Influence of empirically derived filtering parameters, ASV, and OTU pipelines on assessing rumen microbial diversity.

Microbes play an important role in human and animal health as well as animal productivity. The host microbial interactions within ruminants play a critical role in animal health and productivity and provide up to 70% of the animal's energy need in the form of fermentation products. As such, many studies have investigated microbial community composition to understand microbial community changes and factors that affect microbial colonization and persistence. The advances in next generation sequencing (NGS) technologies and low cost of sequencing have gravitated many studies to utilize 16S rDNA-based analysis tools for interrogation of microbiomes at a much finer scale than traditional culturing. However, such methods that rely on single base pair differences for bacterial taxa clustering may inflate or underestimate diversity leading to inaccurate identification of bacterial diversity. Therefore, in this study, we sequenced mock communities of known membership and abundance to establish filtration parameters to reduce inflation of microbial diversity due to PCR and sequencing errors. Additionally, we evaluated the effect of the resulting filtering parameters proposed using established bioinformatic pipelines on a study consisting of Holstein and Jersey cattle to identify bread and treatment effects on the bacterial community composition and the impact of the filtering on global microbial community structure analysis and results. Filtration resulted in a sharp reduction in bacterial taxa identified, yet retain most sequencing data (retaining > 79% of sequencing reads) when analyzed using 3 different microbial analysis pipelines (DADA2, Mothur, USEARCH). After filtration, conclusions from α and ß-diversity tests show very similar results across all analysis methods. The mock community-based filtering parameters proposed in this study help provide a more realistic estimation of bacterial diversity. Additionally, the filtration reduced the variation between microbiome analysis methods and help identify microbial community differences that could have been missed due to large animal to animal variation observed in the unfiltered data. As such, we believe, the new filtering parameters described in this study will help obtain diversity estimates closer to realistic values and will improve the ability of detecting microbial community differences and help better understand microbial community changes in 16S rDNA-based studies.

Use of indirect calorimetry to evaluate utilization of energy in lactating Jersey dairy cattle consuming diets with increasing inclusion of hydrolyzed feather meal.

A study using indirect calorimetry and 12 lactating multiparous Jersey cows (53 ± 23 d in milk at the beginning of the experiment; mean ± standard deviation) was conducted to evaluate the utilization of energy in cattle consuming diets containing increasing hydrolyzed feather meal (HFM). A triplicated 4 × 4 Latin square design with 35-d periods (28-d adaption and 4-d collections) was used to compare 4 different dietary treatments. Treatments contained (DM basis) HFM at 0% (0HFM), 3.3% (3.3HFM), 6.7% (6.7MFM), and 10.0% (10HFM). Diets were formulated such that HFM replaced blood meal and nonenzymatically browned soybean meal. With increasing HFM, linear increases were observed for dietary NEL content (1.61, 1.64, 1.69, and 1.70 ± 0.042 Mcal/kg of DM for 0HFM, 3.3HFM, 6.7MFM, and 10HFM, respectively), and the efficiency of converting ME to NEL (0.708, 0.711, 0.717, and 0.719). Apparent total-tract digestibility of CP linearly decreased with increasing HFM (63.4, 61.1, 59.9, and 58.6 ± 1.46% for 0HFM, 3.3HFM, 6.7MFM, and 10HFM, respectively), whereas long-chain fatty acid digestibility increased with increasing HFM (77.2, 77.7, 78.5, and 80.6 ± 1.30%). With increased inclusion of HFM, fecal N excretion increased (199, 230, 239, 237 ± 12.1 g/d for 0HFM, 3.3HFM, 6.7MFM, and 10HFM, respectively), whereas urinary N excretion decreased (166, 151, 155, and 119 ± 14.8 g/d). Increasing the concentration of HFM resulted in a quadratic effect on DMI (19.6, 20.2, 20.3, and 19.1 ± 0.79 kg/d for 0HFM, 3.3HFM, 6.7MFM, and 10HFM, respectively) and milk yield (31.7, 32.0, 31.9, and 29.7 ± 1.32 kg/d). Increasing HFM linearly decreased the milk protein concentration (3.34, 3.29, 3.23, and 3.23 ± 0.158 for 0HFM, 3.3HFM, 6.7MFM, and 10HFM, respectively) and yield (1.05, 1.05, 1.02, and 0.96 ± 0.040 kg). The inclusion of HFM did not affect energy-correct milk yield (average of 39.3 ± 1.54). Results of this study suggest that HFM can increase dietary NEL content compared with blood meal and nonenzymatically browned soybean meal and maintained energy-corrected milk yield; however, feeding HFM at greater than 6.7% of diet DM decreased DMI, and protein availability may have been reduced with increased HFM, leading to a linear decrease in milk protein concentration and yield.

