In vitro evaluation of Saccharomyces cerevisiae cell wall fermentability using a dog model.

Six Saccharomyces cerevisiae cell wall samples were tested by the in vitro gas production technique using dog faeces as inoculum. In particular, the substrates resulted from three different production processes (alcoholic_A, bakers_BA and brewers_BR) and were characterized by two different carbohydrates (mannans + glucans) concentrations nitrogen-free extract (NFE high and low). Gas production of fermenting cultures was recorded for 72 hr to estimate the fermentation profiles. The organic matter degradability (OMD), fermentation liquor pH, short-chain fatty acids (SCFA) and ammonia (NH3 ) productions were also measured. All substrates presented a high percentage of OMD (>92%) and moderate fermentability in terms of cumulative volume of gas related to incubated OM (OMCV >50 ml/g) and short-chain fatty acids production (>25 mmol/g), proving their functional properties. Comparing the substrates, it seems evident that the production process affects the chemical composition of the yeast cell wall in terms of crude protein, ether extract and ash content. Consequently, the in vitro fermentation process was significantly different among substrates for volume of gas, SCFA and ammonia production. Regarding the fermentation rate profiles, the production process influenced mainly the curve shape, whereas the NFE concentration affected the quantity of gas produced per hour. In particular, both S. cerevisiae_BR showed very high percentage of OM degradability, gas and SCFA productions and a fast fermentation process due to their high content of fermentable carbohydrates. On the contrary, both S. cerevisiae_BA yeast cell walls appeared to be less degradable and fermentable, probably due to their high content of ether extract. Regarding both S. cerevisiae_A, the high protein content of these substrates could explain the contrasting in vitro results (high degradability with low gas and SCFA production).

Nutritional management of search and rescue dogs.

Dogs used for search and rescue (SAR) may experience continuous micro-traumas that predispose them to skeletal disorders. The aim of the present study was to evaluate the effect of diet on osteo-articular apparatus in healthy SAR dogs. A total of sixteen SAR dogs were divided into two groups (low supplementation (LS) and high supplementation (HS)) and were fed for 3 months with two experimental diets, characterised by the same protein and energy density, but different in n-3 PUFA (6·2 v. 8·4 % of metabolisable energy), chondroitin sulfate (219·8 v. 989·0 mg/kg DM) and glucosamine (769·2 v. 1318·7 mg/kg DM) in the LS and HS groups, respectively. At recruitment all dogs showed no joint inflammation signs, except four that showed mild symptoms. Haematology and serum biochemistry were performed every 30 d. Joint status was scored by physical and lameness evaluations. The sampling effect analysis showed potential beneficial effects by a decrease in a specific marker of membrane integrity (creatine kinase; CK). Comparing groups, glucose was significantly higher and CK was significantly lower in the HS group; however, in both cases the levels of these parameters fell in the normal range. At the end of the experiment, erythrocytes, Hb and packed cell volume were significantly higher in the HS compared with the LS group. These could result in an improvement in dogs' performance, even if this aspect was not investigated in the present study. Concerning joint evaluation (pain on manipulation, lameness and range of motion), no statistically significant differences were detected between the groups and within the experimental period.

Comparison of nutritional and antinutritional traits among different species (Lupinus albus L., Lupinus luteus L., Lupinus angustifolius L.) and varieties of lupin seeds.

