Considerable interest has focused on the influence of dietary fat quality on cardiovascular disease (CVD) risk. Increasingly, novel dietary oils rich in oleic acid and alpha-linolenic acid (ALA) are being developed and marketed with an aim to improve fatty acid intakes and reduce CVD risk. The objective of this research was to investigate the efficacy of high-oleic canola oil (HOCO) alone, or blended with flaxseed oil (FXCO), on traditional and emerging clinical biomarkers of CVD risk. An additional aim was to study the influence of dietary and genetic factors on metabolism of 13C-ALA to long-chain PUFA. Using a diet-controlled randomized crossover design, thirty-six hypercholesterolaemic subjects consumed three isoenergetic diets for 28 days each containing ~36% energy from fat, of which 70% was provided by HOCO, FXCO, or a Western dietary fat blend (WD; control). Endpoint measures revealed reductions (P<0.001) in serum lipid concentrations, including a 7.4% and 15.1% decrease in LDL-cholesterol after HOCO and FXCO diets, respectively, as compared with the WD control. Moreover, a reduction (P=0.023) in plasma E-selectin concentration was found after the FXCO diet compared with the WD control. Consumption of the dietary oils failed to alter whole-body fat oxidation or energy expenditure, nor lead to alterations in body composition. FXCO diet increased (P<0.001) plasma ALA ~5-fold, EPA ~3-fold, and DPA ~1.5-fold, but did not modulate DHA levels compared with the WD control. At 24 and 48 hours the amount of administered 13C-ALA recovered as plasma 13C-EPA and 13C-DPA was lower (P<0.001) after FXCO diet compared with HOCO and WD diets, suggesting decreased ALA conversion efficiency with very high intakes of dietary ALA. No difference in plasma 13C-DHA enrichment was observed across diets. Moreover, minor alleles of selected single nucleotide polymorphisms in the FADS1/FADS2 gene cluster were associated with reduced (P<0.05) plasma fatty acid compositions and apparent conversion of 13C-ALA. However, increased consumption of ALA in the FXCO diet compensated for lower levels of EPA in minor allele homozygotes. Taken together, substitution of dietary fats common to WD with both HOCO and FXCO represents an effective strategy to target several biomarkers for CVD risk reduction.

It is becoming increasingly evident that the development of atherosclerotic coronary heart disease (CHD) can largely be regulated by lifestyle and dietary choices. The type of fatty acids regularly consumed may promote or prevent atherogenesis. Flaxseed, the richest plant source of the omega-3 fatty acid alpha-linolenic acid (ALA) is thought to protect against atherosclerotic disease. However, the mechanism(s) by which flaxseed exerts these anti-atherogenic effects requires further investigation. Alternatively, there are dietary fatty acids that are thought to induce significant deleterious effects upon our cardiovascular health. Epidemiological evidence associates dietary trans fatty acids (TFAs) with atherosclerotic CHD. This evidence has largely focused on the main source of TFAs in the North American diet, industrially hydrogenated vegetable shortening (iTFAs). It is assumed that TFAs stimulate atherosclerosis but the only studies to date have shown no effect of TFAs on atherosclerosis. Even less is known of the impact of naturally occurring TFAs from dairy and meat products of ruminant animals (rTFAs) on atherosclerotic disease. We investigated the effects of flaxseed supplementation on atherosclerosis and vascular function in two animal models, the hypercholesterolemic rabbit and the cholesterol fed, low density lipoprotein receptor (LDLr-/-) deficient mouse. New Zealand White rabbits and LDLr-/- mice were fed a diet containing flaxseed in the absence or presence of dietary cholesterol for a period of 6 to 24 weeks. We found that dietary flaxseed inhibits the atherogenic effects of a high cholesterol diet in both animal models. The anti-atherogenic effect was achieved in the mouse model through a capacity to lower circulating cholesterol levels and at a cellular level by inhibiting cell proliferation and inflammation. This reduction is also associated with an improved vascular relaxation response as demonstrated in the rabbit model. We also investigated the effects of consuming TFAs from two sources, industrially hydrogenated iTFAs rich in elaidic TFA (C18:1t-9) or naturally-occurring ruminant rTFAs rich in vaccenic TFA (C18:1t-11), on atherosclerotic development in the LDLr-/- mouse in the presence or absence of elevated dietary cholesterol. Our results