Objetivou-se avaliar o uso de nitrato de cálcio (NC) na alimentação de ruminantes com vistas à mitigação da emissão de metano. Foram utilizadas quatro fêmeas de cada subespécie, Bos taurus taurus (Holandês) e vBos taurus indicus (Nelore), com peso médio inicial de 909,0 kg ± 72,0 e 387,0 kg ± 25,5, respectivamente, alimentadas com diferentes níveis de nitrato de cálcio na dieta (0%, 1%, 2% e 3%), sendo utilizado delineamento quadrado latino 4X4 replicado. O experimento teve duração de 4 períodos de 28 dias cada. Foram avaliados, a ingestão, excreção e digestibilidade de nutrientes, por meio de marcador externo dióxido de titânio, o comportamento ingestivo, com monitoramento visual por 24 horas, parâmetros sanguíneos, dinâmica ruminal, com esvaziamento total do rumen, contagem de protozoários, fermentação ruminal, pela técnica ex-situ, parâmetros urinários e balanço de nitrogênio, com coleta manual de urina tipo spot, e a biodigestão dos dejetos, por biodigestores do tipo batelada. Os taurinos apresentaram valores superiores para CMS, ingestão e digestibilidade de nutrientes e excreção de MS, PB, FDN, FDA, N, MO e EB, e inferiores para CMS em relação ao PV e PM. Os zebuínos apresentaram maiores taxas de ingestão, ruminação e mastigação de MS e FDN em min/kg, tendo valores inferiores para as mesmas taxas em kg/min. A genética e a dose de nitrato influenciaram as variáveis séricas, porém sem valores fora da normalidade. Quanto à dinâmica ruminal, os taurinos apresentaram valores absolutos superiores, e valores em relação ao PV e PM inferiores. Os zebuínos apresentaram maiores pH médio e mínimo e menos tempo de pH abaixo de 6,2, e maior contagem de protozoários. As concentrações de N-NH3, volume e produção de CH4 foram maiores para os zebuínos. Os taurinos apresentaram valores mais elevados para N microbiano (g/dia) e balanço de N retido (g/kg N ingerido), e menores valores para balanço de N nas fezes (g/kgN ingerido) e N microbiano (mg/kgPM.d-1). O aumento no nível de NC na dieta aumentou a digestibilidade de EE e ENN, tempo médio ruminando e taxa de mastigação de MS e FDN (min/kg), e diminuiu o CMS em relação ao PV e PM e excreção de MS, EE, ENN, MO e EB. A taxa de ingestão de MS e FDN (kg/min), a concentração de creatinina na urina (mg/kgPV), o tempo total comendo e balanço de nitrogênio amoniacal o rúmen sofreram efeito quadrático com a adição de NC na dieta. A porcentagem de FDN, PB e EE nos afluentes dos bidigestores sofreram influência da dose de nitrato na alimentação das vacas. Os biodigestores contendo os dejetos de taurinos tiveram os afluentes com maiores valores de FDN e FDA e menor teor de sólidos totais (ST), com menor volume de biogás e CH4 e menor relação de CH4/quantidade de fezes. Os taurinos apresentaram melhor aproveitamento do alimento, produzindo menos CH4, com menor rendimento verdadeiro de produção de metano nos biodigestores. A adição de NC afetou o consumo, digestibilidade e utilização do nitrogênio porém, sem afetar a produção de CH4 nem a eficiencia dos biodigestores. / The aim of the present study was evaluate the use of calcium nitrate in the feeding of ruminants to mitigate of methane. Four females cattle from each subspecies of cattle, Bos taurus taurus (Holstein) and Bos taurus indicus (Nellore), with initial average weight of 909,0 kg ± 72,0 e 387,0 kg ± 25,5, respectively, were used, and they were fed with distinct levels of calcium nitrate in the diet (0%, 1%, 2% e 3%) at four periods of 28 days each. Experimental design was composed by a replicated Latin square 4X4. Were avaluated: ingestion, excretion and digestibility of nutrients, by means of external marker titanium dioxide; the observation of behavior parameters during 24 hours; serum analysis; rumen content collection, aiming assessment of fermentation; protozoa count; ruminal fermentation by the ex-situ technique; manual urine collection for measurement of urinary compounds and nitrogen balance; and the anaerobic digestion of feces using batch digesters. Holsteins presented higher values for the DMI, ingestion and digestibility of the nutrients and excretion of DM, CP, NDF, ADF, N, OM e GE, and lower values for DMI at body weight (BW) and metabolic weight (MW) basis. Nellore cattle showed higher rates of ingestion, rumination and chewing of DM e NDF min/kg, having lower values for the same rates in kg/min. Serum variables were influenced by genetics and nitrate levels, but without values beyond normality. As rumen dynamics, Holsteins presented superior absolute values and inferior values when these parameters were calculated in BW and MW basis. The zebu cattle presented medium and minimum pH larger and less time pH below 6.2, having larger count for protozoa count. Nellore had higher concentrations for NH3, CH4 and production of CH4. Holstein presented higher values for microbial N (g/day) and N retained balance (g/kg N ingested) and lower values for N balance in feces (g/kgN ingested) and microbial N (mg/kgMW.d-1). The increase in the level of CN in the diet increased linearly the digestibility of EE and NNE, average time ruminating, chewing rate for DM and NDF (min/kg), and decreased the DMI in BW and MW basis and the excretion of DM, EE, NNE, OM and GE. The intake rate of DM and NDF kg/min, creatinine in urine (mg/kgBW), the total time eating and NH3 balance showed quadratic effect by increasing the addition of CN in the diet. The levels of nitrate on animal feed influenced the percentage of NDF, CP and EE in the batch digesters afluents. The biodigestors containing the taurine feces presented the highest values of NDF/ ADF affluent and lowest values for total solids (TS), lower volume of biogas and CH4 and lower ratio of CH4/amount of feces. Taurines presented better use of the feed, producing lesser CH4, and lower ultimate methane yield at batch digesters. The CN levels affected the intake, digestibility and nitrogen metabolism, however without affecting the production of CH4 or the batch digester efficiency.

