Research conducted for this dissertation had three goals; 1) determine if CLA can induce milk fat depression immediately postpartum, 2) determine if CLA can alter energy availability, 3) determine the mechanism behind the mammary gland's decreased sensitivity to CLA immediately postpartum. The first study provides strong evidence indicating CLA can decrease milk fat synthesis immediately postpartum, but the dose required is approximately 3x greater than in established lactation. This trial also provided evidence that CLA can alter energy status, as CLA decreased days to EBAL nadir by nearly 5 days. This is relevant as recovery of EBAL from its lowest point provides an important signal for initiating ovarian activity and days to nadir is highly correlated with days to first ovulation. Study two was designed to determine if CLA induced milk fat depression could improve energy status during heat stress. Rumen-inert CLA reduced milk fat synthesis, and was able to improve energy availability, but did not increase milk yield or yield of other milk components. Although production was unchanged in this study, the study did provide further evidence that rumen-inert CLA can alter energy availability. Study three utilized intravenous infusion of CLA in cows in mid and early lactation to determine the mechanism for the mammary gland's decreased sensitivity in early lactation. It is postulated that increased fatty acid oxidation and subsequent enhanced levels of circulating NEFA present during the transition period competitively prevent adequate CLA uptake by the mammary gland. In the current study, trans-10, cis-12 CLA concentration in milk was not different between early and established lactation, while milk fat yield was drastically reduced on d 4 and 5 of trans-10, cis-12 CLA infusion in mid lactation cows, but unaltered in early lactation. Further, NEFA levels were nearly 3 fold higher in early lactation than in mid lactation, providing further evidence that increased circulating NEFAs in early lactation are unlikely to be the source of the mammary gland's decreased sensitivity during this time. Do to the variation in gene expression observed in this trial, we were unable to make any definitive conclusions as to the sensitivity of the expression of genes involved in milk lipid synthesis to CLA in early vs. mid lactation.

The objective of these studies was to investigate the milk fat synthesis regulation by transcription factors. In the first study, bovine mammary epithelial (MAC-T) cells were treated with sterol regulatory element binding protein-1 (SREBP-1) specific siRNA. The mRNA and protein expression of SREBP-1 were decreased by more than 90% by siRNA. Fatty acid (FA) synthesis, uptake, and selected lipogenic enzyme expression were reduced in cells treated with SREBP-1 siRNA. Therefore, SREBP-1 plays an important role in integrated regulation of lipid synthesis in MAC-T cells through regulation of key enzymes. In the second study, MAC-T cells treated with hormones or FA were transfected with luciferase reporter constructs containing response elements for SREBP-1, peroxisome proliferator-activated receptor γ (PPARγ), or liver X receptor (LXR). The activation of PPARγ and SREBP-1 were stimulated by insulin and insulin combined with leptin, respectively. Trans-10, cis-12 conjugated linoleic acid (CLA) inhibited SREBP-1 activation, and this inhibition was not attenuated by insulin and leptin. Neither trans-10 nor cis-12 double bond inhibited SREBP-1 activation. Taken together, trans-10 and cis-12 double bonds need to be conjugated in CLA to reduce SREBP-1 activation and this inhibition cannot be overcome by insulin and leptin combination in MAC-T cells. In the third study, lactating dairy cows were intravenously infused with 0.625 g/h trans-10, cis-12 CLA for 14 h. We confirmed the appearance of trans-10, cis-12 CLA in the milk of CLA treated cows. Milk and component yield were not affected by the CLA treatment. The desaturation of stearic acid was reduced by CLA. The mRNA and protein expression of transcription factors or lipogenic enzymes were not affected by trans-10, cis-12 CLA. DNA-binding activities for PPARγ and LXR and the activation of SREBP-1 to its mature form were not changed by the treatment. The infusion time in this study was probably too short to induce any changes in transcription factors and lipogenic enzymes. We confirmed DNA-binding activities of PPARγ and LXR in bovine mammary gland. Overall, a prominent role for SREBP-1 in mammary epithelial cell lipid synthetic pathways was described and regulation of transcription factor activation by trans-10, cis-12 CLA was specific to SREBP-1. / Ph. D.

