Diversity and community structure of anaerobic gut fungi in the rumen of wild and domesticated herbivores.

The rumen houses a diverse community that plays a major role in the digestion process in ruminants. Anaerobic gut fungi (AGF) are key contributors to plant digestion in the rumen. Here, we present a global amplicon-based survey of the rumen AGF mycobiome by examining 206 samples from 15 animal species, 15 countries, and 6 continents. The rumen AGF mycobiome was highly diverse, with 81 out of 88 currently recognized AGF genera or candidate genera identified. However, only six genera (Neocallimastix, Orpinomyces, Caecomyces, Cyllamyces, NY9, and Piromyces) were present at >4% relative abundance. AGF diversity was higher in members of the families Antilocapridae and Cervidae compared to Bovidae. Community structure analysis identified a pattern of phylosymbiosis, where host family (10% of total variance) and species (13.5%) partially explained the rumen mycobiome composition. As well, diet composition (9%-19%), domestication (11.14%), and biogeography (14.1%) also partially explained AGF community structure; although sampling limitation, geographic range restrictions, and direct association between different factors hindered accurate elucidation of the relative contribution of each factor. Pairwise comparison of rumen and fecal samples obtained from the same subject (n = 13) demonstrated greater diversity and inter-sample variability in rumen versus fecal samples. The genera Neocallimastix and Orpinomyces were present in higher abundance in rumen samples, while Cyllamyces and Caecomyces were enriched in fecal samples. Comparative analysis of global rumen and feces data sets revealed a similar pattern. Our results provide a global view of AGF community in the rumen and identify patterns of AGF variability between rumen and feces in herbivores Gastrointestinal (GI) tract.IMPORTANCERuminants are highly successful and economically important mammalian suborder. Ruminants are herbivores that digest plant material with the aid of microorganisms residing in their GI tract. In ruminants, the rumen compartment represents the most important location where microbially mediated plant digestion occurs, and is known to house a bewildering array of microbial diversity. An important component of the rumen microbiome is the anaerobic gut fungi (AGF), members of the phylum Neocallimastigomycota. So far, studies examining AGF diversity have mostly employed fecal samples, and little is currently known regarding the identity of AGF residing in the rumen compartment, factors that impact the observed patterns of diversity and community structure of AGF in the rumen, and how AGF communities in the rumen compare to AGF communities in feces. Here, we examined the rumen AGF diversity using an amplicon-based survey targeting a wide range of wild and domesticated ruminants (n = 206, 15 different animal species) obtained from 15 different countries. Our results demonstrate that while highly diverse, no new AGF genera were identified in the rumen mycobiome samples examined. Our analysis also indicate that animal host phylogeny, diet, biogeography, and domestication status could play a role in shaping AGF community structure. Finally, we demonstrate that a greater level of diversity and higher inter-sample variability was observed in rumen compared to fecal samples, with two genera (Neocallimastix and Orpinomyces) present in higher abundance in rumen samples, and two others (Cyllamyces and Caecomyces) enriched in fecal samples. Our results provide a global view of the identity, diversity, and community structure of AGF in ruminants, elucidate factors impacting diversity and community structure of the rumen mycobiome, and identify patterns of AGF community variability between the rumen and feces in the herbivorous GI tract.

Temporal progression of anaerobic fungal communities in dairy calves from birth to maturity.

Establishment of microbial communities in neonatal calves is vital for their growth and overall health. While this process has received considerable attention for bacteria, our knowledge on temporal progression of anaerobic gut fungi (AGF) in calves is lacking. Here, we examined AGF communities in faecal samples from six dairy cattle collected at 24 different time points during the pre-weaning (days 1-48), weaning (days 48-60), and post-weaning (days 60-360) phases. Quantitative polymerase chain reaction indicated that AGF colonisation occurs within 24 h after birth, with loads slowly increasing during pre-weaning and weaning, then drastically increasing post-weaning. Culture-independent amplicon surveys identified higher alpha diversity during pre-weaning/weaning, compared to post-weaning. AGF community structure underwent a drastic shift post-weaning, from a community enriched in genera commonly encountered in hindgut fermenters to one enriched in genera commonly encountered in adult ruminants. Comparison of AGF community between calves day 1 post-birth and their mothers suggest a major role for maternal transmission, with additional input from cohabitating subjects. This distinct pattern of AGF progression could best be understood in-light of their narrower niche preferences, metabolic specialisation, and physiological optima compared to bacteria, hence eliciting a unique response to changes in feeding pattern and associated structural GIT development during maturation.

Energy balance affects pulsatile secretion of luteinizing hormone from the adenohypophesis and expression of neurokinin B in the hypothalamus of ovariectomized gilts.

The pubertal transition of gonadotropin secretion in pigs is metabolically gated. Kisspeptin (KISS1) and neurokinin B (NKB) are coexpressed in neurons within the arcuate nucleus of the hypothalamus (ARC) and are thought to play an important role in the integration of nutrition and metabolic state with the reproductive neuroendocrine axis. The hypothesis that circulating concentrations of luteinizing hormone (LH) and expression of KISS1 and tachykinin 3(TAC3, encodes NKB) in the ARC of female pigs are reduced with negative energy balance was tested using ovariectomized, prepubertal gilts fed to either gain or lose body weight. Restricted feeding of ovariectomized gilts caused a rapid and sustained metabolic response characterized by reduced concentrations of plasma urea nitrogen, insulin, leptin, and insulin-like growth factor-1 and elevated concentrations of free fatty acids. The secretory pattern of LH shifted from one of low amplitude to one of high amplitude, which caused overall circulating concentrations of LH to be greater in restricted gilts. Nutrient-restricted gilts had greater expression of follicle-stimulating hormone and gonadotropin-releasing hormone receptor, but not LH in the anterior pituitary gland. Expression of KISS1 in the ARC was not affected by dietary treatment, but expression of TAC3 was greater in restricted gilts. These data are consistent with the idea that hypothalamic expression of KISS1 is correlated with the number of LH pulse in pig, and further indicate that amplitude of LH pulses may be regulated by NKB in the gilt.

