Les lipases sont des enzymes présentant un grand intérêt industriel. L’intérêt de ces enzymes a conduit à caractériser ces enzymes, à mieux comprendre leur mécanisme réactionnel et leur cinétique, et à établir des méthodes efficaces de production en système d’expression homologue et hétérologue. Plus récemment, l’ingénierie enzymatique permet d’améliorer les caractéristiques des enzymes. Ce thèse s’est fixé deux objectifs principaux: premièrement, la purification et la fonctionnalisation d’acides gras poly-insaturés de type Omega-3 (PUFAs), et spécialement l’acide cis-4, 7, 10, 13, 16, 19-docosahexaénoique (DHA) et deuxièmement la production de lipides structurés (SL). Un premier objectif fut de produire une molécule pharmaceutique, le nicotinyl DHA ester. Le co-substrat du DHA est le nicotinol, un alcool qui après absorption, il est rapidement converti en acide nicotinique (Vitamine B3). La trans-esterification enzymatique entre l’ester éthylique du DHA et le nicotinol a été optimisée dans le but de synthétiser un ester présentant les propriétés cumulatives des deux réactants. Après la sélection de l’enzyme optimale (lipase immobilisée de Candida antarctica; Novozyme 435) et le choix du milieu réactionnel (milieu sans solvant), le procédé a été optimisé. Une conversion supérieure à 97 % a été obtenu en 4 heures avec 45 g.L-1 d’enzyme. Dans ces conditions, une productivité de 4.2 g de produit .h-1.g d’enzyme-1 a été obtenue. Ce projet nécessite une haute pureté en DHA. Un procédé de purification enzymatique a été choisi. Les lipases sont capables de discriminer entre les acides gras en fonction de la longueur de chaine et du degré d’insaturation. Les lipases agissent par résolution cinétique, en réagissant plus efficacement avec les acides gras saturés et mono-insaturés qu’avec les PUFAs résistants. La lipase YLL2 de Yarrowia lipolytica apparait comme un bon candidat car elle est homologue à une des lipases les plus efficaces, la lipase de Thermomyces lanuginosus. YLL2 a permis d’obtenir une discrimination très efficace. Les raisons de la sélectivité de l’enzyme ont été identifiées : il s’agit du positionnement de la double liaison la plus proche de la fonction carboxylique. La concentration en DHA la plus élevée a été obtenue avec YLL2 (73%) avec un pourcentage de récupération du DHA-EE de 89%. YLL2 est par conséquent l’enzyme décrite la plus efficace pour la purification du DHA.La mutagénèse ciblée dans le site actif de YLL2 a été utilisée pour améliorer la sélectivité de cette enzyme. L’analyse de la structure 3D et les alignements avec des lipases homologues a permis de choisir les cibles de mutagénèse dirigée. Les acides aminés cibles ont été changés de manière à restreindre ou élargir le site actif. De ce premier screening de variantes deux positions ont permis d’améliorer la spécificité de l’enzyme, les positions I100 et V235. Finalement la saturation de ces 2 positions a été réalisée. Le dernier objectif de la thèse était la production de SL par acidolysis enzymatique entre l'huile d'olive vierge et les acides caprylic ou capric utilisant la lipase YLL2 immobilisé. Le SL obtenu devrait être riche en acide oléique à la position sn-2 tandis que les C8:0 et C10:0 devraient être principalement estérifiés aux positions sn-1,3. YLL2 immobilisé sur Accurel 1000 a été testé dans un système sans solvant. La réaction d’acidolysis d'huile d'olive avec C8:0 ou C10:0 a été optimisée avec la méthodologie de surface de réponse (RSM). / Lipases are enzymes with applications extended to a wide variety of industries. The variety of lipases applications led to increased research to characterize them and better understand their kinetics and reaction mechanisms and to establish methods for lipase production in homologous and heterologous expression systems. Lately enzymatic engineering allowed the improvement of lipase characteristics. This thesis project studies the use of lipases for two main objectives: lipase-catalyzed purification and functionalization of Omega-3 polyunsaturated fatty acids (PUFAs), especially cis-4, 7, 10, 13, 16, 19-docosahexaenoic acid (DHA) and production of structured lipids (SL). DHA was used for the synthesis of a pharmaceutical molecule, the nicotinyl DHA ester. The co-substrate of the reaction was nicotinol, an alcohol from the group B pro-vitamin, which after absorption is rapidly converted into nicotinic acid (Vitamin B3). The enzymatic trans-esterification of DHA ethyl esters with nicotinol was optimised to synthesise an ester presenting the cumulative properties of the two reactants. After enzyme (immobilized lipase from Candida antarctica; Novozym 