Effects of full-time v. part-time grazing on seasonal changes in milk coagulation properties and fatty acid composition.

For this research communication our objective was to investigate to what extent milk coagulation properties and milk fatty acid (FA) composition were affected by different feeding systems, season and their interaction. Eighteen cows in total were subjected to one of three different feeding system treatments: full-time grazing or part-time grazing combined with indoor feeding of fresh grass with low or high concentrate supplementation. Milk was sampled in spring, summer and autumn. Milk coagulation time was 15.0, 19.0 and 17.7 min, coagulation dynamics 1.67, 3.41 and 1.79 min, and curd firmness 52.7, 32.4 and 47.0 mm in spring, summer and autumn, respectively. Thus, milk coagulation properties of the milk were lower during summer. There were strong seasonal effects on milk FA proportions, but there were not always changes with progressing season, or changes were different with respect to the impact of the feeding systems (system × season interaction). The milk fat was favourably rich in oleic acid, conjugated linoleic acid and α-linolenic acid and had a low n-6/n-3 fatty acid ratio in all systems. Factors like seasonal variations in grass composition and the energy balance of the cows were considered relevant for the milk FA composition. Overall, seasonal variations in milk quality were less pronounced with part-time grazing with fresh grass indoors as compared to full-time grazing without concentrate.

Grazing Allometry: Anatomy, Movement, and Foraging Behavior of Three Cattle Breeds of Different Productivity.

Modern breeding has formed a multitude of cattle breeds ranging from undemanding, low-productive breeds to high-productive, specialized dairy, or beef cattle. The choice of breed has important implications for farm management, but its impact on pasture vegetation is underestimated. We hypothesized (i) that anatomy, movement, and foraging behavior of cattle are allometrically related on the individual level, (ii) that differences among cattle are not explained by individual variation alone but also by breed, and (iii) that anatomy, movement, and foraging behavior of a cattle breed is related to its productivity. In order to test these hypotheses, we conducted a controlled grazing experiment in which three cattle breeds simultaneously grazed three types of heterogenous, alpine pastures: low-productive Highland cattle (average weight: 358 kg); local, dual-purpose Original Braunvieh (582 kg); and high-productive Angus × Holstein crossbreed (679 kg). We measured body weight and claw base of nine cows per breed after 10 weeks of grazing alpine pastures. Over a period of 9 days, we recorded the step frequency and lying time by pedometer and space use by GPS. Moreover, we visually observed foraging behavior on three occasions per cow. Forage selectivity and quality were calculated for every cow's diet. Allometric relationships were analyzed on the individual level by fitting standardized major axes. For most parameters measured, we detected strong allometric relationships and clear differences among breeds that depended on the level of productivity. The claws of Highland cattle were relatively large compared to their body weight and thus they exerted less static pressure than other breeds. Moreover, the more productive a breed was, the higher its selectivity and step frequency were. For example, Highland cattle foraged shrubs and thistles more frequently than high-productive Angus × Holstein. The latter walked longer distances to select higher-quality forage, while Highland cattle used the space more evenly, visited steeper slopes, and moved further away from water points. Irrespective of breed, vegetation composition influenced cattle behavior: On pastures of low forage quality, animals walked more, foraged more selectively, and used space less evenly. In conclusion, the observed breed-specific differences can be used to improve pasture management and grassland conservation.

Preferential Partitioning of Rumen-Protected n-3 and n-6 Fatty Acids into Functionally Different Adipose Tissues.

Lipids are stored at various sites inside the body as adipose tissue (AT). These include subcutaneous, abdominal, and intermuscular locations. The AT substantially differ in their metabolic function. It is, however, unclear whether AT have specific requirements for individual essential n-3 and n-6 fatty acids (FA). If so, control mechanisms would partition FA from the blood. To investigate the hypothesis of a selective FA incorporation, 18 beef heifers were fed diets supplemented with 60 g/kg diet with lipids from either fish oil (FO) or sunflower oil (SO). The lipids had partially been rumen-protected to ruminal biohydrogenation of n-3 and n-6 FA. The AT analyzed for n-3 and n-6 FA by gas chromatography were obtained from pericardial, longissimus thoracis (LT) intermuscular, perirenal, and subcutaneous sites. The greatest proportions of n-3 and n-6 FA were found in the pericardial AT. Despite generally low abundance, n-3 FA proportions increased with FO compared to SO supplementation in all AT, but to a different extent. No such partitioning was found for the n-6 FA when supplementing SO. Concomitantly, the n-6/n-3 FA ratio was reduced with FO in all AT, except in the pericardial AT. The latter has specific metabolic functions and thus appears to be quite resistant to diet-induced changes in FA profile in order to maintain its function. The present findings showed the special role of specific n-3 and n-6 FA in bovine AT.

