Absence of a functional gut microbiome impairs host amino acid metabolism in the Pacific spiny dogfish (Squalus suckleyi).

Nitrogen recycling and amino acid synthesis are two notable ways in which the gut microbiome can contribute to host metabolism, and these processes are especially important in nitrogen-limited animals. Marine elasmobranchs are nitrogen limited as they require substantial amounts of this element to support urea-based osmoregulation. However, following antibiotic-induced depletion of the gut microbiome, elasmobranchs are known to experience a significant decline in circulating urea and employ compensatory nitrogen conservation strategies such as reduced urea and ammonia excretion. We hypothesized that the elasmobranch gut microbiome transforms dietary and recycled nutrients into amino acids, supporting host carbon and nitrogen balance. Here, using stable isotope analyses, we found that depleting the gut microbiome of Pacific spiny dogfish (Squalus suckleyi) resulted in a significant reduction to the incorporation of supplemented dietary 15N into plasma amino acids, notably those linked to nitrogen handling and energy metabolism, but had no effect on gut amino acid transport. These results demonstrate the importance of gut microbes to host amino acid pools and the unique nitrogen handling strategy of marine elasmobranchs. More broadly, these results elucidate how the gut microbiome contributes to organismal homeostasis, which is likely a ubiquitous phenomenon across animal populations.

Dose response to postruminal urea in lactating dairy cattle.

Inclusion of urea in dairy cattle diets is often limited by negative effects of high levels of feed urea on dry matter intake (DMI) and efficiency of rumen N utilization. We hypothesized that supplying urea postruminally would mitigate these limitations and allow greater inclusion of urea in dairy cattle diets. Four rumen-fistulated Holstein-Friesian dairy cows (7 ± 2.1 lactations, 110 ± 30.8 d in milk; mean ± standard deviation) were randomly assigned to a 4 × 4 Latin square design to examine DMI, milk production and composition, digestibility, rumen fermentation, N balance, and plasma constituents in response to 4 levels of urea continuously infused into the abomasum (0, 163, 325, and 488 g/d). Urea doses were targeted to linearly increase the crude protein (CP) content of total DMI (diet plus infusion) by 0%, 2%, 4%, and 6% and equated to 0%, 0.7%, 1.4%, and 2.1% of expected DMI, respectively. Each 28-d infusion period consisted of a 7-d dose step-up period, 14 d of adaptation, and a 7-d measurement period. The diet was fed ad libitum as a total mixed ration [10.9% CP, 42.5% corn silage, 3.5% grass hay, 3.5% wheat straw, and 50.5% concentrate (dry matter basis)] and was formulated to meet 100%, 82%, and 53% of net energy, metabolizable protein, and rumen-degradable protein requirements, respectively. Linear, quadratic, and cubic effects of urea dose were assessed using polynomial regression assuming the fixed effect of treatment and random effects of period and cow. Dry matter intake and energy-corrected milk yield responded quadratically to urea dose, and milk urea content increased linearly with increasing urea dose. Apparent total-tract digestibility of CP increased linearly with increasing urea dose and ruminal NH3-N concentration responded quadratically to urea dose. Mean total VFA concentration was not affected by urea dose. The proportion of N intake excreted in feces decreased linearly and that excreted in urine increased linearly in response to increasing urea dose. The proportion of N intake excreted in milk increased linearly with increasing urea dose. Urinary urea excretion increased linearly with increasing urea dose. Microbial N flow responded cubically to urea dose, but the efficiency of microbial protein synthesis was not affected. Plasma urea concentration increased linearly with increasing urea dose. Regression analysis estimated that when supplemented on top of a low-CP diet, 179 g/d of postruminal urea would maximize DMI at 23.4 kg/d, corresponding to a dietary urea inclusion level of 0.8% of DMI, which is in line with the current recommendations for urea inclusion in dairy cattle diets. Overall, these results indicate that postruminal delivery of urea does not mitigate DMI depression as urea dose increases.

Nitrogen utilization, whole-body urea-nitrogen kinetics, omasal nutrient flow, and production performance in dairy cows fed lactose as a partial replacement for barley starch.

