubasiemedium bygevoeg wat uit 20 mL rumenvloeistof en 80 mL van ‘n buffermedium bestaan het. In die eerste proef is 100 mg MEL by die substraat gevoeg, sodat die aanvanklike MEL konsentrasie in die flessies 1000 mg/L was. Die flessies is by 39° C geïnkubeer vir 0 (Kontrole), 6, 24 of 48 ure, onder streng anaerobiese kondisies. Met die beïndiging van die inkubasieperiode is 100 mL van ‘n 0.2 M perchloorsuuroplossing bygevoeg om enige melamien wat nie gedegradeer was nie, op te los. In al die proewe is melamienbepalings by wyse van LC/MSMS gedoen. Melamiendegradering was laag na 6 en 24 h inkubasie (3.2 en 5.5%, respektiewelik) en teen 48 h inkubasie het dit toegeneem tot 13.6%. In die tweede proef het die flessies 0 (Kontrole), 0.2 (T1) of 0.4 mg (T2) melamien bevat. Behandeling het geen invloed op individuele of totale VVS-konsentrasies by enige van die inkubasietye gehad nie en ook nie op NH3-konsentrasies by 6 en 48 h nie. Om een of ander onverklaarbare rede het die T2-behandeling gelei tot hoër NH3-konsentrasies by 24 h. Die gevolgtrekking is gemaak dat die byvoeging van MEL geen effek op rumen NH3-konsentrasies het nie en dat die mikroorganismes in die rumen nie daartoe in staat sal wees om MEL as ‘n stikstof-bron sal kan benut nie. In die derde proef is die uitskeiding van MEL in melkkoeie ondersoek. Vyf lakterende Holsteinkoeie is ewekansig aan vyf behandelings toegeken in ‘n 5 x 5 Latynsevierkantontwerp. Gedurende elke periode het koeie die behandelings vir 7 d ontvang, gevolg deur ‘n 8 d MEL-onttrekkingsperiode. Die eksperimentele diëte is geformuleer om ‘n daaglikse MEL-inname van 0 (M0), 500 (M1), 1000 (M2), 5000 (M3) of 10000 mg (M4) per koei/dag te verseker, toegedien via 15 kg/d van ‘n suiwelkonsentraat in pilvorm. Berekeninge gebasseer op die werk van Newton & Utley (1978) stel voor dat ‘n MEL inname van 0.16 g/kg lewende massa, geen negatiewe effek op herkouers se gesondheid sal hê nie. Dus, ‘n koei wat 600 kg weeg, sal geen skade lei deur die inname van 100 g MEL nie. In hierdie proef was die hoogste MEL behandeling (M4 = 10g/d) tien keer laer as die voorgestelde veiligheidsvlak van Newton & Utley (1978). Behandeling het geen invloed op DMI, melkopbrengs of melksamestelling gehad nie. Melamien is so gou as 8 h na eerste inname in die melk waargeneem, waarna die konsentrasie vinnig toegeneem het en ‘n piek na 3 d bereik het. Behandeling het geen invloed op die uitskeidingsdoeltreffendheid van melamien in melk gehad nie en waardes het gewissel van 1.5 tot 2.1%. Die gemiddelde skynbare verteerbaarheid van MEL was 78%. Die gemiddelde mis- en uriene-MEL-konsentrasies was 22 en 54%, onderskeidelik. Hoër melk-, mis- en uriene-MEL-konsentrasies is waargeneem namate die MEL-inhoud van die diëte gestyg het. Die gevolgtrekking is gemaak dat MEL spoedig na eerste inname in die melk verskyn en dat ‘n onttrekkingsperiode van 8 d benodig word voordat melk-, mis- en uriene-MEL onwaarneembare vlakke bereik. Uriene en mis is die primêre uitskeidingsroetes van ingenome MEL. Die vierde proef is onderneem om MEL-absorpsie in die melkklier met behulp van arterio-veneuse (A-V) verskille te ondersoek. Vyf koeie het elk 10 g MEL/d vir drie agtereen-volgende dae ontvang. Dag 3 van die proef is gekies vir bloedkolleksies aangesien vorige studies (Cruywagen et al., 2009; Shen et al., 2010; Sun et al., 2011) gewys het dat melk MEL op dag 3 van MEL inname, piek konsentrasies beryk. Vroeg gedurende die oggend van d 3 is kateters in die kaudale oppervlakkige epigastriese