Esse trabalho foi conduzido com o objetivo de avaliar os efeitos da inclusão de óleo de soja ou de teores de caroço de algodão (CA) em dietas contendo alto teor de concentrado para bovinos confinados. No experimento I foram utilizados seis bovinos machos, castrados, da raça Nelore (407 ± 24 kg de peso corporal), distribuídos em um delineamento em Quadrado Latino 6 x 6. Foram avaliados 5 teores de inclusão (0, 8, 16, 24 e 32%) de CA em dietas contendo 80% de concentrado (tratamentos CA0, CA8, CA16, CA24 e CA32, respectivamente). Além disso, foi avaliada uma dieta contendo o mesmo teor de lipídios totais da dieta CA32, porém sem a inclusão de caroço de algodão, utilizando óleo de soja (tratamento OS). A inclusão de caroço de algodão na dieta reduziu (efeito linear, P < 0,01) o consumo de matéria seca e o consumo de matéria orgânica dos animais. A digestibilidade aparente total da MS (P = 0,019) e da MO (P = 0,020) reduziu linearmente devido à inclusão de CA na dieta dos animais. A inclusão de óleo de soja na dieta não afetou o digestibilidade da MS (P = 0,462) e da MO (P = 0,469) dos animais em comparação aos animais alimentados com a dieta controle, sem CA, assim como também não foram observados efeito da fonte de lipídios sobre a digestibilidade da MS (P = 0,124) e da MO (P = 0,110). O tempo de ruminação aumentou linearmente (P = 0,002) em resposta a inclusão de CA. O pH ruminal dos animais aumentou (efeito quadrático, P < 0,05) em resposta à inclusão de caroço de algodão nas dietas. O inclusão de óleo de soja na dieta não afetou (P = 0,329) o pH ruminal dos animais em comparação com a dieta controle sem CA. A produção total de ácidos graxos de cadeia curta reduziu (P < 0,001; efeito linear) devido à inclusão de CA. A síntese de Pmic reduziu linearmente (P = 0,011) em resposta a inclusão de CA na dieta. No experimento II, foram utilizados 280 bovinos machos, da raça Nelore e peso médio inicial de 349 ± 33 kg, distribuídos em 40 baias. O delineamento utilizado foi blocos incompletos casualizados. O máximo peso final foi obtido com inclusões de 7,6% de CA. A inclusão de CA na dieta reduziu o CMS dos animais (P < 0,001). A inclusão de óleo de soja reduziu o CMS dos animais (P < 0,05) em comparação com os animais alimentados com a dieta controle sem caroço de algodão. O máximo GPD foi observado com 8% de inclusão de CA na dieta. A máxima EA ocorreu com 14,5% de CA na dieta. Os dados referentes as característica da carcaça (PQC, RC, AOL e EGS) também apresentaram resposta quadrática (P < 0,05) à inclusão de CA na dieta. Comparado ao tratamento controle, sem CA na dieta, a inclusão de óleo de soja não afetou nenhuma das características da carcaça avaliadas (P > 0,05). As características químicas da carne não diferiram (P > 0,05) entre os animais alimentados com dietas contendo 32% de CA daqueles alimentados com óleo de soja. A oxidação dos lipídios (TBARS) apresentou redução (efeito quadrático; P = 0,035) devido à inclusão de CA na dieta. Na avaliação sensorial da carne assada realizada através do painel consumidor, não foram observados efeitos dos tratamentos sobre o sabor, odor e aceitabilidade geral da carne (P > 0,05). Na avaliação das características organolépticas da carne realizada pelos provadores treinados também não foram observados efeitos dos tratamentos sobre o odor, sabor e aceitabilidade geral (P > 0,05). No experimento III, avaliou-se a cinética de fermentação ruminal do milho e do caroço de algodão através da produção cumulativa de gases. Foram utilizados cinco frascos (repetições) contendo 1g de substrato (milho ou CA) além de 5 frascos “brancos” (sem substrato). As leituras de pressão e volume dos gases acumulados nos frascos de incubação foram realizadas com transdutor de pressão nos tempos 0, 2, 4, 6, 8, 10, 12, 15, 19, 24, 30, 36, 48, 72 e 96 horas. O milho apresentou um maior potencial de produção de gases quando comparado ao caroço de algodão (P < 0,001). O tempo de colonização não apresentou diferença entre as duas fontes de energia (P = 0,646). O caroço de algodão apresentou uma maior taxa de degradação comparado com o milho (P = 0,001) e, consequentemente, para a redução (P < 0,001) do tempo necessário para atingir a máxima produção de gás. O milho apresentou uma maior potencial de degradação quando comparado com o caroço de algodão, resultado esse que pode ser observado pelo maior