In order to promote the use of lupin in pig nutrition, in this research the nutritional characteristics (i.e. dietary fibre, alkaloid and fatty acid profile) and the in vitro gas production of 12 lupin varieties grown in the Mediterranean basin and belonging to three lupin species (Lupinus albus, Lupinus angustifolius and Lupinus luteus) were assessed. Four varieties of L. albus (Asfer, Lublanc, Lutteur and Multitalia) were grown in South Campania. Three varieties of L. luteus (Dukat, Mister and Taper), three of L. angustifolius (Jindalee, Sonet and Wonga) and two of L. albus (Rosetta and Luxor) were grown in Eastern Sicily. Lupinus albus varieties showed interesting nutritional and dietetic characteristics (i.e. high protein and low fibre content); the lipid fraction, rather elevated, is well represented by monounsaturated fatty acids (544 g/kg), whereas saturated fatty acids (SFAs) are less represented (167 g/kg) and the n-3/n-6 ratio (0.510) is the most favourable. Lupinus luteus varieties presented the most remarkable dietetic aspects, in terms of polyunsaturated fatty acid (PUFA) content (569 g/kg), n-6 PUFA series (490 g/kg), UFA/SFA (5.24) and PUFA/SFA (3.56) ratios and atherogenic (0.059) and thrombogenic (0.100) indices and very low alkaloid content (1.07 mg per 100 g). Lupinus angustifolius varieties showed the least interesting nutritional and dietetic characteristics: low protein and fat content, high fibre level, high SFA amount (248 g/kg) and the lowest favourable nutritional indices (IA: 0.164 and IT: 0.334). Regarding the fermentation process, in L. albus, the tendency to increase the rate of gas production during the early stages of fermentation suggests that the high presence of alkaloids did not affect the in vitro degradability, production of short-chain fatty acids and fermentation process, probably due to their concentration and/or water solubility. Lupinus angustifolius and L. luteus showed intermediate and slightly worse in vitro fermentation patterns respectively. From a nutritional and dietetic point of view, lupin may represent an interesting alternative to soya bean in pig feeding.

Drug survival of biologic therapy in a large, disease-based registry of patients with psoriasis: results from the Psoriasis Longitudinal Assessment and Registry (PSOLAR).

BACKGROUND: Drug survival is a marker for treatment sustainability in chronic diseases such as psoriasis. OBJECTIVE: The aim of these analyses was to assess survival of biologic treatments in the PSOriasis Longitudinal Assessment and Registry (PSOLAR). METHODS: PSOLAR is a large, prospective, international, disease-based registry of patients with psoriasis receiving (or eligible for) systemic therapy in a real-world setting. Drug survival is defined as the time from initiation to discontinuation (stop/switch) of biologic therapy on registry. The number of patients who discontinued each treatment and the duration of therapy were recorded. Using Kaplan-Meier survival curves and Cox-regression analyses [hazard ratios (HR) and 95% confidence intervals (CIs)], time to discontinuation was compared across cohorts undergoing first-, second- or third-line treatment with ustekinumab, infliximab, adalimumab or etanercept. RESULTS: As of the 2013 data cut, 12 095 patients with psoriasis were enrolled in PSOLAR. Of the 4000 patients initiating any new biologic therapy, approximately 3500 started a first-line, second-line or third-line biologic therapy during the registry. Lack of effectiveness was the most common reason for discontinuation across biologic therapies. Based on the multivariate analysis, significantly shorter times to discontinuation were observed for infliximab [HR (95%CI) = 2.73 (1.48-5.04), P = 0.0014]; adalimumab [4.16 (2.80-6.20), P < 0.0001]; and etanercept [4.91 (3.28-7.35) P < 0.0001] compared with ustekinumab [reference treatment]) for first-line biologic use; results were similar for treatment effects for second/third-line therapies. Although limited in power, analyses in patients with concurrent psoriatic arthritis confirmed by a rheumatologist reflect observations in the overall psoriasis population. CONCLUSION: Drug survival was superior for ustekinumab compared with infliximab, adalimumab and etanercept in patients with psoriasis.

Regulation of Stearoyl Coenzyme A Desaturase 1 Gene Promoter in Bovine Mammary Cells.

Stearoyl-Coenzyme A desaturase 1 (SCD1) belongs to the fatty acid family of desaturases. In lactating ruminants, the SCD1 protein is highly expressed in the mammary gland and is relevant for the fatty acid composition of milk and dairy products. Bovine mammary epithelial cells (BME-UV1), cultured in vitro, have been proposed as a model to reproduce the biology of the mammary gland. The present study was designed to investigate the responsiveness of bovine SCD1 promoter to serum, insulin, oleic acid, and NFY transcription factor in BME-UV1 cells. A luciferase-based reporter assay was used to monitor the transcriptional activity of the SCD1 promoter region in BME-UV1 cells treated or not with insulin and/or oleic acid. The level of endogenous SCD1 mRNA was evaluated by Real time PCR. Insulin (20 ng/mL) induced a 2.0 to 2.5-fold increase of SCD1 promoter activity. Additionally, the effect of insulin was inhibited by oleic acid, serum components, and NFY enforced expression. Serum and NFY showed no synergistic or additive effect on SCD1 promoter activity suggesting that they repress SCD1 transcription through the same responsive element.