Association of glucose metabolism and insulin resistance with feed efficiency and production traits of finishing beef steers.

Increasing nutrient utilization efficiency is an important component of enhancing the sustainability of beef cattle production. The objective of this experiment was to determine the association of glucose metabolism and insulin resistance with dry matter intake (DMI), average daily gain (ADG), gain:feed ratio (G:F), and residual feed intake (RFI). Steers (n = 54; initial body weight = 518 ±â€…27.0 kg) were subjected to an intravenous glucose tolerance test (IVGTT) where glucose was dosed through a jugular catheter and serial blood samples were collected. Three days after the last group's IVGTT, steers began a 63-d DMI and ADG test. Body weight was measured on days 0, 1, 21, 42, 62, and 63, and DMI was measured using an Insentec Roughage Intake Control system (Hokofarm Group, Emmeloord, the Netherlands). To examine relationships between DMI, ADG, G:F, and RFI with IVGTT measurements, Pearson correlations were calculated using Proc Corr of SAS 9.4 (SAS Inst. Inc., Cary, NC). Additionally, cattle were classified based on DMI, ADG, RFI, and G:F, where the medium classification was set as mean ±â€…0.5 SD, the low classification was < 0.5 SD from the mean, and the high classification was > 0.5 SD from the mean. No associations between DMI and IVGTT parameters were observed, and no differences were detected when classifying cattle as having low, medium, or high DMI. Peak insulin concentration in response to the IVGTT tended to be correlated with ADG (r = 0.28; P = 0.07), indicating cattle with greater ADG tend to have a greater insulin release in response to glucose. Glucose nadir concentrations tended to be positively correlated with ADG (r = 0.26; P = 0.10). Additionally, the glucose nadir was greater in high-ADG steers (P = 0.003). The association of greater glucose nadir with high-ADG could indicate that high-ADG steers do not clear glucose as efficiently as low-ADG steers, potentially indicating increased insulin resistance. Further, RFI was not correlated with IVGTT measurements, but low RFI steers had a greater peak glucose concentration (P = 0.040) and tended to have a greater glucose area under the curve (P = 0.09). G:F was correlated with glucose area under the curve (r = 0.33; P = 0.050), glucose nadir (r = 0.35; P = 0.011), and insulin time to peak (r = 0.39; P = 0.010). These results indicate that glucose metabolism and insulin signaling are associated with growth and efficiency, but the molecular mechanisms that drive these effects need to be elucidated.

Feed efficiency is an important component of improving the sustainability of beef production. There is a need to understand how metabolism influences feed efficiency. This experiment aimed to explore the association of glucose metabolism and insulin resistance with feed intake, growth, and efficiency of finishing beef cattle. The results indicate that there is a relationship between insulin resistance and improved efficiency measured as the ratio of growth to feed intake. The findings of this experiment are novel as they show a relationship between insulin resistance and feed efficiency and indicate further research is needed to determine the mechanisms of insulin resistance in ruminants that contribute to improved feed efficiency.

Genes involved in the cholecystokinin receptor signaling map were differentially expressed in the jejunum of steers with variation in residual feed intake.

The jejunum is a critical site for nutrient digestion and absorption, and variation in its ability to take up nutrients within the jejunum is likely to affect feed efficiency. The purpose of this study was to determine differences in gene expression in the jejunum of beef steers divergent for residual feed intake (RFI) in one cohort of steers (Year 1), and to validate those genes in animals from a second study (Year 2). Steers from Year 1 (n = 16) were selected for high and low RFI. Jejunum mucosal tissue was obtained for RNA-seq. Thirty-two genes were differentially expressed (PFDR≤0.15), and five were over-represented in pathways including inflammatory mediator, cholecystokinin receptor (CCKR) signaling, and p38 MAPK pathways. Several differentially expressed genes (ALOX12, ALPI, FABP6, FABP7, FLT1, GSTA2, MEF2B, PDK4, SPP1, and TTF2) have been previously associated with RFI in other studies. Real-time qPCR was used to validate nine differentially expressed genes in the Year 1 steers used for RNA-seq, and in the Year 2 validation cohort. Six genes were validated as differentially expressed (P < 0.1) using RT-qPCR in the Year 1 population. In the Year 2 population, five genes displayed the same direction of expression as the Year 1 population and 3 were differentially expressed (P < 0.1). The CCKR pathway is involved in digestion, appetite control, and regulation of body weight making it a compelling candidate for feed efficiency in cattle, and the validation of these genes in a second population of cattle is suggestive of a role in feed efficiency.

Impact of endophyte-infected tall fescue seed consumption on endocrine changes associated with intake regulation and post-absorptive metabolism in growing steers.

