Effect of synthetic and bovine milk conjugated linoleic acid (CLA) on immune function : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Nutrition Science at Massey University

CLA is a collective name for a mixture of positional and geometrical isomers of linoleic acid (c-9, c-12-octadecadioenic acid) which possess conjugated double bonds. CLA occurs in a variety of foods, but is present at higher concentrations in products from ruminants. Milk fat is the richest natural source of CLA. The objective of this research was to examine the immunomodulatory properties of CLA (both synthetic and natural CLA derived from bovine milk fat). Two experiments were conducted at the Milk and Health Research Centre, Massey University, Palmerston North, New Zealand. The aim of the first experiment was to investigate the dose effect of different concentrations of synthetic CLA (Tonalin) on immune function. Mice were fed either skim milk powder based diet or the same diet supplemented with 0.1, 0.25, 0.5, 1.0 or 2.0% synthetic CLA (Tonalin) by weight. Animals were immunised orally with a mixture of polio vaccine in sodium bicarbonate (25μl) and subcutaneously with Fluvax and Tetanus toxoid vaccine on days 7 and 21. After 4 weeks feeding, mice were euthanased by isoflurane overdose. Various immune parameters were measured and the results showed that synthetic CLA (Tonalin) enhanced a range of immune functions. Synthetic CLA stimulated PHA induced T lymphocyte proliferation at 0.25, 0.5 and 1.0% as compared with the control group (p < 0.05). Synthetic CLA enhanced macrophage phagocytosis in a dose dependent manner. Synthetic CLA enhanced antibody responses (mucosal and systemic) to vaccines (polio vaccine, Fluvax and Tetanus toxoid). Natural killer cell activity was significantly enhanced in mice fed 0.25 and 0.5% CLA. In general, 0.25% CLA was regarded as the best CLA level which achieved optimal immunoregulating effects. The aim of the second experiment was to examine the effect of natural CLA derived from milk fat on immune responses in mice. Mice were fed a skim milk powder (SMP) based diet. The control diet was skim milk powder only, without any CLA or milk fat supplementation. The dietary treatments were: ordinary milk fat, fractionated milk fat (1st stage), 0.2% synthetic CLA (Tonalin) and CLA enriched milk fat. Animals were fed these diets for 28 days. Mice were immunised orally with a mixture of polio vaccine/ovalbumin/cholera toxin in sodium bicarbonate on days 7, 14 and 21 and subcutaneously with Fluvax and ADT (Diptheria and Tetanus toxoid vaccine) on days 7 and 21. Natural CLA was found to stimulate PHA and Con A induced T lymphocyte blastogenesis. Supplementation with natural CLA also led to increased antibody responses to vaccines and increased CD25+ populations in peripheral blood in mice. Natural CLA also enhanced macrophage phagocytosis. Synthetic CLA enhanced a range of immune functions which is consistent with the results in the first experiment. It is noted that although the CLA content is low in milk fat, the natural CLA derived from milk fat expressed potent effects in enhancing the growth of immune cells and promoting a range of immune functions in mice. Key words: conjugated linoleic acid (CLA), lymphocyte, macrophage, immunity, milk fa

Comparison of conjugated linoleic acid (CLA) content in milk of ewes and goats with the same dietary regimen

Milk fat is an important source of potential anticarcinogens named conjugated linoleic acid (CLA). The c9, t11-CLA is the major isomer and it is produced by ruminal hydrogenation of linoleic acid that leads first to vaccenic (11t-18:1) and finally to stearic acid (18:0). An alternative CLA pathway is related to the action of the mammary Δ9-desaturase enzyme on 11t-18:1. Diet is considered the main factor that influence the CLA concentration in milk fat. Differences in CLA content of sheep (Antongiovanni et al., 2002) and goats milk (Nudda et al., 2002) have been hypothesized, but results are difficult to compare due to different experimental condition in which the trials are carried out. The aim of this work is to compare the composition of milk fatty acids and CLA content in goats and sheep maintained in the same environmental condition and dietary regimen

The effect of strategic supplementation with trans-10,cis-12 conjugated linoleic acid on the milk production, estrous cycle characteristics, and reproductive performance of lactating dairy cattle

