The Gram-negative bacterium 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 can live on long chain fatty acids as its sole carbon source. However, the rate of fatty acid uptake was reduced after starvation (Mangroo, 1992). In this study, it was found that this starvation effect of reducing oleate uptake rate could only be observed in the initial minute post starvation. The starvation effect was not due to the depletion of its substrates ATP or reduced coenzyme A. Neither was the effect caused by a lowered acyl-CoA synthetase level. This reduction in oleate uptake rate can be reversed by incubation in oleate. It has been reported that lactate activated oleate uptake in 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 (Mangroo and Gerber, 1993). In the present study, it was discovered that when [9, 10-³H]oleate was used as the radioactive tracer in uptake assay, the majority of the radioactivity effluxed from the cells. It was found that lactate did not have an effect on the overall oleate uptake. Its apparent activation on oleate uptake was due to the reduction in the efflux rate of radioactive probes, instead of increasing the uptake rate of oleate. Acyl-CoA synthetase in 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 has always been described as a protein of around 45 kDa (Kameda and Nunn, 1981; Kameda 𝘦𝘵 𝘢𝘭., 1986). However, the 𝘧𝘢𝘥𝘋 gene that encodes the 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 acyl-GoA synthetase predicts a 62 kDa protein (Black 𝘦𝘵 𝘢𝘭., 1992; Fulda 𝘦𝘵 𝘢𝘭., 1994). This new form of acyl-CoA synthetase has been partially purified. This form, which has a size of around 62 kDa, can be converted to its 45 kDa counterpart in a Triton X-1 00 and temperature dependent manner. This induced processing of the 62 kDa enzyme was found to be inhibited by oleate, a natural substrate of acyl-CoA synthetase. The two forms of acyl-CoA synthetase have also been shown to be immunologically related. A much simpler purification scheme was developed for the 45 kDa acyl-CoA synthetase using the membrane bound form of acyl-CoA synthetase. The selective extraction of the 62 kDa acyl-CoA synthetase from the membrane, together with the induced processing into the 45 kDa form has provided the basis for this comparatively simple process. The availability of the expression system made the low yield associated with this simple process acceptable. Using oleate as substrate, the Kₘ and Vₘₐₓ for the 45 kDa acyl-CoA synthetase were determined to be 85 ± 18μM and 1550 ± 165 nmole/min/mg protein respectively, whereas the Kₘ and Vₘₐₓ for the 62 kDa enzyme were determined to be 38 ± 12μM and 633 ± 79 nmole/min/mg protein respectively. This suggested that the 62 kDa enzyme has higher affinity towards oleate than its 45 kDa counterpart under standard assay conditions while the ratio of Vₘₐₓ /Kₘ remained relatively constant. Evidence suggesting Triton X-1 00 activates acyl-CoA synthetase by providing a surface for catalysis was presented. This interaction between acyl-CoA synthetase and the Triton X-1 00 I oleate mixed micelle could be affected by the surface charge of the mixed micelle. / Thesis / Master of Science (MS)

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The objective of two studies was to manipulate the essential fatty acid content of turkey semen by enhancing the dietary levels of either n-3 polyunsaturated fatty acids (PUFA) or n-6 PUFA and determine the effect on fertility. In 1999 (Trial 1), and again in 2000 (Trial 2), Large White tom turkeys, 37 weeks of age, were fed one of three diets substituted with chicken fat, soybean oil, or menhaden fish oil. Chicken fat provided the industry's standard ratio of n-6 to n-3, soybean oil provided a greater ratio of n-6 to n-3, and fish oil provided a lower ratio of n-6 to n-3. Contemporary hens were inseminated weekly with semen collected from each group of toms. The effects of dietary lipids on tom body weights, fertility, motility, perivitelline layer sperm penetration percent, and live vs. dead sperm were analyzed. Whereas body weight increased linearly from 31 to 56 weeks of age (WOA), there was no effect of dietary treatment. As measured by the Accudenz® procedure, there were differences in sperm motility due to dietary treatment during 48 and 51 WOA during Trial 1. During