Fescue toxicosis is a syndrome occurring from the consumption of endophyte-infected tall fescue and results in substantial economic losses to the beef industry primarily from reduced growth accompanied by decreased dry matter intake (DMI); however, the associations characterizing this reduction in DMI have yet to be elucidated. The objective of this experiment was to identify endocrine changes associated with intake regulation post-consumption of endophyte-infected tall fescue seed (E+). Twelve Holstein steers were stratified by body weight and assigned to 1 of 3 treatments (n=4): 0 ppm ergovaline (ERV), 1.8 ppm ERV, or 2.7 ppm ERV. Treatments were achieved by combining differing proportions of ground E+ and non-endophyte-infected tall fescue seed. Steers were adapted to their diets for 7 d followed by a 7 d DMI collection period. Within treatment, steers were assigned to a sampling day (d 16 or d 17). Blood samples were collected every 20 min for 8 h, beginning 1 h before feeding. Intake data was analyzed using the MIXED procedure of SAS 9.4 (SAS Inst. Inc., Cary, NC) with treatment, day, and the interaction as fixed effects. Hormone and metabolite data were analyzed with the fixed effect of treatment, time, and the interaction including time as a repeated measure and orthogonal contrasts. Dry matter intake was linearly decreased with increasing ERV in the diet (P < 0.001). Insulin and leptin concentrations exhibited a quadratic effect (P = 0.018 and P = 0.005) with insulin concentrations highest for the 2.7 ppm treatment and leptin concentrations highest for the 1.8 ppm treatment. No differences were detected for active ghrelin or ß-hydroxybuytrate concentrations among treatment groups. Further, steers consuming both the 1.8 and 2.7 ppm ERV treatments had lower prolactin concentrations compared to the 0 ppm treatment (quadratic, P= 0.019). Glucose concentrations had a tendency for a linear increase as ERV concentrations increased (P = 0.091). A treatment × time interaction (P = 0.002) was noted in NEFA concentrations, with the 1.8 ppm ERV treatment showing increased pre-feeding concentrations, and the 2.7 ppm ERV treatment exhibiting elevated NEFA concentrations as time post-feeding progressed. The results suggest that E+ consumption reduces intake likely through alterations in intake-related hormones and post-absorptive metabolism and contributes to our current understanding of E+ effects on intake reduction while providing avenues for future research.

Technical note: Validation of energy dispersive X-ray fluorescence for determination of indigestible markers in ruminant fecal and rumen fluid samples.

Determination of digestibility and passage rate is important for further understanding of nutrient utilization and thereby aids in improving nutrient utilization efficiency. Titanium dioxide and chromium ethylenediaminetetraacetic acid are commonly used as indigestible markers for determining passage rates of diets to aid in determination of digestibility. Analyzing Ti and Cr involves the use of procedures such as acid digestions, inductively coupled plasma spectroscopy, and atomic absorption. These commonly used methodologies involve hazardous chemicals, destruction of samples, and low sample throughput. The objective of this experiment was to develop and validate an accurate and precise method for measuring both Ti and Cr using energy dispersive X-ray fluorescence (ED-XRF). Energy dispersive X-ray fluorescence is an analytical technique used for analyzing elements in various sample types. The samples were added to the ED-XRF machine and irradiated with X-rays. The intensity of the X-rays emitted (termed fluorescent X-rays) was used for calculation of the concentration of the element. The method for Ti was constructed using fecal samples from cattle consuming three different diet types (finishing diet, dairy lactation diet, and grazing native range pasture). The Cr method was developed for rumen fluid analysis. We compared the machine-calculated concentrations of each element to the concentration calculated by a standard curve. For both the Ti and Cr, the standard curve-calculated value had a lower percent difference overall at 4.56% and 12.59%, respectively, compared to the machine percent difference of 8.35% and 16.38% for Ti and Cr, respectively. To determine accuracy and precision of the method, samples were spiked with various amounts of Ti or Cr and measured for their respective compounds with percent recovery and inter- and intra-assay CV-calculated thereafter. The average recovery for Ti across all diet types was 100.3%, and the recovery for Cr in rumen fluid was 95.7%. The average inter- and intra-assay CV for Ti, across all diet types, were 9.70% and 2.16%, respectively. For Cr, the average inter- and intra-assay CV were 5.42% and 8.45%, respectively. The ED-XRF method requires minimal additional chemicals, is cost-effective, and allows for sample preservation as well as a high throughput of samples. Our results indicate utilization of ED-XRF is an accurate and precise method for determination of Ti in feces and Cr in rumen fluid.

Markers are elements supplied to an animal to help determine the digestibility of various feedstuffs. Studying digestibility allows us to improve how nutrients are used by animals and reduce production costs and environmental impact. The method proposed here uses an X-ray fluorescence machine to analyze the markers with no hazardous chemicals, low labor input, and cost. Even though this equipment utilizes radiation, the construction of the equipment eliminates the exposure of operators to radiation. Training and monitoring will differ between institutions. Traditional methods use hazardous chemicals and have higher cost and labor use. We determined that this is a valid method for analyzing titanium in fecal samples and chromium in rumen fluid.

Dam parity affects fetal growth, placental size, and neonatal metabolism in spring-born beef calves.

To determine effects of dam parity on perinatal nutrient availability in beef cattle, data and samples were collected from 18 primiparous and 35 multiparous spring-calving Sim-Angus dams and their calves. Time to stand was recorded and neonatal vigor assessed. Jugular blood was collected from a subset of calves at 0 (post-standing and pre-suckling) 6, 12, 24, 48, and 72 h of age, and blood chemistry panels were completed. Expelled placentas were dissected, dried, and weighed. Prepartum maternal circulating glucose, non-esterified fatty acids (NEFA), triglycerides, and urea N were analyzed. All statistical models included the fixed effect of dam parity, and calf sex (when P ≤ 0.25) was included for calf and placental variables. Effects of sampling hour, and parity × hour were included for calf metabolites over time using repeated measures. Multiparous dams had greater body weight prepartum (P < 0.001) but similar (P = 0.25) body condition score. Maternal circulating urea N and triglycerides were greater (P ≤ 0.05) in multiparous dams pre-calving. Calves born to primiparous dams weighed 10% less (P ≤ 0.04) at birth with smaller (P ≤ 0.01) heart and abdominal girths. Cotyledonary, intercotyledonary, and total placental masses were less (P ≤ 0.05) for primiparous dams. Dam parity did not affect (P ≥ 0.58) calf time to stand, vigor score at 10 min, or rectal temperature. Serum glucose was greater (P = 0.03) at 0 h but less (P ≤ 0.04) at all other hours in calves from primiparous dams. Calves from primiparous dams had greater (P ≤ 0.02) serum NEFA at 6, 12, and 24 h although plasma triglycerides were greater (P < 0.001) at 6 h. Calves from primiparous dams had greater (P ≤ 0.04) serum urea N at 12 h and creatinine at 12 and 24 h. Plasma insulin was greater (P ≤ 0.04) in calves from multiparous dams at 12, 48, and 72 h, but parity did not affect (P ≥ 0.18) serum total protein or plasma cortisol. Serum aspartate aminotransferase was greater (P ≤ 0.04) at 6 and 24 h, creatine kinase was greater at 24 h, and gamma-glutamyl transpeptidase was less (P ≤ 0.04) at 6, 12, and 24 h, for calves from primiparous dams. Calves born to primiparous dams had greater (P ≤ 0.02) total bilirubin and direct bilirubin at 12 and 24 h. Data indicate that calves born to first-parity heifers had decreased perinatal nutrient availability, resulting in reduced fetal and placental growth, as well as greater energy reserve mobilization and metabolic indicators of stress as neonates.

