The influence of casein and urea as nitrogen sources on in vitro equine caecal fermentation

To access the fermentative response of equine caecal microbial population to nitrogen availability, an in vitro study was conducted using caecal contents provided with adequate energy sources and nitrogen as limiting nutrient. Two nitrogen (N) sources were provided, protein (casein) and non-protein (urea). Caecal fluid, taken from three cannulated horses receiving a hay–concentrate diet, was mixed with a N-free buffer–mineral solution. The influence of four N levels (3.7, 6.3, 12.5 or 25 mg of N in casein or urea) was studied using the gas production technique. Total volatile fatty acids (VFA), NH₃-N and gas production were measured after a 24-h incubation period. Microbial biomass was estimated using adenine and guanine bases as internal markers, and ATP production was estimated stoichiometrically. Microbial growth efficiency (Y_ATP) and gas efficiency (E_gas) were estimated. Fermentation with casein as the sole N source was generally characterized by lower total VFA, NH₃-N, total gas production and higher acetate : propionate (A : P) ratio and Y_ATP than with urea. Results herein presented indicate that, under these in vitro conditions, caecal microbial population does in fact use urea N, but less efficiently than casein in terms of microbial growth.     

Teladorsagia circumcincta: Survival of adults in vitro is enhanced by the presence of a mammalian cell line

Adult Teladorsagia circumcincta survival and motility in vitro was examined in a range of different cell culture media, supplements and gas mixes. Under optimum conditions, worms survived for 14 days, exhibiting high motility for 9 days and egg production for 72 h. Optimum conditions involved co-culture of worms with a HeLa cell line in a supplemented cell medium (CEM) and an atmosphere containing 10% CO₂, 5% O₂ 85% N₂, 65% humidity at 37 °C. The incubation medium consisted of Minimum Essential Medium with 10% fetal calf serum, 1% non-essential amino acids, 1% glutamax and 1% penicillin-neomycin-streptomycin cocktail mix. Compared with optimum conditions, incubation in CEM alone, cell conditioned CEM, RPMI alone, Medium 199 alone, reduced CO₂ or O₂, or when cells were replaced with Escherichia coli, both survival and motility were reduced. Optimum conditions for adult T. circumcincta maintenance for culture, anthelmintic testing or generation of excretory/secretory products are described.     

深圳市前海桂湾公园土壤蒸发及植被蒸腾模拟

蒸散发（ET）是连接水文循环和地表能量平衡的重要组成部分,准确的估算土壤蒸发（LE_s）和植被蒸腾（LE_v）对城市水资源分配和管理具有重要意义。针对城市林地蒸散发双源模型的缺失,本研究基于MOD16模型提出了应用于城市林地区域改进的MOD16双源模型。改进的MOD16双源模型更加准确的描述城市林地区域复杂下垫面的能量分配过程。结合高时空分辨率的Sentinel-2遥感卫星影像,对2020年深圳市前海桂湾公园20个无云日的LE_s和LE_v进行反演研究。用Shuttleworth-Wallace （S-W）模型和双作物系数法（FAO dual-K_c）验证模型性能,同时分析改进的MOD16模型对输入变量的敏感度,结果表明：改进的MOD16模型与S-W模型和FAO dual-K_c之间的均方根误差（RMSE）分别为21.39 W/m²和20.41 W/m²,平均绝对误差（MAE）分别为18.81 W/m²和19.05 W/m²,R²分别为0.801和0.634。改进的MOD16模型可以应用在城市林地LE_s和LE_v估算中。桂湾公园地区总蒸散月均值在85-165 W/m²之间。研究区域的LE_s和LE_v呈现出明显的季节变化,春夏季蒸散量升高,秋冬季蒸散量降低,LE_s的变化范围为0-50 W/m²,LE_v的变化范围为0-120 W/m²。LE_v高值在研究区呈现零散分布,多集中在前海桂湾沿岸,LE_s高值集中在研究区的西北、东北和东南部。敏感性分析结果表明,改进后的MOD16模型对植被覆盖、太阳辐射和湿度更敏感。其中,LE_v模拟结果受净辐射和植被覆盖影响最大,LE_s的模拟结果受湿度和植被覆盖的影响最大。因此,应用改进的MOD16模型时需要优先确保这些参数的准确输入。修正后的MOD16模型极大地提高了高分辨率、小尺度区域城市林地LE_s和LE_v模拟的准确性,为准确获取植被耗水量信息从而科学指导城市林地灌溉、解决城市水资源分配与管理提供有力工具。     