produção de gases. Apesar não suportar um grande potencial de degradação, parte da fração fribrosa do caroço de algodão apresentou alta degradabilidade, o que pode ser observado na sua maior taxa de degradação quando comparado com o milho / Three trials were conducted to evaluate the effects of the inclusion of levels of whole cottonseed (WC) or soybean oil (SO) in diets for Nellore feedlot cattle. In the experiment I six rumen-cannulated Nellore steers (407 ± 24 kg BW) were used in a 6 x 6 Latin square design experiment. Were evaluated 5 levels of inclusion of WC (0, 8, 16, 24 and 32% DM basis) in diets containing 87% concentrate (treatments WC0, WC 8, WC16, WC24 and WC32 respectively). Also, it was evaluated a diet containing the same amount of total fat content of WC32 but with no WC and using soybean oil (SO treatment). Increasing WC levels linearly decreased DM and OM intake (P < 0.01). CP and NDF intakes were not affected by the treatments. Total fat digestibility linearly increased due the WC increase in the diets (P < 0.001). Total DM (P = 0.019) and OM (P = 0.020) digestibilities linearly decreased with increasing levels of WC. SO inclusion did not affect DM (P = 0.462) and OM (P = 0,469) digestibilities compared with CA0 and there was no effect of fat source over DM (P = 0.124) and OM (P = 0.110) digestibilities. Rumination time linearly increased due the WC inclusion (P = 0,002). Increasing WC levels quadratically increased ruminal pH (P < 0.05) but no effect was observed with the SO addiction in the diet (P = 0.329) compared with CA0. Total VFA was linearly decreased with increasing levels of WC (P < 0.001). Increasing levels of WC linearly decreased the microbial protein synthesis (P = 0.011). In the experiment II a total of 280 Nellore bulls (initial BW = 349 ± 33 kg) were allotted in 40 pens. The statistical design was randomized incomplete blocks. The maximum final BW was obtained with 7.6% of inclusion of WC. Increasing levels of WC decreased DMI (P < 0.001). Adding SO also decreased DMI (P < 0.05) compared with WC0. Increasing the inclusion rate of WC quadractically increased the ADG, G:F, HCW, dressing percent, LM area and 12-rib fat (P < 0.05). The maximum ADG were obtained with 8% of inclusion of WC. On the other hand, the maximum G:F was obtained with 14.5% of WC. Compared with WC0, the SO inclusion did not affect carcass characteristics (P > 0.05). The inclusion of WC quadractically affect meat composition (P < 0.05). Compared with CA32, the inclusion of SO in the diet did not affect the meat composition. Increasing WC in the diets quadratically decrease (P = 0.035) lipid oxidation. No effects were observed in the sensorial evaluation flavor, taste and overall acceptability by the costumers (P > 0.05). Training sensory panelists also did not find differences between the treatments for flavor and taste. Increasing WC linearly decreased the tenderness (P = 0.045). Compared with CA32, the inclusion of SO increased meat tenderness (P = 0.031). In the experiment III was evaluated the ruminal fermentation kinetics of corn and cottonseed through the in vitro gas production technique. Five bottles for each energy source (corn and cottonseed), plus five additional bottles containing buffered medium and rumen fluid inoculum only (blanks) were used. The volume of gas produced was recorded at 0, 2, 4, 6, 8, 10, 12, 15, 19, 24, 30, 36, 48, 72 e 96 h of incubation using the pressure reading technique. Gas values were corrected for blank incubations. The corn had greater gas production than cottonseed (P < 0.001). Lag time was not different between the two energy sources (P = 0.646). Cottonseed had higher FDR than corn (P = 0.001) and shorter time to half asymptote (P < 0.001). Corn had higher potential of degradation compared with cottonseed observed by the higher gas production. Moreover, even with lower potential of degradation, the cottonseed fibrous fraction had a high degradability compared to corn