Alpine and lowland grazing differentially alter the reproductive tract redox milieu and amino acid composition in cattle.

An alpine environment is unique due to pasture biodiversity, with an abundant content of natural antioxidant polyphenols. The present study investigated the effects of lowland and alpine grazing on the oviduct and uterine tissue redox status and amino acid concentrations in plasma and reproductive fluids. In the first experiment, heifers grazed on lowland (H-LOW: n = 13) and on alpine (H-ALP: n = 15) pastures. In the second experiment, heifers grazed on the same lowland (HS-LOW: n = 6) and on a different alpine (HS-ALP: n = 6) pasture. The abundance of mRNA transcripts for antioxidant enzymes in the oviduct (glutathione S-transferase alpha 2, glutathione synthetase (GSS)) and the endometrium (catalase, glutathione-disulfide reductase, GSS) was less (P <  0.05), and for glutathione peroxidase 4 in the endometrium greater (P =  0.006) in the H-LOW than in the H-ALP group. The abundance of mRNA transcript for catalase was less in the endometrium in the H-LOW than in the H-ALP (P =  0.001) group. Catalase and NAD(P)H quinone dehydrogenase 1 concentrations in the oviduct were greater in the HS-LOW than in the HS-ALP group (P <  0.05). Of 32 amino acids analysed, there were differences in concentrations in the H-LOW and H-ALP group of 13, seven and 15 in plasma, oviduct and uterine fluids, respectively (P <  0.05). Comparing the HS-LOW to the HS-ALP groups, there were 13, one and three amino acids in the plasma, oviduct and uterine fluids, respectively, that were differentially abundant (P <  0.05). The grazing systems had some effect on the redox status and amino acid patterns in reproductive tissues.

Little Difference in Milk Fatty Acid and Terpene Composition Among Three Contrasting Dairy Breeds When Grazing a Biodiverse Mountain Pasture.

In the mountains, autochthonous and robust breeds are often used to valorize biodiverse grasslands. Along with their lower nutrient requirements, compared to specialized dairy breeds, they are expected to be better adapted to complex environments and valorize grasslands into dairy products of high quality. Therefore, the aim of the present study was to investigate the grazing selection of three contrasting dairy breeds on a biodiverse mountain pasture, and its consequences on milk fatty acid (FA) profile and prevalence of individual terpenes. A dual-purpose breed from the Italian Alps, the Valdostana Red Pied (Va), was compared to Montbéliardes (Mo), more specialized in milk production, and the highly specialized Holsteins (Ho). Diet selection was measured by scan-sampling, calculating selectivity indexes, and collecting simulated bites during two consecutive days in June (end of first grazing cycle) and July (second grazing cycle). Milk samples were collected at each milking during these experimental periods. Yield of milk and its fat and protein contents were measured. Milk FA and terpenes were analyzed by gas chromatographic methods. We tested the effects of breed, period and their interaction in a repeated mixed model, and calculated Pearson's correlations between behavioral data and milk FA as well as terpenes. The Va grazed less mature vegetation than Ho, but this difference was not sufficient to lead to a major breed effect on milk FA profile and prevalence of terpenes. However, the proportion of α-linolenic acid (C18:3 n-3) was always higher in the milk fat of Va than Ho (Mo were intermediary), but this without any correlation to grazing selection. This could be a consequence from a different metabolism concerning ruminal biohydrogenation, but must be further investigated. Finally, we confirmed previous studies that highlighted a link between milk quality and cows' grazing behavior, but here without differences among breeds. All cows adapted their behavior to the herbage evolution during the season, leading to higher proportions of unsaturated FA in July than June milks. Our study suggests that under mountain grazing conditions (biodiverse pasture and cows in late lactation), milk quality depends more on herbage composition than on cow breed.

Iodine bioavailability from cow milk: a randomized, crossover balance study in healthy iodine-replete adults.