Effect of fibrolytic enzymes added to a Andropogon gayanus grass silage-concentrate diet on rumen fermentation in batch cultures and the artificial rumen (Rusitec)

In vitro batch cultures were used to screen four fibrolytic enzyme mixtures at two dosages added to a 60 : 40 silage : concentrate diet containing the C₄ tropical grass Andropogon gayanus grass ensiled at two maturities – vegetative stage (VS) and flowering stage (FS). Based on these studies, one enzyme mixture was selected to treat the same diets and evaluate its impact on fermentation using an artificial rumen (Rusitec). In vitro batch cultures were conducted as a completely randomized design with two runs, four replicates per run and 12 treatments in a factorial arrangement (four enzyme mixtures×three doses). Enzyme additives (E1, E2, E3 and E4) were commercial products and contained a range of endoglucanase, exoglucanase and xylanase activities. Enzymes were added to the complete diet 2 h before incubation at 0, 2 and 4 μl/g of dry matter (DM). Gas production (GP) was measured after 3, 6, 12, 24 and 48 h of incubation. Disappearance of DM (DMD), NDF (NDFD) and ADF (ADFD) were determined after 24 and 48 h. For all four enzyme mixtures, a dosage effect (P<0.05) was observed for NDFD and ADFD after 24 h and for DMD, NDFD and ADFD after 48 h of incubation of the VS diet. For the FS diet, a dosage effect was observed for GP and NDFD after 24 h and for GP, DMD, NDFD and ADFD after 48 h of incubation. There was no difference among enzyme mixtures nor was there an enzyme×dose interaction for the studied parameters. Because of the greatest numerical effect on NDF disappearance and the least cost price, enzyme mixture E2 at 4 µl/g of diet DM was selected for the Rusitec experiment. The enzyme did not impact (P>0.05) DM, N, NDF or ADF disappearance after 48 h of incubation nor daily ammonia-N, volatile fatty acids or CH₄ production. However, enzyme application increased (P<0.05) microbial N production in feed particle-associated (loosely-associated) and silage feed particle-bound (firmly associated) fractions. With A. gayanus silage diets, degradation may not be limited by microbial colonization, but rather by the ability of fibrolytic enzymes to degrade plant cell walls within this recalcitrant forage.     

温带混交林碳水通量模拟及其对冠层分层方式的响应——耦合的气孔导度-光合作用-能量平衡模型

利用Leuning建立的耦合的光合作用、气孔导度和能量平衡方程,以将冠层分成多层的方式,包括Gaussian五点积分法、将冠层平均分为多层的方法,逐层计算温带混交林的碳水通量,最后累加至冠层尺度,以模拟CO2和H2O通量。该模型以常规气象观测数据作为驱动变量,计算出冠层与大气之间的碳水交换,与涡动相关系统的通量观测数据进行比较,分析了不同的冠层分层方式对多层模型模拟结果的影响。从3个温带混交林通量站涡动相关系统的能量平衡闭合度来看,中国长白山站CBS、韩国GDK和日本MMF站点的能量平衡比率(EBR)分别为0.76、0.66和1.07,居于国际同类观测范围(0.34—1.2)的中上水平,因此,涡动相关系统的观测数据较为可靠。从碳水通量的日变化来看,用Gaussian五点积分法将冠层分为五层的模型能较好的模拟碳水通量的"单峰形"日变化趋势。夜间Fc为负值且变化趋势较为平缓,表明生态系统进行呼吸作用释放CO2,从日出开始Fc逐渐变为正值,表明生态系统进行光合作用吸收CO2,Fc在中午时分达到最大值,下午Fc逐渐减小,日落之后又回复到夜间的负值。H2O通量的日变化曲线与CO2通量相似,且模拟值与涡动相关实测值具有较好的一致性。在多层模型中,对冠层采用不同的分层方法,对碳水通量模拟结果有一定的影响。以Gaussian五点积分法将冠层分为五层的方法作为对照,分别将冠层平均分为2、5、10、20层的方法得到的碳水通量与其进行比较。从平均值来看,分层越多,H2O通量模拟值越低,而CO2通量模拟值越高。不同的分层方法产生的差异,主要来自于不同层的辐射吸收、温湿度、风速等环境要素的垂直廓线差异,且叶片光合作用对光的响应是非线性的。     

Effect of harvest date and nitrogen fertilization rate on the nutritive value of amaranth forage (Amaranthus hypochondriacus)

