K.F. Knowlton

Effect of manure application on abundance of antibiotic resistance genes and their attenuation rates in soil: field-scale mass balance approach.

The development of models for understanding antibiotic resistance gene (ARG) persistence and transport is a critical next step toward informing mitigation strategies to prevent the spread of antibiotic resistance in the environment. A field study was performed that used a mass balance approach to gain insight into the transport and dissipation of ARGs following land application of manure. Soil from a small drainage plot including a manure application site, an unmanured control site, and an adjacent stream and buffer zone were sampled for ARGs and metals before and after application of dairy manure slurry and a dry stack mixture of equine, bovine, and ovine manure. Results of mass balance suggest growth of bacterial hosts containing ARGs and/or horizontal gene transfer immediately following slurry application with respect to ermF, sul1, and sul2 and following a lag (13 days) for dry-stack-amended soils. Generally no effects on tet(G), tet(O), or tet(W) soil concentrations were observed despite the presence of these genes in applied manure. Dissipation rates were fastest for ermF in slurry-treated soils (logarithmic decay coefficient of -3.5) and for sul1 and sul2 in dry-stack-amended soils (logarithmic decay coefficients of -0.54 and -0.48, respectively), and evidence for surface and subsurface transport was not observed. Results provide a mass balance approach for tracking ARG fate and insights to inform modeling and limiting the transport of manure-borne ARGs to neighboring surface water.

Invited review: Sustainability of the US dairy industry.

The US dairy industry has realized tremendous improvements in efficiencies and milk production since the 1940s. During this time, farm and total cow numbers have decreased and average herd size has increased. This intensification, combined with the shift to a largely urban public, has resulted in increased scrutiny of the dairy industry by social and environmental movements and increased concern regarding the dairy industrys sustainability. In response to these concerns, a group of scientists specializing in animal welfare, nutrient management, greenhouse gas emissions, animal science, agronomy, agricultural engineering, microbiology, and economics undertook a critical review of the US dairy industry. Although the US dairy system was identified as having significant strengths, the consensus was that the current structure of the industry lacks the resilience to adapt to changing social and environmental landscapes. We identified several factors affecting the sustainability of the US dairy industry, including climate change, rapid scientific and technological innovation, globalization, integration of societal values, and multidisciplinary research initiatives. Specific challenges include the westward migration of milk production in the United States (which is at odds with projected reductions in precipitation and associated limitations in water availability for cattle and crops), and the growing divide between industry practices and public perceptions, resulting in less public trust. Addressing these issues will require improved alignment between industry practices and societal values, based upon leadership from within the industry and sustained engagement with other interested participants, including researchers, consumers, and the general public.

Effects of Milk Replacer Composition on Growth, Body Composition, and Nutrient Excretion in Preweaned Holstein Heifers