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-09-20T12:26:13Z No. of bitstreams: 1 francianecedrolavale.pdf: 8805942 bytes, checksum: 47ea510c699cff3c39f83d726954737a (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-09-22T15:11:47Z (GMT) No. of bitstreams: 1 francianecedrolavale.pdf: 8805942 bytes, checksum: 47ea510c699cff3c39f83d726954737a (MD5) / Made available in DSpace on 2017-09-22T15:11:47Z (GMT). No. of bitstreams: 1 francianecedrolavale.pdf: 8805942 bytes, checksum: 47ea510c699cff3c39f83d726954737a (MD5) Previous issue date: 2016-02-12 / O presente estudo versa sobre taxonomia e morfologia de protozoários ciliados (Litostomatea, Trichostomatia) endossimbiontes de ruminantes domésticos no Brasil. O trabalho encontra-se dividido em quatro seções. A Seção 1 revisa os principais métodos em microscopia e biologia molecular utilizados no estudo taxonômico de protistas ciliados do rúmen. A Seção 2 apresenta um inventário de espécies de protozoários ciliados do rúmen de ovinos no Brasil, no qual foram inventariadas 28 espécies de ciliados, sendo que, Entodinium alces, Metadinium esalqum e Metadinium rotundatum foram observados pela primeira vez em ovinos e outras 12 espécies foram observadas pela primeira vez em ovinos no Brasil. Ainda, a sessão apresenta a redescrição da espécie Entodinium contractum com base na morfologia geral e descrição da infraciliatura oral; a Seção 3 faz a caracterização morfológica de seis morfotipos da espécie Diplodinium anisacanhum e breve revisão taxonômica do gênero Diplodinium. A caracterização morfológica e morfométrica dos morfotipos de D. anisacanthum demonstraram grande similiaridade entre os mesmos sugerindo que tais ciliados constituam uma única espécie dotada de polimorfismos. A breve revisão realizada destaca as incongruências taxonômicas existentes no gênero Diplodinium e aponta a necessidade de estudos recentes com os representantes deste gênero de modo a reformular a taxonomia do grupo. Por fim, a Seção 4 descreve uma nova espécie de ciliado ofrioscolecídeo, Diploplastron dehorityi e redescreve a espécie Diploplastron affine com base em dados morfológicos, morfométricos e detalhes da infraciliatura oral. A nova espécie difere em alguns aspectos da espécie D. affine. As placas esqueléticas em D. dehoryti não são justapostas e possuem formato recurvado. Ainda, a nova espécie possui o corpo em formato oval, diferindo do formato elíptico de D. affine. Além das semelhanças existentes entre a nova espécie e a espécie congênere, D. dehorityi possui grande similaridade morfológica com espécies pertencentes ao gênero Eremoplastron, diferindo apenas no número de placas esqueléticas. / This study is about taxonomy and morphology of endosymbiotic ciliated protozoa (Litostomatea, Trichostomatia) of Brazilian domestic ruminants. The work is divided into four sections. Section 1 reviews the main methods in microscopy and molecular biology used in the taxonomic study of rumen ciliates. Section 2 presents an inventory of species of ciliated protozoa Brazilian sheep, which were inventoried 28 species of ciliates, and, Entodinium alces, Metadinium esalqum and Metadinium rotundatum were first observed in sheep and other 12 species were first observed in Brazilian sheep. Still, the session shows the redescription of Entodinium contractum Kofoid & Christenson (Entodiniomorphida, Ophryosocolecidae) based on general morphology and description of oral infraciliature; Section 3 is the morphological characterization of a polymorphic species of ophryoscolecid ciliate Diplodinium anisacanhum da Cunha and a brief taxonomic revision of the genus Diplodinium Schuberg. The morphological and morphometric characterization of D. anisacanthum showed great similiarities among morphotypes suggesting that such ciliates constitute a single species endowed with polymorphism. A brief review conducted highlights the inconsistencies in the taxonomy of the genus Diplodinium and demonstrates the need for recent studies with representatives of the genus in order to improve the taxonomy of it. Finally, Section 4 describes a new species of ophryoscolecid ciliate Diploplastron dehorityi and redescribe the species Diploplastron affine (Dogiel & Fedorowa) based on morphological and morphometric characterization and details of oral infraciliature. The new species differs in some aspects of the species D. affine. The skeletal plates in D. dehorityi are not juxtaposed and have curved shape. Still, the new species has the oval body, differing from the elliptical body of D. affine. In addition to the similarities between the new species and congener species, D. dehorityi has great morphological similarity to species of the genus Eremoplastron, differing only in the number of skeletal plates.