This study was conducted to assess effects of harvest date (i.e., 40 and 60 d after planting) and N fertilization rate (i.e., 120, 180, 240 kg N/ha) on the nutritive value of amaranth forage (Amaranthus hypochondriacus) using a factorial experiment with a randomized complete block design. The content of dry matter (DM), crude protein (CP), true protein (TP), ether extract (EE), water soluble carbohydrates (WSC), ash-free neutral detergent fiber (NDFom), ash-free acid detergent fiber (ADFom), lignin(sa), ash, Ca, P, Na, K, oxalic acid and nitrate were determined. Soluble CP (SP) and protein fractions non-protein N (A), true protein rapidly degraded in the rumen (B₁), true protein degraded in the rumen at a moderate rate (B₂), true protein associated with the cell wall and slowly degraded in the rumen (B₃) and acid detergent insoluble CP (C) were measured according to the Cornell Net Carbohydrate and Protein System. In vitro gas production (IVGP), OM disappearance (OMD) and NDFom disappearance (NDFD) were determined using a gas production technique. Results showed that the later harvest date increased (P<0.05) DM, EE, WSC, NDFom, ADFom, lignin(sa), B₃ and C; while CP, TP, ash, Ca, P, K, SP, A, B₁, B₂, nitrate, total and soluble oxalic acid, IVGP, b (i.e., gas production from the insoluble fermentable fractions at 120 h), c (i.e., rate of gas production during incubation), OMD and NDFD decreased (P<0.05). With increasing N fertilization rate, CP, TP, EE, P, nitrate, oxalic acid, SP, A, b, OMD and NDFD increased (P<0.05), however B₂ declined (P<0.05). Increasing N fertilization increased yield, CP concentration and nutrient digestibility. At 40 d after planting use of amaranth forage as a ruminant feed is limited due to its high nitrate content. However, at 60 d, although a depression in digestibility and CP content occurred, this forage has the potential as a ruminant feed due to the much lower nitrate levels.     

Screening of plants from diversified natural grasslands for their potential to combine high digestibility,and low methane and ammonia production

A total of 156 plant species from 35 botanical families collected from diversified grasslands in the French Massif Central were screened in vitro for their potential to combine high nutritive value for ruminants and a reduced impact on the environment. The vegetative part of plants were analyzed for their chemical composition and incubated in a batch system containing buffered rumen fluid at 39°C for 24 h. The gas production and composition were recorded, and the fermentation end-product concentrations in the incubation medium and the in vitro true organic matter digestibility (IVTOMD) were determined. The results were expressed relative to perennial ryegrass (PRG) values used as a reference. We observed that no relationship between methane (CH₄) and volatile fatty acids (VFA) was evidenced for 12 plants, the fermentation of these plants producing significantly less CH₄ for a similar level of VFA production. In all, 13 plants showed 50% less CH₄ production per unit of organic matter truly digested (OMTD) than PRG. Among these plants, two reduced CH₄ by more than 80% and four species had an IVTOMD higher than 80%. The underlying modes of action seem to be different among plants: some result in an accumulation of H₂ in the fermentation gas, but others do not. In terms of nitrogen (N) use efficiency, the fermentation of 37 plants halved the ratio between ammonia (N–NH₃) and plant N content compared with PRG, of which six showed a complete absence of N–NH₃ in the medium. Among these plants, four maintained the IVTDMO at values not significantly different from PRG (P>0.05). Considering the multi-criteria selection, 16 plants showed simultaneously a reduction of more than 80% in N–NH₃ production and 30% in CH₄ emission per unit of OMTD compared with PRG, including three with an IVTOMD higher than 80%. Overall, the botanical families that reduced simultaneously CH₄ and N–NH₃ most efficiently were the Rosaceae, Onagraceae, Polygonaceae and Dipsacaceae. The Onagraceae also gave high values for IVTOMD.     