demonstrate that consuming iTFAs dose dependently initiates atherosclerotic development but not beyond the effects of dietary cholesterol alone. However, consuming rTFAs rich in vaccenic acid protects against hyperlipidemia and atherosclerosis in the presence or absence of dietary cholesterol. The effects of combining dietary flaxseed and iTFAs in the diet were also examined in this model. Adding whole ground flaxseed or flaxseed oil (ALA) to diets containing low and high doses of iTFAs completely prevented atherosclerotic development in the absence of dietary cholesterol. Flaxseed was also able to partially prevent atherosclerosis caused by iTFAs and cholesterol. Our results suggest that the omega-3 ALA fatty acid rich content of flaxseed is mainly responsible for the anti-atherogenic effects of flaxseed. Our results highlight potential mechanisms for the beneficial effects of dietary flaxseed and the mixed effects of TFAs on cardiovascular health and underscore the need for further basic and clinical investigations.

Dietary flaxseed has cardioprotective effects that may be achieved through its rich content of the omega-3 fatty acid, alpha linolenic acid (ALA). We investigated the effects of dietary flaxseed both with and without an atherogenic cholesterol-enriched diet to determine the effects of dietary flaxseed on the expression of the adipose cytokines leptin and adiponectin. Rabbits were fed one of four diets: a regular (RG) diet, or a regular diet with added 0.5% cholesterol (CH), or 10% ground flaxseed (FX), or both (CF) for 8 weeks. Levels of leptin and adiponectin expression were assessed by RT-PCR in visceral adipose tissue. Consumption of flaxseed significantly increased plasma and adipose levels of ALA. Leptin, but not adiponectin, mRNA expression was lower in CH animals and was elevated in CF animals. Changes in leptin expression were strongly and positively correlated with adipose ALA levels and inversely correlated with levels of en face atherosclerosis. Our data demonstrate that the type of fat in the diet as well as its caloric content can specifically influence leptin expression. The findings support the hypothesis that the beneficial cardiovascular effects associated with flaxseed consumption may be related to a change in leptin expression.

Considerable interest has focused on the influence of dietary fat quality on cardiovascular disease (CVD) risk. Increasingly, novel dietary oils rich in oleic acid and alpha-linolenic acid (ALA) are being developed and marketed with an aim to improve fatty acid intakes and reduce CVD risk. The objective of this research was to investigate the efficacy of high-oleic canola oil (HOCO) alone, or blended with flaxseed oil (FXCO), on traditional and emerging clinical biomarkers of CVD risk. An additional aim was to study the influence of dietary and genetic factors on metabolism of 13C-ALA to long-chain PUFA. Using a diet-controlled randomized crossover design, thirty-six hypercholesterolaemic subjects consumed three isoenergetic diets for 28 days each containing ~36% energy from fat, of which 70% was provided by HOCO, FXCO, or a Western dietary fat blend (WD; control). Endpoint measures revealed reductions (P<0.001) in serum lipid concentrations, including a 7.4% and 15.1% decrease in LDL-cholesterol after HOCO and FXCO diets, respectively, as compared with the WD control. Moreover, a reduction (P=0.023) in plasma E-selectin concentration was found after the FXCO diet compared with the WD control. Consumption of the dietary oils failed to alter whole-body fat oxidation or energy expenditure, nor lead to alterations in body composition. FXCO diet increased (P<0.001) plasma ALA ~5-fold, EPA ~3-fold, and DPA ~1.5-fold, but did not modulate DHA levels compared with the WD control. At 24 and 48 hours the amount of administered 13C-ALA recovered as plasma 13C-EPA and 13C-DPA was lower (P<0.001) after FXCO diet compared with HOCO and WD diets, suggesting decreased ALA conversion efficiency with very high intakes of dietary ALA. No difference in plasma 13C-DHA enrichment was observed across diets. Moreover, minor alleles of selected single nucleotide polymorphisms in the FADS1/FADS2 gene cluster were associated with reduced (P<0.05) plasma fatty acid compositions and apparent conversion of 13C-ALA. However, increased consumption of ALA in the FXCO diet compensated for lower levels of EPA in minor allele homozygotes. Taken together, substitution of dietary fats common to WD with both HOCO and FXCO represents an effective strategy to target several biomarkers for CVD risk reduction.

