Tea crude extracts effectively inactivate severe acute respiratory syndrome coronavirus 2.

It is well known that black and green tea extracts, particularly polyphenols, have antimicrobial activity against various pathogenic microbes including viruses. However, there is limited data on the antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged rapidly in China in late 2019 and which has been responsible for coronavirus disease 2019 (COVID-19) pandemic globally. In this study, 20 compounds and three extracts were obtained from black and green tea and found that three tea extracts showed significant antiviral activity against SARS-CoV-2, whereby the viral titre decreased about 5 logs TCID50 per ml by 1·375 mg ml-1 black tea extract and two-fold diluted tea bag infusion obtained from black tea when incubated at 25°C for 10 s. However, when concentrations of black and green tea extracts were equally adjusted to 344 µg ml-1 , green tea extracts showed more antiviral activity against SARS-CoV-2. This simple and highly respected beverage may be a cheap and widely acceptable means to reduce SARS-CoV-2 viral burden in the mouth and upper gastrointestinal and respiratory tracts in developed as well as developing countries.

Fighting antimicrobial resistance (AMR): Chinese herbal medicine as a source of novel antimicrobials - an update.

Antimicrobial resistance (AMR) has now emerged as a global public health crisis, requiring the discovery of new and novel antimicrobial compounds, that may be precursors of future therapeutic antibiotics. Chinese Herbal Medicine (CHM) comes with a rich pedigree of holistic and empirical usage in Asia for the last 5000 years. Extracts of Anemarrhena asphodeloides Bunge, Angelica sinensis (Oliv.) Diels, Dianthus superbus L. Forsythiae fructus (Lian Qiao), Lonicerae flos (Jin Yin Hua), Naemorhedi cornu, Platycladus orientalis Franco, Polygonum aviculare, Polygonum cuspidatum, Poria cocos (Schw.), Rehmannia glutinosa (Gaertn.) DC, Rheum palmatum, Salvia miltiorrhiza Bunge, Scutellaria barbata, Scutellariae radix (Huang Qin) and Ursi fel (Xiong Dan) have shown to have antimicrobial properties against clinically significant Gram-negative and Gram-positive bacterial pathogens, as well as the mycobacteria (TB and non-tuberculous mycobacteria). Evidence is now beginning to emerge through systematic reviews of the outcomes of clinical studies employing CHM to treat infections. Of the 106 Cochrane systematic reviews on CHM, 16 (ca 15%) reviews examine CHM in the context of treating a specific infection disease or state. This update examines direct antimicrobial effect of CHM on bacterial pathogens, as well as synergistic effects of combining CHM with conventional antibiotics.

Imaging stress and magnetism at high pressures using a nanoscale quantum sensor.

Pressure alters the physical, chemical, and electronic properties of matter. The diamond anvil cell enables tabletop experiments to investigate a diverse landscape of high-pressure phenomena. Here, we introduce and use a nanoscale sensing platform that integrates nitrogen-vacancy (NV) color centers directly into the culet of diamond anvils. We demonstrate the versatility of this platform by performing diffraction-limited imaging of both stress fields and magnetism as a function of pressure and temperature. We quantify all normal and shear stress components and demonstrate vector magnetic field imaging, enabling measurement of the pressure-driven [Formula: see text] phase transition in iron and the complex pressure-temperature phase diagram of gadolinium. A complementary NV-sensing modality using noise spectroscopy enables the characterization of phase transitions even in the absence of static magnetic signatures.

Spaceflight-Relevant Challenges of Radiation and/or Reduced Weight Bearing Cause Arthritic Responses in Knee Articular Cartilage.

