CAPES / O objetivo do presente trabalho foi identificar e quantificar os protozoários ciliados e bactérias do rúmen de bovinos cruzados (Europeu x Nelore), com média de 2,5 anos, alimentados em pastagens de inverno (Azevém e Aveia + Azevém) e verão (Brachiaria spp. e Brachiaria spp. + Esrela Africana). Amostras de conteúdo ruminal foram obtidas do centro da massa ruminal, após o abate dos animais. A contagem de bactérias totais (CBT), para as frações sólida e para líquida, foi realizada em placas com auxílio de contador de colônias manual. Para a análise morfológica empregou-se a técnica de coloração de Gram e a leitura das lâminas foi feita em microscópio óptico com objetiva de 100x, sendo as bactérias classificadas em cocos e bacilos, gram positivos e gram negativos. A avaliação da atividade fermentativa foi evidenciada pelo crescimento de colônias com coloração rósea atravéz de uma fonte de carboidrato. A quantificação e identificação dos gêneros de ciliados foram realizadas em câmara Sedgewick-Rafter em microscopia ótica. Foi determinado o teor de Matéria Seca, Proteína Bruta, Matéria Mineral, Fibra em Detergente Neutro e Fibra em Detergente Ácido, Digestibilidade In Vitro na Matéria Seca, Lignina, Celulose, Hemicelulose. O delineamento experimental foi inteiramente casualizado, com quatro tratamentos e 10 repetições para cada tratamento. Os dados foram submetidos à análise por meio da metodologia de Modelos Lineares, generalizados, com distribuição Poisson (1%) e foi utilizado o procedimento GENMOD. Foram observadas bactérias Gram positivas e negativas, nas formas cocos e bacilos e 11 gêneros de protozoários ciliados, sendo Diplodinium o gênero predominante. A maior concentração de bactérias foi encontrada nas forrageiras de inverno (494,8 x1010 mL-1), com maior intensidade na pastagem de Azevém. Nos protozoários ciliados, maior concentração foi observada no verão (200,70 x104 mL-1), principalmente na pastagem de Brachiaria spp. / The objective of the present work was to identify and quantify the ciliate protozoa and crossbovine rumen bacteria (European x Nellore), with an average of 2.5 years, fed on winter pastures (Azevém and Aveia + Azevém) and summer (Brachiaria spp And Brachiaria spp. + Esrala Africana). Samples of ruminal contents were obtained from the center of the ruminal mass, after the slaughter of the animals. The total bacterial count (CBT), for solid and liquid fractions, was performed in plates with the aid of a manual colony counter. For the morphological analysis the Gram staining technique was used and the slides were read under an optical microscope with objective of 100x, being the bacteria classified in cocci and bacilli, gram positive and gram negative. The evaluation of the fermentative activity was evidenced by the growth of colonies with pink coloration through a carbohydrate source. The quantification and identification of ciliate genera were performed in Sedgewick-Rafter chamber under optical microscopy. The content of Dry Matter, Crude Protein, Mineral Matter, Neutral Detergent Fiber and Acid Detergent Fiber, In Vitro Digestibility in Dry Matter, Lignin, Cellulose and Hemicellulose were determined. The experimental design was completely randomized, with four treatments and 10 replicates for each treatment. The data were submitted to analysis using the methodology of Linear Models, generalized with Poisson distribution (1%) and the GENMOD procedure was used. Gram positive and negative bacteria were observed, in the forms cocci and bacilli and 11 genera of ciliate protozoa, being Diplodinium the predominant genus. The highest concentration of bacteria was found in winter forages (494.8 x1010 mL-1), with higher intensity in the Azevém pasture. In the ciliate protozoa, higher concentration was observed in the summer (200.70 x 10 4 mL-1), mainly in Brachiaria spp.