This report describes a new group of anaerobic bacteria that degrade oxalic acid. The new genus and species, Oxalobacter formigenes, are inhabitants of the rumen and also of the large bowel of man and other animals where their actions in destruction of oxalic acid may be of considerable importance to the host. Isolates from the rumen of a sheep, the cecum of a pig, and from human feces were all similar Gram-negative, obligately anaerobic rods, but differences between isolates in cellular fatty acid composition and in serologic reaction were noted. Measurements made with type strain OxB indicated that 1 mol of protons was consumed per mol of oxalate degraded to produce approximately 1 mol of CO2 and 0.9 mol of formate. Substances that replaced oxalate as a growth substrate were not found.

Rumen protected Methionine (MET) and Lysine (LYS) are critical for milk protein synthesis in dairy cows. N-acetyl-L-methionine (NALM) is a MET derivative that consists of L-Met protected with an acetyl group that is attached to the α-amino group.N-acetyl-L-lysine (NALL) is a LYS derivative that is similarly protected. The objectives of these studies were to quantify the gastrointestinal availability of NALM and NALL. Three experiments were run as 3 × 3 Latin square using 3 second lactation Holstein cows that have been fitted with cannulas in the rumen and duodenum. The cows were fed diets containing the supplements for two weeks prior to each experiment so that the rumen microbes had time to adjust to the supplement. Each period consisted of 10 d of adaptation followed by 2 d of sampling. A dose of 0, 30, or 60 g of NALM was placed under the rumen mat at the time of feeding every day during experiment 1. The cows were similarly supplied with 0, 60, or 120 g of ƐNALL during experiment 2. The cows were supplemented with 0 g, 120 g ofƐNALL, or 120 g of diNALL during experiment 3. On day one of sampling, a liquid marker (Co-EDTA) was also administered at the time of the protected AA administration. Blood, ruminal, and duodenal samples were taken at hours 0, 1, 3, 6, 9, 12, and 24 post-feeding. There were no differences for milk production, milk protein, milk fat, or DMI for NALM or either NALL. There were no differences for ruminal escape (69.1% and 46.2% respectively) and duodenal appearance (2.16% and 3.40% respectively). The ruminal escape of ƐNALL was not different between the 120 g dose (32.7%) and the 60 g dose (27.2%). Duodenal appearance was higher (P < 0.01) for the 60 g dose (2.86%) than for the 120 g dose (1.19%) of ƐNALL. Acetate, propionate, butyrate, and valerate were higher (P < 0.01) for the supplemented cows during experiment 1 with NALL. There were no differences between ƐNALL and diNALL for rumen escape, duodenal appearance, VFA production, or blood LYS AUC. Results of the experiment verify significant protection of the N-acetyl MET and LYS from rumen degradation.

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / During one of our preliminary studies in 2015, metagenomic DNA extracted from the goat rumen was sequenced and the in silico mining of the biorefining enzyme showed the presence of significant number of different biocatalysts, such as amylases (E.C 3.2.1.1), xylanases (E.C 3.2.1.8), pectinases (E.C 3.2.1.15) and cellulases (E.C 3.2.1.4). Hence, a subsequent study was conducted which is aimed at extracting metagenomic DNA from the goat rumen, constructing the metagenomic library using pCC2-FOS™ plasmid vector (Epicentre®), and eventually screening the constructed library for potential novel amylases using soluble starch as a substrate. Accordingly, rumen digesta was aseptically collected from four compartments of each goat and pulled before extraction of metagenomic DNA. The conventional CTAB protocol was modified to extract the metagenomic DNA from the rumen digesta. As a result, high molecular weight DNA was obtained and used to construct the metagenomic fosmid library. Since the host (Escherichia coli EPI 300-T1r) supplied with CopyControlHTP Fosmid Library Production Kit has background amylase expression we opted for a knockout E. coli strain with deleted starch hydrolysis (amylase expression) pathway. The library was subsequently screened for the presence of amylase isoforms using soluble starch as a substrate. Therefore, for the purpose of this study, four fosmids clones showing amylase activity were selected, recombinant vector isolated and MiSeq-sequenced. Out of four recombinant proteins, only one (pET30a(+)-amy-vut12) was successfully expressed. Subsequently, pET30a(+)-amyvut12 was further characterize physicochemically. Interestingly, the recombinant enzyme showed maximum activity in the pH and temperature ranges of 6.0 - 8.0 and 70 - 90oC, respectively. Hence, this implies that novel recombinant protein has sound activity from acidic to alkaline pH range and potently thermostable. Further work should be done to optimize and improve the solubility of three other recombinant proteins (pET30a(+)-amy-vut2, pET30a(+)- amy-vut9 and pET30a(+)-amy-vut14) studied, which might harbour important traits. Most importantly, immobilization as well as crystallographic studies of pET30a(+)-amy-vut12 and downstream applications should further be investigated.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20434号 / 農博第2219号 / 新制||農||1049(附属図書館) / 学位論文||H29||N5055(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 廣岡 博之, 教授 松井 徹, 教授 久米 新一 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Sustainable farming is an integrated practice of crop and livestock production system (integrated crop-livestock system; ICLS) that aims to reduce the environmental impacts of agricultural practices while maintaining the productivity and profitability. The use of one step's byproducts by another is a crucial component of this practice. The continuity and effectiveness of sustainable farming greatly rely on deep understanding of each component and good management strategy. One essential aspect involved in all farming components is the role of microorganisms in mediating the biological processes therein. Thus, understanding the composition and activities of these communities would open up ways to engineer them and optimize the respective processes for better sustainable farming practices. The research presented in this dissertation aimed to characterize the microbial metabolism involved in the ICLS with a broader goal of manipulating these systems to improve sustainable agriculture. We focused on two systems that are widely used in the United States, and employed the analysis of 16S rRNA-V4 element for this purpose. In our first system, we characterized the rumen