Michael Derno

Methane production in cattle calculated by the nutrient composition of the diet.

Abstract In this study data originating from complete metabolic trials with cattle of both sexes, fed 337 rations at feed intake levels between one to three times maintenance energy requirement were used to regress the total CH4 emission to the level of DM intake and to the nutrient composition, respectively. A major component of the measured CH4 emission cannot be explained by DM intake but is rather due to differences in dietary nutrient composition. The amount of digestible nutrients especially of the carbohydrate fraction (starch, sugar, N-free residuals) are reliable to estimate CH4 release with high precision (r 2 = 0.885). Its production rate increased to 1.9-fold higher levels (range 1.8 – 2.1) per g of N-free residuals compared to that induced per g of protein, starch or sugar, respectively. Furthermore, diets rich in fat reduced CH4 formation in the rumen. The regression equations fit a wide range of diets and diet compositions, and more importantly, they are applicable to various types of production systems.

Calorimetric validation of 13 C bicarbonate and doubly labeled water method for determining the energy expenditure in goats

SummaryThe purpose of the present study was to validate the13C bicarbonate method (13C-M) and the doubly labeled water method (DLWM) for the estimation of the CO2 production R(CO2) in goats as a ruminant model. Indirect calorimetry was chosen as the reference method. Studies were carried out in 2 male African dwarf goats at 3 different developing stages (age: 5, 10, and 14 months, body mass: 14.6, 20.3, and 21.7 kg). Animals were fed a balanced feed 14 days before and during the studies. The isotope tracers (4 mg/kg NaH13CO3, 120 mg/kg2H2O, and 75 mg/kg H218O; 99 At.-%) were simultaneously given as a single pulse injection into the jugular vein. Thereafter, the animals were kept for 8 days in two respiration chambers (volume of chamber: 2.85 m3, air flow rate: 25 1 / min) for the estimation of CO2 production and O2 consumption. For the determination of R(CO2) using the13C-M samples of exhaled breath were drawn from the respiration chambers. The13C enrichment and CO2 concentration of breath samples were measured by means of an infrared isotope analyzer. In order to determine R(CO2) by means of the DLWM, blood serum was used. The2H and18O enrichments were measured by an isotope ratio mass spectrometer. Urine samples were collected over 24 h to quantify renal water losses.The R(CO2) was calculated by means of the13C-M using the area under the13C enrichment-time curve. The determination of R(CO2) by means of the DLWM was based on the slopes of the2H and18O disappearance curves and the body water pool obtained from the zero time intercept of the isotope curves.The values of R(CO2) resulting from the13C-M were found to be comparable with those from the calorimetric measurement. Smaller (not statistically significant) values of R(CO2) - 92% from13C-M and 87% from DLWM - compared to the indirect calorimetry could indicate the incorporation of13C and2H into metabolites other than CO2 and H2O, respectively. The body water contents calculated from the zero time intercepts of the2H and18O disappearence curves amounted to 66% and 63%, respectively. The body water content was found to be not related to the age of animals. The renal water loss was calculated to be 35% of the total water loss (0.76 l/d).ZusammenfassungZweck der vorliegenden Untersuchung war die Validierung der13C-Bicarbonat-Methode (13C-M) und der doppeltmarkierten Wassermethode (DLWM) zur Bestimmung der CO2-Produktion R(CO2) bei Ziegen als Wiederkäuermodell. Die indirekte Kalorimetrie war als Referenzmethode ausgewählt worden. Die Untersuchungen wurden an 2 Afrikanischen Zwergziegen bei 3 unterschiedlichen Entwicklungsstadien (Alter: 5, 10 und 14 Monate, Körpermasse: 14,6, 20,3 und 21,7 kg) durchgeführt. Die Tiere wurden 14 Tage vor und während der Untersuchung bilanziert gefüttert. Die Isotopentracer (4 mg/kg NaH13CO3, 120 mg/kg2H2O and 75 mg/kg H218O; 99 At.-%) wurden simultan als einmalige impulsförmige Dosis in die Vena jugularis injiziert. Danach befanden sich die Tiere für 8 Tage in zwei Respirationskammern (Kammervolumen: 2,85 m3, Luftdurchsatz: 25 l/ min), um die CO2-Produktion und den O2-Verbrauch zu bestimmen. Für die Bestimmung von R(CO2) mit Hilfe der13C-M wurden Proben der Ausatemluft aus den Respirationskammern gezogen. Die Messung der13C-Anreicherung und der CO2-Konzentration der Atemproben erfolgte mittels eines Infrarot-Analysators. Für die Bestimmung von R(CO2) mittels der DLWM wurde Blutserum verwendet. Die2H- und18O-Anreicherungen wurden massenspektrometrisch gemessen. Urinproben wurden zur Quantifizierung des renalen Harnverlusts über 24h gesammelt. R(CO2) wurde mittcls der13C-M unter Verwendung der Fläche unter13C-Anreicherung-Zeit-Kurve berechnet. Die Bestimmung von R(CO2) mittels der DLWM basierte auf den Anstiegen der2H- und18O-Kurven und dem Körperwasser-Pool. Die Werte von R(CO2) aus der13C-M war vergleichbar mit denen aus kalorimetrischen Messungen. Die im Vergleich zur indirekten Kalorimetrie kleineren (nicht statistisch signifikant) Werte von R(CO2) — 92% aus13C-M und 87% aus DLWM — deuten auf die Inkorporation von13C und2H in andere Metaboliten als CO2 und H2O hin. Die Körperwasser-Pools (bezogen auf die Körpermasse), berechnet aus den Ordinatenabschnitten der2H- und18O-Kurven (bei t=0), betrugen 66% bzw. 63%. Die Körperwasser-Pools zeigten keine Altersabhängigkeit. Der renale Wasserverlust wurde zu 35% des totalen Wasserverlusts (0,76 1 / d) berechnet.