435) and reaction medium (solvent-free system) selection, the process was optimised. A conversion to nicotinyl-DHA superior to 97 % was obtained in 4 hours using 45 g.L-1 of enzyme. With a productivity of 4.2 g of product .h-1.g of enzyme-1.This project requires DHA of high purity. Enzymatic purification was chosen for the production of DHA concentrates. Lipases can discriminate between fatty acids in function of their chain length and saturation degree. Lipases react more efficiently with the bulk of saturated and mono-unsaturated fatty acids than with the PUFAs. The objective was the discovery of more specific enzymes for DHA purification. The lipase Lip2 from Yarrowia lipolytica (YLL2) appears as a good candidate since it is homologous to one of the most efficient lipase, the lipase from Thermomyces lanuginosus. YLL2 enables a high discrimination to be obtained, enzyme selectivity being principally due to the positioning of the double-bond the closest from the carboxylic group. The highest concentration of DHA was obtained with YLL2 (73%) with a recovery percentage of DHA-EE of 89%. YLL2 is the most efficient described lipase for DHA purification.Site directed mutagenesis was used to improve YLL2 from Y. lipolytica. Using its three dimensional structure and alignment with homologous lipases, targets for site directed mutagenesis were chosen. Chosen amino acids were substituted by two amino acids of different sizes. From the screening of variants two positions with promising specificities where chosen, positions I100 and V235. Finally saturation of both positions and the analysis of their performances in the selected reactions were carried out. The last objective was the production of SL by enzymatic acidolysis between virgin olive oil and caprylic or capric acids using immobilized Lip2 from Y. lipolytica. The SL obtained should be rich in oleic acid at the sn-2 position while C8:0 and C10:0 should be mainly esterified at the sn-1,3 positions. Lip2 from Y. lipolytica immobilized on Accurel MP 1000 was tested in a solvent-free system. The acidolysis reaction of olive oil with C8:0 or C10:0 was optimized by response surface methodology (RSM)

Mehrfach ungesättigte omega-3 Fettsäuren (n-3 PUFAs), wie Eicosapentaensäure (EPA) und Docosahexaensäure (DHA), schützen vor kardiovaskulären Erkrankungen, wie tödlichen Arrhythmien. In vitro Untersuchungen belegen, dass rekombinante Cytochrom P450 (CYP) Enzyme nicht nur die n-6 PUFA Arachidonsäure (AA), sondern auch die n-3 PUFAs EPA und DHA als alternative Substrate verwenden. Dabei entstehen bioaktive regio- und stereoisomere Epoxy- und Hydroxymetaboliten, CYP-Eicosanoide, die als sekundäre Botenstoffe bei der Regulation von Gefäß-, Nieren- und Herzfunktionen fungieren. Die genauen molekularen Mechanismen dieser Metabolite sind noch weitgehend unerforscht. In der vorliegenden Arbeit wurde zunächst der ernährungsbedingte Einfluss auf das endogene CYP-Eicosanoidprofil im Menschen untersucht. Die Ergebnisse der klinischen Studie zeigten, dass n-3 PUFAs auch in vivo alternative Substrate von CYP-Enzymen darstellen und wenn verfügbar sogar effektiver zu ihren Metaboliten umgesetzt wurden als AA. Als ein wichtiger Metabolit entsteht nach EPA/DHA-Supplementation 17,18-EEQ, welcher womöglich der eigentliche Vermittler der kardioprotektiven Effekte von n-3 PUFAs ist. Unter Verwendung eines etablierten Zellmodells mit spontan schlagenden neonatalen Rattenkardiomyozyten (NRKMs) wurde der anti-arrhythmische Effekte von 17,18-EEQ genauer untersucht. Der negativ chronotrope Effekt von EPA auf NRKMs wurde tatsächlich durch 17,18-EEQ vermittelt, insbesondere dem R,S-Enantiomer. Mittels Strukturfunktionsanalyse wurden synthetische Analoga mit gleicher Wirksamkeit wie dem 7,18-EEQ gefunden, wobei strikte strukturelle Merkmale für die biologische Funktion identifiziert wurden. Die Suche nach einem molekularen Ziel für CYP-Epoxyeicosanoide führte zu einem möglichen Rezeptorkandidaten, der hinsichtlich seiner Ligandenspezifität untersucht wurde. Dieser oder zukünftige andere Rezeptorkandidaten stellen ein mögliches neues zelluläres Ziel zur Behandlung kardialer Arrhythmien dar. / The n-3 polyunsaturated fatty acids (n-3 PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), protect from cardiovascular disease, especially from fetal arrhythmia. Moreover, in vitro studies proved that recombinant cytochrome P450 (CYP) enzymes not only accept the physiologically most important n-6 PUFA arachidonic acid (AA), but also EPA and DHA as alternative substrates, thereby generating regio- and stereospecific biologically active epoxy- and hydroxymetabolites, CYP-eicosanoids. These