O objetivo deste trabalho foi avaliar o efeito de um preparado de anticorpos policlonais (PAP) contra bactérias ruminais específicas, Streptococcus bovis e Fusobacterium necrophorum, em parâmetros ruminais da fermentação, em vacas canuladas, adaptadas ou não a uma dieta de alta proporção de carboidratos prontamente fermentescíveis, após indução à acidose. O delineamento experimental utilizado foi o quadrado latino 3X3 replicado em arranjo fatorial de tratamentos 3X2, sendo 2 aditivos alimentares (PAP na apresentação em pó - PAPP e PAP na apresentação líquida - PAPL) mais um grupo controle (CON) e dois manejos de adaptação à dieta, resultando em seis tratamentos. O primeiro quadrado latino foi submetido a um protocolo de adaptação à dieta do tipo gradual ou step-up: dos dias D0 a D4 os animais receberam 100% de forragem; do D5 ao D9, 30% de concentrados e do D10 ao D14, 60% de concentrados. O segundo quadrado latino recebeu 100% de forragem do D0 ao D14 (sem adaptação). Nos D15 e D16, todos os animais receberam dieta com 80% de concentrados. Para as análises foram coletadas amostras de líquido ruminal a cada 3 horas a partir da 0h antes da alimentação até as 36h (D15 e D16) durante o desafio com uma dieta de 80% de concentrados. Os dados foram analisados pelo procedimento Mixed do SAS com nível de significância de 0,05. Foi observada interação entre tempo e adaptação (P<0,05) para pH ruminal com diferença entre método de adaptação nas 0, 3, 6, 9, 12 e 36 horas pós alimentação, quando o grupo não adaptado teve valores maiores que o grupo adaptado, sendo que na hora 24 ocorreu o contrário. Para a concentração de ácidos graxos de cadeia curta (AGCC), nas horas 0, 3, 6, 9 e 36 pós alimentação o grupo adaptado obteve maiores valores comparado ao grupo não adaptado. Para proporção molar de acetato, a 0 hora o grupo sem adaptação obteve valores maiores comparado ao grupo adaptado. Já nas horas 24, 27 e 30 o grupo com adaptação que obteve maiores valores. Para a proporção molar de propionato o grupo sem adaptação teve valores mais altos em comparação ao outro grupo das 3 às 36 horas pós alimentação. Quanto à proporção acetato:propionato (Ac:Pr) às 6, 12, 24, 27, 30 e 36 horas pós alimentação, o grupo de animais adaptados teve valores mais altos que o grupo não adaptado. Na proporção molar de butirato, o grupo de animais adaptados obteve maiores valores nas horas 0, 3, 6, 9, 12, 33 e 36. Para os valores de nitrogênio amoniacal (N-NH3), às 6 horas pós alimentação, o grupo não adaptado obteve maiores valores que o grupo adaptado (26,1 vs. 19,3, respectivamente). Nas horas 9, 30, 33 e 36 ocorreu o contrário. Observou-se também interação entre tempo e aditivo (P=0,0430) para a proporção molar de butirato. Porém, quando a análise foi realizada por tempo, nenhum efeito foi observado. Para os valores relativos de protozoários mensurados (Dasytricha, Isotricha, Epidinium, Diplodinium e Entodinium) apenas o Entodinium apresentou efeito de adaptação (P<0,0236) tendo sua proporção maior no grupo adaptado. Os valores de haptoglobina também não foram influenciados nem por aditivo nem por adaptação. O preparado de anticorpos policlonais não foi tão eficaz quanto a adaptação gradual à dieta de alto concentrado para controlar alterações dos parâmetros ruminais. / The objective of this trial was to evaluate the effects of polyclonal antibodies preparation (PAP) against specific rumen bacteria Streptococcus bovis and Fusobacterium necrophorum on rumen fermentation parameters in ruminally cannulated cows adapted or not to highly fermentable carbohydrates diets (HFC) after an acidosis challenge. The experimental design was two 3X3 Latin squares in a factorial arrangement of treatments 3X2 regarding two feed additives (PAP in powder presentation - PAPP and PAP in liquid presentation - PAPL) plus control group (CON) and two managements of diets adaptation, resulting in six treatments. The first Latin square had a step-up diet adaptation: from D0 to D4 100% forage; D5 to D9 30% of concentrates and D10 to D14 60% of concentrates. The second Latin square received 100% forage from D0 to D14. On D15 and D16, all animals received a diet with 80% of concentrates. For analysis, rumen fluid was sampled at 0 and every 3 h posfeeding totaling 36 h (D15 and D16) of challenge with a diet with 80% of concentrates. Data were analyzed by MIXED procedure with a significance level of 0.05. An interaction between time and adaptation (P<0,05) was observed for ruminal pH. At 0, 3, 6, 9, 12 and 36 h postfeeding, the non-adapted group had higher values compared to the adapted group and at 24 h postfeeding, the inverse was observed. For total short-chain fatty acids concentration, at 0, 3, 6, 9 and 36 h postfeeding, the adapted group had higher values compared to non-adapted group. For molar proportion of acetate at 0h postfeeding, the non-adapted group had higher values than the adapted group, and at 24, 27 and 30h, the adapted group had greater values than the non-adapted group. For molar proportion of propionate the non-adapted group had greater values compared to the adapted group from 3 to 36h postfeeding. For acetate:propionate (Ac:Pr) ratio at 6, 12, 24, 27, 30 and 36 h postfeeding, the adapted group had greater values compared to the nonadapted group. For butyrate molar proportion at 0, 3, 6, 9, 12, 33 and 36h postfeeding the adapted group had greater values than the non-adapted group. For ammonia nitrogen (NH3- N) concentration at 6h, the non-adapted group had greater values than the adapted group (26.1 vs. 19.3, respectively), however at 9, 30, 33 and 36h postfeeding, the adapted group had higher values compared to the non-adapted group. It was also observed an interaction between time and additive (P=0.0430) for butyrate molar proportion, but when the analysis was performed by time no effect was observed. For the relative values of protozoa measured (Dasytricha, Isotricha, Epidinium, Diplodinium and Entodinium) only Entodinium presented adaptation effect (P<0.0236) with a higher proportion in the adapted group. Haptoglobin values was also not influenced (P>0.05) by additive or adaptation effect. Polyclonal antibodies preparation was not as effective as the gradual adaptation to the diet high concentrate to control changes of ruminal parameters.