Peer-reviewedThis is the author’s version of a work that was accepted for publication in Journal of Dairy Science. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Journal of Dairy Science, Volume 95, Issue 5, May 2012, Pages 2442-2451: DOI 10.3168/jds.2011-4632The objective was to determine the effects of a protected (lipid-encapsulated) conjugated linoleic acid (LE-CLA) supplement on milk production, estrous cycle characteristics, and reproductive performance in lactating dairy cows on a pasture-based diet. Spring calving dairy cows (n = 409) on a single pasture-based commercial dairy farm were used in a completely randomized block design. Cows were assigned to 1 of 2 dietary supplements [LE-CLA (n = 203) or no supplement (control, n = 206)]. The LE-CLA cows received 51 g/d of a lipid supplement containing 5 g of both trans-10,cis-12 and cis-9,trans-11 CLA from 0 to 60 d in milk. Milk samples were collected 3 times weekly, and each sample was analyzed for progesterone to determine the interval to first ovulation and estrous cycle characteristics. Milk yield and concentrations of fat, protein, and lactose were measured every 2 wk. Cows were inseminated following visual observation of estrus. The breeding season commenced on April 8, 2009 and continued for 16 wk. Transrectal ultrasonography was carried out at 30 to 36 d and 60 to 66 d post-AI to diagnose pregnancy. The LE-CLA treatment resulted in a decrease in milk fat concentration (36.9 ± 0.06 g/kg vs. 30.7 ± 0.06 g/kg for control and LE-CLA, respectively) and yield (0.91 ± 0.02 kg/d vs. 0.84 ± 0.02 kg/d for control and LE-CLA, respectively); however, milk yield was increased by LE-CLA supplementation (24.7 ± 0.7 kg/d vs. 27.2 ± 0.7 kg/d for control and LE-CLA, respectively), resulting in no overall difference in milk energy output. No effect of LE-CLA was observed on any estrous cycle characteristics or measures of reproductive performance. These results support that in pasture-based systems of dairy production, where energy intake limits milk production, energy spared by CLA-induced milk fat depression is partitioned toward increasing milk yield rather than toward body reserves

Enzymatic approach of linoleic acid ruminal biohydrogenation

Ruminal biohydrogenation (BH) corresponds to a microbial reduction of dietary unsaturated fatty acid. The linoleic acid (C18:2) BH is divided into three steps: first an isomerisation into conjugated linoleic acids (CLA), then a reduction producing mainly trans-octadecenoic acids (trans-C18:1), and a final reduction producing stearic acid (C18:0). Isomerisations of CLA and trans-C18:1 can lead to a number of positional and geometrical isomers. The control of BH reactions is of interest for researchers because BH directly affects the composition of fatty acids of milk and meat. In order to better understand C18:2 BH and its variations, the development of an enzymatic approach is necessary to ascertain if the action of modulators affects the bacterial enzyme activity or ruminal bacteria. The aim of this study was to investigate the C18:2 BH capacity of ruminal content after inactivation of bacteria by chloramphenicol (Cm), an inhibitor of protein synthesis in prokaryotes. The BH of C18:2 produced mainly cis9,trans11-CLA and trans10,cis12-CLA, and trans11-C18:1 and trans10-C18:1 isomers, as previously described (Jouany et al., 2007). The increase in cis12-C18:1 came from reduction of trans10,cis12-CLA, that of trans6+7+8-C18:1 from the reduction of minor CLA isomers not quantified in this study, like trans8,trans10-CLA (Shingfield et al., 2008). The trans11 pathway was rapid: the cis9,trans11-CLA production was maximal at about 1h of incubation while trans11-C18:1 accumulated throughout incubation. On the other hand, trans10 pathway was slow: trans10,cis12-CLA regularly increased during incubation, so that it was more abundant than cis9,trans11-CLA after 3h incubation, and trans10-C18:1 only began to increase after 2h of incubation. The amount of C18:0 began to increase in the media when trans11-C18:1 concentration was over 0.05 mg/mL. Such evolution of fatty acids involved in C18:2 BH was similar to that reported in vitro with living ruminal microorganisms by Harfoot et al. (1973) and Jouany et al. (2007). So, this enzymatic approach using Cm could be an interesting and valid method to study C18:2 BH, however 3h of incubation were not sufficient to study the final reduction

A serological investigation of caseous lymphadenitis in four flocks of sheep

A double antibody sandwich ELISA developed by ID-DLO, Lelystad to detect Corynebocterium pseudotuberculosis infection was used on 329 sheep from four pedigree Suffolk flocks in which clinical cases of caseous lymphadenitis (CLA) had occurred. At subsequent necropsy, typical CLA lesions were seen in 133 sheep, and the diagnosis was confirmed on culture. Lesions were most commonly seen in lungs (n = 46), parotid lymph nodes (n = 44), prescapular lymph nodes (n = 38) and mediastinal lymph nodes (n = 31). The sensitivity of the ELISA test for detecting culture-positive sheep was 0.88, while the specificity of the test was 0.55. The antibody ELISA detected 87.5 per cent of sheep that had CLA lesions restricted to internal organs only. It was concluded that the ELISA test has a valuable role in detecting sheep with both clinical and subclinical CLA