aar (melkaar) en die kaudale aurikulêre slagaar geplaas. Die bloedtrekkingsperiode het ‘n aanvang geneem direk nadat die koeie volledig uitgemelk is na toediening van oksitosien om te verseker dat soveel as moontlik residuele melk verwyder word. Monsters van veneuse-, sowel as arteriële bloed, is 9-uurliks geneem. Na die finale bloedtrekking is oksitosien weer toegedien, die kateters is versigtig verwyder en die koeie is direk daarna weer gemelk. Al die bloedmonsters is gesentrifugeer en plasmamonsters is ontleed vir MEL, asook vir aminosuursamestelling ten einde bloedtoevoer na die uier te bereken. Die positiewe fluks (bereken van A-V verskil) het bevestig dat netto MEL absorpsie in die melkklier plaasvind, met ‘n doeltreffendheid van 0.29%. Melamienuitskeiding in die melk was teen ‘n tempo van 5.63 mg/h. Die gemiddelde plasma- en melk-MEL konsentrasies was 5.2 en 3.9 mg/kg, onderskeidelik. Die uitskeidingsdoeltreffendheid van MEL na melk, uitgedruk as persentasie van ingenome MEL, was 1.47%. Die gevolgtrekking is gemaak dat MEL wat deur koeie ingeneem word, tot netto MEL-absorpsie in die melkklier sal lei, maar dat die absorpsiedoeltreffendheid baie laag is. In die finale proef is daar gepoog om die invloed van fermentasieprosesse gedurende die vervaardiging van kaas, joghurt en kefir op die produkte se melamieninhoud na te gaan indien die produkte van melamienbevattende melk gemaak sou word. ‘n Tweede doel van hierdie proef was om te bepaal of MEL in kaas gedegradeer kan word tydens rypwording. Kaas, joghurt en kefir is gemaak van melk wat ‘n MEL-inhoud van 6.77 mg/kg gehad het. Die kaas is vervolgens vir twee weke by 6° C rypgemaak. Die MEL-inhoud van die joghurt en kefir was 6.76 en 6.78 mg/kg, onderskeidelik, wat daarop dui dat die onderskeie fermentasieprosesse wat tydens die bereiding van joghurt en kefir plaasvind, geen invloed op hul MEL-inhoud gehad het nie en dat MEL nie gedurende hierdie kort fermentasieperiodes gedegradeer is nie. Die persentasie MEL na wei en kaas versprei was 97.4 en 6.5%, onderskeidelik. Die gevolgtrekking is gemaak dat die verskillende fermentasieprosesse betrokke tydens die vervaardiging van joghurt en kefir wat van melamienbesmette melk gemaak word, nie die MEL-konsentrasie verlaag het nie. Tydens die vervaardiging van kaas, word die MEL hoofsaaklik na die weikomponent versprei en baie min na kaas. Melamien word ook nie in kaas afgebreek gedurende ‘n verouderingsproses van twee weke nie. Die finale gevolgtrekkings is gemaak dat MEL maklik deur melkkoeie geabsorbeer word en dat die hoof uitskeidingsroete via urine is. Die uier het ‘n lae affiniteit vir MEL absorpsie en ongeveer 2% van ingenome MEL is in die melk uitgeskei. Wanneer kaas van MEL besmette melk gemaak word, sal die meerderheid van die MEL in die weifraksie konsentreer, met slegs 6.5% teenwoordig in die kaas. / The Hennie Steenberg Trust Fund, the Ernst and Ethel Erickson Trust and the National Research Foundation (NRF) for their financial support