Approximately two-thirds of beef calf deaths prior to weaning occur within the first 3 wk after birth. The goal to have heifers produce their first calf by 2 yr of age likely contributes to factors that limit nutrients available for fetuses and calves immediately after birth. However, little is known about differences in heifers (first parity) and cows (later parities) regarding factors affecting calf resilience, such as fetal growth and calf metabolism shortly after birth. Our data show that calves born to first-parity heifers had altered nutrient availability, demonstrated through smaller placentas, lower birth weights, and altered metabolites in early life. Although calves had similar vigor and ability to maintain body temperature, calves born to first-parity heifers had to mobilize more energy and had lower insulin during the first 3 d post-birth. Calves born to first-parity heifers had greater indicators of stress during the first 72 h of life not associated with calving difficulties. Overall, these effects may have increased morbidity and mortality of calves born to first-parity heifers if they were in a less intensively-managed system. Better understanding of challenges faced by calves born to first-parity dams provides opportunities for their improved management.

Association of maternal temperament and offspring disposition on growth performance.

Animal behavior is complex and varies in definition, depending upon specific traits under observation. Temperament is one component of behavior, that in cattle, is described as the level of fearfulness to a novel or threatening environment. Temperament is a heritable trait which is important since aggressiveness and docility contribute to reproductive success, growth, and carcass quality. We observed maternal temperament at calving and the subsequent influence, if any, on offspring disposition at weaning and their effects collectively on growth performance and carcass traits. Maternal behaviors at calving were observed at four locations within the University of Arkansas system. Cows were assigned a maternal disposition score (MDS) at calving; a scale from 1 to 5 in which aggression decreases. At weaning, calves were assigned a chute score (CS); a scale from 1 to 6 in which aggression increases. Both scoring systems have been previously established. Blood was collected during the 56-d backgrounding period postweaning for blood glucose analysis. Data were analyzed using GLIMMIX procedures of SAS (α = 0.05). The relationship between the two scoring systems was determined with a Pearson correlation (P = 0.22). Animal was the experimental unit and blocked by location for all dependent variables. Location, sex, diet, and MDS were included in the class as covariables for all growth performance and carcass data related to CS. Cows that were more aggressive birthed heavier calves (P < 0.01) compared to indifferent cows. Calves born to cows with either very aggressive or very attentive (MDS of 2 or 3, respectively) scores were heavier upon feedlot entry (P = 0.03) compared to those from indifferent or apathetic cows (MDS of 4 or 5, respectively). Calves defined as nervous and restless (CS of 3 and 2, respectively) were heavier at weaning compared to docile calves (P < 0.01). Restless calves were heavier compared to nervous calves upon arrival and exiting the feedlot (P ≤ 0.01). Calves that were docile at weaning had greater marbling compared to calves that were restless (P ≤ 0.01). Calves that were restless at weaning had greater lean muscle area compared to calves that were nervous (P = 0.05). No definitive relationship was determined between dam and calf temperament. However, the results suggest temperament does impact growth performance and carcass traits but whether the influence comes from the dam or calf temperament, specifically, remains unanswered.

Effects of adding ruminal propionate on dry matter intake and glucose metabolism in steers fed a finishing ration.

The objective of this experiment was to determine if supplying additional propionate to the rumen alters dry matter intake (DMI), feeding behavior, glucose metabolism, and rumen fluid metabolites in steers fed a finishing diet. Ruminally cannulated steers (n = 6) were fed a finishing diet ad libitum. Steers were randomly assigned to one of three treatments in a 3 × 6 Latin rectangle design with three 15 d periods. Treatments of no Ca propionate (Control), 100 g/d (Low), or 300 g/d (High) were ruminally dosed twice daily. Individual intake was measured using an Insentec feeding system. Pre-feeding blood samples were collected on day 7 and rumen fluid samples were collected on day 13. An intravenous glucose tolerance test (IVGTT) was conducted on day 14 and liver biopsies were collected on day 15. Liver samples were analyzed for expression of genes involved in gluconeogenesis. Data were analyzed using a mixed model with period, treatment, day, and their interaction included, with day and minute within period as a repeated measure and steer as a random effect. Meal size (P = 0.049), meal frequency (P = 0.046), and DMI (P < 0.001) were decreased in High steers. Day 7 plasma glucose (P = 0.23) and lactate (P = 0.47) were not affected by treatment, but insulin was decreased (P = 0.008) and non-esterified fatty acids were increased (P = 0.044) in the High treatment compared with the Control. Rumen fluid lactate was decreased (P = 0.015) in the High treatment compared with the Low treatment. Total VFA concentrations did not differ (P = 0.88) between treatments. There was treatment × time interaction for proportions of acetate and propionate (P < 0.001) and the acetate:propionate ratio (P = 0.005). The effect on acetate was due to a decrease in the High treatment 2 h after dosing the treatment. Propionate proportions were greater in the High treatment than the Control at all time points and differed from the Low except at 0 h. Propionate treatments had no major effects on the glucose and insulin parameters observed in the IVGTT other than a tendency (P = 0.09) for an increased insulin time to peak. These data indicate that exogenous propionate decreases DMI but the decrease in propionate from fermentation due to reduced DMI might negate the supply of exogenous propionate in VFA supply to the animal. Mechanisms other than hepatic oxidation of propionate might be responsible for DMI regulation.

