Effects of graded levels of dietary pomegranate peel on methane and nitrogen losses, and metabolic and health indicators in dairy cows.

This study aimed to quantify the effects of dietary inclusion of tannin-rich pomegranate peel (PP) on intake, methane and nitrogen (N) losses, and metabolic and health indicators in dairy cows. Four multiparous, late-lactating Brown Swiss dairy cows (796 kg body weight; 29 kg/d of energy corrected milk yield) were randomly allocated to 3 treatments in a randomized cyclic change-over design with 3 periods, each comprising 14 d of adaptation, 7 d of milk, urine, and feces collection, and 2 d of methane measurements. Treatments were formulated using PP that replaced on a dry matter (DM) basis 0% (control), 5%, and 10% of the basal mixed ration (BMR) consisting of corn and grass silage, alfalfa, and concentrate. Gaseous exchange of the cows was determined in open-circuit respiration chambers. Blood samples were collected on d 15 of each period. Individual feed intake as well as feces and urine excretion were quantified, and representative samples were collected for analyses of nutrients and phenol composition. Milk was analyzed for concentrations of fat, protein, lactose, milk urea N, and fatty acids. Total phenols and antioxidant capacity in milk and plasma were determined. In serum, the concentrations of urea and bilirubin as well as the activities of alanine aminotransferase (ALT), aspartate aminotransferase, glutamate dehydrogenase, alkaline phosphatase, and γ-glutamyl transferase were measured. The data were subjected to ANOVA with the Mixed procedure of SAS, with treatment and period as fixed and animal as random effects. The PP and BMR contained 218 and 3.5 g of total extractable tannins per kg DM, respectively, and thereof 203 and 3.3 g of hydrolyzable tannins. Total DM intake, energy corrected milk, and methane emission (total, yield, and intensity) were not affected by PP supplementation. The proportions of C18:2n-6 and C18:3n-3 in milk increased linearly as the amount of PP was increased in the diet. Milk urea N, blood urea N, and urinary N excretion decreased linearly with the increase in dietary PP content. Total phenols and antioxidant capacity in milk and plasma were not affected by the inclusion of PP. The activity of ALT increased in a linear manner with the inclusion of PP. In conclusion, replacing up to 10% of BMR with PP improved milk fatty acid composition and alleviated metabolic and environmental N load. However, the elevated serum ALT activity indicates an onset of liver stress even at 5% PP, requiring the development of adaptation protocols for safe inclusion of PP in ruminant diets.

Prediction of nitrogen excretion from data on dairy cows fed a wide range of diets compiled in an intercontinental database: A meta-analysis.

Manure nitrogen (N) from cattle contributes to nitrous oxide and ammonia emissions and nitrate leaching. Measurement of manure N outputs on dairy farms is laborious, expensive, and impractical at large scales; therefore, models are needed to predict N excreted in urine and feces. Building robust prediction models requires extensive data from animals under different management systems worldwide. Thus, the study objectives were (1) to collate an international database of N excretion in feces and urine based on individual lactating dairy cow data from different continents; (2) to determine the suitability of key variables for predicting fecal, urinary, and total manure N excretion; and (3) to develop robust and reliable N excretion prediction models based on individual data from lactating dairy cows consuming various diets. A raw data set was created based on 5,483 individual cow observations, with 5,420 fecal N excretion and 3,621 urine N excretion measurements collected from 162 in vivo experiments conducted by 22 research institutes mostly located in Europe (n = 14) and North America (n = 5). A sequential approach was taken in developing models with increasing complexity by incrementally adding variables that had a significant individual effect on fecal, urinary, or total manure N excretion. Nitrogen excretion was predicted by fitting linear mixed models including experiment as a random effect. Simple models requiring dry matter intake (DMI) or N intake performed better for predicting fecal N excretion than simple models using diet nutrient composition or milk performance parameters. Simple models based on N intake performed better for urinary and total manure N excretion than those based on DMI, but simple models using milk urea N (MUN) and N intake performed even better for urinary N excretion. The full model predicting fecal N excretion had similar performance to simple models based on DMI but included several independent variables (DMI, diet crude protein content, diet neutral detergent fiber content, milk protein), depending on the location, and had root mean square prediction errors as a fraction of the observed mean values of 19.1% for intercontinental, 19.8% for European, and 17.7% for North American data sets. Complex total manure N excretion models based on N intake and MUN led to prediction errors of about 13.0% to 14.0%, which were comparable to models based on N intake alone. Intercepts and slopes of variables in optimal prediction equations developed on intercontinental, European, and North American bases differed from each other, and therefore region-specific models are preferred to predict N excretion. In conclusion, region-specific models that include information on DMI or N intake and MUN are required for good prediction of fecal, urinary, and total manure N excretion. In absence of intake data, region-specific complex equations using easily and routinely measured variables to predict fecal, urinary, or total manure N excretion may be used, but these equations have lower performance than equations based on intake.