Made available in DSpace on 2016-12-08T16:24:04Z (GMT). No. of bitstreams: 1 PGCA12MA037.pdf: 918485 bytes, checksum: 8954d3d831facc72cc305f43c58a9a17 (MD5) Previous issue date: 2012-06-25 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The supplementation with rumen protected conjugated linoleic acid (CLA) has demonstrated to change milk composition and also milk fatty acid profile. However, few studies have fed this supplement in a rumen unprotected form. The objective of this study was to evaluate the effects of a rumen unprotected CLA supplement (29.8% of cis-9, trans-11 CLA and 29.9% of trans-10, cis-12 CLA) on productive parameters, milk fatty acid profile and energy balance of lactating goats. Twenty Toggenburg lactating goats were used in a cross-over design and received the following treatments: a) Control: 30 g/d of Megalac-E®; b) CLA: 30 g/d of a CLA supplement. Treatments were mixed into a concentrate (1 kg/d) which was fed in two equal meals after the morning and afternoon milking. Experimental periods lasted 14 d and were separated by a 6 d interval. There was a 13.9 and 13.3% reduction in milk fat content and yield, respectively, in CLA-fed goats. Moreover, the CLA treatment induced a 6.6% reduction in the forage intake, a 4.8 and 4.6% reduction in the concentration and yield of total solids in milk, respectively. Milk yield and body weight were unaffected by the CLA treatment and averaged 2.6 ± 0.1 kg/d and 39.9 ± 0.5 kg (mean ± SE), respectively. When fatty acids were grouped according to origin, there was a 13% decrease in de novo synthesized fatty acids (<C16), a 15.5% increase in preformed fatty acids taken up from blood circulation (>C16), and a 9.5% decrease in those fatty acids (C16) derived from both sources. Furthermore, on desaturase indexes, supplementation with CLA reduced by 31.76% the C14:1/14:0 ratio, by 5.9% the C16:1/C16: ratio, by 25.2% the C17:1/C17:0 ratio, by 25.56% the C18:1/C18:0 and by 5.22% the CLA cis-9, trans-11/ C18:1 trans-11 ratio. The inclusion of CLA also increased the concentration of cis-9, trans-11 CLA and trans-10, cis-12 CLA by 29.2 and 1376%, respectively, which resulted in a 993.4% increase in the trans-10, cis-12 CLA secretion in milk fat. Likewise, the concentration of trans-10 C18:1 and trans-11 C18:1 was 134.1 and 39.9% higher in CLA-fed goats, respectively. The non-esterified fatty acids (NEFA) were reduced in 22.4% in CLA-fed goats. The energy balance improved in 123.8% of CLA-treatment when compared to control treatment / O fornecimento de ácido linoleico conjugado (CLA) em uma forma inerte no rúmen tem demonstrado efeitos consistentes sobre o teor de gordura e perfil de ácidos graxos do leite. Contudo, poucos estudos utilizaram esse suplemento na forma desprotegida da biohidrogenação ruminal. Os objetivos deste estudo foram avaliar os efeitos do fornecimento de um suplemento de CLA desprotegido da bio-hidrogenação ruminal (29,8% de cis-9, trans-11 e 29,9% de trans-10, cis-12) sobre parâmetros produtivos, perfil de ácidos graxos do leite e no balanço energético de cabras leiteiras. Vinte cabras Toggenburg foram utilizadas em um delineamento cross-over e receberam os seguintes tratamentos: a) Controle: 30 g/d de Megalac-E® e; B) CLA: 30 g/d de um suplemento de CLA. Os suplementos lipídicos foram misturados a um concentrado à base de milho (1 kg/d) fornecidos duas vezes ao dia em porções iguais, logo após as ordenhas da manhã e da tarde. Cada período experimental consistiu de 14 dias, separados por um período de seis dias para minimizar efeitos residuais. O tratamento CLA reduziu a concentração e a produção da gordura do leite em 13,9 e 13,3%, respectivamente.O tratamento reduziu ainda a concentração e