Propionate metabolism by the liver is thought to be a key regulator of appetite and feed intake of animals, including cattle. Previous research has shown that providing propionate to the rumen of cattle decreases feed intake. Propionate is also a major contributor to glucose for cattle to use as an energy source for growth and maintenance. In this experiment, it was hypothesized that increasing ruminal propionate would depress feed intake and decrease insulin sensitivity. Supplying 300 g of propionate a day to the rumen decreased feed intake and increased the proportion of propionate in the rumen fluid of steers. However, when propionate production was calculated based on feed intake, there was likely no difference in propionate supply to the animal. The lack of increase in propionate supply to the animal could explain the lack of effect on glucose metabolism, insulin sensitivity, and liver gene expression. The lack of an increase in propionate also indicates that the effect of propionate on feed intake could be due to alternative mechanisms than liver metabolism of propionate.

Patterns and determinants of the global herbivorous mycobiome.

Despite their role in host nutrition, the anaerobic gut fungal (AGF) component of the herbivorous gut microbiome remains poorly characterized. Here, to examine global patterns and determinants of AGF diversity, we generate and analyze an amplicon dataset from 661 fecal samples from 34 mammalian species, 9 families, and 6 continents. We identify 56 novel genera, greatly expanding AGF diversity beyond current estimates (31 genera and candidate genera). Community structure analysis indicates that host phylogenetic affiliation, not domestication status and biogeography, shapes the community rather than. Fungal-host associations are stronger and more specific in hindgut fermenters than in foregut fermenters. Transcriptomics-enabled phylogenomic and molecular clock analyses of 52 strains from 14 genera indicate that most genera with preferences for hindgut hosts evolved earlier (44-58 Mya) than those with preferences for foregut hosts (22-32 Mya). Our results greatly expand the documented scope of AGF diversity and provide an ecologically and evolutionary-grounded model to explain the observed patterns of AGF diversity in extant animal hosts.

TECHNICAL NOTE: Analysis of volatile fatty acids in rumen fluid by gas chromatography mass spectrometry using a dimethyl carbonate extraction.

Analysis of rumen fluid volatile fatty acids (VFA) is typically conducted by injecting acidified aqueous rumen fluid into a gas chromatograph (GC) with a flame ionization detector (FID). Aqueous samples are highly problematic because of the large vapor volume that can lead to poor peak shape and contamination of inlets, potentially causing sample carryover. Methods using aqueous samples are not well suited for use in a mass spectrometer (MS) detector system. The objective of this project was to validate a dimethyl carbonate (DMC) extraction process and GCMS method for rumen VFA analysis. To perform the extraction, 100 µL of sample, KHSO4 (500 g/L), and 2-ethylbutyrate (internal standard; 8.5 mM) were added to a microcentrifuge tube (in order) followed by 1 mL of DMC. The mixture was thoroughly vortexed and centrifuged. The organic layer (top) was removed and placed in a GC vial. The DMC extract was injected (0.5 µL) into an Agilent 5977B GCMS (8:1 split injection) with a polar DB-FFAP column. The column was held at 105 °C for 5 min, increased at 10 °C/min to 150 °C, then 65 °C/min to 240 °C, and held constant for 10 min. The peak area of acetate relative to the internal standard is linear from approximately 2 mM to at least 130 mM and encompasses the expected values of rumen concentrations for the other VFA. Recovery of VFA from spiked rumen fluid was tested at three concentrations in rumen fluid from steers fed a finishing diet or grazing wheat pasture. Recovery was not affected by the diet of the animals (P > 0.10) or the amount of VFA spiked (P > 0.19) for acetate, propionate, isobutyrate, or butyrate. There was an interaction of amount of VFA spiked and the diet of the animal (P = 0.021) for valerate and a tendency for an interaction (P = 0.051) for isovalerate, due to the recovery of the VFA being lower in the medium spike amount in rumen fluid from cattle on wheat pasture. Overall, recovery was greatest for propionate (101.9 ± 1.67%) and lowest for valerate (95.7 ± 1.95%). Including the 10-min hold at 240 °C at the end of each run prevented carryover from sample to sample. This method appears to perform well in a GCMS system and accurately and precisely quantifies rumen fluid VFA.

Scientists need to measure the end-products of microbial digestion called volatile fatty acids in the gastrointestinal tract of ruminants for many reasons. The methods currently available for the analysis of volatile fatty acids have evolved over time along with new technology. Many of the methods available are either not compatible with a mass spectrometer detector or require hazardous chemicals to prepare the samples. The method described here uses a less-hazardous chemical to prepare the samples for analysis with a mass spectrometer detector. The method tested in this manuscript was adequate in preparing samples for volatile fatty acid analysis.

Retained energy in lactating beef cows; effects on maintenance energy requirement and voluntary feed intake.

The objectives of these experiments were to determine the relationship between maintenance requirements and energy partitioned to maternal tissue or milk production in limit-fed Angus cows and to determine the relationship between retained energy during the lactation period to dry-period voluntary forage intake (VDMI). Twenty-four mature fall-calving Angus cows were used in a 79-d study during late lactation to establish daily metabolizable energy required for maintenance (MEm). Cows were individually fed daily a mixed diet (2.62 Mcal MEl/kg, 18.2% crude protein) to meet energy and protein requirements of 505 kg beef cows producing 8.2 kg milk daily. If cow BW changed by ±9 kg from initial BW, daily feed intake was adjusted to slow BW loss or reduce BW gain. Milk yield and composition were determined on 3 occasions throughout the study. Maintenance was computed as metabolizable energy intake minus retained energy assigned to average daily maternal tissue energy change, average daily milk energy yield, and average daily energy required for pregnancy. After calves were weaned, cows were fed a low-quality grass hay diet (8.2% crude protein, 65% NDF) and VDMI was measured for 21 days. Lactation maintenance energy was 83% the default value recommended by NASEM (2016. Nutrient Requirements of Beef Cattle: Eighth Revised Edition.) for lactating Angus cows. Increasing lactation-period retained energy (decreasing BW loss and increasing milk energy yield) was associated with lower maintenance energy requirements (P < 0.01; R 2 = 0.92). Increased residual daily gain during lactation was associated with lower lactation maintenance energy requirements (P = 0.05; R 2 = 0.17). Post-weaning VDMI was not related to late-lactation milk energy production, although sensitive to lactation period BCS and BW loss. These results contradict previous reports, suggesting that maintenance requirements increase with increasing milk yield.