Methane emission, metabolism, and performance of Holstein dairy cows with low, medium, and high lymphocyte proliferation during transition.

This study aimed to identify interactions between state of lactation (dry or early lactating) and immune responder group (low, medium, or high) for energy metabolism traits as well as metabolic and immunological traits in dairy cows. In early lactation, when the energy priority of cows shifts toward the mammary gland, the energy available to be partitioned toward the immune system may differ among individuals. The equilibrium between energy supply from feed, digestion, and body reserve mobilization and energy expenditure with milk, immune system, methane, and heat production is delicate in this stage. Seventeen Holstein cows entering their second to fifth lactation were kept under comparable feeding, housing, and management conditions and were studied from 14 ± 6 d before calving to 11 ± 3 d after calving. Feed intake, milk yield, body condition, blood metabolites, and cortisol as well as gaseous exchange in respiration chambers were measured. The latter was used to quantify methane emission and to calculate resting metabolic rate and heat production. Subsets of blood leukocytes and peripheral blood mononuclear cells (PBMC) were monitored. Activation and proliferation of the PBMC in response to the mitogen phytohemagglutinin ante- and postpartum were assessed using the oxygen consumption rate (24-h cell culture assay) and the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay (72-h cell culture assay). Cows were classified based on the in vitro proliferative response of the PBMC measured postpartum in low (n = 6), medium (n = 5), and high (n = 6) responders. We found no interaction of state of lactation with responder group for feed intake, milk yield, efficiency, metabolic traits, and immune cell activation ante- and postpartum. However, after calving, low-responder cows produced less methane per unit of body weight and per unit of energy-corrected milk compared with the other cows. This might be indicative of a low rumen fermentation intensity. Low responders might therefore suffer from a lower availability of digestible energy in early lactation and not be able to sustain the shift from immune cell activation to proliferation. If so, the selection of environmentally friendly low-methane emitters could promote phenotypes with a compromised immune response in the critical early lactation.

Accuracy of methane emissions predicted from milk mid-infrared spectra and measured by laser methane detectors in Brown Swiss dairy cows.

Since heritability of CH4 emissions in ruminants was demonstrated, various attempts to generate large individual animal CH4 data sets have been initiated. Predicting individual CH4 emissions based on equations using milk mid-infrared (MIR) spectra is currently considered promising as a low-cost proxy. However, the CH4 emission predicted by MIR in individuals still has to be confirmed by measurements. In addition, it remains unclear how low CH4 emitting cows differ in intake, digestion, and efficiency from high CH4 emitters. In the current study, putatively low and putatively high CH4 emitting Brown Swiss cows were selected from the entire Swiss herdbook population (176,611 cows), using an MIR-based prediction equation. Eventually, 15 low and 15 high CH4 emitters from 29 different farms were chosen for a respiration chamber (RC) experiment in which all cows were fed the same forage-based diet. Several traits related to intake, digestion, and efficiency were quantified over 8 d, and CH4 emission was measured in 4 open circuit RC. Daily CH4 emissions were also estimated using data from 2 laser CH4 detectors (LMD). The MIR-predicted CH4 production (g/d) was quite constant in low and high emission categories, in individuals across sites (home farm, experimental station), and within equations (first available and refined versions). The variation of the MIR-predicted values was substantially lower using the refined equation. However, the predicted low and high emitting cows (n = 28) did not differ on average in daily CH4 emissions measured either with RC or estimated using LMD, and no correlation was found between CH4 predictions (MIR) and CH4 emissions measured in RC. When individuals were recategorized based on CH4 yield measured in RC, differences between categories of 10 low and 10 high CH4 emitters were about 20%. Low CH4 emitting cows had a higher feed intake, milk yield, and residual feed intake, but they differed only weakly in eating pattern and digesta mean retention times. Low CH4 emitters were characterized by lower acetate and higher propionate proportions of total ruminal volatile fatty acids. We concluded that the current MIR-based CH4 predictions are not accurate enough to be implemented in breeding programs for cows fed forage-based diets. In addition, low CH4 emitting cows have to be characterized in more detail using mechanistic studies to clarify in more detail the properties that explain the functional differences found in comparison with other cows.

Persistence of differences between dairy cows categorized as low or high methane emitters, as estimated from milk mid-infrared spectra and measured by GreenFeed.