There is little known about the effect of both reduced weight bearing and exposure to radiation during spaceflight on the mechanically-sensitive cartilage lining the knee joint. In this study, we characterized cartilage damage in rat knees after periods of reduced weight bearing with/without exposure to solar-flare-relevant radiation, then cartilage recovery after return to weight bearing. Male Sprague Dawley rats (n = 120) were either hindlimb unloaded (HLU) via tail suspension or remained weight bearing in cages (GROUND). On day 5, half of the HLU and GROUND rats were 1 Gy total-body X-ray irradiated during HLU, and half were sham irradiated (SHAM), yielding 4 groups: GROUND-SHAM; GROUND-IR; HLU-SHAM; and HLU-IR. Hindlimbs were collected from half of each group of rats on day 13. The remaining rats were then removed from HLU or remained weight bearing, and hindlimbs from these rats were collected on day 62. On day 13, glycosaminoglycan (GAG) content in cartilage lining the tibial plateau and femoral condyles of HLU rats was lower than that of the GROUND animals. Likewise, on day 13, immunoreactivity of the collagen type II-degrading matrix metalloproteinase-13 (MMP-13) and of a resultant metalloproteinase-generated neoepitope VDIPEN was increased in all groups versus GROUND-SHAM. Clustering of chondrocytes indicating cartilage damage was present in all HLU and IR groups versus GROUND-SHAM on day 13. On day 62, after 49 days of reloading, the loss of GAG content was attenuated in the HLU-SHAM and HLU-IR groups, and the increased VDIPEN staining in all treatment groups was attenuated. However, the increased chondrocyte clustering remained in all treatment groups on day 62. MMP-13 activity also remained elevated in the GROUND-IR and HLU-IR groups. Increased T2 relaxation times, measured on day 62 using 7T MRI, were greater in GROUND-IR and HLU-IR knees, indicating persistent cartilage damage in the irradiated groups. Both HLU and total-body irradiation resulted in acute degenerative and pre-arthritic changes in the knee articular cartilage of rats. A return to normal weight bearing resulted in some recovery from cartilage degradation. However, radiation delivered as both a single challenge and when combined with HLU resulted in chronic cartilage damage. These findings suggest that radiation exposure during spaceflight leads to and/or impairs recovery of cartilage upon return to reloading, generating long-term joint problems for astronauts.

Comparison of antibiotic susceptibility in viridans group streptococci in low and high antibiotic-prescribing General Practices.

WHAT IS KNOWN AND OBJECTIVE: Antibiotic resistance has become a global public health issue. Most antibiotics are prescribed in the community, although there is less stewardship of such agents in the community compared to secondary and tertiary care. Few studies have attempted to examine the prescribing practices in General Practice and its impact on antibiotic resistance and, therefore, a study was performed in order to compare antibiotic susceptibilities of commensal viridans group streptococci (VGS) obtained from patient cohorts in General Practices (GP), who were high and low prescribers of oral antibiotics. METHOD: Sixty-five patients (<1 month-81 years; 77% female: 23% male) were enrolled onto the study, and viridans group streptococci (n = 5/patient) were collected from each patient's nasal passages and oropharynx region and tested for antibiotic susceptibility against (i) tetracyclines (doxycycline); (ii) macrolides (erythromycin); (iii) ß-lactams (penicillin G); and (iv) fluoroquinolones (ofloxacin & levofloxacin). RESULTS AND DISCUSSION: There were no significant differences in MICs between high and low GP prescribers with doxycycline (P = 0·094), erythromycin (P = 0·122), ofloxacin (P = 0·193) and levofloxacin (P = 0·058). However, there was a significant difference between high and low GP practices with regard to penicillin G (P = 0·031). This finding is important as the ß-lactams are the most commonly prescribed oral antibiotic in the community. WHAT IS NEW AND CONCLUSION: This study demonstrates that high prescribing practices may lead to an altered (higher) level of resistance to these agents in the commensal VGS population, which may be important as reservoirs of antibiotic resistance determinants in subsequent horizontal gene transfer events, particularly with newly colonizing pathogens, including pneumococci. Primary care physicians should be aware that increased prescribing of antibiotics may led to increased level of penicillin resistance.

Antibacterial effects on acinetobacter species of commonly employed antineoplastic agents used in the treatment of haematological malignancies: an in vitro laboratory evaluation.