microbiomes of beef cattle alternately grazing nontoxic MaxQ and toxic KY-31 tall fescue pasture, to understand how these cultivars shape the rumen microbiome and identify microbial species potentially capable of degrading ergot alkaloids for better feed utilization. We found that KY-31 grazing remodeled the rumen microbiome substantially at the cellulolytic and saccharolytic guilds. It suppressed the abundances of Fibrobacter, a major ruminal cellulolytic bacterium, as well as those of Pseudobutyrivibrio and Butyrivibrio, and these losses were compensated by increased occurrences of Eubacterium species. Parts of these new communities lingered once developed, and a different guild composition surfaced upon transfer to MaxQ. We also discovered that most of the observations were not evident at the whole microbiome levels but was identified by analyzing the sessile and planktonic fractions separately. Thus, it showcased the need for analyzing sessile and planktonic segments separately while interrogating a heterogenous microbiome. Finally, we identified several potential ergovaline degrading bacteria such as Paraprevotella and Coprococcus. In our second system, we studied the microbiome composition and associated transformation pathways mediating nitrogen loss in two dairy manure storage systems, the clay-lined Earthen Pit (EP) and aboveground concrete storage tank (CS) on two commercial dairy farms, to develop strategies to minimize these losses. We first developed a catalog of the archaea and bacteria that were present therein based on the 16S rRNA-V4 amplicons from manure samples collected from several locations and depths of the storages. Then, we inferred the respective metabolic capabilities via PICRUSt2 and literature curation, and developed schemes for nitrogen and carbon transformation pathways operating at various locations of EP and CS. Our results showed that the stored manure microbiome composition was more complex and exhibited more location-to-location variation in EP compared to CS. Further, the inlet and a location with hard surface crust in EP had unique consortia. With regards to nitrogen transformation, the microbiomes in both storages had the potential to generate ammonia but lacked the organisms for oxidizing it to nitrate and further to gaseous compounds such as anammox and autotrophic nitrifiers. However, microbial conversion of nitrate to gaseous N2, NO, and N2O via denitrification and to stable ammonia via dissimilatory nitrite reduction (DNRA) seemed possible. Minor quantity of nitrate was present in manure, potentially originating from oxidative processes occurring on the barn floor. Higher prevalence of nitrate-transforming microbes at the near-surface locations and all depths of the inlet were found as a result of this instance. These findings suggested that ammonia oxidation to nitrate started on the barn floor and as manure is being stored in EP and CS, nitrate was lost to the environment via denitrification. For carbon transformation, hydrogenotrophic Methanocorpusculum species were the primary methane producers, and it exhibited higher abundance in EP. / Doctor of Philosophy / Sustainable farming is an integrated practice of crop and livestock production systems that aims to reduce the environmental impacts of agricultural practices while maintaining the productivity and profitability. The use of one step's byproducts by another such as the utilization of arable land to grow forages for livestock grazing or the use of manure as organic nitrogen amendments for crops is a crucial component of this practice. The continuity and effectiveness of sustainable farming greatly rely on deep understanding of each component and good management strategy. One essential aspect involved in all farming components is the role of microorganisms in mediating the biological processes therein. Thus, understanding the composition and activities of these communities would open up ways to engineer them and optimize the respective processes for a better sustainable farming practice. The research presented in this dissertation aimed to characterize the microbial metabolism involved in the integrated crop-livestock system with a broader goal of manipulating these to improve sustainable agriculture. We focused on two systems that are widely used in the United States, and employed bioinformatic analysis of a genetic marker for this purpose. In our first system, we characterized the rumen microbiomes of beef cattle grazing alternately on KY-31 tall fescue, a major grass used in Virginia that carry a toxin-producing fungi, and nontoxic MaxQ tall fescue pasture, to understand how these cultivars shape the rumen microbiome and identify potential microbial species capable of degrading the toxin for better feed utilization. We found that KY-31 grazing remodeled the rumen microbiome substantially, especially affecting microbes responsible for degrading cellulose and starch. Some of these communities lingered once developed, and a different microbial population surfaced upon transfer to MaxQ. Several potential toxin-degrading bacteria were also identified. In our second system, we studied the microbiome composition and associated transformation pathways mediating nitrogen loss in two dairy manure storage systems, the clay-lined Earthen Pit (EP) and aboveground concrete storage tank (CS), to develop strategies to minimize these losses. We first develop a catalog of the archaea and bacteria that were present in the manure samples collected from several locations and depths of the storages based on a genetic marker. Then, we inferred the respective metabolic capabilities and developed schemes for nitrogen and carbon transformation pathways operating at various locations of EP and CS. Our results showed that the stored manure microbiome exhibited more location-to-location variation in EP compared to CS. Oxygen exposure, continuous addition of fresh manure, and the presence of crust at the storage surface gave rise to these unique populations. With regards to nitrogen transformation, the microbiomes in both storages had the potential to generate ammonia but lacked the organisms for oxidizing it to nitrate and further to gaseous compounds. However, microbial conversion of nitrate to gaseous N2, NO, and N2O seemed possible. These observations showcased that ammonia is stable during storage. Nitrate, on the other hand, can be converted into volatile nitrogen compounds via various processes. Thus, it is imperative to limit the level of nitrate in manure prior to placement in the storage, which is potentially originating from oxidative processes occurring on the barn floor.

