Mirko Cattani

Technical note: In vitro total gas and methane production measurements from closed or vented rumen batch culture systems

This study compared measured gas production (GP) and computed CH4 production values provided by closed or vented bottles connected to gas collection bags. Two forages and 3 concentrates were incubated. Two incubations were conducted, where the 5 feeds were tested in 3 replicates in closed or vented bottles, plus 4 blanks, for a total of 64 bottles. Half of the bottles were not vented, and the others were vented at a fixed pressure (6.8 kPa) and gas was collected into one gas collection bag connected to each bottle. Each bottle (317 mL) was filled with 0.4000 ± 0.0010 g of feed sample and 60 mL of buffered rumen fluid (headspace volume = 257 mL) and incubated at 39.0°C for 24 h. At 24 h, gas samples were collected from the headspace of closed bottles or from headspace and bags of vented bottles and analyzed for CH4 concentration. Volumes of GP at 24 h were corrected for the gas dissolved in the fermentation fluid, according to Henrys law of gas solubility. Methane concentration (mL/100mL of GP) was measured and CH4 production (mL/g of incubated DM) was computed using corrected or uncorrected GP values. Data were analyzed for the effect of venting technique (T), feed (F), interaction between venting technique and feed (T × F), and incubation run as a random factor. Closed bottles provided lower uncorrected GP (-18%) compared with vented bottles, especially for concentrates. Correction for dissolved gas reduced but did not remove differences between techniques, and closed bottles (+25 mL of gas/g of incubated DM) had a greater magnitude of variation than did vented bottles (+1 mL of gas/g of incubated DM). Feeds differed in uncorrected and corrected GP, but the ranking was the same for the 2 techniques. The T × F interaction influenced uncorrected GP values, but this effect disappeared after correction. Closed bottles provided uncorrected CH4 concentrations 23% greater than that of vented bottles. Correction reduced but did not remove this difference. Methane concentration was influenced by feed but not by the T × F interaction. Corrected CH4 production was influenced by feed, but not by venting technique or the T × F interaction. Closed bottles provide good measurements of CH4 production but not of GP. Venting of bottles at low pressure permits a reliable evaluation of total GP and CH4 production.

True dry matter digestibility of feeds evaluated in situ with different bags and in vitro using rumen fluid collected from intact donor cows

The aim of the study was to determine the comparability of the true dry matter digestibility (TDMd) achieved in situ with either traditional nylon bags (IS nylon) or synthetic filter bags (IS filter), and in vitro with either conventional bottles (CB) or the DaisyII incubation technique, using rumen fluid collected by oro-ruminal suction from intact cows. The four TDMd results were compared by linear regression. For each method, 11 feeds were incubated for 48 h in two separate incubations. The reproducibility of TDMd measurements was 97.9%, 95.1%, 98.8% and 96.0% for IS nylon, IS filter, CB and DaisyII, respectively. The determination coefficient, the slope (b), and the significant difference of the slope from unity, of the linear relationship between TDMd values (g/kg DM) were as follows: IS nylon v. IS filter, R2 = 0.97, b = 0.86, P = 0.02; IS nylon v. CB, R2 = 0.90, b = 1.02, P = 0.27; IS nylon v. DaisyII, R2 = 0.90, b = 1.06, P < 0.01; and IS filter v. DaisyII, R2 = 0.95, b = 0.98, P = 0.13. In situ filter bags and DaisyII underestimated the TDMd values compared with IS nylon bags and CB, respectively. In situ, the replacement of nylon with filter bags could simplify the procedure of analysis, with less manipulation of residuals and less labour, and offers the possibility to increase the number of samples analysed simultaneously. The close relationships found among methods suggest that rumen fluid collected from intact cows can produce in vitro estimates of TDMd at 48 h well correlated with those obtained in situ.