Although about 75-80% of neutropenic fevers are thought to be caused by infections, a causal organism can be confirmed microbiologically or suspected clinically in only 30-50%, and even fewer of these cases (16%) have a documented bacteraemia. The cause of neutropenic fever in the remaining cases remains elusive. The reasons for this failure may be due to the difficulty in recovering low numbers of organisms, fastidious organisms which fail to grow using conventional culture media, the presence of non-culturable organisms, or the presence of inhibitory substances in specimens. Previously, the authors showed the presence of Acinetobacter in peripheral blood of febrile neutropenic patients with a haematological malignancy, using 16S rDNA polymerase chain reaction (PCR) and sequencing techniques. However, conventional culture was unable to detect these organisms. Hence, it was felt necessary to examine the antibacterial properties of four antineoplastic agents used in the treatment of haematological malignancy, namely bleomycin, cisplatin, doxorubicin and vincristine. A total of 56 wild-type Acinetobacter including seven species (A. calcoaceticus [n=17], A. septicus [n=11], A. baumannii [n=10], A. johnsonii [n=7], A. lwoffii [n=8] A. haemolyticus [n=2] and A. radioresistens [n=1]) were examined for their susceptibility to the four antineoplastic agents at therapeutic concentration. No inhibition was observed, but inhibition was seen at higher concentrations of both bleomycin and doxorubicin. Time to detection of blood culture bottles containing separate antineoplastic agents (i.e., bleomycin and doxorubicin) was compared to that containing saline using a paired t-test. Samples containing doxorubicin at 1 pg/mL were shown to have a mean time to detection of 21.8 h (range: 15.6-31.4 h). Bottles containing saline had a mean time to detection of 22.9 h (range: 18.2-31.3 h). Statistical analysis showed no significant difference (P=0.3361) between time to detection for blood culture bottles containing doxorubicin at achievable plasma concentration and corresponding negative controls. With regard to bleomycin (300 miu/mL), the mean time to detection was 27.29 h (range: 20.2-38.4 h) in the test bottles, with mean time to detection in the saline negative controls of 22.56 h (range: 17.0-30.1 h). Paired t-test gave P=0.000451, hence a significant difference in time to detection for blood cultures containing therapeutic levels of bleomycin. Overall, the antineoplastic agents vincristine, cisplatin or doxorubicin did not have any inhibitory effects on the Acinetobacter organisms examined. At worst, therapeutic concentrations of bleomycin may delay automated detection of an Acinetobacter bacteraemia by a mean time of 5.9 h.

Differential hypothalamic secretion of neurocrines in male common marmosets: parental experience effects?

Pregnancy and lactation produce a plethora of hormonal changes in females that promote maternal care of offspring. Males in the biparental marmoset species (Callithrix jacchus) demonstrate high levels of parenting behaviour and express enhanced circulating reproductive hormones. Furthermore, these hormonal changes are influenced by paternal experience. To determine whether the paternally experienced male marmoset has altered neurocrine hypothalamic release, as the maternal females does, we examined the release of several reproductive neurocrines, dopamine (DA), oxytocin (OT), vasopressin (AVP) and prolactin (PRL), in cultured explants of the hypothalamus of paternally experienced male marmosets compared to naïve, paternally inexperienced males. DA levels secreted from the isolated hypothalamus were significantly lower in the experienced males, whereas OT and PRL levels were significantly higher than levels found in inexperienced males. PRL levels decreased rapidly in the hypothalamic media, suggesting that PRL production occurs elsewhere. AVP levels did not change. Stimulation of the cultured explants with oestradiol significantly decreased DA levels in the inexperienced males but did not alter the other neurocrines, suggesting a direct effect of oestradiol on DA suppression in the hypothalamus. Although other factors such as age and rearing experience with siblings may play a role in hypothalamic neurocrine levels, these results demonstrate that paternal experience may impact upon the secretion of neurocrines in a male biparental primate.

Grazing management and supplementation effects on forage and dairy cow performance on cool-season pastures in the southeastern United States.

Cool-season annual forages provide high-quality herbage for up to 5 mo in the US Gulf Coast states, but their management in pasture-based dairy systems has received little attention. Objectives of this study were to evaluate pasture and animal responses when lactating Holstein cows (n=32, mean DIM=184±21) grazed either N-fertilized rye (Secale cereale L.)-annual ryegrass (Lolium multiflorum Lam.) mixed pastures or rye-annual ryegrass-crimson clover (Trifolium incarnatum L.)-red clover (Trifolium pratense L.) pastures at 2 stocking rates (5 vs. 2.5 cows/ha) and 2 rates of concentrate supplementation [0.29 or 0.40 kg of supplement (as is)/kg of daily milk production]. Two cows paired by parity (one multiparous and one primiparous) were assigned randomly to each pasture. The 2 × 2 × 2 factorial arrangement of treatments was replicated twice in a completely randomized design. Forage mixture and supplementation rate did not affect milk production during three 28-d periods. Greater milk production occurred at the low (19.7 kg/d) than the high (14.7 kg/d) stocking rate during periods 2 and 3, but production was similar during period 1. Despite lower production per cow, milk production per hectare was generally greater at the high stocking rate (81.6 vs. 49.5 kg/ha). Generally, greater pregraze herbage mass on pastures at the lower stocking rate (1,400 vs. 1,150 kg/ha) accounted for greater herbage allowance. Both forage (8.0 vs. 5.9 kg/d) and total (14.1 vs. 11.6) organic matter intake were greater at the low stocking rate. Cows fed less supplement had greater forage organic matter intake (8.0 vs. 6.1 kg/d). Greater herbage mass was associated with the greater intake and subsequent greater milk production. Differences in forage nutritive value, blood metabolites and milk composition, although showing some response to treatments, may not be of sufficient magnitude to affect choice of pasture species or other management practices. Animal performance was not improved by adding clovers to mixed cool-season grass pastures like those in this study. Stocking rate had a major effect on pasture and animal performance. During the cool season, supplementation with concentrates should be planned based on estimated energy intake from forages to achieve optimum milk production and ensure maintenance of body condition.