Increasing the concentration of linolenic acid in diets fed to Jersey cows in late lactation does not affect methane production.

Although the inclusion of fat has reduced methane production in ruminants, relatively little research has been conducted comparing the effects of source and profile of fatty acids on methane production in lactating dairy cows. A study using 8 multiparous (325 ± 17 DIM; mean ± SD) lactating Jersey cows was conducted to determine effects of feeding canola meal and lard versus extruded byproduct containing flaxseed as a high-C18:3 fat source on methane production and diet digestibility in late-lactation dairy cows. A crossover design with 32-d periods (28-d adaptation and 4-d collections) was used to compare 2 different fat sources. Diets contained approximately 50% forage mixture of corn silage, alfalfa hay, and brome hay; the concentrate mixture changed between diets to include either (1) a conventional diet of corn, soybean meal, and canola meal with lard (control) or (2) a conventional diet of corn and soybean meal with an extruded byproduct containing flaxseed (EXF) as the fat source. Diets were balanced to decrease corn, lard, and canola meal and replace them with soybean mean and EXF to increase the concentration of C18:3 (0.14 vs. 1.20% of DM). Methane production was measured using headbox-style indirect calorimeters. Cattle were restricted to 95% ad libitum feed intake during collections. Milk production (17.4 ± 1.04 kg/d) and dry matter intake (15.4 ± 0.71 kg/d) were similar among treatments. Milk fat (5.88 ± 0.25%) and protein (4.08 ± 0.14%) were not affected by treatment. For methane production, no difference was observed for total production (352.0 vs. 349.8 ± 16.43 L/d for control vs. EXF, respectively). Methane production per unit of dry matter intake was not affected and averaged 23.1 ± 0.57 L/kg. Similarly, methane production per unit of energy-corrected milk was not affected by fat source and averaged 15.5 ± 0.68 L/kg. Heat production was similar, averaging 21.1 ± 1.02 Mcal/d. Digestibility of organic matter, neutral detergent fiber, and crude protein was not affected by diet and averaged 69.9, 53.6, and 73.3%, respectively. Results indicated that increasing C18:3 may not affect methane production or digestibility of the diet in lactating dairy cows.

Reducing methane production with corn oil and calcium sulfate: Responses on whole-animal energy and nitrogen balance in dairy cattle.