Currently, various attempts are being made to implement breeding schemes aimed at producing low methane (CH4) emitting cows. We investigated the persistence of differences in CH4 emission between groups of cows categorized as either low or high emitters over a 5-mo period. Two feeding regimens (pasture vs. indoors) were used. Early- to mid-lactation Holstein Friesian cows were categorized as low or high emitters (n = 10 each) retrospectively, using predictions from milk mid-infrared (MIR) spectra, before the start of the experiment. Data from MIR estimates and from measurements with the GreenFeed (GF; C-Lock Technology Inc., Rapid City, SD) system over the 5-mo experiment were combined into 7-, 14-, and 28-d periods. Feed intake, eating and ruminating behavior, and ruminal fluid traits were determined in two 7-d measurement periods in the grazing season. The CH4 emission data were analyzed using a split-plot ANOVA, and the repeatability of each of the applied methods for determining CH4 emission was calculated. Traits other than CH4 emission were analyzed for differences between low and high emitters using a linear mixed model. The initial category-dependent differences in daily CH4 production persisted over the subsequent 5 mo and across 2 feeding regimens with both methods. The repeatability analysis indicated that the biweekly milk control scheme, and even a monthly scheme as practiced on farms, might be sufficient for confirming category differences. However, the relationship between CH4 data estimated by MIR and measured with GF for individual cows was weak (R2 = 0.26). The categorization based on CH4 production also generated differences in CH4 emission per kilogram of milk; differentiation between cow categories was not persistent based on milk MIR spectra and GF. Compared with the high emitters, low emitters tended to show a lower acetate-to-propionate ratio in ruminal volatile fatty acids, whereas feed intake and ruminating time did not differ. Interestingly, the low emitters spent less time eating than the high emitters. In conclusion, the CH4 estimation from analyzing the milk MIR spectra is an appropriate proxy to form and regularly control categories of cows with different CH4 production levels. The categorization was also sufficient to secure similar and persistent differences in emission intensity when estimated by MIR spectra of the milk. Further studies are needed to determine whether MIR data from individual cows are sufficiently accurate for breeding.

Invited review: Nitrogen in ruminant nutrition: A review of measurement techniques.

Nitrogen is a component of essential nutrients critical for the productivity of ruminants. If excreted in excess, N is also an important environmental pollutant contributing to acid deposition, eutrophication, human respiratory problems, and climate change. The complex microbial metabolic activity in the rumen and the effect on subsequent processes in the intestines and body tissues make the study of N metabolism in ruminants challenging compared with nonruminants. Therefore, using accurate and precise measurement techniques is imperative for obtaining reliable experimental results on N utilization by ruminants and evaluating the environmental impacts of N emission mitigation techniques. Changeover design experiments are as suitable as continuous ones for studying protein metabolism in ruminant animals, except when changes in body weight or carryover effects due to treatment are expected. Adaptation following a dietary change should be allowed for at least 2 (preferably 3) wk, and extended adaptation periods may be required if body pools can temporarily supply the nutrients studied. Dietary protein degradability in the rumen and intestines are feed characteristics determining the primary AA available to the host animal. They can be estimated using in situ, in vitro, or in vivo techniques with each having inherent advantages and disadvantages. Accurate, precise, and inexpensive laboratory assays for feed protein availability are still needed. Techniques used for direct determination of rumen microbial protein synthesis are laborious and expensive, and data variability can be unacceptably large; indirect approaches have not shown the level of accuracy required for widespread adoption. Techniques for studying postruminal digestion and absorption of nitrogenous compounds, urea recycling, and mammary AA metabolism are also laborious, expensive (especially the methods that use isotopes), and results can be variable, especially the methods based on measurements of digesta or blood flow. Volatile loss of N from feces and particularly urine can be substantial during collection, processing, and analysis of excreta, compromising the accuracy of measurements of total-tract N digestion and body N balance. In studying ruminant N metabolism, nutritionists should consider the longer term fate of manure N as well. Various techniques used to determine the effects of animal nutrition on total N, ammonia- or nitrous oxide-emitting potentials, as well as plant fertilizer value, of manure are available. Overall, methods to study ruminant N metabolism have been developed over 150 yr of animal nutrition research, but many of them are laborious and impractical for application on a large number of animals. The increasing environmental concerns associated with livestock production systems necessitate more accurate and reliable methods to determine manure N emissions in the context of feed composition and ruminant N metabolism.

Symposium review: Uncertainties in enteric methane inventories, measurement techniques, and prediction models.