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology / Lipases have been one of the important biocatalysts that catalyse the transformation of lipids to yield very important products that can be of beneficial in food, agriculture, pharmaceutical medicine and for the biodiesel production. In the search for novel biocatalysts, notably lipases, the conventional culture-based techniques were used but it can only address sourcing the biomolecule from 1-10% of the microbial population leaving the wealth of the biomolecules packed in 90-99% of the microbial community unaccounted for. Metagenomic technique, which is culture-independent, was developed as a comprehensive approach to address literally 100% of the microbial population thereby maximizing the chances of obtaining novel biocatalysts with superior physico-chemical and catalytic traits. In principle, any biomolecule including lipase could be sourced from any biologically-active environment, of which animal rumen is one. However, among the rumenant animals goat has diverse feeding habit, thereby laying ground for increased microbial diversity in its gastro-intestinal tract. It was thus, postulated that goat rumen could be potential source of novel lipase isoforms. Therefore, the aim of the study was to extract metagenomic DNA from goat rumen and construct a metagenomic fosmid library and screen the library for lipase isoforms. The fosmid clones were functionally screened using 1% tributyrin as a substrate and five positive clones were selected. From the five clones, two fosmids were selected for further study. Following nucleotide sequencing and in-silico analysis of the insert of the two selected clones, one lipase encoding open reading frame (Lip-VUT3 and Lip-VUT5) from each fosmid clones of approximately 212 and 248 amino acids, respectively, was identified. The amino acid sequences of the Lip-VUT3 ORF contained a classical conserved lipase GSDL sequence motif while the amino acid sequences of the Lip-VUT5 ORF contained a classical G-L-S-L-G conserved lipase/esterase motif sequence. The two genes (Lip-VUT3 and Lip-VUT5) were successfully expressed in Escherichia coli BL21 (DE3) and the purified enzymes exhibited respective temperature optima of 60 °C and 70 °C, and respective pH optima of 6.0 and 10.0. Further biochemical characterisation indicated that Lip-VUT3 and Lip-VUT5 lipases showed tolerance towards a wide concentration (50%-100%) of methanol. Lip-VUT3 had a Km value of 0.287 mM while Lip-VUT5 had a Km value of 0.556 Mm. This shows that Lip-VUT3 lipase has a higher affinity for olive oil than Lip-VUT5. Lip-VUT3 and Lip-VUT5 were characterised to be true lipases that have been recovered from the rumen environment through metagenomic approach. Therefore, the study proved that metagenomic approach helps in recovering novel lipase isoforms with potential down stream industrial and therapeautic applications from goat rumen metagenome, a rich but untapped source.