produção de sólidos totais em 4,8 e 4,6%, respectivamente. O tratamento CLA reduziu o consumo de forragem em 6,6%.O peso vivo e a produção de leite não diferiram entre tratamentos, com médias de 39,9 ± 0,5 kg e 2,6 ± 0,1 kg/dia, respectivamente. Quando os ácidos graxos foram agrupados conforme a origem, houve uma redução de 13% naqueles derivados da síntese de novo (<C16), um aumento de 15,5% nos pré-formados oriundos da circulação (>C16) e uma redução de 9,45% para os originários de ambas a fontes (C16:0 e C16:1). A suplementação com CLA reduziu os índices de dessaturase do C14:1/C14:0 em 31,7%, do C16:1/C16:0 em 5,9%, do C17:1/C17:0 em 25,2%, do C18:1/C18:0 em 25,5% e do CLA cis-9, trans-11/trans-11 C18:1 em 5,2%. As concentrações de CLA trans-10, cis-12 e CLA cis-9, trans-11 foram 1376 e 29,2%maiores nas cabras que receberam CLA, respectivamente. As concentrações de C18:1 trans-10 e C18:1 trans-11 foram elevadas em 134,1 e 39,9%, respectivamente. A secreção de CLA trans-10, cis-12 aumentou 993,42% no tratamento CLA. O tratamento CLA reduziu a concentração de ácidos graxos não esterificados (AGNE) no plasma sanguíneo em 22,4%. O balanço energético dos animais melhorou no tratamento CLA em 123,8% quando comparado ao tratamento controle

Made available in DSpace on 2016-12-08T16:24:10Z (GMT). No. of bitstreams: 1 PGCA12MA089.pdf: 1289246 bytes, checksum: aeab19a1d5b6ebb9c5e6219d765deed7 (MD5) Previous issue date: 2012-04-10 / Feeding trans-10, cis-12 CLA has resulted in milk fat depression in dairy cows, ewes and goats. However, its effects in goats are less understood in comparison with other ruminant species. Therefore, the objective of the present study was to evaluate the effect of increasing doses of a rumen unprotected trans-10, cis-12 CLA supplement on milk production, secretion of milk components and milk fatty acid profile in dairy goats. Eight Toggenburg non pregnant goats (135 ± 21 DIM) were used in a duplicate 4 x 4 Latin Square design (12-d treatment periods separated by 6-d intervals) according to the order of lactation (primiparous and multiparous). The animals within each group were randomly assigned to the following dietary treatments: CLA0: 45 g/d of Megalac-E; CLA15: 30 g/d of Megalac-E+15 g/d CLA; CLA30: 15 g/d of Megalac-E+30 g/d of CLA; CLA45: 45 g/d of CLA. The lipid supplements were mixed in the concentrate and fed individually to the animals after morning and afternoon milkings. The CLA supplement (Luta-CLA 60) had 29.9% of trans-10, cis-12 CLA as methyl esters, resulting in doses of 0, 4.48, 8.97 and 13.45 g/d of this CLA isomer for CLA0, CLA15, CLA30 and CLA45 treatments, respectively. Dry matter intake, milk yield, content and secretion of milk protein and lactose, body condition score and body weight were unaffected by the dietary treatments., The comparison of milk fat content and yield observed on the last day of each treatment period (d 12) with those found on day 0 showed that CLA0 treatment increased the milk fat content. Thus, milk fat content and yield on day 0 were considered as 100%. The CLA15, CLA30 and CLA45 treatments reduced milk fat yield by 8.1, 26.1 e 32.7% and milk fat content by 4.5, 21.5 e 28.3%, respectively. The increase in dietary trans-10, cis-12 CLA dose reduced the concentration of milk fatty acids arising from de novo synthesis and increased the concentration of those derived from blood circulation. However, the milk secretions of both classes of fatty acids were reduced linearly as the CLA dose increased. The increase in dietary trans-10, cis-12 CLA also caused a linear reduction in milk fat C14:1/C14:0, C16:1/C16:0, C17:1/C17:0 and C18:1/C18:0 dessaturase indexes. Milk fat trans-10, cis-12 CLA content and secretion