Effects of exogenous enzymes on in vitro gas production kinetics and ruminal fermentation of four fibrous feeds

This study was conducted to investigate effects of increasing doses: 0 (control), 6 (low), 12 (medium) and 24 (high) mg/g DM of ZADO^® enzyme preparation mixture (ENZ) on in vitro gas production (GP) and some ruminal fermentation parameters of the fibrous feeds Saccharum officinarum (leaves), Andropogon gayanus (leaves), Pennisetum purpureum (leaves) and Sorghum vulgare (straw). Rumen liquor was obtained from two Brown Swiss cows fitted with permanent rumen cannulae fed a total mixed ration of a 500:500 commercial concentrate and alfalfa hay ad libitum. The GP was recorded at 2, 4, 6, 8, 10, 12, 24, 48, 72 and 96 h of incubation. After 96 h, the incubation was stopped and the pH of the mixture was determined and filtrate used to determine dry matter degradability (DMD), partitioning factor (PF₉₆), gas yield (GY₂₄), in vitro organic matter digestibility (OMD), metabolizable energy (ME), short chain fatty acids (SCFA), and microbial crude protien production (MCP). In general, the crude protein (CP) content of the fibrous feeds was low and ranged from 23 g/kg DM (S. officinarum) to 44 (A. gayanus). The fibre contents (i.e., NDFom and ADFom) were highest (P<0.05) in S. officinarum. Increasing ENZ dose linearly increased (P<0.05) GP of all fibrous feeds and had a quadratically increased (P<0.05) asymptotic gas production in P. purpureum and S. vulgare and rate of gas production in S. officinarum and S. vulgare. Addition of ENZ also quadratically increased (P<0.05) GP at all incubation times in S. officinarum and S. vulgare, and A. gayanus, but only at 72 h in A. gayanus. The parameters of ruminal fermentation of OMD, ME, GY₂₄ and SCFA linearly increased (P<0.05) and MCP linearly decreased (P<0.05) with the ENZ addition. Addition of enzyme affected ruminal fermentation of our feeds differently, mainly dependent on their fibre content, although dosage of enzyme was also important as impacts generally increased at higher dosages of ENZ.     

In vitro ruminal methanogenesis of a hay-rich substrate in response to different combination supplements of nitrocompounds; pyromellitic diimide and 2-bromoethanesulphonate

Sixteen combinations of 5 treatments at 4 levels were designed in a L₁₆(4⁵) orthogonal experimental design to evaluate associative effects of five methanogenesis inhibitors at four dose levels: nitroethane (NE, 0 mM, 5 mM, 10 mM and 15 mM), 2-nitroethanol (NEOH, 0 mM, 5 mM, 10 mM and 15 mM), 2-nitro-1-propanol (NPOH, 0 mM, 5 mM, 10 mM and 15 mM), pyromellitic diimide (PMDI, 0 mM, 0.02 mM, 0.05 mM and 0.07 mM) and 2-bromoethanesulphonate (BES, 0 mM, 0.01 mM, 0.03 mM and 0.05 mM) on in vitro ruminal methane production of the mixed substrate (Chinese wildrye hay:maize meal = 4:1) using a cumulative gas production technique. After 48 h incubation, in vitro dry matter disappearance (IVDMD), total gas production (GP₄₈, ml/g DM) and total volatile fatty acids (VFA) production in various combinations of these inhibitors were decreased by 10.6-56.0, 26.5-44.5 and 20.3-47.6%, respectively (P<0.05). The molar proportion of acetate in the inhibitor combination groups was decreased by 6.6-12.5% while those of propionate and butyrate were increased by 7.0-19.2 and 21.9-56.5% (P<0.01), respectively. Methane proportion (MP) in total gas production was reduced by 79.4-98.5% (P<0.01), and the highest inhibition occurred in the combination of 10 mM NE, 10 mM NPOH, 0.07 mM PMDI and 0.01 mM BES in cultures. The partial correlation coefficients between NE, NEOH, NPOH, PMDI or BES and CH₄ proportion were −0.465 (P<0.01), −0.417 (P<0.01), −0.355 (P<0.05), −0.408 (P<0.01) and −0.345 (P<0.05), respectively, indicating that NE was the most potent inhibitor, followed by NEOH and PMDI, and finally NPOH and BES. In general, VFA production in the inhibitor combinations was substantially shifted to produce much more butyrate and propionate and less acetate. The combination of 15 mM NE, 10 mM NEOH, 5 mM NPOH, 0.07 mM PMDI and 0.01 mM BES in cultures, leading to >95% methane inhibition, may be the optimal application of these inhibitors with less depression of total VFA production. Further feeding trials to validate these combinations is still required on rumen function, methane production, growth performance and milk production.     