BACKGROUND: Milk and dairy products are considered important dietary sources of iodine in many countries. However, to our knowledge, iodine bioavailability from milk has not been directly measured in humans. OBJECTIVE: The aim of this study was to compare iodine bioavailability in iodine-replete adults from: 1) cow milk containing a high concentration of native iodine; 2) milk containing a low concentration of native iodine, with the addition of potassium iodide (KI) to assess a potential matrix effect; and 3) an aqueous solution of KI as a comparator; with all 3 containing equal amounts of total iodine (263 µg/250 mL). We also speciated iodine in milk. DESIGN: We conducted a 3-wk, randomized, crossover balance study in adults (n = 12) consuming directly analyzed, standardized diets. During the 3 test conditions - high intrinsic iodine milk (IIM), extrinsically added iodine in milk (EIM), and aqueous iodine solution (AIS) - subjects collected 24-h urine over 3 d and consumed the test drink on the second day, with 3- or 4-d wash-out periods prior to each treatment. Iodine absorption was calculated as the ratio of urinary iodine excretion (UIE) to total iodine intake. Milk iodine speciation was performed using ion chromatography-mass spectrometry. RESULTS: Iodine intake from the standardized diet was 195 ± 6 µg/d for males and 107 ± 6 µg/d for females; the test drinks provided an additional 263 µg. Eleven subjects completed the protocol. There was a linear relation between iodine intake and UIE (ß = 0.89, SE = 0.04, P < 0.001). There were no significant differences in UIE among the 3 conditions (P = 0.24). Median (range) fractional iodine absorption across the 3 conditions was 91 (51-145), 72 (48-95), and 98 (51-143)% on days 1, 2, and 3, respectively, with day 2 significantly lower compared with days 1 and 3 (P < 0.001). In milk, 80-93% of the total iodine was inorganic iodide. CONCLUSION: Nearly all of the iodine in cow milk is iodide and although fractional iodine absorption from milk decreases with increasing dose, its bioavailability is high. The trial was registered at clinicaltrials.gov as NCT03590431.

Grazing on Upland Pastures Part-Time Instead of Full-Time Affects the Feeding Behavior of Dairy Cows and Has Consequences on Milk Fatty Acid Profiles.

Different grazing management systems are practiced on upland dairy farms during summer, depending on topography, local traditions, and infrastructure. The present experiment compared two distinct management systems with respect to feeding behavior and milk-related properties. Two similar groups of eight Valdostana Red Pied cows originating from two farms were followed during three grazing events in summer on three upland grazing sites. Cows in the full-time grazing group were kept exclusively on pasture and milked twice daily in a mobile milking parlor. Cows in the part-time grazing group had access to pasture for 4 h and 2 h after their morning and evening milkings, respectively. The part-time grazing cows differed markedly in their feeding behavior; they exhibited shorter daily ingestion times and longer durations of ingestion and idling bouts than full-time grazing cows. Part-time grazing cows had lower milk protein and casein contents, but milk yield and milk coagulation properties did not differ from the full-time grazing cows. As a result of the fasting periods in the barn, part-time grazing cows synthesized less fatty acids de novo and mobilized body fat reserves, as evidenced by the higher proportion of oleic acid in their milk fat.

Partitioning of Rumen-Protected n-3 and n-6 Fatty Acids is Organ-Specific in Growing Angus Heifers.

Dietary polyunsaturated fatty acids (PUFA), especially n-3 and n-6 fatty acids (FA), play an important role in the regulation of FA metabolism in all mammals. However, FA metabolism differs between different organs, suggesting a distinct partitioning of highly relevant FA. For the present study in cattle, a novel technology was applied to overcome rumen biohydrogenation of dietary unsaturated FA. Angus heifers were fed a straw-based diet supplemented for 8 weeks with 450 g/day of rumen-protected oil, either from fish (FO) or sunflower (SO). The FA composition in blood and five important organs, namely heart, kidney, liver, lung, and spleen, was examined. In blood, proportions of polyunsaturated FA were increased by supplementing FO compared to SO. The largest increase of eicosapentaenoic acid (EPA) proportion was found with FO instead of SO in the kidney, the lowest in the lung. Docosahexaenoic acid (DHA) was increased more in the liver than in kidney, lung, and spleen. The heart incorporated seven times more EPA than DHA, which is more than all other organs and described here for the first time in ruminants. In addition, the heart had the highest proportions of α-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) of all organs. The proportions of polyunsaturated FA in the lung and spleen were exceptionally low compared to heart, liver, and kidney. In conclusion, it was shown that the response to FO in the distribution of dietary n-3 FA was organ-specific while proportions of n-6 FA were quite inert with respect to the type of oil supplemented.