Ruminant production systems are important contributors to anthropogenic methane (CH4) emissions, but there are large uncertainties in national and global livestock CH4 inventories. Sources of uncertainty in enteric CH4 emissions include animal inventories, feed dry matter intake (DMI), ingredient and chemical composition of the diets, and CH4 emission factors. There is also significant uncertainty associated with enteric CH4 measurements. The most widely used techniques are respiration chambers, the sulfur hexafluoride (SF6) tracer technique, and the automated head-chamber system (GreenFeed; C-Lock Inc., Rapid City, SD). All 3 methods have been successfully used in a large number of experiments with dairy or beef cattle in various environmental conditions, although studies that compare techniques have reported inconsistent results. Although different types of models have been developed to predict enteric CH4 emissions, relatively simple empirical (statistical) models have been commonly used for inventory purposes because of their broad applicability and ease of use compared with more detailed empirical and process-based mechanistic models. However, extant empirical models used to predict enteric CH4 emissions suffer from narrow spatial focus, limited observations, and limitations of the statistical technique used. Therefore, prediction models must be developed from robust data sets that can only be generated through collaboration of scientists across the world. To achieve high prediction accuracy, these data sets should encompass a wide range of diets and production systems within regions and globally. Overall, enteric CH4 prediction models are based on various animal or feed characteristic inputs but are dominated by DMI in one form or another. As a result, accurate prediction of DMI is essential for accurate prediction of livestock CH4 emissions. Analysis of a large data set of individual dairy cattle data showed that simplified enteric CH4 prediction models based on DMI alone or DMI and limited feed- or animal-related inputs can predict average CH4 emission with a similar accuracy to more complex empirical models. These simplified models can be reliably used for emission inventory purposes.

Kinetics of solutes and particles of different size in the digestive tract of cattle of 0.5-10 years of age, and relationships with methane production.

The digestive physiology of cattle is characterised by comparatively long digesta mean retention times (MRTs), a particle sorting mechanism (difference in MRTs of large vs. small particles) and a distinct digesta washing (difference in MRTs between particles and fluids) in the reticulorumen (RR). How these processes mature during ontogeny, and how they link to other digestion characteristics and methane production, is largely unknown. We used a set of passage markers (Co-EDTA for fluids and hay particles of 2, 5 and 8 mm length mordanted with Cr, La and Ce, respectively) to measure MRTs in 12 heifers (0.5-2.1 years; hay only) and two groups of 15 lactating cows (2.4-10.0 years; forage-only vs. forage-concentrate diet). The MRTs differed between markers (Co < Cr < La < Ce) and were longer in heifers than cows, consistent with the lower feed intake in heifers. MRTs were mostly similar between cow groups and increased with age. Digesta washing was not affected by group, age, feed intake and number of chews per unit of feed. The degree of digesta washing was not related to CH4 measures. Particle sorting was more prominent in cows than heifers but did not differ between cow groups or change with age in cows. This could be the consequence of the abrupt increase in intake from heifers to cows at a time when gut capacity is not yet fully developed; particle sorting might then clear smaller particles from the RR sooner allowing a higher intake. Surprisingly, CH4 yield per ingested feed did not correlate with MRTs, and CH4 yield per unit of digested fibre decreased with increasing MRTs and with increasing fibre digestibility. As this pattern occurred in heifers and both cow groups, it appeared independent of age, indicating a mechanism that has not been described in the literature so far and requires further investigation.

Biological implications of longevity in dairy cows: 2. Changes in methane emissions and efficiency with age.

Previous studies indicated that absolute CH4 emissions and CH4 yield might increase and that milk production efficiency might decrease with age in cattle. Both would make strategies to increase longevity in dairy cattle less attractive. These aspects were experimentally determined in Brown Swiss cattle distributed continuously across a large age range. Thirty lactating dairy cows (876-3,648 d of age) received diets consisting of hay, corn silage, and grass pellets supplemented with 0 or 5kg of concentrate per day. Twelve heifers (199-778 d of age) received hay only. Cows and heifers were members of herds subjected to the 2 different feeding regimens (with or without concentrate) for the past 10 yr. Methane emissions were measured individually for 2 d in open-circuit respiration chambers, followed by quantifying individual feed intake and milk yield over 8 d. Additional data on digestibility, rumination time, and passage time of feed of all experimental animals were available. Regression analyses were applied to evaluate effects of age and feeding regimen. Body weight, milk yield, and the hay proportion of forage dry matter intake were considered as covariates. Methane emissions per unit of intake, body weight, and milk yield were significantly related to age. Their development in the cows with age was characterized by an increase to maximum at around 2,000 d of age, followed by a decline. This response was not accompanied by corresponding age-related changes in intake, chewing activity, digesta passage time, and digestibility of organic matter, which would have explained shifts in CH4. However, fiber digestibility showed a similar change with age as methane emissions, resulting in quite stable methane emissions per unit of digestible fiber. As expected, methane emissions intensity per unit of milk produced was greater by 8% without concentrate than with concentrate, but no difference was noted in the response to age when the animals were subjected to different feeding regimens. The efficiency of milk production was only marginally influenced by age and diet, and no different response was observed for age in the 2 dietary regimens. In conclusion, life cycle analyses of milk production systems focusing on longevity should consider changing methane yields with age in addition to the variation in environmental costs for replacements of culled cows.

Biological implications of longevity in dairy cows: 1. Changes in feed intake, feeding behavior, and digestion with age.