The addition of fat and calcium sulfate to diets fed to ruminants has resulted in a reduction in methane production, but the effects on energy balance have not been studied. A study using indirect calorimetry and 16 multiparous (8 Holstein and 8 Jersey; 78 ± 15 d in milk; mean ± standard deviation) lactating dairy cows was conducted to determine how mitigating methane production by adding corn oil or calcium sulfate to diets containing reduced-fat distillers grains affects energy and nitrogen balance. A replicated 4 × 4 Latin square design with 35-d periods (28 d of adaption and 4 d of collections) was used to compare 4 different dietary treatments. Treatments were composed of a control (CON) diet, which did not contain reduced-fat distillers grain and solubles (DDGS), and treatment diets containing 20% (dry matter basis) DDGS (DG), 20% DDGS with 1.38% (dry matter basis) added corn oil (CO), and 20% DDGS with 0.93% (dry matter basis) added calcium sulfate (CaS). Compared with CON, dry matter intake was not affected by treatment, averaging 29.6 ± 0.67 kg/d. Milk production was increased for diets containing DDGS compared with CON (26.3 vs. 27.8 ± 0.47 kg/d for CON vs. DDGS, respectively), likely supported by increased energy intake. Compared with CON, energy-corrected milk was greater in DG and CO (30.1 vs. 31.4, 31.7, and 31.0 ± 0.67 kg/d for CON, DG, CO, and CaS, respectively). Compared with CON, the addition of calcium sulfate and corn oil to diets containing DDGS reduced methane production per kg of dry matter intake (22.3, 19.9, and 19.6 ± 0.75 L/kg per d for CON, CO, and CaS, respectively). Similarly, methane production per kilogram of energy-corrected milk was reduced with the addition of calcium sulfate and corn oil to diets containing DDGS (14.2, 12.5, and 12.4 ± 0.50 L/kg per d for CON, CO, and CaS, respectively). Compared with CON and CaS, the intake of digestible energy was greater for DG and CO treatments (57.7, 62.1, 62.0, and 59.0 ± 1.38 Mcal/d for CON, DG, CO, and CaS, respectively). Intake of metabolizable energy was greater in all treatments containing DDGS compared with CON (50.5 vs. 54.0 ± 1.08 Mcal/d for CON vs. DDGS, respectively). Net balance (milk plus tissue energy) per unit of dry matter was greater in CO (containing DDGS and oil) than CON (1.55 vs. 1.35 ± 0.06 Mcal/kg for CO vs. CON, respectively). Tissue energy was greater in DG and CO compared with CON (6.08, 7.04, and 3.16 ± 0.99 Mcal/d for DG, CO, and CON, respectively. Results of this study suggest that the addition of oil and calcium sulfate to diets containing DDGS may be a viable option to reduce methane production and in the case of oil also improve net energy balance in lactating dairy cows.

Use of indirect calorimetry to evaluate utilization of energy in lactating Jersey dairy cattle consuming common coproducts.

The use of coproducts as an alternative feed source is a common practice when formulating dairy rations. A study using 12 multiparous (79 ± 16 d in milk; mean ± standard deviation) lactating Jersey cows was conducted over 5 mo to evaluate the effects of dried distillers grains with solubles (DDGS) or canola meal on milk and gas production. A replicated 4 × 4 Latin square design was used to compare 4 dietary treatments. Treatments comprised a control (CON) containing no coproducts, a treatment diet containing 10% (dry matter basis) low-fat DDGS (LFDG), a treatment diet containing 10% high-fat DDGS (HFDG), and a 10% canola meal (CM) treatment. The crude fat content of the LFDG, HFDG, and CM treatments was 6.05 ± 0.379, 10.0 ± 0.134, and 3.46 ± 0.085%, respectively. Coproducts were included in partial replacement for corn and soybean meal. Indirect headbox-style calorimeters were used to estimate heat production. Dry matter intake and milk yield were similar between all treatments, averaging 17.4 ± 0.56 kg/d and 24.0 ± 0.80 kg, respectively. Milk urea N was affected by treatment and was highest in CON (20.6 mg/dL; 18.0, 19.9, and 18.1 ± 0.62 mg/dL in LFDG, CM, and HFDG, respectively). Heat production per unit of metabolic body weight tended to be affected by treatment and was lowest for CON, and diets containing coproducts were not different (192, 200, 215, and 204 ± 5.91 kcal/kg of metabolic body weight for CON, LFDG, CM, and HFDG, respectively). The concentration of metabolizable energy was affected by dietary treatment; specifically, HFDG did not differ from CON but was greater than LFDG and CM (2.58, 2.46, 2.29, and 2.27 ± 0.09 Mcal/kg for HFDG, CON, LFDG, and CM, respectively). The concentration of net energy balance (milk plus tissue) tended to be affected by dietary treatment; HFDG did not differ from either CON or LFDG, but it was higher than CM (1.38, 1.36, 1.14, and 1.06 ± 0.11 Mcal/kg for HFDG, CON, LFDG, and CM, respectively). Results of this study indicate that milk production and dry matter intake were not affected by feeding common coproducts and that differences may result in whole-animal energy use; fat content of DDGS is a major factor affecting this.

