In order to verify the research from this laboratory that sheep omasal epithelium contains mRNA encoding for a peptide transporter (s) and to determine di- to octapeptide transport capability, poly(A)+ RNA isolated from sheep omasal epithelium was injected into Xenopus laevis oocytes. Poly(A)+ RNA was functionally expressed in Xenopus oocytes 4 to 7 d post-injection. Peptide (5 di-, 10 tri-, 6 tetra-, 2 penta-, 1 hepta-, 1 septa-, 1 octapeptide) transport capability was measured by impaling oocytes with a microelectrode to monitor membrane potential (Vm). Oocytes were maintained in pH 5.5 buffer. Peptide transport was identified as being expressed when, in the presence of a buffered peptide substrate (1 mM), the oocyte membrane showed persistent depolarization (a more positive Vm). In the absence of peptide transport, the membrane became depolarized with the addition of buffered substrate, but rapidly repolarized to the resting potential. Peptide transport was expressed for some di-, tri-, and tetrapeptides. Measured depolarization ranged from 9.6 mV to 42.1 mV. Larger peptides were not transported by the oocytes. When transport expression was measured with the substrates in a pH 7.5 buffer, no transport occurred indicating that transport was dependent on a proton gradient. The data indicate that sheep omasal epithelium contains mRNA that code for a protein(s) capable of proton-dependent di-, tri-, and tetrapeptide transport. This provides further evidence that absorption of peptides from the ruminant stomach is possible. / Master of Science

Production of ammonia N, a-amino N, and peptide N was investigated following in vitro ruminal incubation of solvent soybean meal (SBM), dehydrated alfalfa, corn gluten feed, fish meal, distillers dried grains with solubles (DDG), cotton seed meal, brewers fried grains, meat and bone meal, blood meal, prolac, and casein (CAS). The influence of milling procedures on the production of ammonia N, a-amino N, and peptide N was also evaluated using different batches of soybean meals and distillers dried grains with solubles. The concentrations of peptide N and ammonia N measured in the cell free media at 0, 2, 4, 6, and 8 h were increased linearly (P < 0.001) with time. The mean concentrations of a-amino N were lower (P < 0.05) than the mean concentrations of peptide N and ammonia N. Production of peptide N, a-amino N, and ammonia N were varied (P < 0.05, time x protein) between proteins and between batches. Irrespective of the protein used, the amino acid composition of peptides (<3,000 MW) that appeared at 8 h had specific patterns suggesting differential utilization of peptides by ruminal microorganisms. Cell-free supernatants obtained following incubation (8 h) of SBM, CAS, and DDG were used as mucosal substrates in parabiotic chambers to quantify absorption of free and peptide-bound amino acids via ruminal and omasal epithelia of sheep. Serosal appearance of amino acids in peptide form was nearly three times higher (P < 0.001) than free amino acids. On tissue dry weight basis, serosal appearance of amino acids was greater (P < 0.01) across omasal than via ruminal tissues. There was a greater serosal appearance of amino acids from CAS than from SBM. Total, total essential (EAA), total nonessential (NEAA), and individual amino acid appearance in serosal fluids varied (P < 0.05, amino acid form x protein source) among SBM, CAS, and DDG. Collectively, these results indicate that the forestomach epithelia of sheep possess the potential to absorb ruminally derived peptides (relatively large amounts) and free amino acids (relatively small amounts). Also, the ruminal microbial degradation of dietary proteins may influence the amounts and types of free and peptide-bound amino acids absorbed via forestomach. / Ph. D.