Twenty-four newborn Holstein heifer calves were fed 1 of 4 milk replacers (MR): control (20% CP, 21% fat; MR fed at 441 g/d); high protein/low fat (HPLF; 28% CP, 20% fat; MR fed at 951 g/d); high protein/high fat (HPHF; 27% CP, 28% fat; MR fed at 951 g/d); and HPHF MR fed at a higher rate (HPHF+; 27% CP, 28% fat; MR fed at 1,431 g/d). Dry calf starter (20% CP, 1.43% fat) composed of ground corn (44.4%), 48% CP soybean meal (44.4%), cottonseed hulls (11.2%), and molasses (1.0%) was offered free choice. Heifers were obtained from a commercial dairy, blocked by groups of 8 in the order acquired, and randomly assigned to treatments within group. Upon arrival at the research farm, heifers were fed the control for 2 feedings. Treatments were imposed when heifers were 4 +/- 1 d of age. Heifers were on study for 61 +/- 1 d. Body weight and body size measures were taken weekly. Four-day total collection of feed refusals, feces, and urine was initiated at 57 +/- 1 d of age. Heifers were slaughtered at the end of the collection period to evaluate body composition. Preplanned contrasts were used to compare control to all, HPLF to HPHF, and HPHF to HPHF+. Heifers fed the control diet consumed more starter than those fed other treatment diets, but their total dry matter intake and apparent dry matter digestibility were lowest. Fecal output was highest in heifers fed the control diet, whereas urine output and urine N excretion were lowest. Nitrogen intake and urine N excretion were greater for heifers fed HPHF+ compared with HPHF but were not affected by MR fat content (HPLF vs. HPHF). Retention (g/d) of N and P was greater in heifers fed all nutrient-dense diets compared with those fed the control diet, but was not improved by increasing fat in the milk replacer (HPLF vs. HPHF) or by increasing the amount fed. Addition of fat to the milk replacer (HPLF vs. HPHF) increased empty body weight fat content without improving average daily gain or frame measures. Increasing the volume fed (HPHF vs. HPHF+) increased growth rate and empty body weight, but HPHF+ heifers were neither taller nor longer and their carcasses contained more fat. Clear improvements in growth and nutrient retention were observed with more nutrient-dense diets, but most of the improvements were seen with the increased protein intake relative to the control MR; adding fat to the high protein MR did not further improve lean tissue gain.

Dietary calcium has little effect on mineral balance and bone mineral metabolism through twenty weeks of lactation in Holstein cows

Calcium and P balance and mobilization from bone were evaluated through 20 wk of lactation to determine the timing and extent of net resorption of bone mineral and mineral balance in lactating dairy cows. Eighteen Holstein cows were blocked by parity and calving date and randomly assigned to 1 of 3 dietary treatments: high (1.03%, HI), medium (0.78%, MED), or low (0.52%, LOW) dietary Ca. Dietary P was 0.34% in all diets. Cows consumed treatment diets from calving to 140 DIM. Total collection of milk, urine, and feces was conducted 2 wk before expected calving and in wk 2, 5, 8, 11, and 20 of lactation. Blood samples were collected at 14 and 10 d before expected calving and 0, 1, 3, 5, 10, 14, 21, 28, 35, 56, 70, 84, 98, and 140 d after calving. Blood samples were analyzed for Ca, P, and parathyroid hormone concentration. Serum concentrations of osteocalcin (OC), a marker of bone formation, and deoxypyridinoline (DPD), a marker of bone resorption, were measured to assess bone mobilization. Rib bone biopsies were conducted within 10 d postcalving and during wk 11 and 20 of lactation. Dietary Ca concentration affected Ca balance, with cows consuming the HI Ca diet in positive Ca balance for all weeks with the exception of wk 11. Interestingly, all cows across all treatments had a negative Ca balance at wk 11, possibly the result of timed estrous synchronization that occurred during wk 11. At wk 20, Ca balances were 61.2, 29.9, and 8.1 g/d for the HI, MED, and LOW diets, respectively. Phosphorus balances across all treatments and weeks were negative. Bone Ca content on a fat-free ash weight basis was least in cows consuming the MED diet, but bone P was not different. Serum Ca and P were not affected by treatment. Dietary Ca concentration did not affect P balance in the weeks examined, but there was a clear effect of parity on balance, markers of bone metabolism, and bone P. Primiparous cows had greater serum OC and DPD concentrations than multiparous cows. Regardless of dietary treatment, serum OC concentration peaked around d 35 of lactation. Simultaneously, DPD concentration began to decrease, which may indicate a switch from net bone resorption to formation after d 35. However, this was not reflected in balance measures. This information may help refine dietary mineral recommendations for lactating dairy cows and suggests that dietary P requirements are independent of dietary Ca.