metabolites serve as second messengers regulating vascular, renal and cardiac function. The precise underlying molecular mechanisms are only partially understood and need further investigation. The first aim of the thesis was to show that the endogenous CYP-eicosanoid profile depends on the availability of the precursor fatty acids. The results of a clinical trial with 20 volunteers, show that n-3 PUFAs serve also in vivo as alternative CYP-dependent substrates and are even preferentially metabolized compared to AA. After EPA/DHA-supplementation 17,18 EEQ was generated as a major metabolite, potentially an important mediator of cardiovascular effects originally attributed to n-3 PUFAs. To test the anti-arrhythmic effect of EPA and 17,18-EEQ, an established cell model with neonatal rat cardiomyocytes (NRKMs) was used. The negative chronotropic effect of EPA was mimicked by 17,18-EEQ, attributed only to the R,S-enantiomer. A structure activity relationship study revealed synthetic analogs, exerting the same biological effect as 17,18-EEQ. Strict structural requirements were found for agonistic function, hinting at a specific interaction with cellular targets, like GPCRs. The search of a molecular target of CYP-eicosanoids led to a putative receptor, which was tested for ligand binding specificity. If the preliminary results on the ligand binding are confirmed in future experiments this receptor might be a novel target for the treatment of cardiac arrhythmia.

Background: Iron deficiency (ID) is the most prevalent nutritional deficiency in the world. In children, both inadequate iron and fatty acid (FA) status have been found to have an effect on cognitive and behavioural function, including physical activity behaviour and attention deficit hyperactivity disorder (ADHD)–related behaviour. Aim: To investigate the effects of supplementation with iron and omega–3 fatty acids (n–3 FAs), alone and in combination, on spontaneous motor activity and ADHD–related behaviour in iron deficient primary school children in KwaZulu–Natal. An additional aim was to evaluate the use of the Actical accelerometer as a tool to assess physical activity behaviour. Methods: The study design was a 2x2 factorial, randomized, double–blind and placebo–controlled trial. Iron deficient school children aged six to ten years with or without mild anaemia were included in the study (n = 321). Subjects were randomly assigned to receive one of the following supplement combinations: (1) 420mg docosahexaenoic acid (DHA)/80 mg eicosapentaenoic acid (EPA) + 50mg of iron as ferrous sulphate (Fe); (2) 420mg DHA/80mg EPA + placebo; (3) 50mg of Fe + placebo; (4) placebo + placebo. Supplements were provided four times a week for a duration of 8.5 months (excluding school holidays). Physical activity of a subgroup of subjects (n=98) was recorded on four random school days at baseline, midpoint and endpoint (12 days in total) during three different time periods namely class time 1 (08h00-10h30), break time (10h30–11h00) and class time 2 (11h00–12h00). Classroom behaviour of study subjects was assessed by teachers at baseline and endpoint using the Conners’ Teacher Rating Scale–Revised: Short Forms (CTRS). Iron status indicators and red blood cell (RBC) FA composition were measured at baseline and endpoint. Treatment effects were assessed for activity and CTRS scores. Furthermore, the relationship between activity, CTRS scores and iron/FA status indicators was determined using bivariate correlation and multivariate linear regression analysis. Results: Overall activity of all subjects varied over time from baseline and midpoint to endpoint. A significant cycle x age interaction (P = 0.005) as well as a significant cycle x time period x gender interaction (P = 0.036) was observed on overall activity. There were no significant interactions of cycle or time period with treatment. However, there was a significant main effect of DHA/EPA supplementation for lower class time 1 activity at endpoint (P = 0.014). Biological markers indicating better or poorer iron status were positively and negatively associated with activity at break time, respectively. Subjects in the group receiving both iron and DHA/EPA supplements showed a significant improvement from baseline to endpoint on the cognitive problems/inattention subscale (P = 0.005) of the CTRS. Hyperactivity scores increased iv significantly from baseline to endpoint in all groups (P = 0.006). DHA (r = –.203; P = 0.040) and EPA (r = –.199; P = 0.044) content of RBC were negatively associated with activity at class time 1. No significant associations were observed