Trial 2, sperm motility differences were observed at 53 WOA with the soybean oil-treated toms having the largest absorbance reading and the chicken fat-treated toms having the largest absorbance reading during 56 WOA. The live vs. dead sperm cells during Trial 1 revealed differences among the toms prior to treatment and post treatment. No dietary effects on percent live vs. dead sperm cells were observed during Trial 2. Once per mo, eggs were collected for a one-week period to analyze for sperm penetration of the perivitelline layer. In Trial 1, sperm from toms fed chicken fat produced more penetrations (holes) during 36, 48, and 52 WOA. In Trial 2, sperm penetration values were lower for toms fed fish oil during 42, 47, and 51 WOA. Whereas there were significant differences in fertility, hatch of total eggs, and hatch of fertile eggs among treatments in Trial 1, a bacterial contamination on the farm during weeks seven through fourteen may have contributed to these findings. No significant differences due to treatment were found in these parameters during the second study. The fatty acid analysis of spermatozoa collected at the conclusion of Trial 2 revealed significant differences in total n-3 and total n-6 content, leading to significant differences in the ratio of total n-6 to total n-3. The mixed results indicated the fertilizing ability of domesticated turkey spermatozoa may not be affected by the n-6 to n-3 ratio in the diet of the tom. / Master of Science

Volatile fatty acids (VFA) are important products of ruminal fermentation. The VFA are not only the major source of energy to the ruminant animals but also influence methane production in the rumen. Therefore it is important to understand mechanism controlling VFA production and to depict VFA production in a model. This will allow us to devise strategies to enhance energy utilization and reduce methane production in ruminant livestock. An evaluation of a mechanistic model in predicting VFA production was conducted and equations were introduced into the model to improve the predictions. Later a continuous culture experiment was conducted to test the hypothesis on which those equations were based on. A mechanistic model -" Molly, was evaluated using a dataset with reported VFA production rates. The results of residual error analysis indicated that the root mean square prediction errors (RMSPE) were 63, 63, and 49% for acetate, propionate and butyrate, respectively. An assessment from two studies reporting VFA production revealed a potential of reducing errors of prediction by representing interconversion among VFA. In the second study, equations based on thermodynamics influence of pH and VFA concentration were introduced in the model to represent interconversion among VFA. The parameters for de novo VFA production and VFA absorption were re derived with (VFAInt) and without (BASE) the new interconversion equations. There were some improvements in the VFA concentration predictions but the improvements were both in VFAInt and BASE models. The RMSPE of VFA production were still above 50% for acetate, propionate and butyrate. The larger errors of predictions were attributed to measurement variation in VFA production literature, or possible incorrect rate constants for interconversion equations. Finally, a third study was conducted to assess the effect of pH, and VFA concentration on VFA and methane production in continuous culture. The treatments consisted of control, 20 mmol/d acetate infusion (INFAC), 7 mmol/d propionate infusion (INFPR), and low pH (LOWPH). Individual isotopes of acetate, propionate and butyrate were infused in the fermenters to estimate interconversions among VFA. With LOWPH treatment methane emission was reduced whereas production of propionate was increased. Hydrogen production was higher in INFAC indicating that some of the acetate could have been degraded to CO2 and H2. It was estimated that around 3 % of de novo acetate was converted to propionate and 9 % to butyrate. Exchange between propionate and butyrate was insignificant and below 1% of de novo production of either VFA. However, treatments did not affect interconversion rates among VFA. These results indicated that pH and VFA concentration do not have thermodynamic influence on VFA interconversion as hypothesized. / Ph. D.