A ring test of a wireless in vitro gas production system

The in vitro gas production (GP) technique has been widely used for feed evaluation. However, variability in results limits useful comparisons. Results from a ring test undertaken in four laboratories (Italy – IT, Spain – SP, Wales – WA and Denmark – DK) using the same wireless equipment (ANKOM Technology), same substrates and same laboratory protocol are presented, including calculation of repeatability and reproducibility according to ISO 5725-2. Hay, maize starch and straw samples and units without sample (blanks) were incubated in five repetitions using rumen inoculum from cows (DK, IT and WA) or sheep (SP). Curves, corrected for blanks, were fitted using an exponential regression model with a lag time. The following variables were considered: (i) GP24 and GP48: raw values at 24 and 48 h (mL/g DM), corrected for blanks; (ii) A: asymptotic GP (mL/g DM); (iii) T1/2: time when half A is produced (h); (iv) GPMR: maximum predicted GP rate (mL/h); (v) L: lag time (h). A mixed model including laboratories as random effect was used. A significant interaction between substrate and laboratories was found for all variables except A. The most repeatable and reproducible results were observed for A and GP48. The results from this ring test suggest the need for more standardisation, particularly in the procedures that occur outside the laboratory.

Recovery of n-3 polyunsaturated fatty acids and conjugated linoleic acids in ripened cheese obtained from milk of cows fed different levels of extruded flaxseed

The aim of the study was to investigate whether the addition of extruded flaxseed (EF) in dairy cow diets had an effect on milk fat and individual fatty acids (FA) recovery in cheese after 90 d of ripening. Eighteen Holstein-Friesian cows, divided into 3 experimental groups (6 cows/group), were fed 3 isonitrogenous and isoenergetic diets with 0 (CTR), 500 (EF500), or 1,000 g/d (EF1000) of EF in 3 subsequent periods (2 wk/each), following a 3 × 3 Latin square design. Dry matter intake (DMI) and milk yield were recorded daily. Individual milk samples were collected on d 7 and 13 of each period to determine proximate and FA composition. Eighteen cheese-making sessions (2 for each group and period) were carried out, using a representative pooled milk sample obtained from the 6 cows of each group (10L). At 90 d of ripening, cheeses were analyzed for proximate and FA composition. Cheese yield was computed as the ratio between the weights of ripened cheese and processed milk. Recoveries of fat, individual FA, and grouped FA were computed as the ratio between the corresponding weights in cheese and in milk. Inclusion of EF did not affect DMI, milk yield, or milk composition. Compared with CTR, the 2 diets containing EF increased the proportion of C18:3n-3 and total n-3 FA, in both milk and cheese. Cheese yield and cheese fat percentage did not differ among diets. Likewise, milk fat recovery in cheese was comparable in the 3 treatments and averaged 0.85. The recoveries of individual FA were, for the most part, not dissimilar from fat recovery, except for short-chain saturated FA (from 0.38 for C4:0 to 0.80 for C13:0), some long-chain saturated FA (0.56 and 0.62 for C20:0 and C21:0, respectively), and for C18:3n-6 (1.65). The recovery of saturated FA was lower than that of monounsaturated FA, whereas recovery of polyunsaturated FA was intermediate. Compared with medium- and long-chain FA, short-chain FA were recovered to a smaller extent in cheese. No differences in recovery were found between n-6 and n-3 FA. In conclusion, FA have different recoveries during cheese-making, with lower values for the short-chain compared with long-chain FA, and for saturated FA compared with unsaturated FA. The addition of EF in dairy cow diets did not influence cheese yield or fat recovery in cheese, irrespective of the inclusion level. The experiment confirmed that feeding cows with EF represents a successful strategy for improving the FA profile of dairy products, through an increase of n-3 FA.

Synthetic and natural polyphenols with antioxidant properties stimulate rumen microbial growth in vitro

This trial compared the effects of two antioxidant phenols, butyl-hydroxyl-toluene (BHT) and a blend of polyphenols extracted from red chicory, on in vitro degradability, gas production (GP), volatile fatty acids, and microbial nitrogen production, using meadow hay and corn grain as fermentation substrates. A batch culture system with automated gas pressure detectors was used. Four replicates of each feed were incubated for 72 h without additive (Control, CTL) or with the addition of low (0.15 mg/g feed) or high (1.5 mg/g feed) dosages of BHT or red chicory. GP curves were fitted to estimate the time at which half of total GP (t1/2) was achieved. The t1/2 values for meadow hay and corn grain were ~16 and 9 h, respectively. A second incubation, conducted using the same experimental design, was stopped at t1/2. Compared with CTL, degradability and GP kinetics were not affected by the two dosages of BHT, except for GP, which increased after 48 h of incubation (P < 0.01). In the second incubation, BHT increased acetate at the expense of butyrate proportion (P < 0.01). Red chicory extract did not influence GP, feed degradability, or volatile fatty acids production in either incubation. Both BHT and red chicory induced a dosage-dependent increase in microbial nitrogen production at t1/2 (P < 0.05), on average from 7.0 to 13.9 mg/g DM. Collectively, our data suggest that increasing dosages of the two additives with antioxidant properties could have induced a shift in the partition of energy, with a higher proportion of nutrients channelled towards microbial protein synthesis.