Effects of Milk Replacer Composition on Selected Blood Metabolites and Hormones in Preweaned Holstein Heifers

We investigated the effects of increasing dietary protein and energy on concentrations of selected blood metabolites and hormones in Holstein heifers. Twenty-four heifers were fed 1 of 4 milk replacer (MR) diets for 9 wk (n = 6/diet): control [20% crude protein (CP), 21% fat MR fed at 441 g of dry matter (DM)/d], HPLF (28% CP, 20% fat MR fed at 951 g of DM/d), HPHF (27% CP, 28% fat MR fed at 951 g of DM/d), and HPHF+ (27% CP, 28% fat MR fed at 1,431 g of DM/d). Heifers were fed twice daily; water and starter (20% CP, 1.43% fat) were offered free choice and starter orts recorded daily. Serum and plasma aliquots from blood samples collected twice weekly after a 12-h fast were analyzed for insulin-like growth factor (IGF)-I, IGF-binding proteins (IGFBP), growth hormone (GH), insulin, glucose, nonesterified fatty acids, triglyceride, and plasma urea nitrogen concentrations. Only plasma glucose, IGFBP-2, and IGFBP-3 were affected by diet. Dietary treatment differences were only noted when the control was compared with the average of the other 3 diets. The addition of fat to the MR (HPLF vs. HPHF) and increased volume of MR (HPHF vs. HPHF+) had no effect on plasma glucose concentration or relative abundance of IGFBP-2 or IGFBP-3. Heifers fed the control diet had less glucose, greater IGFBP-2, and less IGFBP-3 than the average of the other 3 diets. There was a diet by week interaction for IGF-I. Serum IGF-I concentration in control heifers varied in a quadratic manner with a nadir (20 +/- 4 ng/mL) at wk 4, whereas IGF-I increased linearly in heifers on other diets. Both insulin and triglyceride changed over time in a complex pattern (significant linear and quadratic contrast effects). The greatest concentrations were measured at wk 0.5 with nadirs at wk 6 for both insulin and triglyceride. Serum GH concentration decreased in a linear manner from wk 0.5 to wk 9 in all heifers. Relative abundance of IGFBP-2 was quadratic over time with the greatest amount of IGFBP-2 observed at wk 5. With the exception of glucose, IGF-I, IGFBP-2, and IGFBP-3, the blood variables measured were not influenced by treatment. The IGF-I -GH-IGFBP axis requires further study in heifers to deduce effects of nutrition on hypothalamic regulation of metabolism. We expected to see more treatment differences in concentrations of metabolites involved with protein and fat metabolism. It is likely that the diets used in this study were not diverse enough in composition to elicit such changes or that the efficiency of use of absorbed protein and fat was not different in these animals.

A model of phosphorus digestion and metabolism in the lactating dairy cow.

A dynamic, mechanistic, compartmental model of phosphorus (P) digestion and metabolism was constructed in the Advanced Continuous Simulation Language using conservation of mass principles and mass action kinetics. Phosphorus was assumed to exist in 3 forms: inorganic (Pi), phytic acid (Pp), and organic (excluding phytic acid; Po). All 3 forms were assumed to be present in the digestive tract with absorption of Pi into blood. Inputs to the model were total P intake; Pp, Po, and Pi as proportions of total P; milk yield; rate of salivation (fixed at 239 L/d); and rate of liquid passage from the rumen (fixed at 198 L/d). The model was fitted to 2 experiments from the literature. Derived parameters were well defined by the data. With a mean observed P intake of 75 g/d, total tract P digestibility was 38%. Phytic acid P digestibility in the rumen was 74%, with no additional Pp digestion in the lower tract. Inorganic P and Po digestibility in the lower tract were 48 and 89%, respectively. Flows of Po and Pi from the rumen were 2.4 and 3.0 times greater than intake, respectively. The increase in Po was apparently due to microbial growth. The increase in Pi arose primarily from secretion of Pi into the rumen via salivation where 65% of absorbed P was recycled to the rumen. Milk synthesis used 30% of absorbed Pi, and 1% was excreted in urine. This research suggested that the primary regulation points for maintaining blood P were bone deposition and resorption and absorption from the intestine. However, because bone P balance was related to both dietary P intake and ruminal phytase activity, it is critical to achieve a better understanding of phytate digestibility across several feeds if dietary P is to be reduced below current requirements.