We are the first lab to report the occurrence of a diet-induced transsulfuration (TS) blockade associating with elevated S-adenosylmethionine (i.e. SAM) synthesis, enhanced lifespan and enhanced reproductive capacity, in a vertebrate animal model. In this paradoxical study, we used LC-MS/MS-derived metabolomics data to report the effects of flaxseed (Linum Usitatissimum) on one-carbon metabolism in White Leghorn laying hens (Gallus gallus). Flaxseed contains a vitamin B6-antagonizing molecule called ‘linatine’ that is particularly effective at reducing vitamin B6 levels in small rodents and poultry. Linatine reduces TS flux through its inhibition of the vitamin B6-dependent enzymes cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE). In this study, our flaxseed-fed hens displayed decreased 4-pyridoxic acid and decreased pyridoxamine, concomitant with 15-fold elevated cystathionine. Homocysteine levels were stable in flaxseed-fed hens despite such highly elevated cystathionine. This is an astonishing finding, because mammalian models would predict the induction of hyperhomocysteinemia (i.e. elevated homocysteine) when cystathionine is so highly elevated (1). We are therefore reporting a phenomenon that might be unique to birds. Our metabolomics data indicate increased consumption of one-carbon donor molecules (e.g. choline, betaine, dimethylglycine, serine, etc) in flaxseed-fed hens, probably as a means of fueling the betaine homocysteine methyltransferase (BHMT) and methionine synthase-B12 (MS-B12) reactions. This modeling approach provides a rationale that flaxseed-fed hens increase their rate of homocysteine remethylation via BHMT and MS-B12, and in turn this would maintain stable homocysteine levels in the animal. We observed that the culminating outcome is elevated synthesis of SAM and an elevated SAM:SAH ratio. The associated biological outcomes are extended lifespan and increased reproductive capacity (i.e. increased daily egg laying) in flaxseed-fed hens. Our data further indicate that flaxseed tremendously stimulates a glucagon-like phenotype in hens. Specifically, flaxseed-fed hens exhibit 3-fold elevated glycated hemoglobin (HbA1c), 2-fold elevated serum free fatty acids (FFAs), 10 to 14% reduced body weight, and slightly reduced plasma pyruvate. These phenotypic markers suggest that glucagon might also play a large role in extending lifespan and enhancing reproductive capacity in flaxseed-fed hens. We predict that the anti-vitamin B6 effects of flaxseed are responsible for stimulating this glucagon-like phenotype in hens. Therefore, we conclude that the vitamin B6-antagonizing effects of linatine (via flaxseed dieting) improve liver function, body leanness, egg laying and survival, in a manner associated with increased SAM synthesis, increased blood FFA levels and increased blood glucose levels, in laying hens. The second part of this dissertation is used to test the hypothesis that polyunsaturated fatty acids (PUFAs) regulate lipid metabolism, increase mitochondrial respiration and decrease E-cadherin expression, in laying hen ovarian tumors. Researchers are unaware of the mitochondrial effects of PUFAs within laying hen ovarian tumors, and only a few publications exist regarding the analysis of E-cadherin in laying hen ovarian cancer. The results of this present work suggest that dietary PUFAs accumulate within laying hen ovarian tumors, and these same tumors exhibit decreased gene transcripts that govern de novo lipogenesis (i.e. FASN). Simultaneously, these tumors exhibit elevated transcripts for oxidative phosphorylation (OXPHOS) and decreased transcripts for phase 2 antioxidant enzymes. These patterns associated with decreased transcript levels of CDH1 (the gene for E-cadherin) in ovarian tumors, but no effect on full length 120kDa protein was observed by PUFAs versus our control diet. Interestingly, the effect of PUFAs on E-cadherin occurred at the level of the cleaved 37kDa and 80kDa E-cadherin fragments, such that PUFAs reduced their levels in hen ovarian tumors. We therefore developed a system for depleting the 80kDa E-cadherin fragment from hen ascites fluid (i.e. fluid from a hen that had ovarian cancer), so that we could test our hypothesis that the 80kDa E-cadherin fragment is important for supporting mitochondrial respiration in normal ovarian surface epithelial cells (IOSE80s). Mitochondrial membrane potential was decreased in IOSE80s when the 80kDa fragment was removed from the ascites; however, there was no effect on basal oxygen consumption in subsequent tests using extracellular flux analysis with Seahorse XFp. During our analysis of microRNA-200a-3p (miR-200a) we did not observe any effect of diet on miR-200a within hen ovarian tumors; however, we did observe that miR-200a levels increased within the tumor when going from stage 2 to stage 4 disease. Overall, we observed reduced risk of ovarian cancer, stage 4 ovarian cancer, multiple peritoneal tumor involvement and cancer-associated mortality, in laying hens that consumed a diet that was supplemented with PUFAs.

The effect of soy as a natural antioxidant against flaxseed rancidity in yeast breads was tested. Variables included: control (100% bread flour); yeast bread with 15% flax meal in place of part of the total bread flour; yeast bread with 15% flax meal and 5% soy; and yeast bread with 15% flax meal and 10% soy. Objective and sensory tests were used to evaluate breads. Peroxide values indicated that the hydroperoxides in breads increased during the first four weeks of the study, and then decreased, as would be expected as breads are exposed to more elements with time. Moisture content was not significantly different between the breads. Breads containing flax were significantly firmer (p < 0.02) in texture. Breads containing flax were also significantly lower in volume (p < 0.005) and significantly darker in crumb color (p < 0.01). The level of 10% soy contributed to a significantly darker crust color (p < 0.04). Quantitative descriptive analysis (QDA) found the level of 10% soy also contributed to an increased stale taste and aftertaste, firmer texture, coarser crumb, and drier loaf (p < 0.05). Musty aroma was not significantly different among breads and all breads containing flax had an increased grainy taste (p < 0.0001). Soy was found to have no significant antioxidant effect on the prevention of flaxseed rancidity in yeast breads. / Master of Science

There is interest in increasing the content of omega-3 (n-3; e.g., linolenic acid [C18:3n3]) fatty acids and conjugated linoleic acid (CLA) in bovine milk, primarily because of their beneficial effects on human health. One strategy to alter bovine milk fatty acid composition is the dietary inclusion of flaxseed, which is a rich source of C18:3 n-3. The aim of this study was to evaluate the effects of extrusion processing of flaxseed and the inclusion of condensed tannins (CT) in a flaxseed supplement on omasal flow of nutrients, ruminal fermentation characteristics, animal performance, and milk fatty acid profiles in dairy cattle. Eight multiparous Holstein cows (712.7 ± 92.3 kg body weight; 116.5 ± 17.5 days-in-milk at the beginning of the study) were assigned to four dietary treatments in a replicated 4 x 4 Latin square design consisting of 28-d periods with 20 d of dietary adaptation. Four cows in one Latin square were ruminally-cannulated to allow ruminal and omasal sampling. Cows were fed either a control diet (CTL) or one of 3 treatment diets that consisted of the daily substitution of 3 kg (DM basis) of the CTL concentrate pellet with 3 kg (DM basis) of either a non-extruded flaxseed and pea product (55% flax¬seed, 36% peas, 8% alfalfa, 1% antioxidant; designated RAW), a extruded flaxseed and pea product (55% flaxseed, 36% peas, 8% alfalfa, 1% anti¬oxidant; designated LPR), or a extruded flaxseed and high-tannin fava bean product (55% flaxseed, 36% high-tannin faba beans, 8% alfalfa, 1% antioxidant; designated LPF). Diets were fed twice daily as total mixed rations. Omasal flow of nutrients was estimated using the omasal sampling technique using iNDF as the single indigestible marker. Dry matter intake was lower (P = 0.01) in cows fed the flaxseed diets (24.0 kg/d) compared to those fed CTL (25.9 kg/d). Milk yield