between activity and CTRS scores at baseline. At endpoint, class time 1 activity was positively associated with all CTRS subscale scores except for the cognitive problems subscale, which only bordered significance (correlation, P = 0.051; regression, P = 0.073). Conclusions: These findings suggest that n–3 FA supplementation may have an influence on ADHD–related behaviour during class time. During school break time when subjects were allowed to move around freely, iron status was positively associated with spontaneous motor activity. Furthermore, the accelerometer might be a useful complimentary tool for assessing both classroom and break time activity behaviour in school children. / Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2012.

Background: Iron deficiency (ID) is the most prevalent nutritional deficiency in the world. In children, both inadequate iron and fatty acid (FA) status have been found to have an effect on cognitive and behavioural function, including physical activity behaviour and attention deficit hyperactivity disorder (ADHD)–related behaviour. Aim: To investigate the effects of supplementation with iron and omega–3 fatty acids (n–3 FAs), alone and in combination, on spontaneous motor activity and ADHD–related behaviour in iron deficient primary school children in KwaZulu–Natal. An additional aim was to evaluate the use of the Actical accelerometer as a tool to assess physical activity behaviour. Methods: The study design was a 2x2 factorial, randomized, double–blind and placebo–controlled trial. Iron deficient school children aged six to ten years with or without mild anaemia were included in the study (n = 321). Subjects were randomly assigned to receive one of the following supplement combinations: (1) 420mg docosahexaenoic acid (DHA)/80 mg eicosapentaenoic acid (EPA) + 50mg of iron as ferrous sulphate (Fe); (2) 420mg DHA/80mg EPA + placebo; (3) 50mg of Fe + placebo; (4) placebo + placebo. Supplements were provided four times a week for a duration of 8.5 months (excluding school holidays). Physical activity of a subgroup of subjects (n=98) was recorded on four random school days at baseline, midpoint and endpoint (12 days in total) during three different time periods namely class time 1 (08h00-10h30), break time (10h30–11h00) and class time 2 (11h00–12h00). Classroom behaviour of study subjects was assessed by teachers at baseline and endpoint using the Conners’ Teacher Rating Scale–Revised: Short Forms (CTRS). Iron status indicators and red blood cell (RBC) FA composition were measured at baseline and endpoint. Treatment effects were assessed for activity and CTRS scores. Furthermore, the relationship between activity, CTRS scores and iron/FA status indicators was determined using bivariate correlation and multivariate linear regression analysis. Results: Overall activity of all subjects varied over time from baseline and midpoint to endpoint. A significant cycle x age interaction (P = 0.005) as well as a significant cycle x time period x gender interaction (P = 0.036) was observed on overall activity. There were no significant interactions of cycle or time period with treatment. However, there was a significant main effect of DHA/EPA supplementation for lower class time 1 activity at endpoint (P = 0.014). Biological markers indicating better or poorer iron status were positively and negatively associated with activity at break time, respectively. Subjects in the group receiving both iron and DHA/EPA supplements showed a significant improvement from baseline to endpoint on the cognitive problems/inattention subscale (P = 0.005) of the CTRS. Hyperactivity scores increased iv significantly from baseline to endpoint in all groups (P = 0.006). DHA (r = –.203; P = 0.040) and EPA (r = –.199; P = 0.044) content of RBC were negatively associated with activity at class time 1. No significant associations were observed between activity and CTRS scores at baseline. At endpoint, class time 1 activity was positively associated with all CTRS subscale scores except for the cognitive problems subscale, which only bordered significance (correlation, P = 0.051; regression, P = 0.073). Conclusions: These findings suggest that n–3 FA supplementation may have an influence on ADHD–related behaviour during class time. During school break time when subjects were allowed to move around freely, iron status was positively associated with spontaneous motor activity. Furthermore, the accelerometer might be a useful complimentary tool for assessing both classroom and break time activity behaviour in school children. / Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2012.