Demography, baseline disease characteristics and treatment history of patients with psoriasis enrolled in a multicentre, prospective, disease-based registry (PSOLAR).

BACKGROUND: Psoriasis is associated with several comorbidities and behavioural risk factors. OBJECTIVES: To evaluate demographic and disease characteristics in patients enrolled in the Psoriasis Longitudinal Assessment and Registry (PSOLAR). METHODS: PSOLAR is a global, prospective, longitudinal, disease-based registry that includes a postmarketing commitment to evaluate safety in patients with psoriasis. Enrolled patients had to be receiving, or be eligible to receive, conventional systemic or biological agents. Demographic/disease characteristics, medical histories, lifestyle risk factors and previous treatments are collected at enrolment. Efficacy and safety data are collected every 6 months for 8 years, and data are extracted annually. Selected parameters are evaluated by age quartile using post hoc analyses. RESULTS: As of 23 August 2012, 11 900 patients were enrolled at 301 sites in North America, Europe and Latin America. Over half of the PSOLAR population (54·7%) is male, with a mean age of 48·6 years and mean body mass index of 30·9 kg m(-2) at enrolment. Mean duration of disease at enrolment was 17·5 years, and mean Physician's Global Assessment score was 2·0. Psoriatic arthritis (35·5%) and cardiovascular diseases (38·2%) were highly prevalent. Diabetes mellitus type II was reported in 11·4% of patients. Depression and anxiety were noted in 14·7% and 11·1% of patients, respectively; 79·0% reported any alcohol use and 56·7% reported smoking or a history of smoking. The occurrence of most comorbidities, including cardiovascular disease and risk factors, increased with age. CONCLUSIONS: In the PSOLAR population, multiple and age-appropriate comorbidities are associated with psoriasis and may affect the selection of psoriasis treatments.

20 × 960-Gb/s Space-division-multiplexed 32QAM transmission over 60 km few-mode fiber.

We show transmission of 20 wavelength-division-multiplexed (WDM) × 960-Gb/s space-division-multiplexed 32QAM modulated channels (spectral efficiency (SE) of 15 bits/s/Hz) over 60 km of few-mode fiber (FMF) with inline few-mode EDFA (FM-EDFA). Soft-decision FEC was implemented and used to achieve error-free transmission.

Meat quality of buffalo young bulls fed faba bean as protein source.

Sixteen Italian Mediterranean Buffalo young bulls were divided into two groups fed isoprotein and isoenergy diets and only differing for protein source of concentrate: faba bean (FB) vs soybean (SB). Animals were slaughtered at 350 kg BW. Meat from FB group showed significantly lower fat, protein, cholesterol and saturated fatty acids than SB group. Significant differences were also found between the three muscles analysed [Longissimus thoracis (LT), Semitendinosus (ST) and Iliopsoas plus Psoas minor (IP)]. ST showed the most favourable fatty acids profile: lower SFA, higher PUFA, MUFA, ω-3, ω-6, CLA and, consequently, lower values for both atherogenic and thrombogenic indexes. Results showed that faba bean can be used as a protein source alternative to soybean in the diet of young buffalo bulls for the production of high quality meat.

Fermentation kinetics of sainfoin hay with and without PEG.

The in vitro gas production technique was applied to evaluate the biological activity of tannins in sainfoin (Onobrychis viciifolia Scop.) hay cut at four progressive phenological stages. The buffalo (Bubalus bubalis) rumen fluid was used as inoculum, polyethylene glycol (PEG) was added as specific binding agent and an alfalfa hay (AH) was used as control. All samples were analysed for chemical composition and condensed tannin (CTs). According to the phenological stage of sainfoin, crude protein gradually decreased [from 219 to 122 g/kg dry matter (DM)] and neutral detergent fibre (NDF) increased (from 391 to 514 g/kg DM). Condensed tannins showed the highest value at the first phenological stage [31.5 mg catechin equivalents (CE)/g DM], and their level significantly decreased at the subsequent growth stages. Gas production and fermentation rate were significantly affected by substrate type and PEG addition. The first stage of sainfoin showed the lowest value of gas and volatile fatty acids production and the slowest fermentation kinetics compared with the other samples, when incubated without PEG, because of the CTs effect. The incubation with PEG improved the fermentation process of the first stage of sainfoin, but no effect was registered on AH, where CTs were not detected. Among the sainfoin samples, the slowest kinetics with PEG was observed in the hay harvested at the fourth stage, probably due to its chemical composition. The use of PEG allowed to verify the effect of tannins on the in vitro fermentation characteristics of sainfoin hay.