Technical note: A new facility for continuous respiration measurements in lactating cows

An open-circuit indirect calorimetry system consisting of 4 climate-controlled respiration chambers for cattle has been constructed and validated. The system allows for the continuous monitoring of O(2), CO(2), and CH(4) concentrations in chamber air, and the simultaneous determination of feed and water intake, overall physical activity, position changes, standing and lying times, and animal behavior. For complete balance trials, feces, urine, and milk can be collected quantitatively. Most importantly, lactating cows can be milked in the chamber, and blood samples can be drawn from permanent catheters without disruption of the measurements. The investigator, on entering the chamber, wears a facemask connected to the ambient air during the whole milking process. Data are routed to a data acquisition system with appropriate data evaluation software developed in our research unit. Thus, dynamic changes of the above-named parameters during the course of the day or of longer time periods can be monitored. Such data are critical for understanding the complex regulation and interplay of feed intake, energy metabolism, climatic conditions, and milk production.

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 β-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.

EFFECTS OF PROTOZOA ON METHANE PRODUCTION IN RUMEN AND HINDGUT OF CALVES AROUND TIME OF WEANING

Effects of the presence or absence of ciliate protozoa on methanogenesis in the rumen and hindgut were investigated in young calves during a 7-week period. Ten Holstein calves, aged 7 days, were divided in two groups (n = 5) and fed an increasing amount of a commercial milk replacer and small amounts of a calves starter. One group was inoculated with ciliate fauna on two occasions, week 5 and 6, while the second remained ciliate-free. The absence of protozoa in the rumen decreased rumen empty weight ( − 23%, P < 0.01), and rumen pool size of N ( − 36%, P < 0.01) and crude fat ( − 37%, P < 0.05). Rumen bacteria of non-faunated calves contained a higher proportion of total amino acid-N per 16 g N ( + 3%, P < 0.01) and D-alanine-N per 16 g N ( + 13%, P < 0.05) compared to faunated calves. Further results contain a reference for a higher bacterial mass in the ciliate-free rumen with an increased number of bacteria adherent to rumen mucosa. The CH4 production in the rumen increased exponentially with the increase in protozoa population size (R2 = 0.68). In presence of 46 · 104 protozoa per ml rumen fluid, the in vitro CH4 production of rumen fluid per mol total VFA was about 34% higher in faunated than in non-faunated calves (P < 0.001). Hydrogen (2H) recovery of rumen fermentation was positively correlated (R2 = 0.55) to the CH4 production rate. Methanogens were attached on rumen mucosa. Methanogenesis, induced by rumen mucosa attached bacteria, was stimulated by ruminal protozoa. In the absence of protozoa in the rumen, the acetate - propionate ratio and butyrate proportion of VFA were reduced. In vivo in the absence of protozoa not only the whole animal CH4 production ( − 30%, P < 0.05) but also the digestibility of carbohydrates ( − 4%, P < 0.05) was reduced. Thereby no difference was observed in the intake of ME per kg DM between the groups. In conclusion, the methanogenesis in the rumen, but not in hindgut, is associated with the development of the ruminal protozoa population. The level of methanogenesis (mol/mol VFA) in the hindgut amounts to 20% of the ruminal methanogenesis.