Os objetivos deste trabalho foram avaliar os efeitos do teor de concentrado em dietas com óleo de soja ou peixe sobre o consumo, a produção e composição do leite, digestibilidades aparentes total e ruminal e sobre os perfis de ácidos graxos no omaso e na gordura do leite de vacas leiteiras. Foram utilizadas quatro vacas da raça Holandesa, com cânulas ruminais, 109 ± 10 dias em lactação, produção média de 31,24 ± 8,57 kg de leite e peso médio de 652 ± 37 kg, no início do experimento. Foi utilizado o delineamento experimental em Quadrado Latino 4x4, com períodos de 21 dias, sendo os 14 primeiros para adaptação e os sete últimos para colheita de amostras, com arranjo fatorial (2x2) dos tratamentos. Os tratamentos foram dietas totais misturadas, contendo silagem de milho, suplementadas com baixos (40%) ou altos (60%) teores de concentrado e com 2% de óleo de soja ou de peixe na matéria seca (MS). Não houve da interação entre a fonte de óleo e o teor de concentrado para a maioria das variáveis estudadas. A presença do óleo de peixe na dieta reduziu o consumo de MS (18,77 x 14,16 kg/d, respectivamente para óleo de soja e peixe - todos os demais dados estão na mesma ordem), as produções de leite (28,72 x 24,34 kg/d), gordura (0,796 x 0,562 kg/d), proteína (0,806 x 0,630 kg/d) e sólidos totais (2,995 x 2,411 kg/d) e os percentuais de gordura (2,81 x 2,34%) e proteína (2,82 x 2,59%) no leite. O tratamento com óleo de peixe aumentou os ácidos graxos insaturados (30,12 x 35,56%) e os ácidos graxos poliinsaturados (4,43 x 5,85%), e reduziu os ácidos graxos saturados (69,66 x 60,05%), na gordura do leite em relação ao óleo de soja. O tratamento com óleo de peixe promoveu uma redução no teor de C18:0 no leite (18,90 x 6,82%) e omaso (52,35 x 15,81%) e aumentou de C18:1 trans-11 no leite (2,84 x 9,75%) e no omaso (6,72 x 18,42%). Os teores de CLA cis-9, trans-11 (0,61 x 1,41%) foram aumentados no leite, mas não no omaso, com dietas com óleo de peixe. Os teores de C20:5 e C22:6 foram aumentados na gordura do leite e no omaso com as dietas com óleo de peixe. Não houve efeito de óleo para o CLA trans-10, cis-12 (P=0,61) na gordura do leite, mas este foi maior para o óleo de soja no omaso (0,09 x 0,02%). O óleo de peixe diminuiu o consumo e fluxo de nutrientes no canal do omaso, aumentou as digestibilidades aparentes total e ruminal de nutrientes, diminuiu a produção de leite, alterou composição do leite e o perfil de ácidos graxos do leite e do omaso. / The objective of this work was to evaluate the effect of concentrate level and oil source on dry matter intake, milk production and composition, apparent ruminal and total tract digestibilities, omasal and milk fatty acid profiles in dairy cows. Four Holstein cows, fitted with rumen cannula, averaging 109 ± days in milk, producing 31.24 ± 8.57 kg milk per day and live weight 652 ± 37 kg, at start of the trial, were used in a 2 x 2 factorial arrangement of treatments applied to a 4 x 4 Latin square design. The length of periods was 21 days, including 14 days of adjustment to the experimental diets followed by seven days of sampling. The treatments were 4 different diets (total mixed ration) containing maize silage supplemented with low (40%) or high (60%) levels of concentrate and 2% of soybean oil or fish oil on a dry matter (DM) basis. It was found that the level of concentrate and oil source interaction had no effect on almost all the variables. Dry matter intake (18.77 x 14.16 kg/d, respectively for soybean oil and fish oil ? all data is in the same order), milk yield (28.72 x 24.34 kg/day), fat yield (0.796 x 0.562 kg/day), protein yield (0.806 x 0.630 kg/day), total solids yield (2.995 x 2.411 kg/day), fat percentage (2.81 x 2.34%) and protein percentage (2.82 x 2.59%) decreased when fish oil was present. Fish oil increased unsaturated fatty acids (FA) (30.12 x 35.56%) and polyunsaturated FA (4.43 x 5.85%), and decreased saturated FA (69.66 x 60.05%) in milk, when compared to soybean oil. Fish oil decreased C18:0 level in milk (18.90 x 6.82%) and omasum (52.35 x 15.81%) and increased trans-11 C18:1 level in milk (2.84 x 9.75%) and omasum (6.72 x 18.42%). cis-19, trans-11 CLA level (0.61 x 1.41%) was increased in milk for fish oil diet, but did not in omasum. C20:5 and C22:6 levels were increased in milk and omasum for fish oil diet. trans-10, cis-12 CLA had no oil effect (P=0.61) in milk, but it was higher in omasum for soybean oil diet (0.09 x 0.02%). Fish oil decreased nutrients intake and omasal flow, increased apparent ruminal and total tract digestibilities, decreased milk production, changed milk composition and fatty acids profile in milk and omasum.