No description available.

Ph. D.

Thesis (MScAgric (Animal Sciences))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Ruminants have a compound stomach system that enables them to utilize forages more efficiently than monogastric animals. However, forages alone do not contain sufficient nutrients to meet the requirements of high producing dairy cows. Forages are high in fibre and their nutrient availability depends on the degree of cell wall degradability. Improvements in forage fermentation would increase energy intake and subsequently milk production and performance by dairy cows. It is therefore important to find ways to improve forage degradation and utilization in the rumen. The use of different non-fibre carbohydrate (NFC) sources has different effects on animal performance. Supplementing forage based diets with energy sources containing sugar, starch or pectin results in variation in performance measurements such as milk yield, milk composition and dry matter intake (DMI). This thesis reports on two studies in which the effect of energy supplementation on forage fermentation and digestion parameters was investigated. In the first study an in vitro gas production protocol was used to determine the effect of sugar (molasses), starch (maize meal) and pectin (citrus pulp) on total gas production and rate of gas production of different forages. The forage substrates included wheat straw (WS), oat hay, (OH) lucerne hay (LUC), ryegrass (RYE) and kikuyu grass (KIK). The three energy sources, as well as a control (no energy source) were incubated in vitro with each of the above mentioned forages. Rumen fluid was collected from two lactating Holstein cows receiving a diet consisting of oat hay, lucerne, wheat straw and a concentrate mix. Forages alone (0.25 g DM) and/or together (0.125 g DM) with either molasses (0.1412 g DM), citrus pulp (0.1425 g DM) or maize meal (0.125 g DM) were weighed into glass vials and incubated for 72 hours. The weights of the energy sources were calculated on an energy equivalent basis. Blank vials, that contained no substrates, were included to correct for gas production from rumen fluid alone. The substrates were incubated in 40 ml buffered medium, 2 ml of reducing solution and 10 ml rumen fluid. Gas pressure was recorded automatically every five minutes using a pressure transducer system and the method based on the Reading Pressure Technique (Mauricio et al., 1999). Gas pressure was converted to gas volume using a predetermined regression equation. In the first gas production trial, the gas production included gas produced by the energy sources, while in the second gas production trial, the energy source gas production was deducted from the total gas production to determine the effect of energy source on gas production of respective forage substrates per se. Data were fitted to two non-linear models adapted from Ørskov and McDonald (1979). Significant forage x energy interactions were observed for the non-linear parameter gas production (b) in Model 1 and for b and lag phase (L) in Model 2 in both trials. In the first gas production trial, the higher fermentability of the energy sources supplemented to forage substrates, increased b (Model 1 & 2) of the LUC and WS. The gas production rate was affected in different ways for different forages, with the most noticeable effect on WS when it was supplemented with energy sources. All the energy sources increased c of WS irrespective of the model used. Energy sources had no effect on the L of LUC, OH or RYE, but decreased the L of WS and KIK. In the second trial, maize meal had no effect on b for any of the forages (Model 1 & 2), while molasses (Model 1 & 2) decreased b for all forage substrates, and citrus pulp (Model 1 & 2) decreased b of OH and RYE, to lower values than those of the control treatments. Gas production rate was not affected by molasses for any of the forage substrates, while citrus pulp (Model 1 & 2) increased c of OH and maize meal increased c of OH and KIK. Lag phase was only affected by energy sources in WS and KIK, where all the energy sources had lower L values than the control treatment. It was concluded that forage fermentability is affected differently by different energy sources. These observations may have important implications, in practice, on rumen health and milk production, and the data obtained can potentially be used as guidelines in feed formulations. In the second study, in vitro digestibility trials were undertaken to determine the effect of sugar (molasses and sucrose), starch (maize meal and maize starch) and pectin (citrus pulp and citrus pectin) on neutral detergent fibre (NDF) and dry matter (DM) degradability of forages. Forage substrates used included wheat straw, oat hay, lucerne hay, ryegrass and kikuyu grass. Rumen fluid was collected from two lactating Holstein cows receiving a diet consisting