[Truncated abstract] The role of fish consumption and omega-3 polyunsaturated fatty acids (PUFAs) in the prevention of disease has been the subject of much investigation in recent years. A clue that these factors might be of importance was the observation that populations consuming high levels of marine omega-3 PUFAs had lower rates of morbidity and mortality from cardiovascular disease. From there, research in this area has expanded to include a range of chronic diseases and their prevention. An area of particular interest is the role of fish consumption in the prevention of various cancers. In Australia, one in three men and one in four women are expected to be diagnosed with cancer by the age of 75. Identifying preventive factors that can be translated into constructive health promotional messages is of great importance in addressing this group of diseases that has such a large impact on the health and wellbeing of the population. ... Results and conclusions Analysis of the data from the population-based case-control study suggests a protective effect of preserved fish consumption, possibly due to the high oil content of these fish. The developed questionnaire is a valid and reliable tool for measuring fish and seafood consumption as determined by regression analysis with an independent biomarker and reliability analysis using intra-class correlation. Importantly, reliability can be maintained despite asking a high level of detail from participants. For ranking according to overall fish consumption, detailed questioning is probably not necessary, however inclusion of variables representing multiple categories of fish and seafood consumption in a regression model enables us to better account for variation in blood omega-3 PUFA levels than a single variable representing overall consumption. For the purpose of questionnaire validation, plasma phospholipid and erythrocyte membrane levels of EPA and DHA are equivalent biomarkers of fish and seafood consumption. The choice between them by future investigators will be based on more practical aspects such as convenience and the fasting state of subjects. The tangible product of this thesis is additional evidence to support a protective association between fish consumption and prostate and breast cancers, and a valid and reliable questionnaire v for measuring habitual consumption of fish and seafood in a West Australian population, that could also be applied to other populations after minor adjustment for local fish and seafood consumption patterns.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

Un procédé enzymatique sans solvant a été développé permettant la synthèse d'un ester phénolique de DHA. L'optimisation des paramètres réactionnels a permis d'atteindre des rendements élevés (440 g/L) d'ester de DHA et d'alcool vanillique (DHA-VE), dont les activités biologiques et le potentiel applicatif ont été évalués. L'activité inhibitrice du DHA-VE vis-à-vis des radicaux ABTS, DPPH et hydroxyle a été démontrée. Un effet neuroprotecteur de l'ester a également été mis en évidence sur des neurones primaires de rat, exposés aux oligomères du peptide [bêta]-amyloïde. Une étude in vivo a permis de montrer que le greffage d'alcool vanillique conduit à une augmentation du taux de DHA au niveau des globules rouges et des neurones, indiquant une biodisponibilité accrue du DHA lorsque celui-ci est couplé au composé phénolique. Aucune toxicité visible de l'ester n'a été constatée. Par ailleurs, l'incorporation de DHA-VE dans divers systèmes émulsionnés a permis d'accroître leur stabilité à l'oxydation, quelles que soient les conditions de stockage. Ceci montre le potentiel de cet ester pour enrichir diverses matrices alimentaires en DHA, tout en améliorant leur stabilité à l'oxydation. Le procédé enzymatique développé a été appliqué à de l'huile de saumon, utilisée comme source d'acides gras polyinsaturés de la série oméga-3. L'incorporation totale de l’alcool vanillique (50 g/L) a été obtenue après 24 h de réaction, conduisant à la production d'une grande variété d'esters, représentatifs de la composition initiale de l'huile en acides gras. Le milieu réactionnel brut issu de l'alcoolyse de l'huile présente une grande