A simplified approach to calculate slurry production of growing pigs at farm level

Abstract A simplified approach to predict the amount of slurry produced by growing pigs at farm level is proposed. The inputs are initial (LWi) and final (LWf) live weights, production (t) and empty (empty) periods, feed consumption (FC), dry matter (DMD), N digestibilities and farm water consumption per pig (FWC). Estimates of the amount of water required (or arisen) per kg of feed for the various physiological functions were estimated by running a published mathematical model using data representing the ordinary conditions of rearing. Water excretion was estimated in two ways depending on: 1) free access (ad lib) to water; 2) restricted access (forced). In the first case, the proportion of water consumed (wiad lib) and those excreted with the urine (wuad lib) and the faeces (wfec) were quantified to be 2.9, 1.72 and 0.33 kg per kg of feed, respectively. From the urinary excretions of N and minerals, obtained as the difference between the digestible nutrient intakes and the retentions, the model predicted a urinary DM content of 2.1% (by weight). In the second case, for pigs receiving drinking water in forced ratio with the feed (wiforced), the urinary production was calculated as wuforced=(wiforced+wf+wo)-(wd+ws+wg+wfec+we), where wf=water content in feed (0.12 kg/kg), wo=water arising from nutrient oxidation (0.25 kg/kg), wd=water required for digestion (0.08 kg/kg), ws=water demand for protein and lipid synthesis (0.06 kg/kg), wg=water retained in body tissues (0.14 kg/kg) and we=water lost through evaporation (0.96 kg/kg). Estimates of fresh slurry production (faeces+urine) were regressed against the values resulting from empirical literature equations and referred to pigs fed water:feed ratios of 2.5:1, 2.9:1 and 4:1. The resulting regression (R2=0.97), with a slope close to unity (1.05), indicated that the approach can be extended to predict the farm fresh slurry production with pigs having free access to water or kept on different water:feed ratios. In agreement with international literature, but not with the current Italian national standards, estimates of mature slurry productions ranged from 1.5 to 2.9 ton/pig/year with DM contents ranging from 8.3 to 3.7%. At farm level the use water meters is recommended as from FWC minus the estimated water consumption (FWCexp) the farmer can evaluate the extra wastage and adjust the predicted mature slurry production.

In vitro fermentation of key dietary compounds with rumen fluid: A genome-centric perspective.

The anaerobic decomposition of organic substrates leads to the generation of gases, such as methane, which can either be a valuable energy carrier in industrial applications or can be considered as a main greenhouse gas when it is naturally emitted. In this study we investigated in vitro the effect of dietary compounds, such as starch and proteins, on the microbial community present in the rumen fluid. High throughput shotgun sequencing, followed by metagenomic assembly and binning allowed the extraction of 18 genome bins. A composite bioinformatic analysis led to the prediction of metabolic pathways involved in the degradation of dietary compounds and in the biosynthesis of crucial products like propionate, methane and ammonia. The identification of genomes belonging to poorly characterized phyla such as Thermoplasmata and Elusimicrobia shed light on their putative role. The high abundance of methylotrophic archaea in the inoculum suggests a relevant role in methane production.