was higher (P = 0.02) in cows fed the LPR diet ( 44.4 kg/d) compared to those fed the RAW diet ( 42.3 kg/d); and tended to be higher (P = 0.07) in cows fed the flaxseed diets compared to those fed the CTL diet. Milk fat yield was unaffected by dietary treatment (P = 0.94), whereas milk protein yield tended (P = 0.10) to increase in cows fed the flaxseed diets compared to those fed the CTL diet. No detrimental effects of dietary treatments on ruminal fermentation and omasal flow of microbial protein were observed. The omasal flow of C18:3 n-3 was higher in cows fed the flaxseed diets compared to those fed the CTL diet (P = 0.04), with the RAW diet (56.9 g/d) having the highest flow of C18: 3n-3 compared to the other diets (LPR = 14.0 g/d; LPF = 14.8 g/d). The omasal flow of total CLA isomers were higher (P = 0.03) in cows fed the LPF diet (6.06 g/d) compared to those fed the LPR diet (3.70 g/d). The C18:3 n-3 content in milk fat (% of fatty acid methyl esters) was higher in cows fed the LPR diet (0.950%) compared to those fed the RAW diet (0.745%). The level of total CLA isomers in milk was also higher in cows fed the LPR diet (0.845%) compared to those fed the RAW diet (0.308%). These results demonstrated that feeding extruded flaxseed products is more effective than feeding whole flaxseed at improving the fatty acid composition of milk fat without negatively impacting animal performance and ruminal fermentation; however, the inclusion of CT in the extruded flaxseed product had no additional benefit.

O presente trabalho teve como objetivo realizar a extração de lignanas e outros compostos fenólicos do farelo de linhaça desengordurado e avaliar a atividade antifúngica dessas frações frente a fungos leveduriformes do gênero Candida. Obteve-se o extrato bruto através de maceração com solução hidroalcólica. O extrato bruto foi hidrolisado em condições básicas e ácidas, objetivando-se o isolamento de heterosídeos e agliconas, respecitvamete. A lignana secoisolariciresinol diglicosídeo foi quantificada por cromatografia líquida de alta eficiência. O flavonoide herbacetin diglicosídeo e a lignana anidrosecoisolariciresinol foram identificados por espectrometria de massas. A avaliação da atividade antifúngica foi realizada in vitro e foi observado que tanto o extrato bruto como o extrato obtido por hidrólise alcalina inibiram o desenvolvimento de Candida krusei CK 02 e Candida parapsilosis RL 20 na concentração de 500 μg/mL. O extrato obtido por hidrólise ácida, na mesma concentração, apresentou atividade inibitória frente a Candida krusei CK 02, Candida parapsilosis RL 20 e Candida tropicallis ATCC 750. A investigação das condições de hidrólise ácida, a melhor caracterização de seus componentes e a avaliação da ação antifúngica dos extratos frente a outros gêneros de fungos constituem perspectivas para trabalhos futuros. / The aim of the present work was carry out the extraction of lignans and other phenolic compounds from defatted flaxseed meal and evaluate the antifungal activity of these fractions against yeast fungi of Candida genus. The crude extract was obtained by maceration with a hydroalcoholic solution. The crude extract was hydrolyzed in basic and acidic conditions, aiming the isolation of glycosides and aglycones, respectively. Secoisolariciresinol diglucoside was quantified by highperformance liquid chromatography. The flavonoid herbacetin diglucoside and the lignan anhydrosecoisolariciresinol were identified by mass spectrometry. The avaliation of antifungal activity was performed in vitro and it was observed that both the crude extract and the extract obtained by alkaline hydrolysis inhibited the development of Candida krusei CK 02 and Candida parapsilosis RL 20 at a concentration of 500 ug/mL. The extract obtained by acid hydrolysis, in the same concentration, showed inhibitory activity against Candida krusei CK 02, Candida parapsilosis RL 20 and Candida tropicallis ATCC 750. The investigation of acid hydrolysis conditions, a better characterization of its components and the avaliation of antifungal activity against other fungi genera represents good potencial for future work.