stabilité et des propriétés antioxydantes importantes par rapport à l'huile de saumon native. En conclusion, l'approche consistant à assembler des composés phénoliques et des lipides polyinsaturés au sein d'une même structure semble prometteuse pour renforcer le potentiel applicatif de ces deux familles de biomolécules et produire de nouveaux ingrédients bioactifs stables / An efficient solvent-free bioprocess was developed for the synthesis of DHA phenolic ester, using the lipase B from Candida antarctica. The protocol developed here led to high-level production (440 g/L) of DHA vanillyl ester (DHA-VE) that exhibits interesting application potential as food ingredient. DHA-VE was characterized by a high stability and a high radical scavenging activity towards DPPH, ABTS and hydroxyl radicals. Neuroprotective properties of DHA-VE were also demonstrated in rat primary neurons exposed to amyloid-[beta] oligomers. Enzymatic esterification of DHA with vanillyl alcohol (VA) led to increased DHA levels in erythrocytes and brain tissues of mice fed DHA-VE-supplemented diet comparing with DHA. No visible toxicity of the ester was found. Enrichment of emulsions with DHA-VE improved significantly their oxidative stability whatever the conditions of storage, showing the potential of DHA-VE to enrich various food matrices with DHA while protecting them against oxidation. The enzymatic process was applied to salmon oil as a source of omega-3 polyunsaturated fatty acids (PUFA). The total conversion of VA (50 g/L) was achieved after 24 h of reaction, leading to the production of a wide variety of esters that mirror the initial composition of the oil. The crude reaction medium recovered from salmon oil alcoholysis exhibited a high stability together with high antioxidant properties in comparison with native salmon oil. In conclusion, the approach that consists in bringing phenolic compounds and PUFA-rich lipids together within a single structure is expected to provide stable bioactive ingredients that should broaden the scope of application of omega-3 PUFAs whose health benefits are increasingly sought

Master of Science / Department of Animal Sciences and Industry / J. Ernest Minton / It has been well documented that omega-3 PUFA (n-3 PUFA) can confer a wide variety of health benefits to humans and animals. The current study was designed to evaluate the ability of n-3 PUFA to modulate the innate immune response in two diverse breeds of horses. Ten Quarter Horse and 10 American Miniature Horse mares were assigned to either an n-3 PUFA supplemented or control diet (5 full-sized and 5 miniature mares/treatment) for 56 d. The treatment diet was designed to deliver 64.4 mg/kg BW combined eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) daily. Whole blood (20 mL) was collected via jugular veinipuncture into heparinized tubes on 0 d, 28 d, and 56 d. Serum PUFA analysis was conducted by gas chromatography. Peripheral blood mononuclear cell (PBMC) production of tumor necrosis factor-alpha (TNFalpha) in response to toll-like receptor (TLR) ligands lipopolysaccharide (LPS), flagellin, and lipoteichoic acid (LTA) was estimated using an equine-specific ELISA. Peripheral blood samples from d 56 were also analyzed for total and differential leukocyte counts and subjected to flow cytometric analysis. Body type did not affect basal or TLR stimulated TNFα production. Serum PUFA analysis revealed a decrease in linoleic acid (LA) and substantial increases in arachidonic acid (ARA), EPA, DHA, and docosapentaenoic acid (DPA) at both d 28 and 56 in horses fed n-3 PUFA (P less than 0.0001 for all). Dietary n-3 PUFA supplementation reduced (P less than 0.05) un-stimulated basal, but not TLR stimulated TNFalpha production by PBMC’s. Supplementation with n-3 PUFA did not affect total or differential leukocyte counts, nor selected cell surface markers. These results suggest that n-3 PUFA supplementation in the horse can modify circulating PUFA and alter the inflammatory response by reducing basal TNFalpha production. Furthermore, under conditions of the current study and considering the endpoints evaluated, the American Miniature Horse could potentially be used as a model for full-sized horse breeds.