Naproxen-PC: a GI safe and highly effective anti-inflammatory.

UNLABELLED: We have been developing a family of phosphatidylcholine (PC)-associated NSAIDs, which appear to have improved GI safety and therapeutic efficacy in both rodent model systems and pilot clinical trials. As naproxen has been demonstrated to be associated with the lowest cardiovascular adverse events in comparison with both COX-2 selective inhibitors and conventional NSAIDs, we have been developing a Naproxen-PC formulation for evaluation in animal models and clinical trials. We have determined that an oil-based formulation of naproxen and triple strength soy lecithin provides excellent GI protection in both: 1) an acute NSAID-induced intestinal bleeding model in rats pretreated with L-NAME that are intragastrically administered a single dose of naproxen (at a dose of 50 mg/kg) vs the equivalent dose of Naproxen-PC; and 2) a more chronic model (at a naproxen dose of 25 mg/kg BID) in rats that have pre-existing hindpaw inflammation (induced with a intradermal injection of Complete Freund's Adjuvant/CFA). Both models demonstrate the superior GI safety of Naproxen-PC vs naproxen while this novel formulation had significant anti-inflammatory efficacy to reduce hindpaw edema and the generation of PGE(2) in the collected joint synovial fluid. CONCLUSION: Naproxen-PC appears to induce significantly less GI injury and bleeding in two rodent model systems while maintaining anti-inflammatory and COX-inhibitory activity.

Changes in antibiotic susceptibility in staphylococci habituated to sub-lethal concentrations of tea tree oil (Melaleuca alternifolia).

AIMS: To investigate the effect of sub-lethal challenge with tea tree oil (TTO) on the antibiotic resistance profiles of staphylococci. METHODS AND RESULTS: Isolates of methicillin-resistant/-sensitive Staphylococcus aureus (MRSA and MSSA) and coagulase-negative staphylococci (CONS) were habituated to sub-lethal concentrations of TTO (72 h). Following habituation, the minimum inhibitory concentrations (MIC) of antibiotics and TTO were determined. Habituated MRSA/MSSA cultures had higher (P < 0.05) MIC values than control cultures for the examined antibiotics. Habituated MRSA/MSSA cultures also displayed decreased susceptibility to TTO. Although the MIC of habituated MRSA/MSSA for the examined antibiotics reverted to control values after subsequent culture in the absence of TTO, the increased MIC against TTO were maintained. When compared with control cultures, habituated CoNS cultures had higher (P < 0.05) MIC values against three-fifths of the antibiotics examined; no changes in TTO MIC were observed. CONCLUSIONS: TTO habituation 'stress-hardens' MRSA and MSSA, evidenced by transient decreased antibiotic susceptibility and stable decreased TTO susceptibility. Although TTO habituation did not decrease susceptibility of CoNS to TTO, such cultures showed transient decreased antibiotic susceptibility. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of TTO at sub-lethal concentrations may reduce the efficacy of topical antibiotics used with TTO in combination therapies.

Lack of horizontal gene transfer of methicillin-resistance genetic determinants from PBP2a-positive, coagulase-negative staphylococci to methicillin-sensitive Staphylococcus aureus using transcutaneous electrical nerve stimulation (TENS).