increased in goats fed increasing doses of trans-10, cis-12 CLA, which, corresponded to transfer efficiencies from diet to milk of 1.18, 1.17 and 1.21% for CLA15, CLA30 and CLA45 treatments, respectively. The energy balance (EB) increased linearly in goats fed increasing doses of trans-10, cis-12 CLA / O fornecimento de CLA trans-10, cis-12 através da dieta tem resultado na depressão da gordura do leite de bovinos, ovinos e caprinos. No entanto, os seus efeitos em caprinos são menos compreendidos perante as outras espécies de ruminantes. Assim, o objetivo do presente estudo foi avaliar o efeito de doses crescentes de CLA trans-10, cis-12 desprotegido da bio-hidrogenação ruminal sobre a produção, secreção dos componentes e perfil de ácidos graxos do leite de cabras leiteiras. Para isso, foram utilizados 8 animais da raça Toggenburg não prenhes com 135 ± 21 dias em lactação, separadas de acordo com o número de lactações para compor dois Quadrados Latinos 4 X 4 (12 dias de tratamento separados por 6 dias de intervalo), um formado por primíparas e o outro por multíparas. Os animais foram submetidos aleatoriamente aos seguintes tratamentos: CLA0) 45 g/d de Megalac-E; CLA15) 30 g/d de Megalac-E+15 g/d de CLA; CLA30) 15 g/d de Megalac- E+30 g/d de CLA; CLA45) 45 g/d de CLA. Os tratamentos foram inclusos no concentrado fornecido aos animais, assim, todos os tratamentos continham 45 g/d de suplemento lipídico. O suplemento de CLA (Luta-CLA 60) continha 29,9% do isômero trans-10, cis- 12 na forma de ésteres metílicos, perfazendo doses de 0, 4,48, 8,97 e 13,45 g/d deste ácido graxo para os respectivos tratamentos citados anteriormente. O consumo de matéria seca de silagem, a produção de leite, a produção e o teor de proteína e lactose do leite, o escore de condição corporal e o peso vivo não foram afetados pelas doses de CLA trans-10, cis- 12. Ao final de cada período experimental (d 12), a avaliação do teor e produção de gordura do leite em relação ao encontrado no dia 0, demonstrou que o tratamento CLA0 levou ao aumento da gordura do leite. Assim, as comparações de produção e teor de gordura do leite foram feitas considerando os valores obtidos no dia 0 como 100%. Os tratamentos CLA15, CLA30 e CLA45 reduziram, respectivamente, a produção de gordura do leite em 8,1, 26,1 e 32,7% e o teor de gordura do leite em 4,5, 21,5 e 28,3%. O uso de doses crescentes de CLA trans-10, cis-12 na dieta alterou o perfil de ácidos graxos do leite, com reduções da concentração dos ácidos graxos oriundos da síntese de novo e aumento da concentração dos provenientes da circulação sanguínea. Porém, quando a secreção destes mesmos ácidos graxos foi avaliada houve redução da secreção de ambas as classes de ácidos graxos. A suplementação de CLA trans-10, cis-12 também acarretou na redução dos índices de dessaturação C14:1/C14:0, C16:1/C16:0, C17:1/C17:0 e C18:1/C18:0. Tanto a concentração como a secreção do CLA trans-10, cis-12 aumentaram conforme o seu aumento na dieta, e correspondeu a uma eficiência de transferência da 1,18, 1,17 e 1,21% para os tratamentos CLA15, CLA30 e CLA45 (respectivamente), sendo que as mesmas não diferiram entre si. Quando o balanço energético (BE) dos animais foi avaliado, as inclusões crescentes do CLA trans-10, cis-12 na dieta aumentaram linearmente o BE