Milk production strategies focusing on longevity and limited use of concentrate are receiving increasing attention. To evaluate such strategies, knowledge of the development with age of animal characteristics, particularly digestion, is indispensable. We therefore investigated the development of feed intake, chewing activity, and digestion in 30 lactating Brown Swiss cows (876-3,648 d old) and 12 heifers (199-778 d old). We also studied whether age effects were exhibited differently in animals selected from herds subjected for 11 yr either to a forage-only or to a forage-concentrate feeding regimen. Forages consisted of grass hay (the only feed for heifers), corn silage, and grass pellets. Measurements lasted for 8 d, where amounts and composition of feeds, feces, and milk were recorded and analyzed. Ruminal pH data and eating and rumination activity were assessed by pH sensors put into the rumen and halter-mounted noseband sensors. The mean retention time of feed particles was assessed using Cr-mordanted fiber and data were used to calculate dry matter gut fill. Data were subjected to regression analyses with age and feeding regimen as explanatory variables, and body weight, milk yield, and proportion of hay in forage as covariates. This allowed separating age-related changes of body weight and milk yield from independent age effects and correcting for differences in preference for individual forages. In cows, organic matter intake increased with age (from slightly below to above 20kg/d), as did mean retention time and gut fill. Digestibility of organic matter did not show a clear age dependency, but fiber digestibility had a maximum in cows of around 4 to 6 yr of age. Ruminal pH and absolute eating and rumination times did not vary with cow age. Young and old cows chewed regurgitated boluses more intensively (60-70 times) than middle-aged cows (about 50 times). Effects of feeding regimen were small, except for fiber intake and rumination time per unit of intake, owing to the different fiber content of the diets. No significant interactions between age and feeding regimen were found. Heifers spent more time eating and ruminating per unit of feed than cows, which resulted in a high fiber digestibility. Irrespective of the feeding regimen tested, older cows maintained intake and digestion efficiency with longer retention times and chewing rumination boluses more intensively. The results support efforts to extend the length of productive life in dairy cows.

Influence of pH and the degree of protonation on the inhibitory effect of fatty acids in the ruminal methanogen Methanobrevibacter ruminantium strain M1.

AIMS: To investigate the relationship between the protonation of medium-chain fatty acids (MCFA) and their inhibitory effect on a ruminal methanogen species. METHODS AND RESULTS: Cell suspensions of Methanobrevibacter ruminantium M1 in 1 mg dry matter (DM) ml(-1) were supplemented with lauric acid (C12 ) and myristic acid (C14 ) at a concentration of 8 µg ml(-1) with different pH levels of the potassium-free buffer, where the calculated degrees of protonation of C12 and C14 varied from 0·3 to 50% and from 1 to 76% respectively. Methane formation, ATP efflux, potassium leakage and cell viability were monitored 15, 30 and 45 min after the reaction started. Declining methane formation rate, increasing ATP efflux and potassium leakage, and decreasing survival of M. ruminantium were observed with increasing degrees of protonation, i.e. with decreasing pH. CONCLUSIONS: The inhibition of methanogenesis by C12 and C14 is more efficient at a pH of 5-6 as compared to pH 7. SIGNIFICANCE AND IMPACT OF THE STUDY: Methane mitigation strategies in ruminants which use supplementation of feed with MCFA such as C12 and C14 may be more effective in a low rumen pH environment. This finding is helpful in designing diets to effectively decrease methane emissions by ruminants.

Fuel feeds function: Energy balance and bovine peripheral blood mononuclear cell activation.

A general phenomenon in peripartum mammals is the breakdown of (acquired) immunity. The incidence of parasite load, disease and inflammation often rise during the specific energetically demanding time of pregnancy and lactation. In this period, blood leukocytes display decreased DNA synthesis in response to mitogens in vitro. Leukocyte activation, the phase of the cell cycle preceding the DNA synthetic phase has hardly been investigated, but the few studies suggest that leukocyte activation may also be impaired by the limited energy/nutrient availability. Leukocyte activation is characterized by manifold processes, thus, we used the cellular oxygen consumption rate (OCR) as a measure of ATP turnover to support all these processes. We hypothesized that the activation of peripheral blood mononuclear cells (PBMC) - in terms of oxygen consumed over basal levels after in vitro stimulation - is altered by energy balance around parturition. We studied peripartum high-yielding dairy cows because they undergo substantial fluctuations in energy intake, energy output and body fat mass. We established a fluorescence-based test strategy allowing for long-term (≥24h) quantification of O(2)-consumption and studied the peripartum period from 5 weeks ante partum to 5 weeks postpartum. In addition, we determined cellular lactate production, DNA/RNA synthesis and cell size and zoo-technical parameters such as animal energy intake and milk yield were assessed, as well as selected plasma parameters, e.g. glucose concentration. The basal OCR of PBMC from pregnant, non-lactating cows (n=6, -5 weeks ante partum) was 1.19±0.15 nmol min(-1) (10(7)cells)(-1) and increased to maximum levels of 2.54±0.49 nmol min(-1) (10(7)cells)(-1) in phytohemagglutinin (PHA)-stimulated PBMC. The basal OCR did not change over the peripartum period. Whereas the activation indices, herein defined as the PHA-induced 24h-increase of OCR above baseline, amounted to 1.1±0.3, 4.2±0.3, 4.1±1.1, 2.1±0.3, and 2.7±0.5 at weeks -5, -1, +1, +2, and +5 relative to parturition, respectively. Because the activation index was positively correlated to plasma glucose levels and to energy balance during late pregnancy (week -5/week -1) and transition to lactation (week -1/week +2), we conclude that PBMC activation is modulated by energy/nutrient availability. In future studies, the activation index should aid the identification of causal mechanisms of disparity in PBMC activation, such as attenuated ion transport or macromolecule synthesis.