Effects of nitrate adaptation by rumen inocula donors and substrate fiber proportion on in vitro nitrate disappearance,methanogenesis, and rumen fermentation acid

A study was conducted to evaluate the main effects of dietary nitrate adaptation by cattle and alfalfa cell wall to starch ratio in in vitro substrates on nitrate disappearance and nitrite and volatile fatty acid (VFA) concentrations, as well as hydrogen (H₂) and methane (CH₄) accumulations. Rumen fluid from steers fed diets containing urea or nitrate was added into in vitro incubations containing sodium nitrate as the sole nitrogen source and 20 cell wall : 80 starch or 80 cell wall : 20 starch as the carbohydrate source. The results showed that during 24 h incubation, rumen fluid inoculums from steers adapted to dietary nitrate resulted in more rapid nitrate disappearance by 6 h of incubation (P < 0.01), no significant effect on nitrite concentration and diminished CH₄ accumulation (P < 0.05). Cell wall to starch ratio did not affect nitrate disappearance, CH₄ accumulation and total VFA concentration. The higher cell wall ratio had the lower total gas production and H₂ concentration (P < 0.05). Ammonia-N (NH₃-N) concentration increased because of adaptation of donors to nitrate feeding (P < 0.05). Nitrate adaptation did not alter total VFA concentration, but increased acetate, and decreased propionate and butyrate molar proportions (P < 0.01).     

Multiscale model intercomparisons of CO2 and H2O exchange rates in a maturing southeastern US pine forest

We compared four existing process‐based stand‐level models of varying complexity (physiological principles in predicting growth, photosynthesis and evapotranspiration, biogeochemical cycles, and stand to ecosystem carbon and evapotranspiration simulator) and a new nested model with 4 years of eddy‐covariance‐measured water vapor (LE) and CO₂ (Fc) fluxes at a maturing loblolly pine forest. The nested model resolves the ‘fast’ CO₂ and H₂O exchange processes using canopy turbulence theories and radiative transfer principles whereas slowly evolving processes were resolved using standard carbon allocation methods modified to improve leaf phenology. This model captured most of the intraannual variations in leaf area index (LAI), net ecosystem exchange (NEE), and LE for this stand in which maximum LAI was not at a steady state. The model comparisons suggest strong linkages between carbon production and LAI variability, especially at seasonal time scales. This linkage necessitates the use of multilayer models to reproduce the seasonal dynamics of LAI, NEE, and LE. However, our findings suggest that increasing model complexity, often justified for resolving faster processes, does not necessarily translate into improved predictive skills at all time scales. Additionally, none of the models tested here adequately captured drought effects on water and CO₂ fluxes. Furthermore, the good performance of some models in capturing flux variability on interannual time scales appears to stem from erroneous LAI dynamics and from sensitivity to droughts that injects unrealistic flux variability at longer time scales.     

Reducing agent can be omitted in the incubation medium of the batch in vitro fermentation model of the pig intestines

Over the past decade, in vitro methods have been developed to study intestinal fermentation in pigs and its influence on the digestive physiology and health. In these methods, ingredients are fermented by a bacterial inoculum diluted in a mineral buffer solution. Generally, a reducing agent such as Na₂S or cysteine-HCl generates the required anaerobic environment by releasing metabolites similar to those produced when protein is fermented, possibly inducing a dysbiosis. An experiment was conducted to study the impact of two reducing agents on results yielded by such in vitro fermentation models. Protein (soybean proteins, casein) and carbohydrate (potato starch, cellulose) ingredients were fermented in vitro by bacteria isolated from fresh feces obtained from three sows in three carbonate-based incubation media differing in reducing agent: (i) Na₂S, (ii) cysteine-HCl and (iii) control with a mere saturation with CO₂ and devoid of reducing agent. The gas production during fermentation was recorded over 72 h. Short-chain fatty acids (SCFA) production after 24 and 72 h and microbial composition of the fermentation broth after 24 h were compared between ingredients and between reducing agents. The fermentation residues after 24 h were also evaluated in terms of cytotoxicity using Caco-2 cell monolayers. Results showed that the effect of the ingredient induced higher differences than the reducing agent. Among the latter, cysteine-HCl induced the strongest differences compared with the control, whereas Na₂S was similar to the control for most parameters. For all ingredients, final gas produced per g of substrate was similar (P>0.10) for the three reducing agents whereas the maximum rate of gas production (R_max) was reduced (P<0.05) when carbohydrate ingredients were fermented with cysteine-HCl in comparison to Na₂S and the control. For all ingredients, total SCFA production was similar (P>0.10) after 24 h of fermentation with Na₂S and in the control without reducing agent. Molar ratios of branched chain-fatty acids were higher (P<0.05) for protein (36.5% and 9.7% for casein and soybean proteins, respectively) than for carbohydrate (<4%) ingredients. Only fermentation residues of casein showed a possible cytotoxic effect regardless of the reducing agent (P<0.05). Concerning the microbial composition of the fermentation broth, most significant differences in phyla and in genera ascribable to the reducing agent were found with potato starch and casein. In conclusion, saturating the incubation media with CO₂ seems sufficient to generate a suitable anaerobic environment for intestinal microbes and the use of a reducing agent can be omitted.     

