Comparison of methods to estimate ruminal degradation and intestinal digestibility of protein in hydrolyzed feather meal with or without blood.

Hydrolyzed feather meal (HFM) is a feedstuff high in rumen undegraded protein (RUP) that can be used as an effective source of metabolizable protein for dairy cattle. Because the production process may vary, the rumen degradability and intestinal digestibility of HFM may also vary. Additionally, some processes may incorporate additional blood into the final product to result in feather meal with poultry blood. To determine the rumen degradability and intestinal digestibility of these products, several laboratory assays can be used; the common assays are the mobile bag (MOB), modified three-step (MTS), and Ross (ROS) assays. Although all 3 assays determine RUP digestibility, they vary in whether they are performed in situ, in vitro, or both. The objective of this study was to evaluate the ruminal degradability and intestinal digestibility of HFM originating from processes that differ in their inclusion of blood, and to compare the MOB, MTS, and ROS assays. Ten samples of HFM, which were identified by the suppliers as HFM with little blood (n = 5) and with more blood (n = 5), were spot-sampled, collected from 10 production plants across the United States, and subjected to all 3 assays. Assay type had an effect on RUP, total-tract crude protein (CP) digestibility, and the amount of RUP digested. A significant effect was observed on RDP and RUP concentrations for blood inclusion; no effect was detected for total-tract CP digestibility. We found no difference in RUP digestibility for assay or blood inclusion. There was also no interaction of the effect of assay or blood inclusion. Results suggest that even though there are differences in chemical composition in HFM associated with the inclusion of blood, such as ash and crude fat, few if any differences are observed in intestinal digestion of protein. Although the assays varied in their estimates of rumen undegraded protein, MOB and MTS yielded the most similar values. However, all 3 assays resulted in similar estimates of RUP digestibility.

Relationships between protein and energy consumed from milk replacer and starter and calf growth and first-lactation production of Holstein dairy cows.

The objective was to determine relationships between protein and energy consumed from milk replacer and starter and calf growth and first-lactation production of Holstein heifer calves. Milk replacer and starter protein intake and metabolizable energy (ME) intake data were collected from 4,534 Holstein heifer calves for growth and 3,627 Holstein cows for production from birth year of 2004 through 2014. Calves from 3 commercial dairy farms were assigned to 45 different calf research trials at the University of Minnesota Southern Research and Outreach Center, Waseca, Minnesota, from 3 to 195 d of life. Calves were moved to heifer growers at 6 mo of age, and calves were returned to their farm of birth a few weeks before calving. Most calves (85%) were fed a 20% crude protein and 20% fat milk replacer at a rate of 0.57 kg/calf daily. Metabolizable energy and protein consumed from milk replacer and starter were calculated for each individual calf for 6 and 8 wk of age. Mixed model analyses were conducted to determine the effect of protein and energy consumed from both milk replacer and starter on calf growth and first-lactation 305-d production of milk, fat, and protein, adjusting for herd, season of birth, year, average daily gain (ADG), and calf trial. Calves with ADG >0.80 kg/d consumed more combined protein and ME than calves with lower ADG. Protein and ME intake from calf starter affected growth more than protein and ME intake from milk replacer because most calves were fed the same fixed amount of milk replacer. Calves born during the fall and winter had greater combined protein and ME intake than calves born during the spring and summer. Milk replacer protein and ME intake did not have a relationship with first-lactation 305-d milk, fat, and protein production. However, starter protein and ME intake during the first 6 and 8 wk of age had a significant positive relationship with first-lactation 305-d milk, fat, and protein production. Consequently, combined protein and combined ME intake had a positive effect on 305-d milk, fat, and protein production. Variance in protein and ME intake was high, suggesting that additional factors affect calf growth during the first 8 wk of life and milk production in first lactation.

Eukaryotic inhibitors or activators elicit responses to chemosensory compounds by ruminal isotrichid and entodiniomorphid protozoa.

Our objectives were to evaluate potential signaling pathways regulating rumen protozoal chemotaxis using eukaryotic inhibitors potentially coordinated with phagocytosis as assessed by fluorescent bead uptake kinetics. Wortmannin (inhibitor of phosphoinositide 3-kinase), insulin, genistein (purported inhibitor of a receptor tyrosine kinase), U73122 (inhibitor of phospholipase C), and sodium nitroprusside (Snp, nitric oxide generator, activating protein kinase G) were preincubated with mixed ruminal protozoa for 3h before assessing uptake of fluorescent beads and chemosensory behavior to glucose, peptides, and their combination; peptides were also combined with guanosine triphosphate (GTP; a chemorepellent). Entodiniomorphids were chemoattracted to both glucose and peptides, but chemoattraction to glucose was increased by Snp and wortmannin without effect on chemoattraction to peptides. Rate of fluorescent bead uptake by an Entodinium caudatum culture decreased when beads were added simultaneously with feeding and incubated with wortmannin (statistical interaction). Wortmannin also decreased the proportion of mixed entodiniomorphids consuming beads. Isotrichid protozoa exhibited greater chemotaxis to glucose but, compared with entodiniomorphids, were chemorepelled to peptides. Wortmannin increased chemotaxis by entodiniomorphids but decreased chemotaxis to glucose by isotrichids. Motility assays documented that Snp and wortmannin decreased net swimming speed (distance among 2 points per second) but not total swimming speed (including turns) by entodiniomorphids. Wortmannin decreased both net and total swimming behavior in isotrichids. Results mechanistically explain the isotrichid migratory ecology to rapidly take up newly ingested sugars and subsequent sedimentation back to the ventral reticulorumen. In contrast, entodiniomorphids apparently integrate cellular motility with feeding behavior to consume small particulates and thereby stay associated and pass with the degradable fraction of rumen particulates. These results extend findings from aerobic ciliate models to explain how rumen protozoa have adapted physiology for their specific ecological niches.