Thesis (M. Sc. (Animal Production)) -- University of Limpopo / The objectives of this study were: 1) To determine the effect of oocyte retrieval techniques (slicing and aspiration) on the quality and quantity of cattle oocytes, 2) To evaluate the effect of different concentrations of hormones on the maturational rate of cattle oocytes selected by brilliant cresyl blue staining, 3) To evaluate fertilization rate and cleavage/embryonic development of oocytes with or without cumulus cells, and 4) To compare the effect of fresh and frozen thawed semen on the fertilization rate of cattle oocytes. In Experiment 1: oocytes were recovered from abattoir derived ovaries using slicing and aspiration. The recovered oocytes were exposed for 90 minutes to 26μM of brilliant cresyl blue (BCB) stain and classified according to the colour of their cytoplasm: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). There was no difference (P>0.05) in the quality of oocytes recovered using slicing (60.7 %) or aspiration (53.7 %) techniques. In experiment 2: The BCB selected and the non-selected immature oocytes were randomly allocated into medium 199 + 10 % fetal bovine serum (FBS) maturation media. The media was supplemented with three different concentrations of hormones as treatments (T). The T1 (0.5 μg/ml of follicle stimulating hormone (FSH), 5mg/ml of luteinizing hormone (LH) and 2 μg/ml of estradiol (E2) as the control group. Then, T2 (1 μg/ml of FSH, 6 mg/ml of LH and 2.5 μg/ml of E2) and T3 (1.5 μg/ml of FSH, 7 mg/ml of LH and 4.5 μg/ml of E2). Maturation rate of oocytes was determined by the protrusion of the first polar bodies 24 hours following in vitro maturation. Treatment 2 yielded higher (P<0.05) maturation rate for both BCB+ (65.6 %) and without BCB (60.3 %) oocytes with T1 giving lower (P<0.05) maturation rate for BCB+ (22 %) and without BCB (16 %) oocytes. However, BCB- oocytes had lower (P<0.05) polar body extrusion (3.03 %, 8.1 % and 2.2 %) for T1, T2 and T3, respectively. In Experiment 3: one group of the presumptive zygotes was denuded of cumulus cells and the other group was cultured with cumulus cells. The presumptive zygotes were in vitro cultured in SOF-BSA and changed to SOF-FBS after 48 hours. High fertilization/cleavage rate was observed in oocytes cultured with cumulus cells (29.0 %) compared to the denuded oocytes (20.0 %) for 2-4 cells stage. Day 7 blastocysts were more (P<0.05) on oocytes cultured with cumulus cells (32 %) compared to denuded oocytes (13 %). In experiment 4: The matured oocytes were fertilized using fresh and frozen thawed semen. The oocytes fertilized with frozen thawed semen obtained a better number of 2-4 cell cleavage (23 %) when compared to fresh semen (19 %). Oocytes that were fertilized with frozen thawed semen also obtained higher morula (13 %) and blastocyst (8 %) compared to fresh semen with morula (3.4 %) and blastocyst (2 %). In conclusion, immature oocytes that were exposed to BCB+ and cultured in M199 supplemented with 10 % FBS, 0.5 μg/ml of FSH, 5 mg/ml of LH and 2 μg/ml of E2 had a higher (P<0.05) number of matured oocytes (extrusion of first polar body) compared to those that were not exposed to BCB (no BCB). Oocytes that were cultured with cumulus cells yielded a higher (P<0.05) number of cleaved embryos compared to the denuded oocytes. Slicing yielded a higher (P<0.05) number of oocytes, however the quality of oocytes recovered was similar compared to those recovered by the aspiration technique (P>0.05). Oocytes fertilized with frozen thawed semen yielded higher (P<0.05) number of 2-4 cell, morula and blastocyst when compared with oocytes that were fertilized using fresh semen. Keywords: ovaries, oocytes, slicing, aspiration, COCs, BCB, polar body and cattle