Effects of supplementation rate of an extruded dried distillers' grains cube fed to growing heifers on voluntary intake and digestibility of bermudagrass hay.

Our objectives were to 1) investigate the difference in chemical composition and disappearance kinetics between loose dried distillers' grains (DDG) and extruded DDG cubes and 2) evaluate the effects of supplementation rate of extruded DDG cubes on voluntary dry matter intake (DMI), rate and extent of digestibility, and blood parameters of growing beef heifers offered ad libitum bermudagrass (Cynodon dactylon) hay. To characterize the changes in chemical composition during the extrusion process, loose and extruded DDG were evaluated via near-infrared reflectance spectroscopy, and dry matter (DM) disappearance kinetics were evaluated via time point in situ incubations. Extruded DDG cubes had greater (P ≤ 0.01) contents of fat, neutral detergent insoluble crude protein, and total digestible nutrients, but lower (P ≤ 0.01) neutral and acid detergent fiber than loose DDG. Additionally, the DM of extruded DDG cubes was more immediately soluble (P < 0.01), had greater (P < 0.01) effective degradability and lag time, and tended (P = 0.07) to have a greater disappearance rate than loose DDG. In the 29-d supplementation rate study, 23 Charolais-cross heifers were randomly assigned to one of four supplemental treatments: 1) control, no supplement; 2) low, 0.90 kg DDG cubes per d; 3) intermediate, 1.81 kg DDG cubes per d; or 4) high, 3.62 kg DDG cubes per d. Titanium dioxide was used as an external marker to estimate fecal output and particulate passage rate (Kp). Blood was collected from each animal to determine supplementation effects on blood metabolites. Indigestible neutral detergent fiber was used as an internal marker to assess the rate and extent of hay and diet DM digestibility (DMD). Increasing supplementation rate increased Kp and total diet DMI linearly (P < 0.01), yet linearly decreased (P < 0.01) hay DMI. Hay DMD decreased quadratically (P < 0.01), while total diet DMD increased linearly (P < 0.01) with increased DDG cube inclusion. Supplemented heifers had greater (P = 0.07) blood urea nitrogen concentrations than control animals 4 h post-supplementation. Intermediate and high rates of supplementation resulted in lower (P < 0.01) serum nonesterified fatty acid concentrations post-supplementation than control heifers. Concentrations of serum glucose and lactate were greatest (P ≤ 0.06) 8 h post-supplementation. Our results suggest that extruded DDG cubes may be an adequate supplement for cattle consuming moderate-quality forage, and further research is warranted.

Growing cattle are oftentimes provided supplemental concentrate as a source of protein and energy in order to meet performance goals when consuming low-quality forages. The effects of supplemental concentrate on forage intake vary, which may be related to the quality of forage and the characteristics of the supplement being evaluated. Dried distillers' grains (DDG) are a by-product of ethanol production and have become a common supplement for growing cattle due to the increased energy and rumen undegradable protein content. A stable DDG cube made via a novel extrusion process may be advantageous for pasture supplementation due to the reduced risk of loss of product from wind and soil mixing that is common with loose DDG. The effects of supplementation rate of traditional concentrate sources on forage intake are abundant, but research regarding extruded DDG cubes is almost nonexistent. Thus, our objective was to evaluate extruded DDG cube supplementation rate (0, 0.90, 1.81, or 3.62 kg DDG cubes per d) for growing cattle on voluntary intake and digestibility of moderate-quality forage. Although increasing supplementation rate reduced forage intake and digestibility, total diet intake and digestibility were increased. Our results suggested that extruded DDG cubes have potential as a supplement for cattle consuming moderate-quality forage.

Effect of vitamin D source and dietary cation-anion difference in peripartum dairy cows on calcium homeostasis and milk production.

The objective of this experiment was to determine the effects of dietary vitamin D source on serum calcium (Ca), urinary Ca excretion, and milk production when fed in combination with a prepartum acidogenic negative dietary cation-anion difference (DCAD) diet. Nonlactating, pregnant multiparous cows (n = 15), balanced for breed (Holstein n = 9 and Jersey n = 6), and previous mature equivalent milk production, were assigned to one of three treatments (five cows/treatment), consisting of a control (PCH; positive DCAD, 8.9 mEq/100 g DM) and two negative DCAD diets (-15.4 mEq/100 g DM), one with vitamin D3 (cholecalciferol; NCH) and one with 25-hydroxyvitamin D3 (calcidiol; NCA; DSM nutritional products). The treatments were formulated to provide 1.95 mg/d of vitamin D and were fed 28 d prior to expected calving date. Delivery of vitamin D sources was accomplished by manufacture of a pellet and 2 kg of these pellets were individually fed simultaneously each day along with 2 kg of ground corn daily at 0800 hours. Negative DCAD treatments were formulated to provide 0.46 kg/d of Animate (Phibro Animal Health) and, if needed, additional Animate was top-dressed at each feeding to achieve a urine pH between 5.5 and 6.0 based on the previous day's urine pH. Close-up cows had ad libitum access to chopped bermudagrass (Cynodon dactylon L.) hay and hay intake was measured using SmartFeed Pro systems (C-Lock Inc.; Rapid City, SD). Prepartum urine and serum samples were collected weekly and serum was collected 36, 48, and 72 h post-calving. Prepartum dry matter intake (DMI) as a percent of body weight was not (P = 0.66) affected by treatments. Cows fed NCH and NCA had greater (P = 0.02) prepartum serum Ca than PCH and tended to have greater urinary Ca excretions (P = 0.10). Average postpartum serum Ca (mg/dL) was greater (P = 0.05) for cows fed NCH (8.8) compared with PCH (7.8), whereas NCA (8.4) was numerically intermediate and not (P > 0.05) different from either of the other treatments. Postpartum DMI was not affected by treatment (P = 0.39). Daily milk yield (MY) (kg/d) was greatest (P < 0.01) for NCA (37.5) compared with the other treatments and NCH (34.1) was intermediate and greater than PCH (29.9). These results suggest that an acidogenic prepartum diet in combination with vitamin D was effective in maintaining peripartum serum Ca and the 25-hydroxy form of vitamin D improved MY compared with NCH in early lactation.