Effects of NaOH treatment on condensed tannin contents and gas production kinetics of tree leaves

Effects of NaOH treatment on the crude protein (CP), condensed tannin (CT) and in vitro gas production kinetics of leaves of Arbutus andrachne, Glycyrrhiza glabra L. and wheat straw were determined. Wheat straw, which is tannin-free, was used as the standard. The NaOH treatment was completed by pulverization of samples with 0, 20, 40, 60 and 80 g/L of NaOH solution in the proportion of 1 L of solution to 1 kg of sample. Gas production was determined at 3, 6, 12, 24, 48, 72 and 96 h of incubation. NaOH treatment linearly decreased (P<0.001) the CT contents of leaves of Arbutus andrachne and Glycyrrhiza glabra L. whereas NaOH treatment had no effect on the CP contents of Arbutus andrachne, Glycyrrhiza glabra L. and wheat straw. The 80 g/L NaOH treatment reduced the CT content of leaves of Arbutus andrachne and Glycyrrhiza glabra L. by 59.6% and 86.7%, respectively. NaOH treatment linearly decreased (P<0.01) gas production rate of Arbutus andrachne although it linearly increased (P<0.01) gas production rate of wheat straw. In contrast, NaOH treatment had no effect on gas production rate of leaves of Glycyrrhiza glabra L. NaOH treatment linearly decreased (P<0.001) potential gas production of leaves of Arbutus andrachne and Glycyrrhiza glabra L. whereas NaOH treatment linearly increased (P<0.001) potential gas production of wheat straw. Treatment with NaOH can be used to improve the nutritive value of tannin-free forages such as straw, but not for tannin-containing leaves.     

In vitro digestion and fermentation characteristics of canola co-products simulate their digestion in the pig intestine

Canola co-products are sources of amino acid and energy in pig feeds, but their fermentation characteristics in the pig intestine are unknown. Thus, we determined the in vitro fermentation characteristics of the canola co-products Brassica juncea solvent-extracted canola meal (JSECM), Brassica napus solvent-extracted canola meal (NSECM), B. napus expeller-pressed canola meal (NEPCM) and B. napus cold-pressed canola cake (NCPCC) in comparison with soybean meal (SBM). Samples were hydrolysed in two steps using pepsin and pancreatin. Subsequently, residues were incubated in a buffer solution with fresh pig faeces as inocula for 72 h to measure gas production. Concentration of volatile fatty acids (VFA) per gram of dry matter (DM) of feedstuff was measured in fermented solutions. Apparent ileal digestibility (AID) and apparent hindgut fermentation (AHF) of gross energy (GE) for feedstuffs were obtained from pigs fed the same feedstuffs. On DM basis, SBM, JSECM, NSECM, NEPCM and NCPCC contained 15, 19, 22, 117 and 231 g/kg ether extract; and 85, 223, 306, 208 and 176 g/kg NDF, respectively. In vitro digestibility of DM (IVDDM) of SBM (82.3%) was greater (P<0.05) than that of JSECM (68.5%), NSECM (63.4%), NEPCM (67.5%) or NCPCC (69.8%). The JSECM had greater (P<0.05) IVDDM than NSECM. The IVDDM for NSECM was lower (P<0.05) than that for NEPCM, which was lower (P<0.05) than that for NCPCC. Similarly, AID of GE was greatest for SBM followed by NCPCC, JSECM, NEPCM and then NSECM. Total VFA production for SBM (0.73 mmol/g) was lower (P<0.05) than that of JSECM (1.38 mmol/g) or NSECM (1.05 mmol/g), but not different from that of NEPCM (0.80 mmol/g) and NCPCC (0.62 mmol/g). Total VFA production of JSECM was greater (P<0.05) than that of NSECM. Total VFA production of NSECM was greater (P<0.05) than that of NEPCM or NCPCC, which differed (P<0.05). The ranking of feedstuffs for total VFA production was similar to AHF of GE. In conclusion, in vitro fermentation characteristics of canola co-products and SBM simulated their fermentation in the small and large intestine of pigs, respectively. The 30% greater VFA production for JSECM than NSECM due to lower lignified fibre of JSECM indicates that fermentation characteristics differ between canola species. The NSECM had the highest fermentability followed by NEPCM and then NCPCC, indicating that fat in canola co-products can limit their fermentability in the hindgut.     