of oat hay, wheat straw and a concentrate mix. In vitro degradability was done with an ANKOM Daisy II incubator and forage substrates were incubated with or without the respective energy sources for 24, 48 and 72 hours. The substrates were incubated in 1076 ml buffered medium, 54 ml of reducing solution and 270 ml rumen fluid. The residues were washed, dried and analyzed for NDF. In the study with the applied energy sources (molasses, maize meal and citrus pulp) there were a forage x energy source interactions. Supplementation with the applied energy sources all improved dry matter degradability (DMD) of forages (24 and 72 hours), when compared to the control treatment, except for RYE supplemented with maize meal and citrus pulp at 24 hours. Molasses seemed to have had the biggest effect on DMD in all forage substrates. Supplementation with maize meal had no effect on neutral detergent fibre degradability (NDFD) of any forage substrate, except for an improvement in NDFD of LUC at 72 hours. Molasses improved NDFD of LUC at 24h, but had no effect on the other forage substrates. Citrus pulp improved NDFD of OH (72 hours), as well as LUC and WS (24 and 72 hours). It is postulated that the NDF of the energy sources was more digestible than that of the respective forages, and that the improved NDFD values could be ascribed to the contribution of the energy source NDFD. Overall, pasture grasses had a higher NDFD than the hays and straw, and appear to be more readily fermentable by rumen microbes than the low quality hays and straw explaining the higher NDFD. In the study involving the purified energy sources (sucrose, maize starch and citrus pectin), forage x energy source interactions were observed. In general, supplementation with these energy sources improved DMD at 24 and 72 hours except for RYE and KIK (72 hours). Pasture grasses (RYE and KIK) had a higher NDFD than LUC, OH and WS. At 72 hours, NDFD was 37.1% for LUC, 42.5% for OH and 40.3% for WS, compared to 70.5% for KIK and 64.9% for RYE. A possible explanation is that KIK and RYE samples came from freshly cut material, harvested after a 28d re-growth period. In general, sucrose (24 and 72 hours) and citrus pectin (72 hours) had no effect on NDFD of forage substrates. However, supplementing oat hay (24 hours) with starch and citrus pectin, and wheat straw (24 and 72 hours) with starch lowered NDFD, when compared to the control treatment. It is hypothesized that microbes fermented the easily fermentable energy sources first, before attacking forage NDF. The study suggested that forage NDFD values are not fixed, and may be altered by type of energy supplementation. / AFRIKAANSE OPSOMMING: Die meervoudige maagsisteem van herkouers stel hulle in staat om ruvoer meer effektief te benut as enkelmaagdiere. Ruvoere alleen bevat egter nie genoeg voedingstowwe om die behoeftes van hoogproduserende melkbeeste te bevredig nie. Ruvoere is ryk aan vesel en hul voedingstofbeskikbaarheid word bepaal deur die graad van selwand degradeerbaarheid. ‘n Verhoging in ruvoerfermentasie sal energieinname verhoog en gevolglik ook melkproduksie en prestasie. Dit is dus belangrik om maniere te vind om ruvoerdegradeerbaarheid en -verbruik in die rumen te verbeter. Die gebruik van verskillende nie-vesel koolhidraat (NFC) bronne het verskillende uitwerkings op die prestasie van diere. Energie-aanvullings soos suiker, stysel en pektien tot ruvoer-gebasseerde diëte, beïnvloed prestasiemaatstawwe soos melkproduksie, melksamestelling en droëmateriaalinname (DMI) op verskillende maniere. Hierdie tesis lewer verslag oor twee studies waar die invloed van energie-aanvullings op ruvoerfermentasie en verteringsmaatstawwe ondersoek is. In die eerste studie is ‘n in vitro gasproduksieprotokol gebruik om die invloed van suiker (melasse), stysel (mieliemeel) en pektien (sitruspulp) op totale gasproduksie (b) en tempo van gasproduksie (c) van verskillende ruvoersubstrate te bepaal. Ruvoersubstrate wat gebruik is, was koringstrooi (WS), hawerhooi (OH), lusernhooi (LUC), raaigras (RYE) en kikuyugras (KIK). Die drie energiebronne, sowel as ‘n kontrole (geen energiebron), is in vitro geïnkubeer saam met elk van die genoemde ruvoere. Rumenvloeistof is verkry van twee lakterende Holsteinkoeie, wat ‘n dieet ontvang het bestaande uit hawerhooi, koringstrooi en ‘n kragvoermengsel. Ruvoere is alleen en/of in kombinasie met melasse (0.1412 g DM), sitruspulp (0.1425 g DM) of mieliemeel (0.125 g DM) in glasbottels afgeweeg en vir 72 uur geïnkubeer. Die massas van die energiebronne is op ‘n energie-ekwivalente basis bereken. Leë bottels wat geen substraat bevat het nie, is ingesluit om te korrigeer vir gasproduksie afkomstig vanaf rumenvloeistof alleen. Substrate is in 40 ml van ‘n buffermedium, 2 ml reduserende oplossing en 10ml rumenvloeistof geïnkubeer. Gasdruk is elke vyf minute outomaties aangeteken deur gebruik