Chemotaxis toward carbohydrates and peptides by mixed ruminal protozoa when fed, fasted, or incubated with polyunsaturated fatty acids.

In contrast to the well-characterized chemotaxis and migratory behavior between the dorsal and ventral locations of the rumen by isotrichids, we hypothesized that chemotaxis toward soluble nutrients maintains entodiniomorphid protozoa in the particulate fraction. The objectives of these experiments were to compare the dose-responsive chemotaxis (1) toward different glucose concentrations when ruminal samples were harvested from fed versus fasted cows; (2) toward increasing concentrations of glucose compared with xylose when protozoa were harvested from a fed cow; (3) toward peptides of bacterial, protozoal, and soy origin; and (4) toward glucose when mixed ruminal protozoa were previously incubated for 0, 3, or 6h in the presence of emulsified polyunsaturated fatty acids (PUFA; Liposyn II, Hospira, Lake Forest, IL). In experiment 1, isotrichid protozoa decreased chemotaxis toward increasing glucose concentration when cows were fasted. Entodiniomorphids exhibited chemotaxis to similar concentrations of glucose as did isotrichids, but to a lesser magnitude of response. In experiment 2, xylose was chemotactic to both groups. Xylose might draw fibrolytic entodiniomorphid protozoa toward newly ingested feed. In contrast, even though isotrichids should not use xylose as an energy source, they were highly chemoattracted to xylose. In experiment 3, entodiniomorphids were not selectively chemoattracted toward bacterial or protozoal peptides compared with soy peptides. In experiment 4, despite isotrichid populations decreasing in abundance with increasing time of incubation in PUFA, chemotaxis to glucose remained unchanged. In contrast, entodiniomorphids recovered chemotaxis to glucose with increased time of PUFA incubation. Current results support isotrichid chemotaxis to sugars but also our hypothesis that a more moderate chemotaxis toward glucose and peptides explains how they swim in the fluid but pass from the rumen with the potentially digestible fraction of particulates.

Effects of wortmannin, sodium nitroprusside, insulin, genistein, and guanosine triphosphate on chemotaxis and cell growth of Entodinium caudatum, Epidinium caudatum, and mixed ruminal protozoa.

The mechanisms by which ruminal protozoa sense and migrate toward nutrients are not fully understood. Chemotaxis by many diverse eukaryotic cells is mediated by phosphatidylinositol-3-kinase, which is highly conserved in receptor tyrosine kinase (RTK) signaling pathways and consistently inhibited by wortmannin. In experiment 1a, increasing the concentration of wortmannin inhibited cell growth nonlinearly at 24h of a culture of the rumen protozoan Entodinium caudatum, but high variability prevented growth inhibition of Epidinium caudatum from reaching significance. In experiment 1b, increasing the insulin concentration recovered 24-h cell counts for both cultures, depending on wortmannin concentration. In experiment 2, addition of sodium nitroprusside (Snp; activator of protein kinase G for cilial beat reversal in nonrumen ciliate models) at 500µM or wortmannin at 200µM in beakers containing rumen fluid decreased random swimming by mixed entodiniomorphids into capillary tubes (inserted into beakers) containing saline. Both Snp and wortmannin increased chemotaxis into tubes containing glucose compared with the beaker control. For isotrichids, beaker treatments had no response. Glucose increased chemotaxis, but peptides decreased chemotaxis even when combined with glucose. In experiment 3, we assessed preincubation of genistein (a purported RTK blocker in nonrumen ciliate models) at 40 or 400µM in beakers and guanosine triphosphate (GTP; a universal chemorepellent in nonrumen ciliate models, perhaps mediated through an RTK) at 10 or 100µM combined with glucose in capillary tubes. Neither genistein nor GTP affected chemotaxis toward glucose for entodiniomorphids. However, GTP at 100µM reduced chemotaxis toward glucose for isotrichids. After the animal is fed, isotrichids that are depleted in glycogen migrate to the dorsal area of the rumen, and the rapid uptake of sugars is enhanced through strong chemotaxis but can be reversed by peptides or GTP. In contrast, entodiniomorphids are less intensely chemoattracted to glucose than isotrichids but are chemoattracted to peptides. Entodiniomorphids' chemoattraction appears to be integrated with slower but prolonged availability of energy from digesting starch and fiber.