Effects of increasing calcium propionate in a finishing diet on dry matter intake and glucose metabolism in steers.

The objective of this study was to determine whether increasing propionate alters dry matter intake (DMI), glucose clearance rate, blood metabolites, insulin concentrations, and hepatic gene expression in steers fed a finishing diet. Holstein steers (n = 15; BW = 243 ± 3.6 kg) were individually fed a finishing diet ad libitum. Steers were allocated by body weight (BW) to receive: no Ca propionate (Control), 100 g/d Ca propionate (Low), or 300 g/d Ca propionate (High) in the diet. Orts were collected and weighed daily to determine DMI. Blood samples were collected on days 0, 7, and 21, and BW recorded on days 0, 14, and 28. An intravenous glucose tolerance test (IVGTT) was conducted on days 14 and 28 of the trial. Liver biopsies were collected on day 33 for gene expression analysis. Blood samples were analyzed for whole blood glucose and lactate, plasma non-esterified fatty acids (NEFAs), and insulin concentrations. Data were analyzed using a mixed model with treatment, day and their interaction included, with day and minute as a repeated measure. The control treatment had greater (P < 0.01) DMI than low and high steers. Body weight was increased in control steers on days 14 and 28 compared with the steers receiving the High treatment (P = 0.03 for the interaction). Blood glucose concentrations tended (P = 0.09) to be higher on day 21 than days 0 and 7 but was not affected by treatment (P = 0.58). Plasma NEFA concentrations were lower (P = 0.05) for control steers than other treatments, and greater (P = 0.002) on day 0 than days 7 and 21. Blood lactate concentrations were greater (P = 0.05) on day 7, than days 0 and 21, but not affected by treatment (P = 0.13). High steers had greater plasma insulin concentrations in response to the IVGTT than steers on the other treatments (P = 0.001). There was no treatment (P ≥ 0.16) or day effect (P ≥ 0.36) on glucose peak, plateau, or clearance rate. High steers had greater expression of solute carrier family 16 member 1 (SLC16A1; P = 0.05) and tended to have greater hepatic expression of solute carrier family 2 member 2 (SLC2A2; P = 0.07). These data indicate that increased propionate may decrease DMI and insulin sensitivity.

Effects of utilizing cotton byproducts in a finishing diet on beef cattle performance, carcass traits, fecal characteristics, and plasma metabolites.

Increased cotton production in the Southwestern United States has increased the availability of cotton byproducts for use in cattle diets. The objective of this experiment was to evaluate the inclusion of cotton byproducts in feedlot finishing diets on the performance, carcass traits, fecal characteristics, and plasma metabolites of steers. Crossbred beef steers (n = 64; BW = 318 ± 12.3 kg) were assigned to 1 of 2 experimental treatments in a randomized complete block design (8 pens per treatment; 4 steers per pen). Treatments included a control (CON) diet, which included prairie hay, Sweet Bran, rolled corn, and a corn steep and molasses-based liquid fat supplement, and a cotton byproduct (CTN) diet, which included cotton gin trash, whole cottonseed, rolled corn, and water. Both diets contained urea and dry supplement. Over the entire feeding period, DMI (P = 0.04) was greater for CTN steers than CON steers with no difference in the gain to feed ratio (P = 0.86) between treatments. The CTN steers tended to have heavier final BW (P = 0.09) and greater overall average daily gain (P = 0.08). The CTN steers had heavier hot carcass weight (P = 0.02) and greater fat thickness (P = 0.03) than CON steers, but marbling score and rib eye area were not different between treatments (P ≥ 0.64). Steers fed the CON diet tended to have a lower yield grade (P = 0.07), less kidney, pelvic and heart fat (P = 0.09), and decreased dressing percentage (P = 0.10) than CTN steers. Liver scores did not differ (P ≥ 0.17) between treatments. Fecal consistency scores were decreased for CTN steers on day 56 (P = 0.03) and fecal pH tended to be greater for the CTN steers on day 28 (P = 0.09) compared with CON steers, but neither differed during other periods (P ≥ 0.18). A treatment × day interaction (P = 0.04) was detected for plasma urea nitrogen (PUN) concentrations, where PUN concentrations differed between treatments only on days 28 and 56. On both days 28 and 56, CTN steers had lower PUN concentrations (P = 0.03, P = 0.002, respectively). No treatment × day interaction was detected for plasma glucose or lactate concentrations. A day effect was observed for both metabolites (P < 0.01). Results from this experiment suggest that cotton byproducts can be effectively used as a source of fiber, fat, and protein in feedlot rations without adverse effects on performance or carcass characteristics.

Associations of mucosal disaccharidase kinetics and expression in the jejunum of steers with divergent average daily gain.