te maak van ‘n drukmetersisteem en die metode is gebasseer op die Reading gasdruktegniek. Gasdruk is omgeskakel na gasvolume deur gebruik te maak van ‘n voorafbepaalde regressievergelyking. In die eerste proef het totale gasproduksie die gas wat deur die onderskeie energiebronne geproduseer is, ingesluit. In die tweede proef is gasproduksie afkomstig van die energiebronne afgetrek van totale gasproduksie, om sodoende die invloed van die energiebronne per se op die gasproduksie van die onderskeie ruvoersubstrate, te bepaal. Data is met behulp van twee nie-liniëre modelle gepas. Betekenisvolle ruvoer x energie-interaksies is in albei proewe waargeneem vir die nie-liniëre parameter b (gasproduksie) in Model 1, en vir b en L (sloerfase) in Model 2. In die eerste proef het die energiebronne se hoë fermentasie gelei to ‘n verhoging in b (Model 1 & 2) van LUC en WS. Energie-aanvullings het die c-waarde van die onderskeie ruvoere verskillend beïnvloed, met WS wat die mees opvallende effek gehad het. Al die energiebronne het die c-waarde van WS verhoog, ongeag watter model gebruik is. Energiebronne het geen invloed op die L-waarde van LUC, OH of RYE gehad nie, maar het wel die L-waarde van WS en KIK verlaag. In die tweede proef het mieliemeel geen invloed op die b-waarde van enige van die ruvoere gehad nie (Model 1 & 2), terwyl melasse (Model 1 & 2) die b-waarde van alle ruvoere verlaag het, en sitruspulp (Model 1 & 2) OH en RYE se b waardes verlaag het tot laer as die kontroles. Melasse het geen invloed op die c-waarde van die onderskeie ruvoersubstrate gehad nie, terwyl sitruspulp (Model 1 & 2) die c-waarde van OH, en mieliemeel die c-waarde van OH en KIK, verhoog het. Energiebronne het slegs ‘n invloed op die sloerfase in WS en KIK gehad, waar dit L verlaag het tot laer waardes as dié van die kontroles. Daar is gevind dat ruvoer-fermenteerbaarheid verskillend beïnvloed word deur verskillende energiebronne. Bogenoemde resultate kan in die praktyk betekenisvolle invloede hê op rumengesondheid en melkproduksie en die data wat verkry is, kan potensieël gebruik word as riglyne in voerformulerings. In die tweede studie is in vitro verteerbaarheidsproewe gedoen om die effek van suiker (molasse en sukrose), stysel (mieliemeel en mieliestysel) en pektien (sitruspulp en sitrus-pektien) op neutraalonoplosbare vesel (NDF) en droë materiaal (DM) degradeerbaarheid van ruvoere, te bepaal. Ruvoersubstrate wat gebruik is, was WS, OH, LUC, RYE en KIK. Rumen vloeistof is verkry van twee lakterende Holstein koeie, wat ‘n dieet ontvang het bestaande uit hawerhooi, koringstrooi en ‘n konsentraat mengsel. Die in vitro degradeerbaarheidsproef is gedoen met ‘n ANKOM Daisy II inkubator. Ruvoersubstrate is geïnkubeer met of sonder die onderskeie energiebronne vir 24, 48 en 72 uur. Die substrate is geïnkubeer in 1076 ml buffer medium, 54 ml reduserende oplossing en 270 ml rumen vloeistof. Residue is gewas, gedroog en geanaliseer vir NDF. In die proef met toegepaste energiebronne (molasse, mieliemeel en sitruspulp), was daar ruvoer x energiebron interaksies. Toegepaste energiebron aanvullings het almal DMD van ruvoersubstrate (24 en 72 uur) verbeter, uitsluitend vir RYE wat aangevul is met mieliemeel (24 uur) en sitruspulp (24 uur). Van al die ruvoersubstrate het molasse die grootste effek gehad op DMD. Mieliemeel aanvullings het geen effek gehad op neutraal-onoplosbare vesel degradeerbaarheid (NDFD) van ruvoersubstrate nie, behalwe vir ‘n verbetering in NDFD van LUC by 72 uur. Molasse het NDFD van lucern by 24 uur verbeter, maar geen effek gehad op ander ruvoersubstrate nie. Sitruspulp het NDFD van OH (72 uur), asook LUC en WS (24 & 72 uur) verbeter. Daar word beweer dat die NDF van energiebronne meer verteerbaar is as die van ruvoersubstrate, en dat die verbetering in NDFD waardes toegeskryf kan word aan die bydraes van energiebronne se NDFD. Weidingsgrasse (RYE & KIK) het oor die algemeen ‘n hoër NDFD as hooie en strooi gehad. Rumen mikrobes blyk ook om dié grasse vinniger te verteer as lae kwaliteit hooie en strooi, wat gevolglik die hoër NDFD verduidelik. In die proef met suiwer energiebronne (sukrose, mieliestysel en sitrus-pektien) is ruvoer x energiebron interaksies waargeneem. Energiebronaanvullings het DMD by 24 en 72 uur verbeter, buiten vir RYE en KIK (72 uur). Weidingsgrasse het hoër NDFD as LUC, OH en WS. By 72 uur was die NDFD van LUC 37.1%, OH 42.5%, WS 40.3%, in vergelyking met 70.5% vir KIK en 64.9% vir RYE. ‘n Moontlike verklaring vir die hoër NDFD van KIK en RYE, is omdat dit vars gesnyde material is, geoes na slegs 28 dae hergroei. Oor die algemeen het sukrose (24 & 72 uur) en sitrus-pektien (72 uur) geen effek gehad op NDFD van ruvoersubstrate nie, terwyl stysel en pektien aanvullings tot OH (24 uur), en stysel aanvullings tot WS (24 & 72 uur) NDFD verlaag het. Daar word hipotetieseer dat mikrobes eers die vinnig fermenteerbare energiebronne fermenteer, voordat hulle ruvoer NDF aanval. Hierdie studie beweer dat ruvoer NDFD waardes nie vas is nie, en dat dié waardes beïnvloed mag word deur energiebron aanvullings.