Short-term feed intake is regulated by macronutrient oxidation in lactating Holstein cows.

In addition to plasma metabolites and hormones participating as humoral signals in the control of feed intake, oxidative metabolic processes in peripheral organs also generate signals to terminate feeding. Although the degree of oxidation over longer periods is relatively constant, recent work suggests that the periprandial pattern of fuel oxidation is involved in regulating feeding behavior in the bovine. However, the association between periprandial oxidative metabolism and feed intake of dairy cows has not yet been studied. Therefore, the aim of this study was to elucidate possible associations existing between single feed intake events and whole-body net fat and net carbohydrate oxidation as well as their relation to plasma metabolite concentrations. To this end, 4 late-lactating cows equipped with jugular catheters were kept in respiratory chambers with continuous and simultaneous recording of gas exchange and feed intake. Animals were fed ad libitum (AL) for 24h and then feed restricted (RE) to 50% of the previous AL intake for a further 24h. Blood samples were collected hourly to analyze ß-hydroxybutyrate (BHBA), glucose, nonesterified fatty acids (NEFA), insulin, and acylated ghrelin concentrations. Cross-correlation analysis revealed an offset ranging between 30 and 42 min between the maximum of a feed intake event and the lowest level of postprandial net fat oxidation (FOX(net)) and the maximum level of postprandial net carbohydrate oxidation (COX(net)), respectively. During the AL period, FOX(net) did not increase above -0.2g/min, whereas COX(net) did not decrease below 6g/min before the start of the next feed intake event. A strong inverse cross-correlation was obtained between COX(net) and plasma glucose concentration. Direct cross-correlations were observed between COXnet and insulin, between heat production and BHBA, between insulin and glucose, and between BHBA and ghrelin. We found no cross-correlation between FOX(net) and NEFA. During RE, FOX(net) increased with an exponential slope, exceeded the threshold of -0.2g/min as indicated by increasing plasma NEFA concentrations, and approached a maximum rate of 0.1g/min, whereas COX(net) decayed in an exponential manner, approaching a minimal COX(net) rate of about 2.5 g/min in all cows. Our novel findings suggest that, in late-lactating cows, postprandial increases in metabolic oxidative processes seem to signal suppression of feed intake, whereas preprandially an accelerated FOX(net) rate and a decelerated COX(net) rate initiate feed intake.

Dose-response effects of woody and herbaceous forage plants on in vitro ruminal methane and ammonia formation, and their short-term palatability in lactating cows.

Plant secondary compounds (PSC) are prevalent in many woody, temperate-climate plant species and play a crucial role in dietary attempts to mitigate methane emissions in ruminants. However, their application requires sufficient palatability and feeding value. In the present study, leaves from silver birch (Betula pendula), hazel (Corylus avellana), blackcurrant (Ribes nigrum), green grape vine (Vitis vinifera) and the herbs rosebay willow (Epilobium angustifolium) and wood avens (Geum urbanum) were tested in various doses with the Hohenheim gas test method in vitro and their short-term palatability in dairy cows. For the palatability experiment, the plants were pelleted with lucerne in different proportions to obtain the same phenol content, but realised contents differed from expected contents. The pellets were provided separately from a mixed basal ration (0.4 : 0.6) to each cow, in a randomised order, for 3 days per plant. All plants mitigated in vitro methane and ammonia formation, often in a linear dose response. These levels of effects differed among plants. Significant effects were observed at 100 (hazel, rosebay willow) to 400 g/kg of plant material. The test plants had a lower feeding value than the high-quality basal diet. This was indicated by in vitro organic matter digestibility, short-chain fatty acid formation and calculated contents of net energy of lactation. Simultaneously, the linear depression of ammonia formation indicated a dose-dependent increase of utilisable CP. Only blackcurrant and birch were less preferred to lucerne. However, this aversion subsided on day 3 of offer. The rosebay willow pellets had the highest phenol content but were not the least palatable. Accordingly, PSC may not be the main determinants of palatability for the plants tested. Plants did not differ significantly in their short-term effects on milk yield and composition, and all of the plants substantially reduced milk urea content. Overall, the results suggest that hazel and vine leaves, and rosebay willow and wood avens herbs should be tested for their potential to mitigate methane and N emissions in vivo.