Orientadora: Maria Regina Barbieri de Carvalho / Banca: Elisabete Maria Macedo Viegas / Banca: Marco Antonio Trindade / Banca: Dalton José Carneiro / Banca: Hirasilva Borba Alves de Souza / Resumo: O objetivo desse trabalho foi avaliar a qualidade dos filés de tilápias do Nilo alimentadas com dietas contendo óleos de soja e de linhaça e extrato de alecrim, e também verificar a ação antioxidante do extrato de alecrim em filés embalados a vácuo. No primeiro experimento, os peixes foram alimentados com dietas contendo óleo de soja, óleo de linhaça e extrato de alecrim, sendo abatidos, filetados, embalados em filme plástico e armazenados a -18ºC por 90 dias. No segundo experimento, filés de tilápia do Nilo provenientes de um cultivo em represa foram imersos em solução de extrato de alecrim nas concentrações de 0, 400, 800 e 1200 ppm, embalados sob ar atmosférico ou a vácuo e armazenados em gelo por 21 dias. Os filés de tilápias alimentadas com dietas contendo o extrato de alecrim apresentaram menores teores de lipídios (1,52%) em relação aos sem extrato (1,77%). A quantidade de ácidos graxos da série n-3 foi maior (6,22%) nos filés de tilápias alimentadas com dietas contendo óleo de linhaça do que o de soja (5,42%). Os valores de pH e BNVT para ambos os experimentos ficaram abaixo dos propostos pela legislação brasileira que estabelece limites de 6,50 e 30 mg/100g músculo, respectivamente. Todos os tratamentos apresentaram diminuição do NNP nos filés durante o armazenamento. Os filés de tilápias que receberam extrato de alecrim na dieta e na imersão apresentaram minimização da oxidação avaliada pelo TBARS. A força de cisalhamento diminuiu de 1,61 a 0,36 kgf e de 0,81 a 0,59 kgf, respectivamente para os filés do primeiro e segundo experimento. A capacidade de retenção de água diminuiu para os filés de tilápias alimentadas com dietas contendo óleo de soja (71,04%) e de linhaça (69,74%) no final do tempo de armazenamento e para os filés embalados a vácuo os valores mantiveram-se estáveis do 7º ao 21º dia...(Resumo completo, clicar no acesso eletrônico abaixo) / Abstract: The objective of this work was to evaluate the quality of Nile tilapia fillets fed with diets contends soy and flaxseed oil and rosemary extract, and also verify antioxidant action of rosemary extract in fillets vacuum packed. In the first experiment, the fish were fed with diets contend soybean oil, flaxseed oil and rosemary extract, being slaughtered, processed, packing in plastic film and stored at -18ºC for 90 days. In the next experiment, fillets were immersed in rosemary extract solution in the concentrations of 0, 400, 800 and 1200 ppm, packed under atmospheric or the vacuum and ice stored for 21 days. Fillets of tilapias fed with diets contend rosemary extract presented lower content of lipids (1.52%) in relation those without extract (1.77%). The amount of n-3 fatty acids was larger (6.22%) found in fillets of tilapias fed with diets contends flaxseed oil of that of soy (5.42%). The values of pH and BNVT for both the experiments were below of recommending for Brazilian legislation that establishes 6.50 e 30 mg/100g muscle, respectively. All the treatments presented reduction of the NNP in fillets during the storage. Fillets of tilapias that received rosemary extract in the diet and the immersion presented minimized of the oxidation evaluated at TBARS. The shear force decrease of 1.61 for 0.36 kgf and of 0.81 for 0.59 kgf, respectively for the fillets of first and second experiment. The liquid holding capacity decrease for fillets of tilapias fed with diet contends soybean oil (71.04%) and flaxseed oil (69.74%) at end of storage time and for fillets packed vacuum the values maintained stable of 7º to 21º day. The luminosity increased in fillets, for both the experiments, during the storage. It was verified bigger losses for cooking in the end of the storage for fillets immersed in solution of rosemary extract and packed under atmospheric air and the vacuum...(Complete abstract, acess undermentioned eletronic adress) / Doutor