Effects of feed iodine concentrations and milk processing on iodine concentrations of cows' milk and dairy products, and potential impact on iodine intake in Swiss adults.

The contribution of milk and dairy products to daily iodine intake is high but variable in many industrialised countries. Factors that affect iodine concentrations in milk and dairy products are only poorly understood. Our aim was to: (1) assess the effect of feed iodine concentration on milk iodine by supplementing five groups of five cows each with one of five dosages from 0-2 mg iodine/kg DM; (2) quantify iodine losses during manufacturing of cheese and yogurt from milk with varying iodine concentrations and assess the effect of cellar-ripening; and (3) systematically measure iodine partitioning during heat treatment and skimming of milk. Milk iodine reached a near-steady state after 3 weeks of feeding. Median milk iodine (17-302 µg/l for 0-2 mg iodine/kg DM) increased linearly with feed iodine (R2 0·96; P < 0·001). At curd separation, 75-84 % of iodine was lost in whey. Dairy iodine increased linearly with milk iodine (semi-hard cheese: R2 0·95; P < 0·001; fresh cheese and yogurt: R2 1·00; P < 0·001), and cellar-ripening had no effect. Heat treatment had no significant effect, whereas skimming increased (P < 0·001) milk iodine concentration by only 1-2 µg/l. Mean daily intake of dairy products by Swiss adults is estimated at 213 g, which would contribute 13-52 % of the adults' RDA for iodine if cow feed is supplemented with 0·5-2 mg iodine/kg DM. Thus, modulation of feed iodine levels can help achieve desirable iodine concentrations in milk and dairy products, and thereby optimise their contribution to human iodine nutrition to avoid both deficiency and excess.

Bovine embryo elongation is altered due to maternal fatty acid supplementation.

The pre-implantation period is prone to embryonic losses in bovine. Embryo-maternal communication is crucial to support embryo development. Thereby, factors of the uterine fluid (UF) are of specific importance. The maternal diet can affect the UF composition. Since omega 3 fatty acids (omega 3 FA) are considered to be beneficial for reproduction, we investigated if dietary omega 3 FA affected factors in the UF related to embryo elongation. Angus heifers (n = 37) were supplemented with either 450 g of rumen-protected fish oil (omega 3 FA) or sunflower oil (omega 6 FA) for a period of 8 weeks. Following cycle synchronization and artificial insemination, the uteri were flushed post mortem to recover the embryos on day 15 of pregnancy. The UF and tissue samples of endometrium and corpus luteum (CL) were collected. Strikingly, the embryo elongation in the omega 3 group was enhanced compared to the omega 6 group. No differences were observed in uterine prostaglandins, even though the endometrial concentration of their precursor arachidonic acid was reduced in omega 3 compared to omega 6 heifers. The dietary FA neither led to differential expression of target genes in endometrium nor CL nor to a differential abundance of low-density lipoprotein cholesterol, cortisol or amino acids in the UF. Interestingly, the omega 3 group displayed a higher plasma progesterone concentration during luteal growth than the omega 6 group, possibly promoting embryo elongation. Further research should include an ovarian perspective to understand the functional link between dietary omega 3 FA and reproductive outcome.

The main determinants of iodine in cows' milk in Switzerland are farm type, season and teat dipping.

Milk and dairy products are important iodine sources and contribute about 30-40 % of total iodine in the Swiss diet. Information about variation in milk iodine concentration (MIC) in Switzerland is limited. We examined MIC and its potential determinants in milk from organic and conventional farms. We collected bulk milk samples at 3-month intervals over 1 year from thirty-two farms throughout Switzerland and Aosta valley, North-West Italy. We sampled all feed components including tap water, collected information on farm characteristics, feeding and teat disinfection practices by questionnaire and estimated the cows' winter and summer iodine intake. Iodine in milk and feed components was measured using inductively coupled plasma MS. The overall median MIC was 87 (range 5-371) µg/l. In multivariate analysis, predictors of MIC were as follows: (1) farm type: median MIC from organic and conventional farms was 55 and 93 µg/l (P=0·022); (2) season: 53, 97 and 101 µg/l in September, December and March (P<0·002); and (3) teat dipping: 97 µg/l with v. 56 µg/l without (P=0·028). In conclusion, MIC varied widely between farms because of diverse farming practices that result in large differences in dairy cow exposure to iodine via ingestion or skin application. Standardisation of MIC is potentially achievable by controlling these iodine exposures. In order for milk to be a stable iodine source all year round, dietary iodine could be added at a set level to one feed component whose intake is regular and controllable, such as the mineral supplement, and by limiting the use of iodine-containing teat disinfectants.