The selective inhibitory effect of NH4Cl on estrogen-stimulated rat uterine in vitro RNA synthesis

Estrogen-stimulated in vitro RNA synthesis in rat uterine nuclear-myofibrilar fractions, isolated uterine nuclei, and nucleoli was selectively inhibited by NH₄Cl (400 μmoles) when added prior to the start of a 37 °C incubation. This inhibitory effect was not observed if the salt was added after the start (as early as 1 min) of a 37 °C incubation. The removal of NH₄Cl from reaction mixtures not yet incubated at 37 °C further reduced in vitro RNA synthesis in both control and hormone-treated nuclei. The data suggest that NH₄Cl (400 μmoles) added prior to, but not after, the start of a 37 °C incubation inhibited estrogen-stimulated rat uterine nucleolar RNA polymerase activity perhaps by removing a protein component(s) which is necessary for hormonal stimulation.     

Changes in in vitro gas and methane production from rumen fluid from dairy cows during adaptation to feed additives in vivo

The adaptation of dairy cows to methane (CH₄)-mitigating feed additives was evaluated using the in vitro gas production (GP) technique. Nine rumen-fistulated lactating Holstein cows were grouped into three blocks and within blocks randomly assigned to one of three experimental diets: Control (CON; no feed additive), Agolin Ruminant^R (AR; 0.05 g/kg dry matter (DM)) or lauric acid (LA; 30 g/kg DM). Total mixed rations composed of maize silage, grass silage and concentrate were fed in a 40 : 30 : 30 ratio on DM basis. Rumen fluid was collected from each cow at days −4, 1, 4, 8, 15 and 22 relative to the introduction of the additives in the diets. On each of these days, a 48-h GP experiment was performed in which rumen fluid from each individual donor cow was incubated with each of the three substrates that reflected the treatment diets offered to the cows. DM intake was on average 19.8, 20.1 and 16.2 kg/day with an average fat- and protein-corrected milk production of 30.7, 31.7 and 26.2 kg/day with diet CON, AR and LA, respectively. In general, feed additives in the donor cow diet had a larger effect on gas and CH₄ production than the same additives in the incubation substrate. Incubation substrate affected asymptotic GP, half-time of asymptotic CH₄ production, total volatile fatty acid (VFA) concentration, molar proportions of propionate and butyrate and degradation of organic matter (OMD), but did not affect CH₄ production. No substrate×day interactions were observed. A significant diet×day interaction was observed for in vitro gas and CH₄ production, total VFA concentration, molar proportions of VFA and OMD. From day 4 onwards, the LA diet persistently reduced gas and CH₄ production, total VFA concentration, acetate molar proportion and OMD, and increased propionate molar proportion. In vitro CH₄ production was reduced by the AR diet on day 8, but not on days 15 and 22. In line with these findings, the molar proportion of propionate in fermentation fluid was greater, and that of acetate smaller, for the AR diet than for the CON diet on day 8, but not on days 15 and 22. Overall, the data indicate a short-term effect of AR on CH₄ production, whereas the CH₄-mitigating effect of LA persisted.     

Atlantic salmon (Salmo salar) muscle precursor cells differentiate into osteoblasts in vitro: Polyunsaturated fatty acids and hyperthermia influence gene expression and differentiation

The formation and mineralisation of bone are two critical processes in fast-growing Atlantic salmon (Salmo salar). The mechanisms of these processes, however, have not been described in detail. Thus, in vitro systems that allow the study of factors that influence bone formation in farmed Atlantic salmon are highly warranted. We describe here a method by which unspecialised primary cells from salmon white muscle can differentiate to osteoblasts in vitro. We have subsequently used the differentiated cells as a model system to study the effects of two factors that influence bone formation in Atlantic salmon under commercial farming conditions, namely polyunsaturated fatty acids, PUFAs, and temperature. Muscle precursor cells changed their morphology from triangular or spindle-shaped cells to polygonal or cubical cells after 3 weeks in osteogenic medium. In addition, gene expression studies showed that marker genes for osteoblastogenesis; alp, col1a1, osteocalcin, bmp2 and bmp4 increased after 3 weeks of incubation in osteogenic media showing that these cells have differentiated to osteoblasts at this stage. Adding CLA or DHA to the osteoblast media resulted in a reduced PGE₂ production and increased expression of osteocalcin. Further, temperature studies showed that differentiating osteoblasts are highly sensitive to increased incubation temperature at early stages of differentiation. Our studies show that unspecialised precursor cells isolated from salmon muscle tissue can be caused to differentiate to osteoblasts in vitro. Furthermore, this model system appears to be suitable for the study of osteoblast biology in vitro.     