This investigation was undertaken in order to determine the characteristics of crude fatty acids, recovered from the resin soap wastes of kraft pulp manufacture, so that efficient distillation units may be designed for distillation of the crude fatty acids. It was found that the crude fatty acids could be distilled at a maximum absolute pressure of 7 m.m., and at a temperature range of 400° to 520ºF. At this temperature range approximately 70% of the total can be recovered as distillate without impairing the quality of the distillate. The remaining 30% is removed as pitch. It was found that when 70% of the total has been distilled approximately 85% of the fatty acids and 80% of the abietic acid originally present have been recovered Crude fatty acids corroded to a marked extent all of the feasible alloys and metals of construction with the exception of the stainless steel containing not less than 20% chromium and not less than 9% nickel. It was found that the most suitable alloy was a chromium-nickel-iron alloy containing 29% chromium and 9% nickel, the balance being iron. / Master of Science

Increased consumer interest in omega-3 fatty acids (FA) has led to novel foods with added omega-3 FA. Additional information regarding omega-3 FA fate within foods is needed for improving quality and stability. This research modeled DHA, an omega-3 FA, fate and explored means of preventing degradation and oxidation of FA in algal oil and mozzarella cheese. In algal oil, TBHQ (synthetic antioxidant) at 0.0175g/g algal oil prevented DHA degradation for at least 6 weeks, and mixed tocopherols (natural antioxidant) at 400ppm prevented DHA degradation and oxidation for about 4 weeks. DHA degradation in algal oil was modeled by an autocatalytic equation. The fate of DHA from algal oil in mozzarella cheese was also modeled by an autocatalytic equation. In an effort to prevent DHA degradation and oxidation, mixed tocopherols were added. The optimum combination of those tested was found, using a response surface design, to be 3% algal oil with 110ppm mixed tocopherols for maximum DHA and minimum oxidation over 2 weeks. This algal oil/antioxidant combination in mozzarella cheese successfully prevented oxidation and DHA degradation over 3 weeks of storage. Approximately 0.1g DHA may be consumed from a 28g serving of this cheese. Approximately 0.5-18 servings of this cheese are equivalent to DHA consumed from a 3oz serving of fish, depending on fish type. Sensory evaluation tests found consumers could distinguish between mozzarella cheese with/without algal oil. Results from this study improve understanding of omega-3 FA behavior in mozzarella cheese and provide a means for preserving quality and nutrition. / Master of Science

The objective of this study was to determine the effects of dietary supplementation of eicosapentanoic (EPA) and docosahexanoic acids (DHA) on reproduction in beef females. In experiment 1, cows (n = 31) were individually fed rumen protected fish meal (FM) or no fish meal (C) supplements. Estrus was synchronized and ovulation induced on d 37. Ovarian follicular growth and diameter were determined by ultrasound on d 35 and d 37. Serum progesterone (P4) profiles were analyzed on d 37 through d 52. On d 52 cows were cannulated, primed with estradiol-17&#946; at -240 min, and stimulated to release PGF2&#945; by oxytocin injection at 0 min with blood sampled every 15 min from -30 min to 240 min. Supplement type did not affect (P > 0.05) follicular diameter, follicular growth or P4 concentrations. In cows fed FM, prostaglandin metabolite (PGFM) concentrations tended (P &#8804; 0.10) to be reduced at 0, 30, and 60 min. In experiment 2, crossbred heifers (n = 214) received FM or C concentrates with corn silage from 30 d before estrous synchronization until 14 d after artificial insemination (AI). Serum fatty acid profiles were determined in five heifers from each group . Estrus detection and AI were conducted from d 37 through d 39. Dietary treatment increased (P < 0.05) EPA and DHA concentrations. Dietary treatment did not affect estrus response or AI conception rates and pregnancy rate. Supplementation of FM increased EPA and DHA concentrations but did not affect reproductive factors. / Master of Science