Influence of replacing corn silage with barley silage in the diets of buffalo cows on milk yield and quality.

A 150-day trial was carried out on 40 Italian Mediterranean buffalo cows that, immediately after calving, were equally divided into two homogeneous groups (M and O) based on the number of calving events and previous milk yield. The animals were fed (16 kg dry matter (DM)/head) two isoenergy/isoprotein diets (NEl: 6.39 MJ/kg DM; 15.4 CP% DM), composed of corn (diet M) or barley silage (diet O) concentrate, alfalfa hay, and a vitamin-mineral supplement. The fermentation characteristics of both silage diets were evaluated by an in vitro gas production technique, and their nutritional values were calculated as follows: NEl (MJ/kg DM) = 0.54 + 0.0959 GP + 0.0038 CP + 0.0001733 CP(2), where GP is the gas production after 24 h of incubation (ml/200 mg DM) and CP is the protein content of silage (g/kg DM). The nutritional values of the silages were slightly different (4.16 vs. 4.14 MJ/kg DM for M and O, respectively) likely due to the high content of hemicellulose in the O diet (22.0 vs. 16.9%). Average milk yield did not differ between the groups; instead, milk fat (8.39 vs. 9.06%; P < 0.01) and protein (4.41 vs. 4.60%; P < 0.01) levels were significantly higher in the O group. The results elicit great interest in southern Italy where corn cultivation is adversely affected by the high cost of irrigation.

Fate of transgenic DNA and evaluation of metabolic effects in goats fed genetically modified soybean and in their offsprings.

The presence of DNA fragments in blood and milk from goats fed conventional (control) or Roundup Ready® soybean meal solvent extracted (s.e.; treated) was investigated by using a polymerase chain reaction approach. The same investigation was carried out on blood, skeletal muscle and organs from kids of both groups fed only dams' milk until weaning. Moreover, the possible effects on cell metabolism were evaluated by determination of several specific enzymes in serum, heart, skeletal muscle, liver and kidney. Fragments of the multicopy chloroplast (trnL) gene were found in blood and milk samples from goats of both groups. In kids, the chloroplast fragments were found in samples of both groups. In samples, which proved positive for the presence of chloroplast DNA, fragments of the specific soybean single copy gene (lectin) were detected in several blood and milk samples. The same fragment was also found in control and treated groups of kids. Transgenic fragments were not found in those samples, which were found positive for chloroplast fragments of control groups of either goats or kids. On the contrary, in blood and milk of treated goats, fragments both of the 35S promoter and the CP4 epsps gene were detected. These fragments were also found in treated kids with a significant detection of the 35S promoter in liver, kidney and blood, and of the CP4 epsps gene fragment in liver, kidney, heart and muscle. A significant increase in lactic dehydrogenase, mainly concerning the lactic dehydrogenase-1 isoenzyme was found in heart, skeletal muscle and kidney of treated kids, thus suggesting a change in the local production of the enzyme. Finally, no significant differences were detected concerning kid body and organ weight.

In vitro fermentation characteristics of different carbohydrate sources in two dog breeds (German shepherd and Neapolitan mastiff).

Few studies have been published on the normal intestinal biota of canines unlike the wealth of information regarding livestock animal species. The in vitro gas production technique (IVGPT) including measurements of accumulating gas during fermentation and end-product determinations allows obtaining a complete picture of microbial activity kinetics. The aim of this study was to study the in vitro fermentation characteristics of different carbohydrate sources using inocula from two dog breeds (German Shepherd and Neapolitan mastiff). Faeces sampled from rectum of two GS and NM adult dogs, fed the same dry food, were used as inocula. The samples, diluted and filtered, were incubated at 39 degrees C under anaerobic condition with nine substrates different for carbohydrate composition (rice, corn, potato, spelt, pure cellulose, beet pulp, wheat bran, inulin and fructo-oligosaccharide). Gas production was recorded 17 times using a manual pressure transducer. After 48 h, the fermentation was stopped and fermenting liquor was analysed for pH and volatile fatty acids (VFA). Organic matter digestibility (OMD) was calculated as difference after burning the residuals. OMD, gas production and end-products were significantly correlated with chemical composition of substrates, in particular carbohydrate fractions (total dietary fibre and starch), confirming the effectiveness of the IVGPT in evaluating dog feeds. Concerning the comparison between breeds significant differences (p < 0.01) were found for OMD, gas production, fermentation kinetic parameters and end-products, suggesting a different pathway of fermentation and consequently, a different anaerobic population.