Metabolic Heat Stress Adaption in Transition Cows: Differences in Macronutrient Oxidation between Late-Gestating and Early-Lactating German Holstein Dairy Cows

High ambient temperatures have severe adverse effects on biological functions of high-yielding dairy cows. The metabolic adaption to heat stress was examined in 14 German Holsteins transition cows assigned to two groups, one heat-stressed (HS) and one pair-fed (PF) at the level of HS. After 6 days of thermoneutrality and ad libitum feeding (P1), cows were challenged for 6 days (P2) by heat stress (temperature humidity index (THI) = 76) or thermoneutral pair-feeding in climatic chambers 3 weeks ante partum and again 3 weeks post-partum. On the sixth day of each period P1 or P2, oxidative metabolism was analyzed for 24 hours in open circuit respiration chambers. Water and feed intake, vital parameters and milk yield were recorded. Daily blood samples were analyzed for glucose, β-hydroxybutyric acid, non-esterified fatty acids, urea, creatinine, methyl histidine, adrenaline and noradrenaline. In general, heat stress caused marked effects on water homeorhesis with impairments of renal function and a strong adrenergic response accompanied with a prevalence of carbohydrate oxidation over fat catabolism. Heat-stressed cows extensively degraded tissue protein as reflected by the increase of plasma urea, creatinine and methyl histidine concentrations. However, the acute metabolic heat stress response in dry cows differed from early-lactating cows as the prepartal adipose tissue was not refractory to lipolytic, adrenergic stimuli, and the rate of amino acid oxidation was lower than in the postpartal stage. Together with the lower endogenous metabolic heat load, metabolic adaption in dry cows is indicative for a higher heat tolerance and the prioritization of the nutritional requirements of the fast-growing near-term fetus. These findings indicate that the development of future nutritional strategies for attenuating impairments of health and performance due to ambient heat requires the consideration of the physiological stage of dairy cows.

Plasma ghrelin is positively associated with body fat, liver fat and milk fat content but not with feed intake of dairy cows after parturition.

Ghrelin is a gastrointestinal peptide hormone that is present in blood mostly in a non-posttranslationally modified form, with a minor proportion acylated at Ser(3). Both ghrelin forms were initially assigned a role in the control of food intake but there is accumulating evidence for their involvement in fat allocation and utilization. We investigated changes in the ghrelin system in dairy cows, exhibiting differences in body fat mobilization and fatty liver, from late pregnancy to early lactation. Sixteen dairy cows underwent liver biopsy and were retrospectively grouped based on high (H) or low (L) liver fat content post-partum. Both groups had a comparable feed intake in week -6 (before parturition) and week 2 (after parturition). Only before parturition was preprandial total ghrelin concentration higher in L than in H cows and only after parturition was the basal plasma concentration of non-esterified fatty acids higher in H than in L cows. Both before and after parturition, H cows had higher preprandial plasma concentrations of acyl ghrelin, a higher acyl:total ghrelin ratio, lower plasma triacylglyceride concentrations and a lower respiratory quotient compared with L cows. These group differences could not be attributed to an allelic variant of the acyl ghrelin receptor. Rather, the ratio of acyl:total ghrelin correlated with several aspects of fat metabolism and with respiratory quotient but not with feed intake. These results show that endogenous ghrelin forms are associated with fat allocation, fatty liver, and utilization of fat during the periparturient period.

Effects of a 6-wk intraduodenal supplementation with quercetin on energy metabolism and indicators of liver damage in periparturient dairy cows

Periparturient dairy cows experience metabolic challenges that result in a negative energy balance (EB) and a range of postpartum health problems. To compensate for the negative EB, cows mobilize fatty acids from adipose tissues, which can lead to fatty liver disease, a periparturient metabolic disorder. Flavonoids, such as quercetin (Q), are polyphenolic substances found in all higher plants and have hepatoprotective potential and the ability to prevent or reduce lipid accumulation in the liver. In ruminants, few studies on the metabolic effects of Q are available, and thus this study was conducted to determine whether Q has beneficial effects on EB, lipid metabolism, and hepatoprotective effects in periparturient dairy cows. Quercetin was supplemented intraduodenally to circumvent Q degradation in the rumen. Cows (n=10) with duodenal fistulas were monitored for 7wk. Beginning 3wk before expected calving, 5 cows were treated with 100mg of quercetin dihydrate per kilogram of body weight daily in a 0.9% sodium chloride solution for a total period of 6wk, whereas the control cows received only the sodium chloride solution. The plasma flavonoid levels were higher in the Q-treated cows than in the control cows. A tendency for higher postpartum (pp) than antepartum (ap) plasma flavonoid levels was observed in the Q-treated cows than in the controls, which was potentially caused by a reduced capacity to metabolize Q. However, the metabolic status of the Q-treated cows did not differ from that of the control cows. The pp increases in plasma aspartate aminotransferase and glutamate dehydrogenase activities were less in the Q-treated cows than in the control cows. The Q had no effect on energy expenditures, but from ap to pp the cows had a slight decline in respiratory quotients. Irrespective of the treatment group, the oxidation of fat peaked after calving, suggesting that the increase occurred because of an increased supply of fatty acids from lipomobilization. In conclusion, supplementation with Q resulted in lower pp plasma aminotransferase and glutamate dehydrogenase, which indicated reduced liver damage. However, the direct effects of Q on the liver and the implications for animal performance remain to be investigated.