Energy balance and diurnal variation in methane production as affected by feeding frequency in Jersey cows in late lactation.

Methane (CH4) production of ruminants typically increases with increased dry matter intake (DMI). However, few studies have observed the effects of feeding multiple times a day and its effects on diurnal variation in CH4 production and energy balance in late-lactation dairy cattle. A study using headbox-style indirect calorimetry and 12 multiparous (225 ± 16.2 d in milk; mean ± SD) lactating Jersey cows was conducted to determine the effects of feeding twice daily on diurnal variation in CH4 production and total energy balance. A crossover design with 14-d periods (10 d of adaption and 4 d of collection) was used to compare 2 treatments. Treatments consisted of either once a day feeding (1×; 100% of feed given at 1000 h) or twice a day feeding (2×; 50% of feed given at 1000 h and the final 50% at 2000 h) with a common diet fed in both treatments. Dry matter intake was not different between treatments, with a mean of 16.9 ± 0.88 kg/d. Once a day feeding tended to have greater milk yield compared with twice a day feeding (21.2 vs. 20.4 ± 1.59 kg/d, respectively). Milk fat and milk protein percentage were not different, with means of 6.18 ± 0.20% and 3.98 ± 0.08%, respectively. Total CH4 production did not differ between treatments, with a mean of 402.1 ± 20.8 L/d. Similarly, CH4 per unit of milk yield and DMI was not different between treatments, with means of 20.5 ± 1.81 and 23.8 ± 1.21 L/kg, respectively. Feeding frequency did not affect diurnal variation of hourly CH4 production, with a mean of 17.1 ± 0.74 L/h. A trend was observed for a treatment × hour interaction. Methane production per hour increased after the second feeding for cattle fed twice versus once daily. Gross energy, digestible energy, metabolizable energy, and balance (milk plus tissue) per kilogram of DMI did not differ by feeding frequency, with means of 4.41 ± 0.01, 3.05 ± 0.03, 2.63 ± 0.03, and 1.32 ± 0.08 Mcal/kg of DM, respectively. Metabolizable energy for maintenance was 146 kcal/kg of metabolic body weight, with an efficiency of converting metabolizable energy to net energy balance (milk plus tissue) of 76%. Nitrogen balance did not differ among treatments, with a mean balance of 17.3 ± 13.0 g/d. Therefore, total CH4 production and energy maintenance were not affected by feeding frequency. However, CH4 was variable throughout the day, and caution should be exercised when collecting CH4 samples at a limited number of time points because this may under- or overestimate total production.

The influence of fat and hemicellulose on methane production and energy utilization in lactating Jersey cattle.

Feeding fat to lactating dairy cows may reduce methane production. Relative to cellulose, fermentation of hemicellulose is believed to result in less methane; however, these factors have not been studied simultaneously. Eight multiparous, lactating Jersey cows averaging (±SD) 98 ± 30.8 d in milk and body weight of 439.3 ± 56.7 kg were used in a twice-replicated 4 × 4 Latin square to determine the effects of fat and hemicellulose on energy utilization and methane production using a headbox-type indirect calorimetry method. To manipulate the concentration of fat, porcine tallow was included at either 0 or 2% of the diet dry matter. The concentration of hemicellulose was adjusted by manipulating the inclusion rate of corn silage, alfalfa hay, and soybean hulls resulting in either 11.3 or 12.7% hemicellulose (dry matter basis). The resulting factorial arrangement of treatments were low fat low hemicellulose (LFLH), low fat high hemicellulose (LFHH), high fat low hemicellulose (HFLH), and high fat high hemicellulose (HFHH). Neither fat nor hemicellulose affected dry matter intake, averaging 16.2 ± 1.18 kg/d across treatments. Likewise, treatments did not affect milk production, averaging 23.0 ± 1.72 kg/d, or energy-corrected milk, averaging 30.1 ± 2.41 kg/d. The inclusion of fat tended to reduce methane produced per kilogram of dry matter intake from 24.9 to 23.1 ± 1.59 L/kg, whereas hemicellulose had no effect. Increasing hemicellulose increased neutral detergent fiber (NDF) digestibility from 43.0 to 51.1 ± 2.35%. Similarly, increasing hemicellulose concentration increased total intake of digestible NDF from 6.62 to 8.42 ± 0.89 kg/d, whereas fat had no effect. Methane per unit of digested NDF tended to decrease from 64.8 to 49.2 ± 9.60 L/kg with increasing hemicellulose, whereas fat had no effect. An interaction between hemicellulose and fat content on net energy balance (milk plus tissue energy) was observed. Specifically, increasing hemicellulose in low-fat diets tended to increase net energy balance, but this was not observed in high-fat diets. These results confirm that methane production may be reduced with the inclusion of fat, whereas energy utilization of lactating dairy cows is improved by increasing hemicellulose in low-fat diets.