The rumen microbiome constitutes a unique genetic resource of plant fiber degrading microbial enzymes that could be used for agricultural and industrial purposes. Anaeromyces mucronatus is a poorly characterized anaerobic lignocellulolytic fungus in the rumen. This thesis aimed at better understanding A. mucronatus YE505 and the particle associated rumen microbiota based on transcriptomic and metatranscriptomic approaches. High quality RNA was isolated from the fiber-associated rumen sample based on an improved RNA extraction method. A transcriptomic study was performed to investigate the expression of the fiber degrading system of A. mucronatus YE505, and the functional diversity of the fiber-associated eukaryotes from the rumen of muskoxen (Ovibos moschatus) was explored by a metatranscriptomic study. Much carbohydrate degradation related protein modules were detected. This study established effective approaches to characterizing the functional contents of rumen eukaryotic microbiome as well as rumen fungi, and identified several candidate genes that merit further investigation. / xiv leaves : ill. (some col.) ; 29 cm

CAPES / O objetivo do presente trabalho foi identificar e quantificar os protozoários ciliados do rúmen de dois grupos genéticos de bovinos de corte (Nelore e Cruzados Nelore x Europeu), sob três sistemas de alimentação (confinado, à pasto e à pasto com suplemento). Amostras de conteúdo ruminal foram obtidas do centro da massa ruminal, após o abate dos animais. A quantificação e identificação dos gêneros de ciliados foram realizadas em câmara de contagem Sedgewick-Rafter em microscopia ótica. Foi determinado o conteúdo de matéria seca (MS), proteína bruta (PB), extrato etéreo (EE), matéria mineral (MM), fibra em detergente neutro (FDN) e fibra em detergente ácido (FDA) dos alimentos analisados. Os dados foram submetidos a analise por meio da metodologia de Modelos Lineares Generalizados, com distribuição Poisson (1%). Adicionalmente os dados foram submetidos a analise dos Componentes Principais. Houve efeito da interação (P<0,001) para as dietas e as raças analisadas. Verificou-se a ocorrência de 14 gêneros, sendo o gênero Entodinium o predominante em todos os animais analisados. Os ciliados pertencentes à ordem Entodiniomorphida, Eodinium, Epidinium, Eremoplastron, Eudiplodinium, Metadinium e Ostracodinium apresentaram maior prevalência nos animais da raça Nelore, quando comparados com o grupo racial cruzado Nelore x Europeu. Protozoários da família Isotrichidae (Dasytricha e Isotricha) foram observados em maior quantidade nos animais à pasto e a pasto recebendo suplemento. Em relação ao tipo de alimentação, animais alimentados exclusivamente a pasto apresentaram maior densidade de ciliados em relação aos animais confinados e/ou a pasto recebendo suplemento. Registrou-se a ocorrência do gênero Buestchlia, em um animal (prevalência de 1,66%) sendo este um dos poucos registros deste gênero em ruminantes. / The aim of this study was to identify and quantify the rumen ciliated protozoa of two genetic groups cattle (Nellore and crossbred Nellore x European) under three feeding systems (confined, pasture and pasture with supplement). The rumen fluid samples were obtained from the center of the rumen mass, after … The quantification was done in Sedgewick-Rafter counting chambre in light microscopy. It was determined the content of dry matter (DM), mineral matter (MM), ether extract (EE), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF) to determine the chemical quality of the feed supplied to the animals. The data were submitted to analysis by Generalized Linear Models methodology, assuming Poisson distribution with logarithmic link function, with significance level of 1%. In addition, data were submitted to principal component analysis. The first two principal components were analyzed by least squares. There was a significant interaction (P<0.001) for diets and analyzed races. Occurrence of 14 ciliate genera was observed, being Entodinium the predominant in all animals analyzed. The ciliates belonging to order Entodiniomorphida, Eodinium, Epidinium, Eremoplastron, Eudiplodinium, Metadinium e Ostracodinium had a higher prevalence in Nellore cattle, compared with the racial group crossbred Nellore x European. Protozoa of the family Isotrichidae (Dasytricha e Isotricha) were observed in greater quantities in animals to pasture and pasture with supplement. Regarding the type of food, animals on pasture had higher ciliates density compared to confined animals and pasture with supplement. Occurrence of gender Buestchlia only one animal (prevalence 1,66%), which is one of the few records of this kind in ruminants.