Review: Comparative methane production in mammalian herbivores.

Methane (CH4) production is a ubiquitous, apparently unavoidable side effect of fermentative fibre digestion by symbiotic microbiota in mammalian herbivores. Here, a data compilation is presented of in vivo CH4 measurements in individuals of 37 mammalian herbivore species fed forage-only diets, from the literature and from hitherto unpublished measurements. In contrast to previous claims, absolute CH4 emissions scaled linearly to DM intake, and CH4 yields (per DM or gross energy intake) did not vary significantly with body mass. CH4 physiology hence cannot be construed to represent an intrinsic ruminant or herbivore body size limitation. The dataset does not support traditional dichotomies of CH4 emission intensity between ruminants and nonruminants, or between foregut and hindgut fermenters. Several rodent hindgut fermenters and nonruminant foregut fermenters emit CH4 of a magnitude as high as ruminants of similar size, intake level, digesta retention or gut capacity. By contrast, equids, macropods (kangaroos) and rabbits produce few CH4 and have low CH4 : CO2 ratios for their size, intake level, digesta retention or gut capacity, ruling out these factors as explanation for interspecific variation. These findings lead to the conclusion that still unidentified host-specific factors other than digesta retention characteristics, or the presence of rumination or a foregut, influence CH4 production. Measurements of CH4 yield per digested fibre indicate that the amount of CH4 produced during fibre digestion varies not only across but also within species, possibly pointing towards variation in microbiota functionality. Recent findings on the genetic control of microbiome composition, including methanogens, raise the question about the benefits methanogens provide for many (but apparently not to the same extent for all) species, which possibly prevented the evolution of the hosting of low-methanogenic microbiota across mammals.

No easy solution for the fractionation of faecal nitrogen in captive wild herbivores: results of a pilot study.

Faecal nitrogen (FN) concentration is used as a marker for habitat quality and digestive efficiency in free-ranging herbivores. In herbivores, FN can be separated into undigested plant N (analysed as the N concentration of the neutral detergent residue) and metabolic faecal N (MFN). It has been suggested that by differential analysis of the faecal fibre-bound N, the MFN fraction can be further split into a bacterial N and an endogenous N fraction [Hesta et al., Br. J. Nutr. 90 (2003) 1007]. We applied these methods to 96 faecal samples of 48 mammalian herbivore species from zoos. Species were grouped into coprophageous and non-coprophageous hindgut fermenters and ruminating and non-ruminating foregut fermenters. Diet was not controlled. The FN decreased with body mass, possibly reflecting higher proportions of concentrates in diets of smaller animals. The proportion of MFN increased with FN, indicating that higher quality food might enhance the gastrointestinal bacterial flora. The only outlier to this pattern was the lesser panda (Ailurus fulgens), confirming the low relevance of fermentative digestion in this herbivorous 'carnivore'. No relevant differences between the four digestion types were noted. The proportion of endogenous faecal N (32-80% of FN) was always higher than that of bacterial faecal N (7-30%), which contradicts basal understanding of herbivore digestive physiology. Thus, the method of Hesta et al. (2003) does not appear applicable to herbivores. While the results do not exclude the possibility that detailed differences might occur between digestion types, they indicate a high degree of similarity between herbivores that rely on bacterial fermentation, regardless of their digestion type, with respect to metabolic faecal losses.

Evidence for increasing digestive and metabolic efficiency of energy utilization with age of dairy cattle as determined in two feeding regimes.

The changes taking place with age in energy turnover of dairy cattle are largely unknown. It is unclear whether the efficiency of energy utilization in digestion (characterized by faecal and methane energy losses) and in metabolism (characterized by urine and heat energy losses) is altered with age. In the present study, energy balance data were obtained from 30 lactating Brown Swiss dairy cows aged between 2 and 10 years, and 12 heifers from 0.5 to 2 years of age. In order to evaluate a possible dependence of age effects on diet type, half of the cattle each originated from two herds kept at the same farm, which were fed either on a forage-only diet or on the same forage diet but complemented with 5 kg/day of concentrate since their first calving. During 2 days, the gaseous exchange of the animals was quantified in open-circuit respiration chambers, followed by an 8-day period of feed, faeces, urine and milk collection. Daily amounts and energy contents were used to calculate complete energy balances. Age and feeding regime effects were analysed by parametric regression analysis where BW, milk yield and hay proportion in forage as consumed were considered as covariates. Relative to intake of gross energy, the availability of metabolizable energy (ME) increased with age. This was not the result of an increasing energy digestibility, but of proportionately lower energy losses with methane (following a curvilinear relationship with the greatest losses in middle-aged cows) and urine (continuously declining). The efficiency of utilization of ME for milk production (k l) increased with age. Potential reasons include an increase in the propionate-to-acetate ratio in the rumen because of a shift away from fibre degradation and methane formation as well as lower urine energy losses. The greater k l allowed older cows to accrete more energy reserves in the body. As expected, offering concentrate enhanced digestibility, metabolizability and metabolic utilization of energy. Age and feeding regime did not interact significantly. In conclusion, older cows seem to have digestive and metabolic strategies to use dietary energy to a certain degree more efficiently than younger cows.