Characteristics of dairy farms in the North-Eastern part of Italy: rations, milk yield and nutrients excretion

Abstract This survey was aimed to evaluate the characteristics of dairy farms in the North-Eastern part of Po valley in terms of ration composition, milk yield and N and P excretions. Eighty-nine farms, with Italian Holstein Friesian cows, were selected in order to cover different situations in term of farm size and milk yield (MY). MY and quality were obtained from the national database of functional controls. Each farm was visited in order to collect information about ingredients and chemical composition of rations used. Farms were classified in four groups differing for dietary crude protein density (Lcp<15.3% DM; Hcp>15.3% DM) and for MY (Lmy>30 kg/d; Hmy<30 kg/d). N and P excretions were quantified by following a mass balance approach. Dietary crude protein content (CP) was not correlated to milk yield (MY) and quality. The estimated amounts of N excreted, discounted for 28% of N losses in atmosphere, were 78.5, 78.2, 87.2 and 89.1 kg/cow/year, and P excreted were 20.2, 18.6, 18.7 and 19.8 kg/cow/year for the LcpLmy, LcpHmy, HcpLmy, HcpHmy groups, respectively. On corn silage and cereals based rations, a dietary CP of 14.3% DM can support 31 kg MY/cow/day.

Associative effects of poor-quality forages combined with food industry byproducts determined in vitro with an automated gas-production system

Thisexperiment aimedtoinvestigatetheassociativeeffectsamongtwolow-qualityforages(crowndaisy,milk thistle) and three agro-industrial byproducts (apple pomace, citrus pulp, tomato peel), by means of an automated gas production(GP) system.All feedswere incubated alone or as 50:50 mixtures of eachforagewitheach byproduct.Samples (0.500 � 0.0010g)ofsinglefeedsormixtureswereincubatedfor96h,inthreereplicatesinindividualbottles(310mL),with 75 mLof buffered rumen fluid. Bottles were ventedby anopen-close valve whenthe internal pressure reached 3.4kPa.The metabolisable energy content of single feeds and mixtures was computed from GP at 24 h and feed chemical composition. Feed substrates were ranked for GP in the following way: byproducts, mixtures, and forages. The two forages did not differ for GP and metabolisable energy content, although differences were observed among byproducts and among mixtures. Both forages interacted positively with apple pomace from 6 h (P < 0.001) to 24 h (P= 0.029) of incubation and with citrus pulp at 12 h (P = 0.005) and 24 h (P = 0.012), whereas no associative effects were detected when forages were incubated with tomato peels. Results suggest that in vitro fermentability of low-quality forages could be efficiently improved by combining these two forages with apple pomace or citrus pulp. These findings are relevant, because the use of low-quality forages and byproducts in ruminant feeding is considered important for improving the environmental and economic sustainability of forage systems in arid and semi-arid areas.

Effects of total replacement of corn silage with sorghum silage on milk yield, composition, and quality

BackgroundIn the last years, difficulties occurring in corn cultivation (i.e., groundwater shortages, mycotoxin contamination) have been forcing dairy farmers to consider alternative silages. Some experiments conducted on lactating cows have proven that the total replacement of corn silage with sorghum silage did not reduce milk yield. However, this kind of substitution involves supplementing sorghum-based diets with grains, to compensate for the lower starch content of sorghum silage compared to corn silage. Change of silage type and inclusion of starch sources in the diet would influence rumen fermentations, with possible effects on milk composition (i.e., fatty acid profile) and coagulation properties. A worsening of milk coagulation properties would have a negative economic impact in Italy, where most of the milk produced is processed into cheese.This study was designed to compare milk composition and quality, with emphasis on fatty acid profile and coagulation properties, in dairy cows fed two diets based on corn or sorghum silage.ResultsThe sorghum diet reduced milk yield (P = 0.043) but not 4% fat corrected milk (P = 0.85). Feeding sorghum silage did not influence milk contents of protein (P = 0.07) and lactose (P = 0.65), and increased fat content (P = 0.024). No differences emerged for milk concentrations of saturated (P = 0.61) and monounsaturated fatty acids (P = 0.50), whereas polyunsaturated fatty acids were lower (P < 0.001) for the sorghum diet. Concentrations of n-6 (P < 0.001) and n-3 fatty acids (P = 0.017) were lower in milk of cows fed the sorghum diet. Milk coagulation properties did not differ between the two diets, except the “a30” (the curd firmness, expressed in mm, 30 min after rennet addition), that was lower (P = 0.042) for the sorghum diet.ConclusionsFeeding a forage sorghum silage, properly supplemented with corn meal, as total replacement of corn silage maintained milk composition and did not influence negatively milk coagulation properties, which have a great economic relevance for the Italian dairy industry. Thus, silages obtained from forage sorghums could have a potential as substitute of corn silages in dairy cow diets.