Previous research shows that approximately half of the coagulase-negative staphylococci (CNS) isolated from patients in the intensive care unit (ICU) at Belfast City Hospital were resistant to methicillin. The presence of this relatively high proportion of methicillin-resistance genetic material gives rise to speculation that these organisms may act as potential reservoirs of methicillin-resistance genetic material to methicillin-sensitive Staphylococcus aureus (MSSA). Mechanisms of horizontal gene transfer from PBP2a-positive CNS to MSSA, potentially transforming MSSA to MRSA, aided by electroporation-type activities such as transcutaneous electrical nerve stimulation (TENS), should be considered. Methicillin-resistant CNS (MR-CNS) isolates are collected over a two-month period from a variety of clinical specimen types, particularly wound swabs. The species of all isolates are confirmed, as well as their resistance to oxacillin by standard disc diffusion assays. In addition, MSSA isolates are collected over the same period and confirmed as PBP2a-negative. Electroporation experiments are designed to mimic the time/voltage combinations used commonly in the clinical application of TENS. No transformed MRSA were isolated and all viable S. aureus cells remained susceptible to oxacillin and PBP2a-negative. Experiments using MSSA pre-exposed to sublethal concentrations of oxacillin (0.25 microg/mL) showed no evidence of methicillin gene transfer and the generation of an MRSA. The study showed no evidence of horizontal transfer of methicillin resistance genetic material from MR-CNS to MSSA. These data support the belief that TENS and the associated time/voltage combinations used do not increase conjugational transposons or facilitate horizontal gene transfer from MR-CNS to MSSA.

Comparison of three techniques for estimating the forage intake of lactating dairy cows on pasture.

Quantifying DMI is necessary for estimation of nutrient consumption by ruminants, but it is inherently difficult on grazed pastures and even more so when supplements are fed. Our objectives were to compare three methods of estimating forage DMI (inference from animal performance, evaluation from fecal output using a pulse-dose marker, and estimation from herbage disappearance methods) and to identify the most useful approach or combination of approaches for estimating pasture intake by lactating dairy cows. During three continuous 28-d periods in the winter season, Holstein cows (Bos taurus; n = 32) grazed a cool-season grass or a cool-season grass-clover mixture at two stocking rates (SR; 5 vs. 2.5 cows/ha) and were fed two rates of concentrate supplementation (CS; 1 kg of concentrate [as-fed] per 2.5 or 3.5 kg of milk produced). Animal response data used in computations for the animal performance method were obtained from the latter 14 d of each period. For the pulse-dose marker method, chromium-mordanted fiber was used. Pasture sampling to determine herbage disappearance was done weekly throughout the study. Forage DMI estimated by the animal performance method was different among periods (P < 0.001; 6.5, 6.4, and 9.6 kg/d for Periods 1, 2, and 3, respectively), between SR (P < 0.001; 8.7 [low SR] vs. 6.3 kg/d [high SR]) and between CS (P < 0.01; 8.4 [low CS] vs. 6.6 kg/d [high CS]). The period and SR effect seemed to be related to forage mass. The pulse-dose marker method generally provided greater estimates of forage DMI (as much as 11.0 kg/d more than the animal performance method) and was not correlated with the other methods. Estimates of forage DMI by the herbage disappearance method were correlated with the animal performance method. The difference between estimates from these two methods, ranging from -4.7 to 5.4 kg/d, were much lower than their difference from pulse-dose marker estimates. The results of this study suggest that, when appropriate for the research objectives, the animal performance or herbage disappearance methods may be useful and less costly alternatives to using the pulse-dose method.

Pasture forages, supplementation rate, and stocking rate effects on dairy cow performance.

Objectives were to evaluate effects of forage species, stocking rate, and supplementation rate on performance and physiology of grazing lactating Holstein cows under intensive rotational stocking management during summer. Eight treatments were arranged in a 2 x 2 x 2 factorial design. Animals (n = 62) grazed pastures of Tifton 85 bermudagrass or Florigraze rhizoma peanut, a tropical legume. Low and high stocking rates were 7.5 and 10.0 cows/ha for bermudagrass and 5.0 and 7.5 cows/ha for rhizoma peanut. Within each forage-stocking rate combination, cows were fed supplement at 0.33 or 0.5 kg of supplement (as-fed basis)/kg daily milk production. Cows grazing rhizoma peanut pastures produced more milk (16.9 vs. 15.4 kg/d) but had higher rectal temperatures (39.4 vs. 39.1 degrees C). Milk production per cow was improved at the higher stocking rate for bermudagrass but was reduced at the higher stocking rate for peanuts. Increasing supplementation rate boosted plasma glucose, milk production, and milk protein percent. Increased supplementation rate had a greater positive impact on milk production of cows grazing bermudagrass compared to rhizoma peanut (21.9 vs. 10.6% increase) due to a lower substitution of grain for forage intake. Organic matter intakes of forage, supplement, and total diet were greatest by cows grazing rhizoma peanut pastures and averaged 12.4, 6.1, and 18.5 kg/d compared to 9.2, 5.4, and 14.6 kg/d for cows grazing bermudagrass. Despite lower individual feed intake and performance, production per unit land area was 29% greater (112 vs. 90 kg of milk/ha per d) for cows grazing bermudagrass due to the greater stocking rate possible with that forage. Only cows supplemented at the high rate and kept at the high stocking rate on bermudagrass maintained body weight. Cows on other treatments lost body weight. Tifton 85 bermudagrass appears to be an excellent summer forage for dairy cows grazing in the southeastern U.S. given its nutritive value characteristics and high yields. Optimum stocking rate may be as high as 10 cows/ha during times of peak growth of forage for low-to-moderately producing cows fed supplement. Furthermore, the positive milk production response to additional supplement when cows grazed Tifton 85 pastures (0.8 kg/kg of supplement), indicates the value of providing supplement to cows grazing this moderate quality forage.