Prediction of drinking water intake by dairy cows.

Mathematical models that predict water intake by drinking, also known as free water intake (FWI), are useful in understanding water supply needed by animals on dairy farms. The majority of extant mathematical models for predicting FWI of dairy cows have been developed with data sets representing similar experimental conditions, not evaluated with modern cows, and often require dry matter intake (DMI) data, which may not be routinely available. The objectives of the study were to (1) develop a set of new empirical models for predicting FWI of lactating and dry cows with and without DMI using literature data, and (2) evaluate the new and the extant models using an independent set of FWI measurements made on modern cows. Random effect meta-regression analyses were conducted using 72 and 188 FWI treatment means with and without dietary electrolyte and daily mean ambient temperature (TMP) records, respectively, for lactating cows, and 19 FWI treatment means for dry cows. Milk yield, DMI, body weight, days in milk, dietary macro-nutrient contents, an aggregate milliequivalent concentration of dietary sodium and potassium (NaK), and TMP were used as potential covariates to the models. A model having positive relationships of DMI, dietary dry matter (DM%), and CP (CP%) contents, NaK, and TMP explained 76% of variability in FWI treatment means of lactating cows. When challenged on an independent data set (n=261), the model more accurately predicted FWI [root mean square prediction error as a percentage of average observed value (RMSPE%)=14.4%] compared with a model developed without NaK and TMP (RMSPE%=17.3%), and all extant models (RMSPE%≥15.7%). A model without DMI included positive relationships of milk yield, DM%, NaK, TMP, and days in milk, and explained 63% of variability in the FWI treatment means and performed well (RMSPE%=17.9%), when challenged on the independent data. New models for dry cows included positive relationships of DM% and TMP along with DMI or body weight. The new models with and without DMI explained 75 and 54% of the variability in FWI treatment means of dry cows and had RMSPE% of 12.8 and 15.2%, respectively, when evaluated with the literature data. The study offers a set of empirical models that can assist in determining drinking water needs of dairy farms.

Technical Note: Collection and preparation techniques change nutrient composition of masticate collected from esophageally fistulated cattle.