The objective of this study was to determine whether the partial replacement of barley starch with lactose (fed as dried whey permeate; DWP) affects N utilization, whole-body urea kinetics, and production in dairy cows. Eight lactating Holstein cows were used in a replicated 4 × 4 Latin square design with 28-d periods. Four cows in one Latin square were ruminally cannulated and used to determine dietary effects on whole-body urea kinetics and N utilization. Cows were fed a barley-based diet that contained 3.6% (dry matter basis) total sugar (TSG; designated control), or diets that contained 6.6, 9.6, or 12.6% TSG. Dietary TSG content was increased by the replacement of barley grain with DWP (83% lactose). Diets were isonitrogenous (∼17.3% crude protein), and starch contents of the control, 6.6, 9.6, and 12.6% TSG diets were 24.3, 22.2, 21.2, and 19.1%, respectively. Whole-body urea kinetics were measured using 4-d infusions of [15N15N]-urea with concurrent total collections of feces and urine. Dry matter intake (mean = 26.7 kg/d), milk yield (mean = 34.9 kg/d), and milk protein and fat contents were unaffected by diet. Ruminal ammonia-N concentration decreased linearly as TSG content increased, whereas ruminal butyrate concentration increased linearly as TSG content increased. Urinary excretion of total N and urea-N changed quadratically, whereas urinary excretion of total N (% of N intake) tended to change quadratically as TSG content increased. Fecal N excretion linearly increased as TSG content increased. A quadratic response was observed for total N excretion as TSG content increased. Milk N and retained N were not affected by diet. As TSG content increased, we observed quadratic responses in the omasal flow of fluid-associated and total bacterial nonammonia N, endogenous production of urea-N, urea-N recycled to the gastrointestinal tract, and urea-N returned to the ornithine cycle. Dietary TSG content did not affect the anabolic utilization of recycled urea-N or the proportion of recycled urea-N that was used for bacterial growth. Our results indicate that feeding DWP did not influence dry matter intake, milk yield, or milk composition. Feeding DWP decreased ruminal ammonia-N concentration, but this did not result in positive responses in milk protein secretion or N balance. The quadratic response in omasal flow of total bacterial nonammonia N indicated that including TSG beyond 9.6% of diet dry matter might depress ruminal microbial protein synthesis.

Integration of the response to a dietary potassium load: a paleolithic perspective.

Our purpose is to integrate new insights in potassium (K(+)) physiology to understand K(+) homeostasis and illustrate some of their clinical implications. Since control mechanisms that are essential for survival were likely developed in Paleolithic times, we think the physiology of K(+) homeostasis can be better revealed when viewed from what was required to avoid threats and achieve balance in Paleolithic times. Three issues will be highlighted. First, we shall consider the integrative physiology of the gastrointestinal tract and the role of lactic acid released from enterocytes following absorption of sugars (fruit and berries) to cause a shift of this K(+) load into the liver. Second, we shall discuss the integrative physiology of WNK kinases and modulation of delivery of bicarbonate to the distal nephron to switch the aldosterone response from sodium chloride retention to K(+) secretion when faced with a K(+) load. Third, we shall emphasize the role of intra-renal recycling of urea in achieving K(+) homeostasis when the diet contains protein and K(+).

Urea-N recycling in lactating dairy cows fed diets with 2 different levels of dietary crude protein and starch with or without monensin.