Metagenomic Analysis of Antibiotic Resistance Genes in Dairy Cow Feces following Therapeutic Administration of Third Generation Cephalosporin

Although dairy manure is widely applied to land, it is relatively understudied compared to other livestock as a potential source of antibiotic resistance genes (ARGs) to the environment and ultimately to human pathogens. Ceftiofur, the most widely used antibiotic used in U.S. dairy cows, is a 3rd generation cephalosporin, a critically important class of antibiotics to human health. The objective of this study was to evaluate the effect of typical ceftiofur antibiotic treatment on the prevalence of ARGs in the fecal microbiome of dairy cows using a metagenomics approach. β-lactam ARGs were found to be elevated in feces from Holstein cows administered ceftiofur (n = 3) relative to control cows (n = 3). However, total numbers of ARGs across all classes were not measurably affected by ceftiofur treatment, likely because of dominance of unaffected tetracycline ARGs in the metagenomics libraries. Functional analysis via MG-RAST further revealed that ceftiofur treatment resulted in increases in gene sequences associated with “phages, prophages, transposable elements, and plasmids”, suggesting that this treatment also enriched the ability to horizontally transfer ARGs. Additional functional shifts were noted with ceftiofur treatment (e.g., increase in genes associated with stress, chemotaxis, and resistance to toxic compounds; decrease in genes associated with metabolism of aromatic compounds and cell division and cell cycle), along with measureable taxonomic shifts (increase in Bacterioidia and decrease in Actinobacteria). This study demonstrates that ceftiofur has a broad, measureable and immediate effect on the cow fecal metagenome. Given the importance of 3rd generation cephalospirins to human medicine, their continued use in dairy cattle should be carefully considered and waste treatment strategies to slow ARG dissemination from dairy cattle manure should be explored.

Blood mineral, hormone, and osteocalcin responses of multiparous Jersey cows to an oral dose of 25-hydroxyvitamin D3 or vitamin D3 before parturition.

Twenty-seven multiparous Jersey cows were randomly assigned to receive an oral bolus containing corn starch (control, CON), corn starch plus 15 mg of 25-hydroxyvitamin D(3) (25-OH), or 15 mg of cholecalciferol (D(3)) at 6 d before expected parturition. Cows were maintained in individual box stalls from 20 d before expected parturition and fed a common diet. Jugular blood samples were collected at -14, -13, -5, -4, -3, -2, -1 d before expected calving, at calving, and at 1, 3, 5, 7, 9, 11, 13, 28, 56, and 84 d postcalving. After calving, cows were housed in 1 pen in a free-stall barn and consumed a common diet. Colorimetric assays were used to analyze Ca, P, and Mg concentrations in serum. Serum concentrations of osteocalcin (OC), an indicator of bone formation, serum 25-hydroxyvitamin D(3), and parathyroid hormone (PTH) were determined in samples obtained from d -5 through d 13. The 9 control multiparous cows and 5 untreated primiparous cows were used to evaluate the effect of parity on the variables that were measured. There was no effect of parity on Ca, PTH, or 25-OH concentration. Compared with second-lactation cows and older cows (>2 lactations), first-lactation cows had greater serum OC (22.3, 32.0, and 48.3 ng/mL, respectively), indicating that younger animals were forming more bone. Blood Ca, P, and Mg decreased near the time of calving and then increased over time. Serum 25-hydroxyvitamin D(3) was greater for cows dosed with 25-OH (119.0 ng/mL) compared with those dosed with D(3) (77.5 ng/mL) or CON (69.3 ng/mL). Cows dosed with 25-OH tended to have lower serum PTH concentration, but treatments did not affect serum Ca, P, or Mg. Serum OC was greater in second-lactation cows compared with cows entering their third or fourth lactation but OC was unaffected by treatment. Although results indicated a 60% increase in serum 25-hydroxyvitamin D(3) due to a single oral dose of 25-OH before calving, the amount administered in this study apparently was not sufficient for initiation of any improvement in Ca homeostasis at parturition.