Reduced AgRP activation in the hypothalamus of cows with high extent of fat mobilization after parturition

Agouti-related protein (AgRP), produced by neurons located in the arcuate nucleus of the hypothalamus stimulates feed intake. During early lactation dairy cows increase their feed intake and additionally mobilize their fat reserves leading to increased plasma non-esterified fatty acid (NEFA) concentrations. Since cows with a higher extent of fat mobilization exhibit the lower feed intake, it seems that high NEFA concentrations confine hyperphagia. To test the involvement of AgRP neurons, we investigated 18 cows from parturition until day 40 postpartum (pp) and assigned the cows according to their NEFA concentration on day 40pp to either group H (high NEFA) or L (low NEFA). Both groups had comparable feed intake, body weight, milk yield, energy balance, plasma amino acids and leptin concentrations. Studies in respiratory chambers revealed the higher oxygen consumption and the lower respiratory quotient (RQ) in H compared to L cows. mRNA abundance of neuropeptide Y, peroxisome proliferator-activated receptor-gamma, AMP-activated protein kinase, and leptin receptor in the arcuate nucleus were comparable between groups. Immunohistochemical studies revealed the same number of AgRP neurons in H and L cows. AgRP neurons were co-localized with phosphorylated adenosine monophosphate-activated kinase without any differences between groups. The percentage of cFOS-activated AgRP neurons per total AgRP cells was lower in H cows and correlated negatively with oxygen consumption and NEFA, positively with RQ, but not with feed intake. We conclude that AgRP activation plays a pivotal role in the regulation of substrate utilization and metabolic rate in high NEFA dairy cows during early lactation.

Methane emission, digestive characteristics and faecal archaeol in heifers fed diets based on silage from brown midrib maize as compared to conventional maize.

The aim of the present experiment was to compare silage prepared from maize having a brown midrib (BMR) mutation with control (CTR) maize to identify their effects on enteric methane emission, digesta mean retention time (MRT), ruminal fermentation and digestibility. In addition, the utility of archaeol present in faecal samples was validated as a proxy for methane production. Seven German Holstein heifers were fed total mixed rations with a maize-silage proportion (either BMR or CTR) of 920 g/kg dry matter (DM) in a change-over design. Heifers were fed boluses with markers to measure MRT; faeces were collected for 7 days and rumen fluid was collected on the penultimate day. Methane emission was measured in respiration chambers on one day. Data were analysed by t-test and regression analysis. DM intake did not differ between the two diets. The apparent digestibility of DM and most nutrients was unaffected by diet type, but apparent digestibility of neutral and acid detergent-fibre was higher in those heifers fed BMR than in those fed CTR. Comparisons between diets revealed no difference in particle or solute MRT in the gastro-intestinal tract and the reticulorumen. Concentrations of short-chain fatty acid and ammonia in rumen fluid and its pH were not affected by silage type. Independent of the mode of expression [l/d, l/kg DM intake, l/kg digested organic matter], methane emissions were not affected by maize-silage type, but with BMR, there was a trend towards lower methane production per unit of digested neutral detergent fibre than there was with CTR silage. Results of the present study show that feeding heifers BMR silage does not increase methane emissions despite a higher fibre digestibility as compared to CTR silage. Therefore, it is assumed that improvements in animal productivity achieved by feeding BMR silage, as some studies have reported, can be obtained without extra environmental cost per unit of milk or meat. Neither faecal archaeol content [µg/g] nor daily amount excreted [mg/d] is suitable to predict methane production in absolute terms [l per day]. However, faecal archaeol content has a certain potential for predicting the methane yield [l per kg DM intake] of individual animals.