Rumensin (monensin; Elanco Animal Health, Greenfield, IN) has been shown to reduce ammonia production and microbial populations in vitro; thus, it would be assumed to reduce ruminal ammonia production and subsequent urea production and consequently affect urea recycling. The objective of this experiment was to determine the effects of 2 levels of dietary crude protein (CP) and 2 levels of starch, with and without Rumensin on urea-N recycling in lactating dairy cattle. Twelve lactating Holstein dairy cows (107 ± 21 d in milk, 647 kg ± 37 kg of body weight) were fed diets characterized as having high (16.7%) or low (15.3%) CP with or without Rumensin, while dietary starch levels (23 vs. 29%) were varied between 2 feeding periods with at least 7d of adaptation between measurements. Cows assigned to high or low protein and to Rumensin or no Rumensin remained on those treatments to avoid carryover effects. The diets consisted of approximately 40% corn silage, 20% alfalfa hay, and 40% concentrate mix specific to the treatment diets, with 0.5 kg of wheat straw added to the high starch diets to enhance effective fiber intake. The diets were formulated using Cornell Net Carbohydrate and Protein System (version 6.1), and the low-protein diets were formulated to be deficient for rumen ammonia to create conditions that should enhance the demand for urea recycling. The high-protein diets were formulated to be positive for both rumen ammonia and metabolizable protein. Rumen fluid, urine, feces, and milk samples were collected before and after a 72-h continuous jugular infusion of (15)N(15)N-urea. Total urine and feces were collected during the urea infusions for N balance measurements. Milk yield and dry matter intake were improved in cows fed the higher level of dietary CP and by Rumensin. Ruminal ammonia and milk and plasma urea nitrogen concentrations corresponded to dietary CP concentration. As has been shown in vitro, Rumensin reduced rumen ammonia concentration by approximately 23% but did not affect urea entry rate or gastrointestinal entry rate. Urea entry rate averaged approximately 57% of total N intake for cattle with and without Rumensin, and gastrointestinal rate was similar at 43 and 42% of N intake for cattle fed and not fed Rumensin, respectively. The cattle fed the high-protein diet had a 25% increase in urea entry rate and no effect of starch level was observed for any recycling parameters. Contrary to our hypothesis, Rumensin did not alter urea production and recycling.

Whole-body urea kinetics and functional roles of urea transporters and aquaporins in urea secretion into the rumen in sheep fed diets varying in crude protein content and corn grain processing method.

The objectives were to determine the effects of dietary crude protein (CP) content and corn grain processing on whole-body urea kinetics and the functional roles of urea transporter-B (UT-B) and aquaporins (AQP) in serosal-to-mucosal urea flux (Jsm-urea) in ovine ruminal epithelia. Thirty-two Rideau-Arcott ram lambs were blocked by bodyweight into groups of 4 and then randomly allocated within blocks to 1 of 4 diets (n = 8) in a 2 × 2 factorial design. Dietary factors were CP content (11% [LP] vs. 16% [HP]) and corn grain processing (whole-shelled [WSC] vs. steam-flaked [SFC] corn). Whole-body urea kinetics and N balance were determined using 4-d continuous intrajugular infusions of [15N15N]-urea with concurrent collections of urine and feces with four blocks of lambs (n = 4). After 23 d on diets, lambs were killed to collect ruminal epithelia for mounting in Ussing chambers to determine Jsm-urea and the measurement of mRNA abundance of UT-B and AQP. Serosal and mucosal additions of phloretin and NiCl2 were used to inhibit UT-B- and AQP-mediated urea transport, respectively. Lambs fed HP had a greater (P < 0.01) N intake (29.4 vs. 19.1 g/d) than those fed LP; however, retained N (g/d or % of N intake) was not different. As a % of N intake, lambs fed SFC tended (P = 0.09) to have a lower N excretion (72.2 vs. 83.5%) and a greater N retention (27.8 vs. 16.6%) compared to those fed WSC. Endogenous urea-N production (UER) was greater in lambs fed HP compared to those fed LP (29.9 vs. 20.6 g/d; P = 0.02), whereas urea-N secreted into the gut (GER; g/d) and urea-N used for anabolic purposes (UUA; g/d) were similar. Lambs fed LP tended (P = 0.05) to have greater GER:UER (0.78 vs. 0.66) and UUA:GER (0.23 vs. 0.13) ratios, and a greater Jsm-urea (144.7 vs. 116.1 nmol/[cm2 × h]; P = 0.07) compared to those fed HP. Lambs fed SFC tended to have a lower NiCl2-insensitive Jsm-urea (117.4 vs. 178.4 nmol/[cm2 × h]; P = 0.09) and had a lower phloretin-insensitive Jsm-urea (87.1 vs. 143.1 nmol/[cm2 × h]; P = 0.02) compared to those fed WSC. The mRNA abundance of UT-B (0.89 vs. 1.07; P = 0.08) and AQP-3 (0.90 vs. 1.05; P = 0.07) tended to be lower in lambs fed SFC compared to those fed WSC. Overall, reducing CP content tended to increase the GER:UER ratio with no changes in the expression or function of UT-B and AQP. Although corn grain processing had no effects on GER, feeding SFC increased the portion of urea secretion into the rumen that was mediated via UT-B and AQP.