Excretion of Antibiotic Resistance Genes by Dairy Calves Fed Milk Replacers with Varying Doses of Antibiotics

Elevated levels of antibiotic resistance genes (ARGs) in soil and water have been linked to livestock farms and in some cases feed antibiotics may select for antibiotic resistant gut microbiota. The purpose of this study was to examine the establishment of ARGs in the feces of calves receiving milk replacer containing no antibiotics versus subtherapeutic or therapeutic doses of tetracycline and neomycin. The effect of antibiotics on calf health was also of interest. Twenty-eight male and female dairy calves were assigned to one of the three antibiotic treatment groups at birth and fecal samples were collected at weeks 6, 7 (prior to weaning), and 12 (5 weeks after weaning). ARGs corresponding to the tetracycline (tetC, tetG, tetO, tetW, and tetX), macrolide (ermB, ermF), and sulfonamide (sul1, sul2) classes of antibiotics along with the class I integron gene, intI1, were monitored by quantitative polymerase chain reaction as potential indicators of direct selection, co-selection, or horizontal gene transfer of ARGs. Surprisingly, there was no significant effect of antibiotic treatment on the absolute abundance (gene copies per gram wet manure) of any of the ARGs except ermF, which was lower in the antibiotic-treated calf manure, presumably because a significant portion of host bacterial cells carrying ermF were not resistant to tetracycline or neomycin. However, relative abundance (gene copies normalized to 16S rRNA genes) of tetO was higher in calves fed the highest dose of antibiotic than in the other treatments. All genes, except tetC and intI1, were detectable in feces from 6 weeks onward, and tetW and tetG significantly increased (P < 0.10), even in control calves. Overall, the results provide new insight into the colonization of calf gut flora with ARGs in the early weeks. Although feed antibiotics exerted little effect on the ARGs monitored in this study, the fact that they also provided no health benefit suggests that the greater than conventional nutritional intake applied in this study overrides previously reported health benefits of antibiotics. The results suggest potential benefit of broader management strategies, and that cost and risk may be avoided by minimizing incorporation of antibiotics in milk replacer.

Manure nutrient excretion by Jersey and Holstein cows.

The objective of this study was to evaluate feces, urine, and N excretion by Jersey and Holstein cows. Sixteen multiparous cows (n=8 per breed) were fed 2 experimental rations at calving in a switchback experimental design. Diets were 50% forage and based on corn meal (control) or whole cottonseed. Half the cows in each breed started on the control diet and half started on the whole cottonseed diet. Cows were switched to the other diet at 60 d in milk and switched back to their original diet at 165 d in milk. Pairs of cows were moved into open-circuit respiration chambers on d 49, 154, and 271 of lactation for 7-d measurement periods. While in the chambers, total collection of feed refusals, milk, recovered hair, feces, and urine was conducted. No effect of the interaction of diet and breed was observed for measures of nutrient digestibility and manure excretion. Total daily manure excretion was lower in Jersey cows than in Holstein cows, with reductions generally proportional to changes in feed intake. Jersey cows consumed 29% less feed and excreted 33% less wet feces and 28% less urine than Holstein cows. Intake, fecal, and urinary N were reduced by 29, 33, and 24%, respectively, in Jersey cows compared with Holstein cows. Equations from American Society of Agricultural and Biological Engineers underpredicted observed values for all manure measures evaluated (urine, manure solids, N, wet manure), and breed bias was observed in equations predicting excretion of urine, N, and wet manure. Although these equations include animal and dietary factors, intercepts of regression of observed values on predicted values differed between Holsteins and Jerseys for those 3 measures. No breed bias was observed in the prediction of manure solids excretion, however, making that equation equally appropriate for Jerseys and Holsteins. The effect of breed on manure and nutrient excretion has significant nutrient management implications.