Made available in DSpace on 2016-12-08T16:24:18Z (GMT). No. of bitstreams: 1 PGCA14MA138.pdf: 701716 bytes, checksum: ae5be4aae0f8d732a1c72c1f572ee5fa (MD5) Previous issue date: 2014-07-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Feeding trans-10, cis-12 conjugated linoleic acid (CLA) to lactating ewes reduces milk fat by down-regulating gene expression of enzymes involved in lipid synthesis in mammary gland. An example is acetyl-CoA carboxylase alpha (ACC&#945;), a key enzyme in the de novo fatty acid synthesis pathway. ACC&#945; is encoded by mRNAs transcribed from three promoters, PI, PII and PIII, characterized as tissue-specific in the ovine genome. This study evaluated the effects of a rumen-unprotected trans-10, cis-12 CLA supplement fed to crossbred Lacaune/Texel lactating ewes, on gene expression of different mRNA transcripts of ACC&#945;. Twelve ewes arranged in a completely randomized design received for 14 days one of the following treatments, at 15 (early), 70 (mid) and 120 (late) days of lactation: Control (forage + 0.9 kg of concentrate-DM) and CLA [forage + 0.9 kg of concentrate + 28g/d of CLA (29.9% trans-10, cis-12)]. The CLA supplement was orally dosed. Mammary gland and adipose tissue biopsies were taken on day 14. Subsequently RNA was extracted, cDNA synthesized and real time PCR analysis conducted. Data were analyzed by MIXED procedure. Milk fat content was reduced by CLA supplementation in all stages of lactation. Compared to Control, in the mammary gland CLA reduced (P<0.05) transcript from PIII in early and mid lactation, increasing transcript from PI in adipose tissue. In the late lactation there was a higher milk fat depression and transcripts PII-derived were downregulated. Overall, our results suggest that trans-10, cis-12 CLA down-regulates ACC&#945; gene expression by decreasing expression from promoter II and III in mammary tissue and increases ACC&#945; gene expression by increasing expression promoter I in adipose tissue, having influences of stage of lactation on the behavior of mRNA isoforms of ACC&#945; / A Suplementação com ácido linoleico conjugado (CLA trans-10, cis-12) para ovelhas em lactação reduz a gordura do leite regulando a expressão dos genes de enzimas envolvidas na síntese lipídica na glândula mamária. Um exemplo é a acetil-CoA carboxilase alfa (ACC&#945;), uma enzima chave na síntese de novo de ácidos graxos. A ACC&#945; é codificada por mRNAs transcritos a partir dos promotores PI, PII, PIII, caracterizados como tecido-específico no genoma ovino. Este estudo avaliou os efeitos da suplementação com CLA trans-10, cis-12 desprotegido da bio-hidrogenação ruminal para ovelhas em lactação cruzadas Lacaune/Texel, sobre a expressão gênica de diferentes transcritos de mRNA da ACC&#945;. Doze ovelhas arranjadas em um delineamento experimental inteiramente casualizado receberam por 14 dias um dos seguintes tratamentos, durante o início, meio e fim de lactação: Controle (forragem + 1 kg de concentrado) e CLA [forragem + 1kg de concentrado + 30mL/d de CLA (29,9% trans-10, cis-12)]. A suplementação com CLA foi administrada oralmente. Foram feitas biópsias de glândula mamária e tecido adiposo no 14º dia de cada período experimental. O RNA foi extraído, o cDNA sintetizado para posterior análise por PCR real time. Os dados foram analisados pelo procedimento MIXED. Teor de gordura do leite foi reduzido pela suplementação de CLA em todos os períodos de lactação. Comparado ao controle, o CLA reduziu (P<0,05) os transcritos oriundos do PIII na glândula mamária no início e meio da lactação, aumentando transcritos oriundos PI no tecido adiposo. No final da lactação houve uma maior depressão da gordura do leite e transcritos derivados do PII foram reduzidos. Em suma, nossos resultados sugerem que o CLA trans-10, cis-12, regula a expressão gênica da ACC&#945;, diminuindo a expressão a partir do promotor II e III, no tecido mamário e aumentando a expressão gênica ACC&#945; através do PI no tecido adiposo, apresentando influências da fase de lactação sobre o comportamento das isoformas de mRNA da ACC&#945;

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