Prediction of rabbit caecal fermentation characteristics from faeces by in vitro gas production technique: roughages.

To find equations able to estimate the fermentation characteristics of the caecum from that of faeces, caecal content and faeces of 10 hybrid Hyla rabbits were used as inocula for an in vitro gas production trial. About 1 g of 12 roughages, 11 hays (ryegrass, alfalfa, sulla, oat, vetch, sulla-lolium, vetch-oat, sulla-oat, clover, ryegrass-clover, sulla-vetch-oat) and a wheat straw, was weighed, in triplicate per inoculum, in 120-ml flasks; 75 ml of anaerobic medium and 4 ml of reducing solution were added and the flasks were placed at 39 degrees C. Caecal content and faeces were diluted respectively 1:2 (CI) and 1:8 (FI) with anaerobic medium and were introduced into their respective flasks (10 ml). Gas production was recorded 20 times at 2-24 h intervals throughout fermentation (120 h). The fermentation characteristics (i.e. degraded organic matter, OMd; potential gas production, A; maximum fermentation rate, R(max); volatile fatty acid, VFA; ammonia, NH(3)) were studied by inocula and substrates. The two inocula did not differ in OMd but CI produced significantly higher gas (A, 213.1 vs. 199.4 ml/g, respectively, for CI and FI, p < 0.01) in less time (R(max), 3.08 vs. 2.24 ml/h, respectively, for CI and FI, p < 0.01). CI also produced higher levels of total VFA (57.86 vs. 46.70 mmol/g OM, respectively, for CI and FI, p < 0.01) and showed a higher branched chain proportion (0.023 vs. 0.018, respectively, for CI and FI, p < 0.01). For some parameters (as OMd pH and propionate) the equations for the estimation of caecal fermentation characteristics from that of faeces were accurate (R(2) > 0.8828) and reliable (CV < 10.78%) suggesting that faeces can be successfully used for the estimation of these parameters.

Rumen fermentation and degradability in buffalo and cattle using the in vitro gas production technique.

An in vitro trial was conducted to investigate the effect of different inoculum sources (buffalo vs. cattle) on rumen fermentation and degradability. Incubations were carried out using rumen fluid obtained from buffalo or cattle fed the same diet [60% grass hay and 40% concentrate; 18 kg dry matter (DM)/day]. The fermentation kinetics of eight feeds commonly used in ruminant nutrition (alfalfa hay, barley meal, beet pulp, corn meal and silage, ryegrass hay and silage and soya bean meal s.e.) were studied with the in vitro gas production technique and rumen fermentation parameters (substrate disappearance, pH and volatile fatty acids production) were determined after 120 h of incubation. The linear relationship indicates that the microbial metabolic pathways of the two inocula for all the substrates were qualitatively similar, albeit often quantitatively different. In this in vitro study, a significant influence of rumen inoculum (buffalo vs. cow) on fermentation and degradability of the examined substrates was found. The differences in buffalo and cattle rumen fermentation can be explained with a different microbial activity of the two ruminant species, because of different amount of microbial population or microbial population constituted by different species of bacteria and protozoa.

Influence of pasture on fatty acid profile of goat milk.

The effect of pasture on the fatty acid profile of milk was evaluated using 30 pluriparous goats, which were divided into two homogeneous groups (H and P) of 15 goats each after kidding. Group H (control) goats were housed in a stall and received alfalfa hay, while group P was led to pasture. The goats of both the groups received the same concentrate. Average milk yield did not differ statistically between the groups. The goats of the P group had significantly higher fat content in milk than those of group H (53.7 vs. 61.5 g/day, p < 0.01), while lactose content was significantly higher for group H (66.2 and 61.4 g/day, for groups H and P, respectively, p < 0.05). Among milk fatty acids, pasture significantly affected the percentages of C18:1 cis9, C18:1 trans11, octadecadienoic acid, mono-unsaturated fatty acid and polyunsaturated fatty acid. Pasture highly influenced the c9 t11 conjugated linoleic acid (CLA) (0.778 and 0.513 g/100 g of fat, for group P and H, respectively, p < 0.01), t10 c12 CLA (0.046 vs. 0.029 g/100 g of fat: p < 0.01, for group P and H, respectively, p < 0.01) and SigmaCLA (0.84 vs. 0.56 g/100 g of fat for group P and H, respectively, p < 0.01) concentrations of milk.