Two experiments determined effects of collection and preparation techniques on nutrient composition of masticate samples from esophageally fistulated cattle. In Exp. 1, 12 esophageally fistulated cattle were maintained on 2 precollection diets, high CP (24% CP; = 6) or low CP (7.7% CP; = 6), for 8 d. On d 9, the esophageal plug was removed, screen bottom bags were attached, and each cow was offered fresh grass. Immediately after fresh grass sample collection was complete, dry grass (hay) was offered and a sample was collected. Blood samples were collected and analyzed for serum urea nitrogen concentration. Masticate samples of both fresh and dry grass were divided and each was either squeezed by hand until no more saliva could be removed or remained unsqueezed. In Exp. 2, 10 esophageally fistulated cattle were fitted with either screen ( = 5) or solid ( = 5) bottom collection bags after removal of the esophageal plug and presented grass hay, fresh grass, alfalfa hay, or fresh alfalfa. In Exp. 1, the precollection diet did not affect ( = 0.49) CP content of masticate even though serum urea nitrogen tended to be greater ( = 0.08) for high- vs. low-CP precollection diets. Forage harvest type offered (fresh vs. hay) interacted ( = 0.01) with preparation technique (squeezed vs. unsqueezed) for CP, where CP decreased in squeezed fresh samples ( < 0.001) but not in squeezed grass hay samples ( = 0.98). In Exp. 2, ingestion greatly increased levels of ash ( < 0.001). Crude protein was greater ( < 0.004) before ingestion for all samples except grass hay ( = 0.43). Levels of NDF were similar before and after ingestion ( > 0.15) for all samples except fresh alfalfa, which was greater after ingestion ( = 0.002). Ingestion status did not affect in vitro OM disappearance (IVOMD; > 0.34) except for grass hay, which was greater after ingestion ( < 0.001). Bag type (screen vs. solid) did not affect ash and NDF ( > 0.31), except for fresh alfalfa, which were greater ( < 0.03) for solid bottom bags. Bag type did not affect alfalfa CP ( = 0.71) but did affect grass CP, which was lower ( = 0.02) for solid bottom bags. Bag type did not affect IVOMD ( > 0.33). More ( = 0.01) fresh forage than hay was recovered through the esophageal opening. Previous diet did not impact masticate samples but squeezing impacted CP levels of high-quality forage and therefore should not be performed. Nutrient values should be reported on an OM basis.

Effects of stocking rate on forage nutrient composition of Nebraska Sandhills upland range when grazed in early summer.

Twelve 2-ha, Nebraska Sandhills upland range paddocks were used in a 2-yr study to determine effects of stocking rate on grazed forage nutrient value in early summer. Paddocks contain a mixture of native cool- and warm-season grasses with forbs as a minor component. Stocking rates were 0 (control), 0.57 (light), and 0.85 (heavy) animal unit months per ha, respectively. Three esophageally fistulated cows collected diet samples on May 14 (d 0), May 21 (d 7), May 28 (d 14), and June 4 (d 21) in 2013 and 2014. Ten quadrats per paddock were clipped and separated into current or previous year growth on each diet sampling date. All samples were analyzed for CP, NDF, and in vitro OM disappearance, which was adjusted to an in vivo OM basis (). For diet samples, treatment interacted ( < 0.001) with sampling date for CP, NDF, and IVOMD. However, treatment and date did not interact ( ≥ 0.32) for clipped samples. Diets collected in control paddocks had greater ( < 0.05) IVOMD and CP and lower NDF compared with light and heavy stocking rate paddocks on d 7, 14, and 21 but light stocking rate paddocks did not differ ( ≥ 0.26) from heavy stocking rate paddocks on any sampling day except d 21 NDF ( = 0.03). In general, previous year growth IVOMD, CP, and NDF were not affected ( ≥ 0.11) by treatment. Generally, current year growth IVOMD and CP were not affected ( ≥ 0.22) by treatment. Current year growth NDF did not differ ( = 0.23) among stocking rates on d 0, but control paddock had lower ( = 0.02) NDF than light and heavy on d 7, 14, and 21. In stocked paddocks, diet samples had greater ( < 0.01) IVOMD and CP and lower NDF than current and previous year growth except IVOMD on d 0 where diet and current year growth did not differ ( = 0.34). Generally, in control paddocks, current year growth and diet samples had greater ( < 0.01) IVOMD and CP than previous year growth. Control paddocks had greater ( < 0.01) forage accumulation than stocked paddocks on d 7, 14, and 21; however, light and heavy stocked paddocks did not differ ( ≥ 0.29). On d 0 it was estimated that 96% of the diet was current year growth and only 4% previous year growth, whereas on d 21, 70% of the diet was current year growth vs. 30% previous year growth. Stocking pastures decrease current year growth forage mass and therefore diet nutritive value by forcing cattle to consume diets containing previous year growth in early summer.