In ruminants, urea produced in the liver as a nitrogenous waste can be secreted into the rumen where it can be used by rumen microorganisms as a source of nitrogen (N) for their growth. Therefore, urea secretion into the rumen is nutritionally important for ruminants particularly when dietary N intake is deficient. Urea secretion into the rumen occurs via transporter proteins in rumen tissue referred to as urea transporters (UT-B) and aquaporins (AQP). The purpose of this research was to investigate the effects of dietary crude protein (CP) content and corn grain processing on urea secretion into the rumen and the function of UT-B and AQP. Thirty-two Rideau-Arcott lambs were assigned to 1 of 4 diets in a 2 × 2 factorial design. Dietary factors were CP content (11% [LP] vs. 16% [HP]) and corn processing (whole-shelled [WSC] vs. steam-flaked [SFC] corn). When compared to feeding HP, feeding LP tended to increase urea secretion into the rumen, but there were no corresponding changes in UT-B and AQP function. Corn processing did not influence urea secretion into the rumen; however, the portion of urea secretion that was facilitated via UT-B and AQP was greater in lambs fed SFC compared to those fed WSC.

Effects of protein sources and inclusion levels on nitrogen metabolism and urea kinetics of Nellore feedlot steers fed concentrate-based diets.

Urea recycling occurs in all mammalian species and represents an important source of ruminal nitrogen (N) for ruminants fed protein-restricted diets. However, its importance for cattle fed adequate amounts of protein and energy remains unclear. Six Nellore feedlot steers fed concentrate-based diets were used in a 6 × 6 Latin square design with a 3 × 2 factorial arrangement of treatments to evaluate ruminal fermentation, urea kinetics, and N excretion. Treatments consisted of 3 protein sources (PS: soybean meal plus urea [SU], corn gluten meal [CGM], and dry distillers grains [DDG]) and 2 inclusion levels (PL; 11% and 14%). Steers were adapted to the diets for 14 d followed by 8 d of sample collection. Feed intake, fecal output, and urine production were measured from day 18 to day 22 of each period. Blood samples were collected every 6 h on day 18. [15N-15N]-urea was infused into the jugular vein for 82 h over day 19 to day 22, and measurement of 15N in background (day 18) and enriched feces and urine (day 21) were used to evaluate urea kinetics. To evaluate the incorporation of recycled urea N into microbial protein (MICP), ruminal and duodenal fluid were collected on day 22. Steers fed SU diets had lower (P < 0.05) nitrogen use efficiency (NUE), greater (P < 0.05) urea-N entry rate (UER), and tended (P < 0.10) to have greater gastrointestinal entry rate of urea-N (GER) compared with those fed CGM or DDG. In addition, steers fed SU had greater (P < 0.05) urea-N returned to ornithine cycle (ROC) compared with those fed CGM or DDG. Increasing PL tended (P < 0.10) to increase UER. The proportion of total microbial N from recycled urea-N was greater (P < 0.05) for steers fed CGM compared with those fed SU and also greater for steers fed diets with 11% CP than for those fed with 14% CP. Diets with 11% CP can be used for Nellore feedlot cattle fed concentrate-based diets without negatively affecting intake, digestibility, and ruminal fermentation. Moreover, diets containing rumen undegradable protein (RUP) feed sources (CGM or DDG) compared with diets with SU markedly increased NUE, while maintaining microbial protein (MICP) synthesis. Results from this study suggest that the equation adopted by NASEM (NASEM. 2016. Nutrient requirements of beef cattle. 8th revised ed. Washington, DC: The National Academies Press) was not accurate in estimating the urea-N used for anabolism (UUA) in Nellore feedlot cattle fed concentrate-based diets.