Effect of nursing or mentoring by adult cows on physical activity, performance and meat quality of fattening beef calves kept on alpine pastures.

BACKGROUND: Compared with weaned calves, nursed calves benefit from access to milk as nutrient-dense feed and mentoring by their dams. Both may be decisive for growth under difficult conditions such as grazing on alpine pastures, where experience could play an essential role. These factors were investigated separately by comparing nursed calves with calves weaned at 3 months of age and grazing with or without a mentor cow (n = 8 calves). RESULTS: Weaned calves with mentor cows spent less time lying and walked more than nursed calves. Irrespective of whether they had a mentor cow or not, the weaned calves grew slower than the nursed calves (357, 428 and 1324 g day-1 respectively). This also adversely affected slaughter weight, dressing percentage, conformation and fat cover at slaughter. In general, mentoring had no effect. Meat quality did not differ whether weaned calves were mentored or not. Nursed calves were superior in intramuscular fat content, tenderness and water-holding capacity. CONCLUSION: The availability of milk appeared to be much more important to the calves under demanding grazing conditions than did mentoring. The differences in growth rate were accompanied by unexpectedly large differences in meat quality, showing that early weaning was not suitable for this system.© 2016 Society of Chemical Industry.

Carcass and meat quality of finished and non-finished Limousin heifers from alpine livestock systems differing in altitudinal origin of the forage.

Effects of the alpine origin of the forage and of finishing on carcass and beef quality were quantified by modelling different alpine livestock system alternatives. Thirty-five Limousin heifers, initially weighing 383 ± 45 kg, were fed fresh grass at 400 or 2000 m above sea level, or a 1:1 mixture of alpine grass and lowland grass hay at 2000 m. After 9 weeks, the six heaviest and oldest animals per group were slaughtered. The remaining animals were finished for 8 weeks on a silage-concentrate diet in the lowlands to similar age and body weight as the first slaughtered group. Carcass and meat quality (M. longissimus thoracis) were assessed in various respects. The average daily gains achieved were of about 600 g/d and similar between forage-type groups. Dressing percentage was 53.5% in the alpine and 57.2% in the lowland group. Carcass conformation and fat cover scores did not differ between forage-type groups. The meat from the alpine groups had greater ultimate pH and smaller redness, yellowness and protein contents. Still, these differences were of minor practical relevance. There was no forage-type effect on water-holding capacity and shear force of the meat. The alpine systems enhanced the proportion of α-linolenic acid in intramuscular fat and decreased the levels of some volatile compounds in perirenal fat. Finishing resulted in compensatory growth, especially in the animals previously fed lowland grass. There was a trend for the finished compared with the non-finished groups towards greater carcass fat cover and intramuscular fat content. Additionally, ultimate pH was smaller and cooking loss was greater with than without finishing. Meat colour differences were also observed. Shear force was not affected by finishing. The finished animals had a smaller α-linolenic acid proportion in the intramuscular fat. In conclusion, the forage type had small effects on carcass and meat quality. Finishing did not substantially improve carcass and meat quality. The (alpine) grass-specific differences in fatty acid profile found in the unfinished cattle were not present in the finished animals.

Potential sources of early-postnatal increase in myofibre number in pig skeletal muscle.