The objective of this study was to quantify the differences in the activity of jejunal maltase and isomaltase between two groups of steers with average dry matter intake (DMI) and differing average daily gain (ADG). DMI and ADG were measured in crossbred steers (n = 69; initial body weight = 456 ± 5.0 kg) consuming a finishing diet containing 67.8% dry-rolled corn, 20.0% wet distillers grains with solubles, 8.0% alfalfa hay, and 4.2% vitamin/mineral supplement on a dry matter basis for 84 d. Jejunal mucosal samples were collected from eight steers with the greatest (high) or least (low) ADG and average DMI (± 0.55 standard deviation). Homogenates of jejunal mucosa were incubated with increasing amounts of maltose and isomaltose to determine the disaccharidase kinetics. Total mucosal protein concentration (mg protein/g tissue; P = 0.45) of the mucosa and small intestinal weights (P = 0.69) did not differ between the groups. Neither the Michaelis-Menten constant (Km) of isomaltase (P = 0.15) nor maltase (P = 0.21) differed between groups. The isomaltase maximum velocity (Vmax) expressed per gram of protein tended to differ (P = 0.10) between groups of steers but did not differ (P = 0.13) when expressed on a tissue basis. Similarly, neither the maltase Vmax expressed per gram of protein (P = 0.31) nor tissue (P = 0.32) differed between groups. While previous studies have indicated that disaccharidase expression is associated with differences in ADG, data presented here indicate that differences in enzyme activity at the end of the finishing period are minimal.

Maintenance energy requirements and forage intake of purebred vs. crossbred beef cows.

The objective of this study was to investigate the impacts of cow breed type and age on maintenance requirements, feed energy utilization, and voluntary forage intake. The main effect of breed type included Angus (ANG; n = 32) and Hereford × Angus (HA; n = 27) lactating cows. The main effect of age included 2- and 3-yr-old (YOUNG; n = 29) and 4- to 8-yr-old (MATURE; n = 30) cows. Within breed type and age class, cows were randomly assigned to 1 of 2 pens for a total of 8 pens, each housing 7 to 9 cow/calf pairs. To determine maintenance energy requirements, cows and calves were limit-fed for 105 d to body weight (BW) and body condition score (BCS) stasis. There were no differences between breeds in cow hip height, BW, average milk yield (P > 0.31), diet digestibility, or cow maintenance energy requirement (P = 0.54). Crossbred cows had greater BCS (P < 0.05) throughout the experiment. Efficiency of calf growth was not different between breeds when expressed as feed intake of the cow/calf pair nor as energy intake of the pair per unit of calf BW gain (P ≥ 0.31). Young cows produced less milk per day and per unit of BW0.75 (P < 0.01); however, there was no effect of cow age on maintenance energy requirement, diet digestibility, or efficiency of calf growth (P > 0.10). Subsequently, a 45-d experiment was conducted to determine voluntary low-quality forage intake. Cows were housed in dry-lot pens equipped with shade, windbreaks, and feed bunks with free-choice access to clean water and a chopped hay ration was provided ad libitum to determine forage intake. Daily forage intake was lower (P = 0.05) for HA compared with ANG (123 vs. 132 g/kg BW0.75, respectively) although there was no difference in BW. However, HA cows sustained greater BCS (P < 0.01). There was no difference (P = 0.60) in forage intake per unit of BW0.75 due to cow age. Results indicate similar calf growth efficiency among breed types although crossbred cows maintained greater body energy stores and consumed less low-quality forage during the voluntary intake experiment. These differences could not be attributed to lower maintenance energy requirements. Neither maintenance energy requirement nor calf growth efficiency was different between young and mature cows.

Evaluation of ruminal degradability and metabolism of feedlot finishing diets with or without cotton byproducts.

Cotton byproducts can be an economical source of protein, fat, and fiber in cattle finishing diets. The objectives of this study were 1) to assess the effects of including whole cottonseed (WCS) and cotton gin trash (CGT) in finishing diets on in situ ruminal degradability and 2) to determine the effects of including cotton byproducts in a finishing diet on rumen fluid pH, lactate, and volatile fatty acid concentrations. Six ruminally cannulated steers were used in a crossover design. Treatments included a control diet (CON; 7% prairie hay [PH], 15% Sweet Bran, 67.25% rolled corn, and 5% liquid supplement) and a cotton byproduct diet (CTN; 7% CGT, 15% WCS, 72.25% rolled corn, and 5% water). Both diets included 0.75% urea and 5% dry supplement. In situ bags containing individual diet ingredients and whole diet samples were incubated in the rumen for up to 96 h. Rumen fluid samples were collected over a 24-h period. No treatment × substrate interactions were detected for any fraction of dry matter (DM) or organic matter (OM) degradability for individual ingredients or whole diets (P ≥ 0.14). The A, B, and C fractions, disappearance rate (Kd), and effective degradability of DM and OM differed between diet ingredients (P ≤ 0.04) but were not different between CON and CTN substrates (P ≥ 0.25). A treatment × substrate interaction (P = 0.04) was detected for the effective degradability of neutral detergent fiber (NDF) of CGT and PH but there was no interaction for other fractions (P ≥ 0.27). The A fraction of NDF was greater (P < 0.001) for CGT than PH; however, the B fraction of NDF tended to be greater (P = 0.08) for PH than CGT. No differences (P ≥ 0.37) were detected for the % NDF disappearance at 48 h between CON and CTN substrates. A tendency for a treatment × substrate interaction (P = 0.10) was observed for the effective degradability of starch among diets; however, when the CON substrate was incubated in steers consuming the CON diet, effective degradability of starch was not different (P = 0.84) from when the CTN diet was incubated in steers consuming the CTN diet. There was no treatment × time interaction or treatment effect for rumen pH; however, there was a time effect (P = 0.03). Steers consuming the CTN diet had greater molar proportions of acetate and decreased molar proportions of propionate compared with CON steers (P < 0.01). This experiment suggests that there are minimal differences between the digestibility of finishing diets containing cotton byproducts and those comprised of traditional finishing diet ingredients.