Kinetic studies of pyruvate kinase duringin vitro differentiation of GM-CFC haemopoietic precursor and bone marrow cells in mice

Pyruvate kinase studies in the granulocyte-macrophage lineage duringin vitro differentiation have been performed using culture techniques on GM-CFC cells and a study has also been done in bone marrow cells.The enzyme exhibits biphasic behaviour with respect to both of its substrates in cells derived fromin vitro cultures at 5 and 7 days of incubation period. However in bone marrow cells these kinetics are only observed for ADP.The different kinetic behaviour of pyruvate kinase toward Fru-1,6-P₂, Ala, Phe and ATP in the three cellular populations allows us to conclude that the expression of pyruvate kinase is associated with the differentiation of these cells.Abbreviations GM-CFC granulocyte-macrophage colony forming cells - PK pyruvate kinase - CFU-E Colony Forming Units Erythroid - E_w Error weight - PEP phosphoenolpyruvate - Fru-1,6-P₂ fructose 1,6-bisphosphate - Ala L-alanine - Phe L-phenylanine - 5 GM granulocytemacrophage colonies obtained after 5 days incubation - 7 GM granulocyte-macrophage colonies obtained after 7 days incubation - h Hill coefficient - S_0,5 substrate concentration that yields half-maximal velocity     

Dependence of forage quality and methanogenic potential of tropical plants on their phenolic fractions as determined by principal component analysis

This study was conducted to elucidate relationships among various phenolic fractions in, and methane (CH₄) emissions from, tropical plants when incubated in ruminal fluid in vitro. As a second objective, principal component analysis (PCA) was tested for its utility in screening plants for their ability to reduce CH₄ formation at simultaneously acceptable nutritional quality. Leaves from 27 tropical plants were analyzed for their nutritional composition and various phenolic fractions. They were incubated in vitro using the Hohenheim gas test method. Variables measured after 24 h of incubation were total gas and CH₄ production, and pH, ammonia, bacterial and protozoal counts, as well as short-chain fatty acids in the incubation fluid. In vitro organic matter (OM) digestibility was computed by a standard equation. The data obtained was subjected to analysis of variance, correlation, regression and PCA. Among phenolic fractions, total phenols had the closest relationship with CH₄/digestible OM (r = ?0.84, P<0.001). The total tannin fraction contributed strongly to this effect (r = ?0.74, P<0.001) whereas the non-tannin phenol fraction was less important (r = ?0.45, P<0.05). Methane reduction by the influence of non-tannin phenols was not associated with a negative effect on protein degradation, while this was the case with tannins. Condensed (r = ?0.60, P<0.01) and hydrolysable tannins (r = ?0.60, P<0.01) contributed to the decrease in CH₄/digestible OM. The loading plot of PCA showed that dietary crude protein (CP) content and incubation fluid ammonia, total short-chain fatty acids, propionate, valerate, iso-butyrate, iso-valerate as well as in vitro OM digestibility were clustered. They had inverse directions to contents of fiber fractions and incubation fluid acetate proportion and acetate-to-propionate ratio. The methane-to-total-gas ratio had the opposite effect of the contents of any phenolic fraction. Plants possessing a favorable forage quality, based on the corresponding PCA score plot, were Carica papaya, Manihot esculenta, Morinda citrifolia, Sesbania grandiflora and Melia azedarach, whereas CH₄ mitigating plants included Swietenia mahagoni, Acacia villosa, Eugenia aquea, Myristica fragrans and Clidemia hirta. All phenolic fractions studied reduced CH₄ emissions from in vitro incubations with ruminal fluid and PCA seems useful to screen plants for high nutritional quality and low ruminal CH₄ formation. However, high forage quality seemed to be partially associated with high CH₄ emission. The search for plants rich in non-tannin phenols might be promising as these compounds appear to decrease CH₄ while they obviously have less negative effect on protein degradation as compared to the tannin fractions.