Differences between Holstein dairy cows in renal clearance rate of urea affect milk urea concentration and the relationship between milk urea and urinary nitrogen excretion.

Urine and fecal excretions from cattle contribute to global nitrogen (N) emissions. The milk urea nitrogen (MUN) concentration in dairy cows is positively correlated with urinary urea N (UUN) emissions, and both decline with the reduction in crude protein intake. However, MUN concentration may differ between individual cows despite feeding the same ration. Thus, we hypothesized that due to differences in endogenous N utilization cows with high MUN concentration excrete more UUN than cows with a low MUN concentration. The objective of the present study was to elucidate N partitioning and urea metabolism in dairy cows with divergent MUN concentrations fed two planes of crude protein. Twenty Holstein dairy cows with high (HMU; n = 10) and low (LMU; n = 10) milk urea concentrations were fed two isocaloric diets with a low (LP) and normal (NP) crude protein level. Methane and ammonia emissions were recorded in respiration chambers. Feed intake, feces and urine excretions and milk yield were recorded for four days and subsamples were analyzed for total N and N-metabolites. A carbon-13 labeled urea bolus was administered intravenously followed by a series of plasma samplings. Total N and UUN excretions and ammonia emissions from excreta were lower on the LP diet, however, methane emissions, urinary N excretions and ammonia emissions were comparable between groups. Although plasma and salivary urea concentrations, urea pool size and urea turnover were higher, HMU cows had lower renal urea clearance rates. Additionally, HMU cows had lower renal clearance rates for creatinine, uric acid and creatine and excreted less uric acid (on the LP diet only) and creatine with urine. In conclusion, contrary to our hypothesis, HMU cows did not excrete more UUN than LMU cows. The lower urinary creatine excretion of HMU cows suggests that these animals have a lower environmental nitrogen footprint.

Influence of forage level and corn grain processing on whole-body urea kinetics, and serosal-to-mucosal urea flux and expression of urea transporters and aquaporins in the ovine ruminal, duodenal, and cecal epithelia.

The objectives were to determine the effects of forage level and grain processing on whole-body urea kinetics, N balance, serosal-to-mucosal urea flux (Jsm-urea), and messenger ribonucleic acid (mRNA) abundance of urea transporter-B (UT-B; SLC14a1) and aquaporins (AQP) in ovine ruminal, duodenal, and cecal epithelia. Thirty-two wether lambs were blocked by body weight into groups of four and assigned to one of four diets (n = 8) in a 2 × 2 factorial design. Dietary factors were forage level (30% [LF] vs. 70% [HF]) and corn grain processing (whole-shelled [WS] vs. steam-flaked [SF]). Four blocks of lambs (n = 4) were used to determine urea kinetics and N balance using 4-d [15N15N]-urea infusions with concurrent fecal and urine collections. Lambs were killed after 23 d of dietary adaptation. Ruminal, duodenal, and cecal epithelia were collected to determine Jsm-urea and mRNA abundance of UT-B and AQP. Lambs fed LF had greater intakes of dry matter (DMI; 1.20 vs. 0.86 kg/d) and N (NI; 20.1 vs. 15.0 g/d) than those fed HF (P < 0.01). Lambs fed SF had greater DMI (1.20 vs. 0.86 kg/d) and NI (20.6 vs. 14.5 g/d) than those fed WS (P < 0.01). As a percentage of NI, total N excretion was greater in lambs fed HF compared with those fed LF (103% vs. 63.0%; P < 0.01) and was also greater in lambs fed WS compared with those fed SF (93.6% vs. 72.1%; P = 0.02). Retained N (% of NI) was greater in lambs fed LF compared with those fed HF (37.0% vs. -2.55%; P < 0.01). Lambs fed SF had a greater (P = 0.02) retained N (% of NI; 28.0% vs. 6.50%) compared with those fed WS. Endogenous urea production (UER) tended (P = 0.09) to be greater in lambs fed HF compared with those fed LF. As a proportion of UER, lambs fed HF had a greater urinary urea-N loss (0.38 vs. 0.22) and lower urea-N transferred to the gastrointestinal tract (GIT; 0.62 vs. 0.78) or urea-N used for anabolism (as a proportion of urea-N transferred to the GIT; 0.12 vs. 0.26) compared with lambs fed LF (P < 0.01). Ruminal Jsm-urea was unaffected by diet. Duodenal Jsm-urea was greater (P < 0.01) in lambs fed HF compared with LF (77.5 vs. 57.2 nmol/[cm2 × h]). Lambs fed LF had greater (P = 0.03) mRNA expression of AQP3 in ruminal epithelia and tended (P = 0.06) to have greater mRNA expression of AQP3 in duodenal epithelia compared with lambs fed HF. Expression of UT-B mRNA was unaffected by diet. Our results showed that feeding more ruminally available energy improved N utilization, partly through a greater proportion of UER being transferred to the GIT and being used for anabolic purposes.