Changes in cell wall composition and in vitro degradation of corn stover fractions (leaf, upper stem and lower stem) with advancing maturity and in response to NH; treatment were determined, and possible inhibitory mechanisms of lignin were evaluated. With advancing maturity, IVDMD decreased (P<.001), associated with decreases (P<.001) in CP and water soluble carbohydrates (WSC), and increases (P<.001) in NDF and ADF. The IVDMD of leaf was higher (P<.001) than of stems, associated with higher CP, hemicellulose:cellulose, and arabinan:xylan, and lower lignin methoxyl content. A hypothesis of formation of reactive quinone methide intermediates from lignin during rumen fermentation was tested in vitro by incubating corn stover fractions with S-containing reducing agents. Sulphur incorporation into residual fiber occurred (P<.05), indicative of nucleophilic addition to quinone methide intermediates. Degradation of NDF was highly correlated with lignin methoxyl content. The impact of lignin on cellulose degradation was studied using lignocellulosic hydrogels, in which hydroxypropylated or unmodified hardwood lignin was blended with cellulose. In vitro cellulose degradation of lignocellulose blends was higher (P<.01) than of control. Addition of lignin at incubation depressed (P<.01) cellulose degradation. Hydroxypropylation enhanced (P<.001) the increase in cellulose degradation with lignin blending, and reduced (P<.001) the inhibitory effect of lignin addition at incubation. Treatment of drought-stressed corn stover with 3% aqueous NH₃ decreased (P<.05) NDF, compared to isonitrogenous NH₃ addition and control, associated with solubilization of hemicellulose. Esterified phenolic acids were released (P<.05) by NH₃ treatment in upper stem. The IVDMD and NDF degradation increased (P <.001) after ammoniation, with higher (P<.05) values for NH₃ treatment than NH₃ added in leaf. The in vitro response to ammoniation of fractions of drought-stressed and non-drought stressed corn stover harvested in subsequent years was compared, using N-sufficient and N-limiting buffers. Response was highest (P<.001) for non-drought stressed stover fractions, and in N-limiting medium. Response appeared to be affected by high concentration of WSC in lower stalks of drought-stressed stover. / Ph. D.