Use of faeces as an alternative inoculum to caecal content to study in vitro feed digestibility in domesticated ostriches (Struthio camelus var. domesticus).

1. In order to find an alternative source of inoculum to caecal content for studying the in vitro feed digestibility in domesticated ostriches (Struthio camelus var. domesticus), caecal content and faeces of 4 male birds were used as inocula for an in vitro gas production trial. 2. About 1 g of each of 5 substrates (maize silage, CS; alfalfa hay, AH; barley, BG; soybean meal, SM; beet pulp, BP) was weighed, in quadruplicate per inoculum, in 120 ml flasks; 75 ml of anaerobic medium and 4 ml of reducing solution were added and flasks were kept at 39 degrees C. Caecal content and faeces were diluted respectively 1 : 2 (CI) and 1 : 4 (FI) with an anaerobic medium and were injected into the respective flasks (10 ml). 3. Gas production was recorded 22 times up to 120 h of incubation and fermentation characteristics (for instance, degraded organic matter, OMd; potential gas production, A; maximum fermentation rate, Rmax; time at which it is reached, Tmax; pH; volatile fatty acid, VFA; ammonia) were studied for each inoculum and substrate. 4. CI and FI showed significant differences in Tmax (16.37 vs 18.47 h, respectively), propionic (16.47 vs 12.07 mmoles/l) and butyric acid (6.50 vs 7.98 mmoles/l) and ammonia concentration (17.18 vs 19.95 mmoles/l). The substrates, according to their chemical composition, showed different fermentation characteristics. However, the regression equations able to estimate some fermentation characteristics of the caecum from those of faeces were statistically significant and showed R2-values ranging from 0.87 to 0.99. 5. The differences in fermentation pathways of the two inocula did not appear to influence the rate and extent of OM digestion. Faecal fermentation predicted rates and extent of OM digestion by caecal fermentation in ostriches; consequently, the faeces could be considered as an alternative to caecal content to study feed digestibility in the species, although there is a need to undertake further research.

Use of two different dietary energy and protein contents to define nutritive requirements of lactating buffalo cows.

Two total mixed rations, differing in energy and protein concentrations (diet A: 6.1 MJ/kg DM of NE(l), 112.5 g/kg DM of metabolizable protein, MP, and 101.7 g of protein digestible at the intestinal level, PDI, vs. diet B: 6.4 MJ, 95.1 g of MP, 78.9 g of PDI) were administered ad libitum for 1 year to two groups of 12 buffaloes in which the partial monthly turnover of animals allowed the lactation stage to be maintained close to 160 days. The diets were formulated to obtain, with a daily intake of about 15.5 kg of DM, a balanced energy supply but an excess of protein (diet A); the opposite occurred with diet B. The average dry matter intake recorded during the trial was 14.5 kg for diet A and 15.0 kg for diet B. Milk production was similar for both groups (7.94 kg vs. 7.95 kg). The diet with a higher protein concentration brought about a significant increase in protein, casein and non-protein nitrogen contents in the milk. A significant increase in milk fat percentage was observed when the animals were fed the diet with a higher energy concentration. The serum urea concentration (8.62 mm/l) of buffaloes fed diet A was outside the physiological range for lactating buffaloes. The energy and protein conversion efficiencies for milk production were more favourable when their supplies were balanced: 3.58 MJ of NE(l)/kg of FPCM for diet A; 2.50 g of MP or 2.07 g of PDI/g of protein milk for diet B. Moreover, when the requirements were satisfied, greater energy or protein supplies failed to induce significant changes in milk yield and quality. Finally, regarding the great differences between estimated MP requirements and those recorded during the trial, the authors suggest that the equations used in the CPM-Dairy software, formulated for dairy cows, are not applicable in buffaloes.