Master of Science / Department of Animal Sciences and Industry / Joann M. Kouba / The objective of this study was to evaluate the reproductive effects of supplementing normally cycling mares with marine-derived omega-3 (n-3) fatty acids during the estrous cycle. Fifteen mares were assigned to a control diet (CONT, n=7) or a fish oil supplemented diet (FO, n=8) containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The FO mares received 18.48 g EPA/10.08 g DHA/mare/d. At the start of the trial, mares were synchronized using a progesterone and estradiol protocol. Following synchronization, mares were monitored with transrectal ultrasonography throughout the second estrous cycle. Ovarian activity, ovulation, and presence of a corpus luteum were noted. Mares were ultrasounded throughout the third estrous cycle until a 35 mm follicle was detected. Upon detection of the 35 mm follicle, hCG was administered. Within 16 hr, transvaginal ultrasound-guided follicular aspiration (TUGA) was performed on the preovulatory follicle, signifying the end of the trial. Follicular fluid was analyzed for fatty acid and hormone concentrations. Serum fatty acids were measured every 2 wk and serum hormone concentrations were analyzed during the second estrous cycle at 5 d to 1 d prior to ovulation, at ovulation, and 3 and 5 d post-ovulation. Samples were also collected prior to hCG administration and on aspiration day for hormone analysis. Serum estradiol-17β, progesterone, luteinizing hormone (LH), and insulin-like growth factor 1 (IGF-1) were measured. Fish oil supplementation increased (P < 0.01) arachidonic acid (ARA), EPA, docosapentaenoic acid (DPA), and DHA in mare serum and increased (P<0.01) EPA, DPA, and DHA in follicular fluid. No overall treatment effect was found on serum hormone concentrations during the second estrous cycle, but a decrease (P<0.05) in IGF-1 was noted in the FO group on aspiration day. Concentrations of IGF-1 were also lower (P<0.05) in follicular fluid in the FO group compared to controls. No other follicular fluid differences were observed. Supplementation resulted in a smaller diameter follicle (P<0.05, 38.0 ± 0.47 mm) on aspiration day than the CONT group (39.5 ± 0.5 mm). Dietary n-3 fatty acids modify mare serum and follicular fluid fatty acid profiles, with supplementation of EPA and DHA decreasing serum and follicular IGF-1 concentrations.

Eggs enriched with omega-3 polyunsaturated fatty acids (PUFA), particularly the longer chain PUFA (LCPUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) can boost human consumption of these fatty acids implicated in human health. Alpha-linolenic acid (ALA) from plant seeds/oils, primarily serve as the source of omega-3 PUFA for hens, however, the scarcity of ALA-rich plants and the limited conversion of ALA to LCPUFA are challenges for egg enrichment. Two major experiments were conducted to determine potential factors regulating egg enrichment of omega-3 LCPUFA based on detailed assessment of PUFA profiles in different lipid pools of hen tissues. In experiment 1, supplementation of graded levels of hempseed products, provided ~ 0.1 to 1.3% of ALA in the diets. Experiment 2, investigated dietary supplementation of flaxseed oil (ALA-rich) and algal DHA (preformed LCPUFA), each providing similar graded levels of total omega-3 PUFA. Both ALA-containing models demonstrated a plateau in DHA enrichment of eggs at higher ALA intakes. ALA-containing diets led to high concentrations of ALA in the triacylglycerol (TAG) fraction of eggs and plasma, and the adipose tissue of flaxseed oil-fed hens. In total phospholipid (PL), particularly the phosphatidylethanolamine (PE), the levels of EPA and ALA in the yolk were linearly associated with those in the liver. In all tissues, DHA dominated the PE pool, exhibiting a plateau with a strong inverse correlation to the ratio of ALA to EPA in the liver, suggesting limited ALA availability for egg DHA enrichment. The use of algal DHA should therefore permit further accumulation of DHA in the total PL and TAG fractions of yolk. However, enrichment via preformed DHA (at 3.36% algal product) was also limited by hepatic PL resulting in more DHA and EPA being shunted to the adipose TAG, concurrent with elevated hepatic acyl-CoA synthetase (ACSL1) expression. As a function of total omega-3 PUFA intakes (regardless of source), similar levels of stearidonic acid (SDA) and particularly EPA accumulated in liver PE. Therefore, hepatic PL regulation, possibly aimed at maintaining EPA level, may potentially be limiting the amount of ALA accumulation in the same pool, hence limiting the endogenous synthesis of DHA and subsequent enrichment in eggs. / February 2017