Background: Cardiovascular disease (CVD) is the leading global cause of death. CVD risk factors are considered intermediaries for the association between dietary fatty acids and CVD. Raised plasma total cholesterol, low density lipoprotein (LDL) cholesterol, raised triglycerides and decreased levels of high density lipoprotein (HDL) cholesterol, as well as reduced fibrinolytic potential (measured as increased clot lysis time) are known risk factors for CVD. Plasminogen activator inhibitor-1 (PAI-1) is a major inhibitor of the fibrinolytic process and an elevated PAI-1 level is therefore considered to be a potential risk factor for CVD. The growing number of controversies around the role that fat intake (more specifically the type of dietary fat) plays in CVD risk, is making it increasingly difficult for consumers and practitioners alike to form conclusions, and make recommendations and decisions regarding fat intake. Knowledge of the intake of individual fatty acids, fatty acid status (as opposed to subgroups of fat such as polyunsaturated fatty acids) and their associations with blood lipids, PAI-1act and fibrinolytic potential is lacking in black South Africans and other populations. Therefore we aimed to investigate dietary fatty acid intake, as well as plasma phospholipid fatty acid status and their associations with blood lipids, PAI-1act and clot lysis time, as a marker for fibrinolytic potential. Methods: Cross-sectional data analysis within the Prospective Rural Urban Epidemiology (PURE) baseline study of apparently healthy black South African men and women (n=1950, 35– 70 years) from rural and urban areas in the North West Province, from whom dietary data were collected. Blood lipid analyses, as well as laboratory analyses of fibrinolysis markers such as PAI-1act and clot lysis time were also performed. Plasma phospholipid fatty acid extraction and isolation were performed on a random subsample (n = 716). Results: The intake of individual fatty acids was significantly higher in urban than rural dwellers. However, the intake of omega-3 polyunsaturated fatty acids was below recommendations in all groups (rural and urban males, and rural and urban females). Total cholesterol and LDL cholesterol were higher in females than in males, with no rural‒urban differences. Intake of alpha-linolenic acid was positively associated with total cholesterol (β=0.143) and triglycerides (β=0.256) in males. The risk of having elevated LDL cholesterol also increased with increased intake of alpha-linolenic acid (OR 1.49, 95% CI 1.04, 2.14). In females, dietary arachidonic acid and eicosapentaenoic acid (EPA) were positively associated with total cholesterol and LDL cholesterol, whereas docosahexaenoic acid (DHA) was negatively associated with total cholesterol and LDL cholesterol. Dietary alpha-linolenic acid was positively correlated with plasma EPA (males r = 0.19, p = 0.002, females r = 0.25, p < 0.001) and DHA (males r = 0.33, p < 0.001, females r = 0.30, p < 0.001). Plasma DHA was positively associated with triglycerides in males (β = 0.410, p< 0.001) and in females (β = 0.379, p< 0.001). PAI-1act was positively associated with clot lysis time, and plasma myristic acid and DHA were positively associated with PAI-1act in females. However, these fatty acids were not associated with clot lysis time. Different types of plasma fatty acids were associated with PAI-1act than with clot lysis time. Plasma alpha-linolenic acid (β = 0.123, P = 0.037), mead acid (β = 0.176, P = 0.019), arachidonic acid (β = 0.253, 0.025) and omega-3 docosapentaenoic acid (omega-3 DPA) (β = 0.224, P = 0.002) were positively associated with clot lysis time, while both myristic acid (β = - 0.130, P = 0.016) and EPA (β = -0.131, P = 0.021) were negatively associated with clot lysis time in male subjects. Plasma oleic acid (C18:1n9) (β = -0.411, P = 0.001) and omega-6 DPA (C22:5n6) (β = -0.285, P = 0.001) were negatively associated with clot lysis time, while dihomogamma- liolenic acid (DGLA) (C20:3n6) were positively associated (β = 0.178, P = 0.001) with clot lysis time in females. Conclusions: These results suggest that specific individual dietary fatty acids might be associated with blood lipids in males differently than in females, irrespective of rural or urban dwelling. It is not known however, if associations would still be present under conditions of greater intake of alpha-linolenic acid. Our results further suggest that a higher percentage of alpha-linolenic acid might be converted to DHA in this population with low intake of essential and long-chain polyunsaturated fatty acids compared to populations with a high intake of these fatty acids. These results suggest that plasma phospholipid fatty acids should not be used in isolation as biomarkers for intake of fat, without taking dietary intake data into consideration also. Associations between fatty acids and clot lysis time might be independent from PAI-1act. The association between mead acid and clot lysis time indicates that clot lysis time might increase with an essential fatty acid deficiency. This may be of particular concern in this population with a documented lower fat intake. Because the study design of this study is crosssectional, it is not able to determine cause-and-effect, and results should therefore be verified with a randomised controlled trial. / PhD (Nutrition), North-West University, Potchefstroom Campus, 2015