The effect of synthetic and bovine conjugated linoleic acid on energy balance : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Nutritional Science at Massey University, Palmerston North, New Zealand

Conjugated linoleic acid (CLA) is biologically active and has altered body composition in experimental animals. Dietary supplementation with synthetic CLA reduced body fat in mice and rats in a number of studies. The CLA used in previously published research contained mixed isomers, the majority of which were 9cl 1t-CLA and 10t12c-CLA. The biologically active isomer at the time of starting the trials described in this thesis was assumed to be 9cl 1t-CLA, due to the prevalence of this isomer in biological tissues. The two trials in this thesis were designed to investigate the effect of dietary CLA on energy balance. In the first (refer Abstract, section 2.1), synthetic CLA reduced body fat in male BALB/c mice in a dose response manner, over the range 0.25 to 1.0% w/w CLA in the diet. High levels (1.0% and 2.0%) caused a reduction in growth. In the second (refer Abstract, section 3.1) dietary treatments supplemented with synthetic CLA, or bovine CLA in milk fat, at levels similar to the 0.25% w/w synthetic CLA treatment found to be effective in reducing body fat in mice, had no effect on energy balance in female Sprague-Dawley rats. The CLA in milk fat contains approximately 86% of the 9cl 1t-CLA isomer while synthetic CLA contains approximately 37%.9cl 1t-CLA and 46 % 10t12c-CLA. Results from these two trials support recent evidence from research demonstrating 10t12c-CLA is the biologically active isomer, in relation to energy metabolism and body composition. 9cl 1t-CLA is the prevalent isomer of CLA found in the human diet. The CLA used previously published research was chemically synthesised and contained a considerably higher proportion of 10t12c-CLA then found in human food sources. PREVIOUS PUBLICATION: The study described in Chapter 2 has been previously published as an abstract and displayed as a poster presentation at the Pacific Partners in Nutrition Conference, held at Auckland, New Zealand, September, 1999 (Hayman, et al., 1999)

Conjugated linoleic acid reduces permeability and fluidity of adipose plasma membranes from obese Zucker rats

NOTICE: this is the author’s version of a work that was accepted for publication in Biochemical and Biophysical Research Communications. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Biochemical and Biophysical Research Communications. July 2010; 398 (2): 199-204.Conjugated linoleic acid (CLA) is a dietary fatty acid frequently used as a body fat reducing agent whose effects upon cell membranes and cellular function remain unknown. Obese Zucker rats were fed atherogenic diets containing saturated fats of vegetable or animal origin with or without 1% CLA, as a mixture of cis(c)9,trans(t)11 and t10,c12 isomers. Plasma membrane vesicles obtained from visceral adi- pose tissue were used to assess the effectiveness of dietary fat and CLA membrane incorporation and its outcome on fluidity and permeability to water and glycerol. A significant decrease in adipose membrane fluidity was correlated with the changes observed in permeability, which seem to be caused by the incor- poration of the t10,c12 CLA isomer into membrane phospholipids. These results indicate that CLA supple- mentation in obese Zucker rats fed saturated and cholesterol rich diets reduces the fluidity and permeability of adipose membranes, therefore not supporting CLA as a body fat reducing agent through membrane fluidification in obese fat consumers

Effects of fat source and dietary sodium bicarbonate plus straw on the conjugated linoleic acid content of milk of dairy cows

The effects of fat source (0.7 kg of fatty acids from extruded soybeans or palmitic acid), of sodium bicarbonate (0.3 kg) plus straw (1 kg) and the interaction of these treatments on the content of conjugated linoleic acid (CLA) in the milk of dairy cows were examined. During nine weeks a group of 10 cows received a ration with palmitic acid and bicarbonate plus straw (ration PAB). During three periods of three weeks a second group of 10 cows received successively a ration with extruded soybeans and bicarbonate plus straw (ration ESB), a ration with palmitic acid without bicarbonate or straw (ration PA), and a ration with extruded soybeans without bicarbonate or straw (ration ES). Rations ES and ESB increased the content of polyunsaturated fatty acids in milk, but decreased milk fat content, compared to rations PAB and PA. Ration ESB led to the greatest milk CLA content, by a synergy between the high amount of dietary fat, and the action of bicarbonate plus straw, favouring trans11 isomers of CLA and C18:1, presumably via a ruminal pH near neutrality. Ration ES favoured trans10 isomers, not desaturated in the mammary gland, so that the milk CLA content was lower than with ration ESB, and resulted in the lowest milk fat content. In conclusion, a ration supplemented with both extruded soybeans and bicarbonate plus straw, was an efficient way to increase the CLA content in the milk of dairy cows