Incidence of Pseudomonas aeruginosa in recreational and hydrotherapy pools.

Pseudomonas aeruginosa remains an important agent of opportunistic infection in patients, particularly those with respiratory complications and burns. One natural niche of this organism is water and water-associated facilities, hence the aim of this study was to examine specimens from recreational and hydrotherapy pools in Northern Ireland over a two-year period. Water specimens (n = 3,510) were obtained from three amenity categories, namely, 13 hydrotherapy pools (specimen number [n] = 323), 51 Jacuzzis/spas (n = 1,397) and 68 swimming pools (n = 1,790). Specimens (100 ml) were filtered through a cellulose acetate (0.45 micron pore size) gridded filter and the membrane was placed on Pseudomonas CFC agar (Oxoid CM559 + SR103) and incubated at 37 degrees C for 48 +/- 2 h. Colonies that clearly showed pyocyanin production or met other identification criteria were considered P. aeruginosa. Of the amenities examined 4/13 hydrotherapy pools (30.8%), 37/51 Jacuzzis/spas (72.5%) and 26/68 swimming pools (38.2%) were positive for P. aeruginosa. The most heavily contaminated amenity category was the Jacuzzi/spa, where 34.7% and 12% of private and public sites respectively were positive for P. aeruginosa at a level of greater than 1,000 cfu 100 ml-1. Approximately twice as many samples were positive in private Jacuzzis/spas compared to publicly operated facilities. There was a similar trend with respect to public and private hydrotherapy pools, though bacterial counts did not exceed 1,000 cfu 100 ml-1. Recreational and therapeutic amenities involving the use of water may be a potential source of P. aeruginosa for susceptible patient groups, including patients with cystic fibrosis and bronchiectasis. This may vary depending on amenity type and public/private ownership of such amenities.

Effects of supplementation on voluntary forage intake, diet digestibility, and animal performance.

A data base was constructed to describe and estimate supplementation effects in nonlactating cattle consuming forage ad libitum. The data base included 66 publications on 126 forages (73 harvested and 53 grazed) and a total of 444 comparisons between a control, unsupplemented treatment and a supplemented treatment. Daily gains were reported for 301 comparisons and voluntary intake for 258. Direct measures of forage digestibility were reported for 202 comparisons, and total diet digestibility for 150. Supplements did not increase gain in all cases. Change in ADG due to supplement was not related closely to intake of supplemental TDN. Lowest increases in ADG were with native forages supplemented with molasses alone or with low intakes of molasses containing high levels of NPN. Greatest increases in gain were with improved forages, supplements with > 60% TDN, and supplemental CP intake > .05% of BW. Supplements decreased voluntary forage intake (VFI) when supplemental TDN intake was > .7% of BW, forage TDN:CP ratio was < 7 (adequate N), or VFI when fed alone was > 1.75% of BW. When supplements increased VFI, forage TDN: CP ratio was > 7 (N deficit), and VFI when fed alone was often low. There was little relationship between change in VFI and sources of supplemental CP and TDN. Supplements caused total diet TDN concentration to deviate from expected values by -10 to +5% of OM. When supplemental TDN intake was > .7% of BW, diet TDN concentration was always less than expected. There was little relationship between deviation from expected total diet TDN and type or composition of forages or supplements. Empirical multiple regression equations were developed to estimate effects of supplements on VFI and total diet TDN concentration. The most acceptable intake equation estimated VFI when fed with supplement (r2 = .84) That equation included VFI when fed alone, supplement intake, CP and TDN concentrations in forage and supplement, and classification codes describing forages and supplemental energy. The most acceptable equation for estimating total diet TDN concentration included only the expected total diet TDN concentration (r2 = .87). These equations may be used in nutritional models to account for associative effects.