In pigs, myogenesis is a biphasic phenomenon with the formation of primary and secondary fibres. Hyperplasia was reported to be accomplished around 90 days of gestation. However, some studies suggest a substantial increase in the total fibre number (TFN) from birth to weaning by counting fibre number in the muscle cross sections. The aim of this study was to establish in which way TFN increases after birth and whether this increase is imputable to new (tertiary) myofibres and/or fibre elongation. The semitendinosus muscle of 128 piglets was examined at days 1 (n = 63), 7 (n = 12), 21 (n = 12), and 28 (n = 41) of age. TFN was increased at days 7, 21 and 28 of age when compared with day 1 (P < 0.01). From day 1 to 28, TFN increased from 463 × 10(3) to 825 × 10(3). Microscopy of longitudinal and transversal serial sections revealed that at day 7 of age very small fibres expressing the embryonic myosin heavy chain (MyHC) isoform were apparent all over the muscle. In addition, intrafascicular terminations of normal-sized fibres expressed the embryonic MyHC isoform. These data suggest that the TFN in the pig muscle is not fixed at birth and its postnatal increase may be related to both elongation of existing muscle fibres and genesis of tertiary myofibres, mainly between birth and 3 weeks of age.

Computational density functional study of polypyrrolic macrocycles: analysis of actinyl-oxo to 3d transition metal bonding.

Density functional theoretical methods are used to study heterobimetallic compounds of a new form of binucleating Schiff-base polypyrrolic macrocycle, denoted [An(VI)O(2)(1)H(2)L], [An(V)O(2)(1)H(2)L](-), [An(VI)O(2)(TM)L] and [An(V)O(2)(TM)L](-), and containing actinyl ions AnO(2)(n+) (An = U, Np, Pu; n = 1, 2) and 3d transition metals (TM): no TM = 1, Mn = 2, Fe = 3, Co = 4, and Zn = 5. Calculated bond orders (TM-O2 = 0.36 to 0.81) provide evidence for partial bond formation between the transition metal (TM) and the actinyl-endo-oxygen for all 24 cases studied. Redox potentials for [An(VI)O(2)(1)H(2)L]/[An(V)O(2)(1)H(2)L](-) couples were found to have the same Np(VI/V) > Pu(VI/V) > U(VI/V) trend as previously studied for the [AnO(2)(H(2)O)(5)](2+/1+) couples, where Np(VI) is the most easily reduced to Np(V). Extrapolation from the earlier penta-aqua actinyl results is used to predict [An(VI)O(2)(1)H(2)L]/[An(V)O(2)(1)H(2)L](-) redox couples of U = -1.10 eV, Np = 0.25 eV, and Pu = 0.01 eV. The calculated redox potential for [U(VI)O(2)(1)H(2)L]/[U(V)O(2)(1)H(2)L](-) is within 0.08 eV of the value found by cyclic voltammetry (-1.18 eV, in THF/NBu(n)(4)BF(4) solvent).

A density functional study of the various forms of UN4O12 containing uranyl nitrate.

In this paper we report the computational results of a density functional study of 73 UN4O12 isomers containing uranyl nitrate, UO2(NO3)2, as a component. The isomers are grouped into three categories and 19 types. Forty-four isomers of 14 types are dinitrogen tetroxide adducts of uranyl nitrate, UO2(NO3)2.N2O4, 22 are nitrosonium salt adducts of uranyl nitrate, NO+UO2(NO3)3-, NO+UO2(NO3)2O(NO2)-, NO+UO2(NO3)2(ONOO)-, or (NO+)2UO2(NO3)2O22-, and 7 are bis(nitrogen dioxide) adducts of uranyl nitrate, UO2(NO3)2.2NO2. The 22 most stable isomers in solution, representing the 20 most stable gas-phase isomers, were selected for analysis. Of these selected structures only two categories and six types were represented. Structures, frequencies, gas-phase and solution energetics, atomic charges, dipole moments, and the bonding within the N2O4 unit and between NO+ and UO2(NO3)3- components have been analyzed in detail. On the basis of relative Gibbs free energy calculations five isomers (the N2O4 adducts a1, a2, and a3 and the nitrosonium salts b1 and b2) were identified as strong candidates to exist and possibly predominate in the gas phase, with a1 and a2 being the strongest candidates. Similarly, four isomers (a6, a5, a8, and a1, all of them N2O4 adducts) were identified as strong candidates to exist and possibly predominate in a nonaqueous solution of nitromethane/dinitrogen tetroxide. Of these, a6 was determined to be the most likely candidate to predominate in solution. The possibility of dissociation in solution has been addressed briefly. In addition, computational evidence for the existence of four new N2O4 isomers 20, 22, 27, and 28 in both the gas and the solution phases is presented for the first time.