Digestion studies in captive Hippopotamidae: a group of large ungulates with an unusually low metabolic rate.

We performed intake and digestibility studies in four common (Hippopotamus amphibius) and four pygmy (Hexaprotodon liberiensis) hippos from two zoological institutions, using acid detergent lignin as an internal marker for the quantification of faecal output. In the case of one pygmy hippo, where total faecal collection was also possible, there was no distinct difference between the two methods of faecal output quantification. Two animals from each species were tested on a conventional zoo diet of hay and concentrates (diet HC) and on hay only (diet H). The other two animals received fresh grass at two different levels of intake (diets G1 and G2). Dry matter (DM) intake was higher on HC than on H or G diets, and averaged 37 +/- 11 for common and 35 +/- 14 g/kg(0.75) for pygmy hippos. There were no species differences in the average digestibility (aD) coefficients. Non-dietary faecal nitrogen averaged 65 +/- 4% of total faecal nitrogen, aD of crude protein (CP) averaged 67 +/- 9% and true protein digestibility 89 +/- 3%. Average digestibility of DM and crude fibre averaged 54 +/- 11% and 45 +/- 17%, respectively. In comparison with ruminants, hippos generally achieve lower aD for DM, organic matter and fibre parameters, but equal or higher aD CP coefficients. This is most likely due to the absence of significant fermentative activity in the hindgut and the corresponding low metabolic faecal nitrogen losses. Digestible energy intake was higher on HC than on H or G diets and averaged 0.30 +/- 0.11 MJ/kg(0.75) metabolic body mass. This value is extremely low for ungulates, supporting earlier suspicions that hippos have particularly low metabolic rates, and explains the proneness of this species to obesity in captivity when fed energy-dense pelleted feeds.

Intake, ingesta retention, particle size distribution and digestibility in the hippopotamidae.

Although several aspects of the digestive physiology of the hippopotamidae-non-ruminating foregut fermenters-have been described, ingesta kinetics and passage characteristics of these species are not well understood. The most outstanding feature of the hippo digestive physiology reported so far is the very long mean ingesta retention times (MRTs) measured by Foose [Foose, T., 1982. Trophic strategies of ruminant versus nonruminant ungulates. PhD dissertation, University of Chicago, Chicago.]. Since those data had been investigated with animals without water access, we intended to measure MRT in hippos which were allowed to enter water pools during the night. MRT parameters as well as dry matter (DM) digestibility were determined in four common (Hippopotamus amphibius) and four pygmy hippos (Hexaprotodon liberiensis) on two different diets each using cobalt ethylendiamintetraacetate (Co-EDTA) as a fluid, chromium (Cr)-mordanted fibre (<2 mm) as a particle and acid detergent lignin (ADL) as an internal digestibility marker. Four of the animals additionally received cerium (Ce)-mordanted fibres (2-10 mm) as particle markers. Total MRTs for fluids and particles ranged between 20-35 and 48-106 h in the common and between 13-39 and 32-107 h in the pygmy hippos. The difference between fluid and particle retention was greater than usually reported in ruminants. Excretion patterns of the markers differed from those usually observed in ruminants but resembled those reported for macropods (kangaroos), indicating a plug-flow reactor-like physiology in the hippo forestomach (FRST). This finding complements other described similarities between the macropod and the hippo forestomach. The measurements of larger particle retention profiles suggest that in the hippo, larger particles might be excreted either faster or at the same rate as smaller particles, indicating a general difference between ruminants and hippos with respect to differential particle retention. The digestive physiology of hippos is characterised by a generally low food intake, long ingesta retention times and dry matter digestibilities lower than reported in ruminants. Moderate digestibilities in spite of long retention times might be the result of the generally high average ingesta particle size in hippos. The comparatively easy management of pygmy hippos, together with the significant correlations between food intake, MRT and digestibility in the pygmy hippos of this study, recommends this species for further studies on the interplay of these parameters in herbivore digestive physiology.