Ruminant livestock play a vital role in fulfilling the nutrient requirements of humans by providing protein, energy, and essential microminerals. With the increasing demand for meat and dairy products, the ruminant industry must continue to improve the productivity and efficiency of ruminant animals with limited resources while minimizing the environmental impact. Rumen fermentation is the focal point of the productivity and efficiency of the animal and numerous chemical, physical and biochemical interactions make the rumen a complex ecosystem. Therefore, improving the understanding of fermentation dynamics in a holistic manner and characterizing how fermentation varies in response to different nutrient supplies can greatly expand our knowledge on rumen fermentation to develop better engineered rumen manipulation strategies. The central aim of these investigations was to employ alternative analytical strategies for holistic exploration of complex relationships among rumen, animal, and dietary variables and to estimate rumen volatile fatty acid (VFA) dynamics under different nutrient supplies. The objective of the first study was to explore the strengths and limitations of mixed-model meta-analysis, recursive feature elimination (RFE), and additive Bayesian networking (ABN) in identifying relationships among diet, rumen, and milk performance variables. Both mixed-models and ABN agreed upon most of the variables and relationships identified while RFE failed to capture interactions. Given the capacity of mixed models for quantitative inquiry and the potential of ABN to illustrate complex associations in a more intuitive way, future investigations combining both approaches hold potential to explore intercorrelated data in a holistic manner. Followed by the successful use of ABN in the first study, the goal of a follow up study was to investigate the potential of two different network approaches to explore rumen level interactions using data generated in continuous culture experiments. Two network analysis approaches, EBIC-LASSO network (ELN) and Bayesian learning network (BLN) were leveraged to explore the relationships among rumen fermentation parameters in continuous culture experiments. Unidirectional ELN illustrated prominent variables while BLN, which produces a directed acyclic graph, identified directional relationships implying causality. Overall, both networking approaches demonstrate strengths in capturing connectedness and directionality of rumen fermentation variables. In a complementary line of work, the next experiment focused on developing an alternative method for iso-tope based assessments to produce less expensive, and more efficient screening of fermentation conditions driven by diet. Cannulated wethers were used in this study and 4 dietary treatments combining lowly and highly degradable fiber (timothy hay and beet pulp, respectively) and protein (heat-treated soybean meal and soybean meal, respectively) were tested. Results indicated that fluid volume of the rumen and the rate of passage were influenced by protein, but not fiber, source. Higher rumen volumes and lower passage rates were associated with heat-treated soybean meals. The effect of dietary treatments on VFA absorption dynamics was prominent compared to the minimal changes in production dynamics. Overall, heat-treated soybean meal appears to influence VFA disappearance resulting in low concentrations within the rumen, but greater flux of VFA disappearance. In conclusion, this method demonstrated the capacity to estimate VFA dynamics beyond concentrations and molar proportions while being cost effective and more physiologically relevant. In a fourth study, we sought to investigate the growth performance and rumen VFA profile in response to different planes of nutrients and naturally occurring coccidiosis. Coccidiosis infection altered rumen isobutyrate concentrations and tended to alter major VFA concentrations suggesting the need of future work to explore coccidiosis effects on rumen fermentation. The first two investigations highlighted the potential and strength of leveraging alternative analytical tools to complement statistical approaches generally used in ruminant nutrition while concurrently improving ability to explain complex associations in the rumen. The third and fourth projects characterized the rumen VFA dynamics and profile in response to the different nutrient degradability and health status, respectively. Collectively, these investigations contribute to better understanding of rumen dynamics through novel analytical and experimental approaches. / Doctor of Philosophy / With increasing global population, income, urbanization, and changes in dietary habits, the demand for meat and milk continues to grow. The ruminant animal industries (beef cattle, dairy cattle, sheep, goat, and buffalo) carry the burden of increasing production utilizing limited resources while minimizing the negative environmental impact caused by ruminant operations. To achieve this goal the productivity of the animal must be increased, and in order to increase the efficiency of production a better understanding of factors driving the production is critical. Ruminant animals have the unique ability to convert plant fiber into human edible milk and meat through a process that predominantly occurs in the special gut compartment called the rumen. In this process several compounds are produced, and among those volatile fatty acid (VFA) is of utmost importance because it fulfills energy demands for growth, production and reproduction. The rumen is a complex ecosystem consisting of numerous variables and associations. Understanding those relationships is crucial to manipulate rumen mechanisms. The overall objective of this work was to evaluate the potential of alternative statistical approaches, which demonstrated success in other disciplines, for better depiction of complex associations and characterization of production and absorption mechanisms of rumen VFA in response to different nutrient supplies. The objective of first investigation was to evaluate a feature selection method (recursive feature elimination; RFE) and a network approach (Additive Bayesian network; ABN) concurrently with a standard variable selection method (mixed model meta-analysis) commonly used to develop animal nutrition models. We attempted to find out the most important dietary, rumen, and animal variables for milk yield, milk fat and protein content as an example. Results indicate that the network approach was well aligned with the standard tool and can be used as a complementary approach. In our second investigation, we leveraged two networking analyses, a frequentist network which was unidirectional and a Bayesian network which was directional to explore rumen level interactions. The unidirectional network approach highlighted the most important variables in the rumen and numerous relationships among these variables. The directional network was more useful in understanding of causal associations within the system. In the third experiment we estimated the production and absorption of VFA in response to the different protein (heat-treated and regular soybean meal) and fiber (timothy hay and beet pulp) sources. The results revealed that the production of VFA was minimally affected by the diet, but the absorption was higher with heat-treated soybean meal. Our last project investigated the effect of parasitic infection, i.e., coccidiosis, and high and low levels of nutrition on growth and rumen VFA of growing lambs. Infection of coccidiosis altered a minor VFA (isobutyrate) and tended to alter total and major VFA (acetate and propionate). All these findings help to improve our understanding of rumen fermentation and subsequently develop strategies to manipulate rumen fermentation to enhance efficiency and productivity.