Anatomical features in the kidney involved in water conservation through urine concentration in dromedaries (Camelus dromedarius).

The aim of this study was to report some of the morphological characteristics of the kidney involved in urine concentration and hence water conservation in the dromedaries. A total of 20 fresh kidneys of 10 apparently healthy camels were used in this study. The architecture of the renal pelvis was revealed by dissection and polyvinyl chloride corrosion casts. Samples were also processed for histology and for enzyme histochemistry. The camel kidney is bean shaped, smooth, multilobar, unipapillary, in which the fusion of renal papillae is complete forming a common renal papilla or crest, which channel urine into a central renal pelvis. It is more or less similar to equine, caprine, ovine and canine kidney. Under certain anatomical requisites the renal pelvis is known to play a role in urine concentration through recycling of urea to increase the medullary osmotic concentration which favors the counter-current mechanism. One of these requisites is an elaborate renal pelvis which is closely associated with the renal medulla. The renal pelvis of the camel has a main crescentic cavity following the long axis and curvature of the kidney. A thick extensive renal crest projects into the cavity of the pelvis. The thick renal crest contains large numbers of long loops of Henle and vasa recta which are important for urine concentration. The renal crest is formed by convergence of the medullary pyramids before it projects into the cavity of the renal pelvis. The crescentic main cavity of the pelvis forms 20-24 three dimensional radiating collateral recesses which contain the medullary pyramids. This close association of the renal pelvis and medulla provide a large surface area for the recycling of urea and hence urine concentration. This large pelvic-medullary interface is lined by simple low cuboidal epithelium which enhances the recycling of urea and water from the pelvic urine into the medulla and directly contributes to urine concentration. The rest of the wall of the renal pelvis and its recesses facing away from the renal crest and medullary pyramids is lined by impermeable transitional epithelium. Another feature is the intense activity of alkaline phosphatase demonstrated in the proximal convoluted tubules which indicates increased membrane transport. It is concluded that the kidney in dromedaries has the anatomical and histochemical requisites for the production of concentrated urine. These requisites enable the kidney to adequately contribute to the ability of the camel to conserve water and withstand the aridity of its habitat.

Nitrogen digestion and urea recycling in Hokkaido native horses fed hay-based diets.

Nitrogen (N) digestion and urea-N metabolism in Hokkaido native horses fed roughage-based diets containing different types and levels of protein sources were studied. Horses (173 ± 4.8 kg) fitted with an ileum cannula were fed four diets consisting of 100% timothy hay (TH), 88% TH and 12% soybean meal (SBM), 79% TH and 21% SBM, and 51% TH and 49% alfalfa hay at 2.2% of body weight. Dietary protein content varied from 5% to 15% of dry matter. Apparent N digestibilities in the pre-cecum and total tract for the TH diet were lower than those for other diets. However, the proportion of post-ileum N digestion to N intake was not affected by the diets. Urea-N production was linearly related to N intake, but gut urea-N entry was not affected by the diets. The proportion of gut urea-N entry to urea-N production tended to be higher for the TH diet (57%) than the two SBM diets (39%). Anabolic use of urea-N entering the gut was not affected by the diets (20-36% of gut urea-N entry). These results indicate that urea-N recycling provides additional N sources for microbial fermentation in the hindgut of Hokkaido native horses fed low-quality roughages.