The growing demand for meat worldwide by the increasing human population (6.8 billion) calls for an increase in livestock production as well as attention to environmental sustainability. Production increases are critical especially in Africa with the highest annual population growth rate (2.5%), where most communities rely on livestock for protein supply. Attempts by intensive livestock farming to optimize production are limited by fibrous quality feeds (roughages) and their unavailability in both developed and developing countries. The overall objective of this study was to scan both domestic and wild herbivores in search for microbial ecosystems with superior fibrolytic potential that can be used as feed additives. It was hypothesized that microbes from wild herbivore can improve fibrous feed breakdown in domesticated ruminants. Experiment 1 evaluated the use of fresh or in vitro cultured faecal inoculum (FF) from two Jersey cows as a potential substitute for rumen fluid (RF). Cultured FF was a better substitute for fresh RF as demonstrated by percentage differences in exocellulase activity (0.4%) and true degradability (TD) (7%), compared to the differences observed between fresh RF and FF for exocellulase activity (33%) and TD (14%). It was applied in subsequent experimentation because it was cost effective (no surgery and reduced sample collection time). The second experiment compared the fibrolytic competence of cultured faecal inocula from three hindgut fermenters (miniature horse (mH), horse (H) and Zebra (ZB)) in summer and winter grazing in their natural environment. Both cellulase enzyme assays (exocellulase, endocellulase and hemicellulase) and in vitro maize stover digestibility study ranked the herbivores according to their fibrolytic competence as ZB > H > mH. The effect of cultured faecal inocula from H, ZB and wildebeest (WB) and its combined systems (N1=H+WB, N2=H+ZB, N3=WB+ZB and N4=H+WB+ZB) on the fermentation of maize stover were also evaluated in vitro. Both enzyme assays and MS degradability studies showed that the combined systems were higher (P<0.01) in fibrolytic activities compared to the individual systems. The microbial ecosystems were ranked as N1 > N2 > N4 > H > ZB > WB >N3; and N3 > N1 > N4 > WB > N2 > ZB >H by their exocellulase activity and degradability parameters, repetitively. The diversity of microbial ecosystems was confirmed by numerous active carboxymethyl cellulase bands present on a carboxymethy cellulose zymograms in experiment 4. The combined microbial ecosystems contain more active and variable bands of cellulases than in the individual microbial ecosystems. Systems N3 and N1 were considered as the best inocula for rumen transinoculation studies. Experiment 5 assessed the in vivo effect of direct-fed microbials from N1 and N3 on MS degradation, ruminal fermentation characteristics and cellulase enzyme profile in sheep. Feed dry matter intake increased (P<0.03) in N1 but tended to increase when inoculated with N3. The treatments, N1 and N3 increased (P<0.05) rumen exocellulase (9.4 and 33.2%, respectively) and endocellulase (82.1 and 47.1%, respectively) specific activities but not hemicellulase activity. Maize stover degradability parameters for N3 (TD, degradability of the insoluble fraction of MS, effective degradability, total SCFA and propionate) measured after 96 h of incubation tended (P>0.05) to be numerically different (1.1, 5.4, 7.1 and 7.9%, respectively). Increase in propionate for N3 was accompanied by higher total SCFA and lower CH4. A decrease in CH4 and no difference in CO2 allow both systems to be environmentally friendly since they have been associated with global warming. These studies showed that direct-fed microbials from N1 and N3 inocula have the potential of improving the utilization of maize stover feeds in ruminants, particularly in view of its simplicity and availability which allows it to be implemented at a relatively lower cost compared to other specific strains or microbial cultures. However, more research is required to identify, purify and classify the superior fibrolytic microbes in the most active ecosystems. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

[Truncated abstract] In Australia, there is pressure to reduce the amount of methane produced by ruminant livestock because they are the single largest source of methane emitted from anthropogenic sources, accounting for 70.7% of agricultural methane emissions. In addition, methane production represents a loss of gross energy intake to the animal. The organisms that are responsible for methane production in the animal gut are a distinct group of Archaea called methanogens. Methanogens occupy three different niches within the rumen. Some live freely in the rumen digesta (planktonic), others are attached to the outer surface of the rumen ciliates (ectosymbiotic), and some reside within the ciliates (endosymbiotic). The types and number of methanogens, as well as rumen ciliates and their symbiotic interactions, influence the amount of methane produced from the rumen. These factors in turn are affected by many factors, including diet and ruminal retention time. In this thesis, I tested the general hypothesis that increasing the amount of grain in the diet and reducing the retention time would affect the abundance and diversity of methanogens in their different niches, including their association with ruminal ciliates. Twenty-four fistulated sheep were used in a complete factorial design with the sheep randomly divided into four groups. ... The change in DGGE banding patterns and Shannon indices when sheep were fed grain indicated that the types of methanogens changed when sheep were fed low and high grain diets, but their diversity did not. In contrast, the diversity of rumen ciliates decreased when sheep were fed a high grain diet. A total of 18 bands from the DGGE analysis of the ciliates were sequenced. All except one, which was 98% similar to Cycloposthium sp. not found previously in the rumen, matched the sequences for previously identified rumen ciliates. Some of the rumen ciliates identified were not present in sheep fed the high grain diet. On a high grain diet, methanogens associate endosymbiotically with rumen ciliates to get better access to hydrogen. It appears that the association between methanogens and rumen ciliates is dictated by the availability of hydrogen in the rumen and not the generic composition of the ciliate population. Furthermore, endosymbiotic methanogens appear to produce less methane than methanogens in other niches. The pot scrubbers did not change ruminal retention time but they did reduce the acetate/propionate measurements observed in sheep on the high grain treatment. The reason why pot scrubbers had this effect remains unknown, but it is interesting to consider that some physical interaction has occurred between the pot scrubbers, the grain and the sheep that has improved the fermentation parameters in sheep fed a high grain diet. The results from this study have advanced our understanding of the interaction between methanogens and ruminal ciliates, and methanogenesis in the rumen in response to dietary changes and mechanical challenges. Extending this work to look more specifically at the species of methanogens that are most closely linked to high methane production and how they interact with the ruminal ciliates will be critical for manipulating enteric greenhouse gas emissions.