The objectives of this study were to investigate the effects of autoclave heating on the rumen protein degradation characteristics of flaxseed (<i>Linum usitatissimum</i>, cv. Vimy), and to compare them to differences in diffuse reflectance infrared Fourier transform (DRIFT) and Synchrotron based Fourier transform infrared microspectroscopy (S-FTIR) measurements of the protein alpha-helix to beta-sheet ratios. Hierarchical cluster analysis (CLA) and principal components analysis (PCA) were also conducted to identify differences in the DRIFT spectra. Flaxseed samples were kept raw for control or autoclaved in batches at 120°C for 20, 40 or 60 min for treatments 1, 2 and 3, respectively. The rumen degradation kinetics of protein were measured along with the protein sub-fractions of the Cornell net carbohydrate and protein system (CNCPS), and chemical composition. Intestinal digestibility was determined using the three-step procedure outlined by Calsamiglia and Stern (1995). Protein supply to the small intestine was determined using the NRC (2001) and DVE/OEB models. The results showed that heating increased dry matter (DM) and ether extract (EE) content, while reducing neutral detergent fibre (NDF) and acid detergent fibre (ADF), with little numerical difference between the three treatments. Soluble crude protein (SCP) also decreased upon autoclaving with concomitant increases in non-protein nitrogen (NPN), neutral detergent insoluble nitrogen (NDIN) and acid detergent insoluble nitrogen (ADIN). The CNCPS protein sub-fractions with the greatest changes were the buffer-soluble true protein fraction (PB1) and the fraction representing buffer-insoluble true protein which is not bound to NDF (PB2) showing dramatic increases, indicating a decrease in the overall protein degradability. <i>In situ</i>experiments showed a reduction in effective degradable dry matter (EDDM) as well as a reduction in effective degradable crude protein (EDCP) without significant differences between the treatments. Intestinal digestibility of protein as estimated by the three-step procedure showed no changes upon autoclaving. Modeling results, with flaxseed as the only feed source, for absorbable ruminally-undegraded feed protein in the intestines using both the NRC (2001) and DVE/OEB systems showed increases as a consequence of the autoclave treatments but again there were no differences between the treatments. The degraded protein balance results showed for both the NRC (2001) and DVE/OEB models that both were decreased upon autoclave treatment. However, the values for the NRC (2001) model suggested a potential nitrogen (N) deficiency and, therefore potentially impaired microbial crude protein (MCP) production, whereas the values for the DVE/OEB system showed potential N excess and, therefore, possible loss from the rumen. DRIFT analysis of protein secondary structure ratios showed a decrease in the alpha-helix to beta-sheet ratio for the whole seed, whereas results from S-FTIR spot data for cotyledon tissue showed autoclaving had the opposite effect on the ratio. CLA and PCA were successfully used to make distinctions between the different treatment spectra and showed enhanced sensitivity upon selection of a smaller spectral window to include only the amide I and II portion of the IR spectrum. The results failed to demonstrate any differences between the autoclave treatments used in this study, and showed that autoclaving generally decreased effectively ruminal degradability of flaxseed protein. The results further indicated that autoclaving had a significant enough effect on the flaxseed to permit identification of the altered alpha-helix to beta-sheet ratio with the mid-IR spectrum, as well as differentiation between the treatments using PCA and CLA. PCA and CLA results suggest that mid-IR spectral methods are more sensitive than traditional methods when used to identify differences between the heat treatments.