Thesis (M. Sc. (Animal Production)) -- University of Limpopo, 2015 / The objectives of this study were: 1) To determine the effect of oocyte retrieval techniques (slicing and aspiration) on the quality and quantity of cattle oocytes, 2) To evaluate the effect of different concentrations of hormones on the maturational rate of cattle oocytes selected by brilliant cresyl blue staining, 3) To evaluate fertilization rate and cleavage/embryonic development of oocytes with or without cumulus cells, and 4) To compare the effect of fresh and frozen thawed semen on the fertilization rate of cattle oocytes. In Experiment 1: oocytes were recovered from abattoir derived ovaries using slicing and aspiration. The recovered oocytes were exposed for 90 minutes to 26μM of brilliant cresyl blue (BCB) stain and classified according to the colour of their cytoplasm: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). There was no difference (P>0.05) in the quality of oocytes recovered using slicing (60.7 %) or aspiration (53.7 %) techniques. In experiment 2: The BCB selected and the non-selected immature oocytes were randomly allocated into medium 199 + 10 % fetal bovine serum (FBS) maturation media. The media was supplemented with three different concentrations of hormones as treatments (T). The T1 (0.5 μg/ml of follicle stimulating hormone (FSH), 5mg/ml of luteinizing hormone (LH) and 2 μg/ml of estradiol (E2) as the control group. Then, T2 (1 μg/ml of FSH, 6 mg/ml of LH and 2.5 μg/ml of E2) and T3 (1.5 μg/ml of FSH, 7 mg/ml of LH and 4.5 μg/ml of E2). Maturation rate of oocytes was determined by the protrusion of the first polar bodies 24 hours following in vitro maturation. Treatment 2 yielded higher (P<0.05) maturation rate for both BCB+ (65.6 %) and without BCB (60.3 %) oocytes with T1 giving lower (P<0.05) maturation rate for BCB+ (22 %) and without BCB (16 %) oocytes. However, BCB- oocytes had lower (P<0.05) polar body extrusion (3.03 %, 8.1 % and 2.2 %) for T1, T2 and T3, respectively. In Experiment 3: one group of the presumptive zygotes was denuded of cumulus cells and the other group was cultured with cumulus cells. The presumptive zygotes were in vitro cultured in SOF-BSA and changed to SOF-FBS after 48 hours. High fertilization/cleavage rate was observed in oocytes cultured with cumulus cells (29.0 %) compared to the denuded oocytes (20.0 %) for 2-4 cells stage. Day 7 blastocysts were more (P<0.05) on oocytes cultured with cumulus cells (32 %) compared to denuded oocytes (13 %). In experiment 4: The matured oocytes were fertilized using fresh and frozen thawed semen. The oocytes fertilized with frozen thawed semen obtained a better number of 2-4 cell cleavage (23 %) when compared to fresh semen (19 %). Oocytes that were fertilized with frozen thawed semen also obtained higher morula (13 %) and blastocyst (8 %) compared to fresh semen with morula (3.4 %) and blastocyst (2 %). In conclusion, immature oocytes that were exposed to BCB+ and cultured in M199 supplemented with 10 % FBS, 0.5 μg/ml of FSH, 5 mg/ml of LH and 2 μg/ml of E2 had a higher (P<0.05) number of matured oocytes (extrusion of first polar body) compared to those that were not exposed to BCB (no BCB). Oocytes that were cultured with cumulus cells yielded a higher (P<0.05) number of cleaved embryos compared to the denuded oocytes. Slicing yielded a higher (P<0.05) number of oocytes, however the quality of oocytes recovered was similar compared to those recovered by the aspiration technique (P>0.05). Oocytes fertilized with frozen thawed semen yielded higher (P<0.05) number of 2-4 cell, morula and blastocyst when compared with oocytes that were fertilized using fresh semen. Keywords: ovaries, oocytes, slicing, aspiration, COCs, BCB, polar body and cattle