Os objetivos deste trabalho foram avaliar os efeitos do teor de concentrado em dietas com óleo de soja ou peixe sobre o consumo, a produção e composição do leite, digestibilidades aparentes total e ruminal e sobre os perfis de ácidos graxos no omaso e na gordura do leite de vacas leiteiras. Foram utilizadas quatro vacas da raça Holandesa, com cânulas ruminais, 109 ± 10 dias em lactação, produção média de 31,24 ± 8,57 kg de leite e peso médio de 652 ± 37 kg, no início do experimento. Foi utilizado o delineamento experimental em Quadrado Latino 4x4, com períodos de 21 dias, sendo os 14 primeiros para adaptação e os sete últimos para colheita de amostras, com arranjo fatorial (2x2) dos tratamentos. Os tratamentos foram dietas totais misturadas, contendo silagem de milho, suplementadas com baixos (40%) ou altos (60%) teores de concentrado e com 2% de óleo de soja ou de peixe na matéria seca (MS). Não houve da interação entre a fonte de óleo e o teor de concentrado para a maioria das variáveis estudadas. A presença do óleo de peixe na dieta reduziu o consumo de MS (18,77 x 14,16 kg/d, respectivamente para óleo de soja e peixe - todos os demais dados estão na mesma ordem), as produções de leite (28,72 x 24,34 kg/d), gordura (0,796 x 0,562 kg/d), proteína (0,806 x 0,630 kg/d) e sólidos totais (2,995 x 2,411 kg/d) e os percentuais de gordura (2,81 x 2,34%) e proteína (2,82 x 2,59%) no leite. O tratamento com óleo de peixe aumentou os ácidos graxos insaturados (30,12 x 35,56%) e os ácidos graxos poliinsaturados (4,43 x 5,85%), e reduziu os ácidos graxos saturados (69,66 x 60,05%), na gordura do leite em relação ao óleo de soja. O tratamento com óleo de peixe promoveu uma redução no teor de C18:0 no leite (18,90 x 6,82%) e omaso (52,35 x 15,81%) e aumentou de C18:1 trans-11 no leite (2,84 x 9,75%) e no omaso (6,72 x 18,42%). Os teores de CLA cis-9, trans-11 (0,61 x 1,41%) foram aumentados no leite, mas não no omaso, com dietas com óleo de peixe. Os teores de C20:5 e C22:6 foram aumentados na gordura do leite e no omaso com as dietas com óleo de peixe. Não houve efeito de óleo para o CLA trans-10, cis-12 (P=0,61) na gordura do leite, mas este foi maior para o óleo de soja no omaso (0,09 x 0,02%). O óleo de peixe diminuiu o consumo e fluxo de nutrientes no canal do omaso, aumentou as digestibilidades aparentes total e ruminal de nutrientes, diminuiu a produção de leite, alterou composição do leite e o perfil de ácidos graxos do leite e do omaso. / The objective of this work was to evaluate the effect of concentrate level and oil source on dry matter intake, milk production and composition, apparent ruminal and total tract digestibilities, omasal and milk fatty acid profiles in dairy cows. Four Holstein cows, fitted with rumen cannula, averaging 109 ± days in milk, producing 31.24 ± 8.57 kg milk per day and live weight 652 ± 37 kg, at start of the trial, were used in a 2 x 2 factorial arrangement of treatments applied to a 4 x 4 Latin square design. The length of periods was 21 days, including 14 days of adjustment to the experimental diets followed by seven days of sampling. The treatments were 4 different diets (total mixed ration) containing maize silage supplemented with low (40%) or high (60%) levels of concentrate and 2% of soybean oil or fish oil on a dry matter (DM) basis. It was found that the level of concentrate and oil source interaction had no effect on almost all the variables. Dry matter intake (18.77 x 14.16 kg/d, respectively for soybean oil and fish oil ? all data is in the same order), milk yield (28.72 x 24.34 kg/day), fat yield (0.796 x 0.562 kg/day), protein yield (0.806 x 0.630 kg/day), total solids yield (2.995 x 2.411 kg/day), fat percentage (2.81 x 2.34%) and protein percentage (2.82 x 2.59%) decreased when fish oil was present. Fish oil increased unsaturated fatty acids (FA) (30.12 x 35.56%) and polyunsaturated FA (4.43 x 5.85%), and decreased saturated FA (69.66 x 60.05%) in milk, when compared to soybean oil. Fish oil decreased C18:0 level in milk (18.90 x 6.82%) and omasum (52.35 x 15.81%) and increased trans-11 C18:1 level in milk (2.84 x 9.75%) and omasum (6.72 x 18.42%). cis-19, trans-11 CLA level (0.61 x 1.41%) was increased in milk for fish oil diet, but did not in omasum. C20:5 and C22:6 levels were increased in milk and omasum for fish oil diet. trans-10, cis-12 CLA had no oil effect (P=0.61) in milk, but it was higher in omasum for soybean oil diet (0.09 x 0.02%). Fish oil decreased nutrients intake and omasal flow, increased apparent ruminal and total tract digestibilities, decreased milk production, changed milk composition and fatty acids profile in milk and omasum.