Epidermal-specific deletion of CD44 reveals a function in keratinocytes in response to mechanical stress.

CD44, a large family of transmembrane glycoproteins, plays decisive roles in physiological and pathological conditions. CD44 isoforms are involved in several signaling pathways essential for life such as growth factor-induced signaling by EGF, HGF or VEGF. CD44 is also the main hyaluronan (HA) receptor and as such is involved in HA-dependent processes. To allow a genetic dissection of CD44 functions in homeostasis and disease, we generated a Cd44 floxed allele allowing tissue- and time-specific inactivation of all CD44 isoforms in vivo. As a proof of principle, we inactivated Cd44 in the skin epidermis using the K14Cre allele. Although the skin of such Cd44Δker mutants appeared morphologically normal, epidermal stiffness was reduced, wound healing delayed and TPA induced epidermal thickening decreased. These phenotypes might be caused by cell autonomous defects in differentiation and HA production as well as impaired adhesion and migration on HA by Cd44Δker keratinocytes. These findings support the usefulness of the conditional Cd44 allele in unraveling essential physiological and pathological functions of CD44 isoforms.

Rumen ciliate protozoa of domestic sheep (Ovis aries) and goat (Capra aegagrus hircus) in Kyrgyzstan.

Species composition and concentration of rumen ciliate protozoa were investigated in the rumen contents of 14 domestic sheep and 1 goat living in Bishkek, Kyrgyzstan. This is the first report on rumen ciliates from ruminants living in Kyrgyzstan. In sheep 12 genera, 28 species and 12 morphotypes were detected, whereas in goat 8 genera, 12 species and 4 morphotypes were detected. The density of ciliates in sheep was (28.1 ± 20.0) × 10(4) cells mL(-1) and in goat was 37.0 × 10(4) cells mL(-1). Dasytricha ruminantium, Isotricha prostoma, Entodinium simulans and Ophryoscolex caudatus were major species (100%) in sheep, and for the first time, Diplodinium rangiferi was detected in a domestic goat.

Phylogeny of intestinal ciliates, including Charonina ventriculi, and comparison of microscopy and 18S rRNA gene pyrosequencing for rumen ciliate community structure analysis.

The development of high-throughput methods, such as the construction of 18S rRNA gene clone or pyrosequencing libraries, has allowed evaluation of ciliate community composition in hundreds of samples from the rumen and other intestinal habitats. However, several genera of mammalian intestinal ciliates have been described based only on morphological features and, to date, have not been identified using molecular methods. Here, we isolated single cells of one of the smallest but widely distributed intestinal ciliates, Charonina ventriculi, and sequenced its 18S rRNA gene. We verified the sequence in a full-cycle rRNA approach using fluorescence in situ hybridization and thereby assigned an 18S rRNA gene sequence to this species previously known only by its morphology. Based on its full-length 18S rRNA gene sequence, Charonina ventriculi was positioned within the phylogeny of intestinal ciliates in the subclass Trichostomatia. The taxonomic framework derived from this phylogeny was used for taxonomic assignment of trichostome ciliate 18S rRNA gene sequence data stemming from high-throughput amplicon pyrosequencing of rumen-derived DNA samples. The 18S rRNA gene-based ciliate community structure was compared to that obtained from microscopic counts using the same samples. Both methods allowed identification of dominant members of the ciliate communities and classification of the rumen ciliate community into one of the types first described by Eadie in 1962. Notably, each method is associated with advantages and disadvantages. Microscopy is a highly accurate method for evaluation of total numbers or relative abundances of different ciliate genera in a sample, while 18S rRNA gene pyrosequencing represents a valuable alternative for comparison of ciliate community structure in a large number of samples from different animals or treatment groups.

Rumen ciliates in the African (Cape) buffalo (Syncerus caffer caffer) living in the vicinity of the Orpen Gate entrance into Kruger National Park, South Africa.

Samples of rumen contents were obtained from 10 African (Cape) buffalo living in the vicinity of the Orpen Gate entrance into Kruger National Park in South Africa. Total number of ciliate protozoa per animal ranged from 3.15 to 23.25 x 103. Forty three different species and forms were observed, of which 35 are a new host record. The total number of species and forms per animal varied from 10 to 17. Eudiplodinium maggii occurred in all 10 animals, followed by Dasytricha ruminantium in nine animals. Diplodinium posterovesiculatum, Eudiplodinium magnodentatum and Ostracodinium mammosum were present in seven animals with all other species and forms occurring in five or less animals. 

Studies on the in vitro cultivation of ciliate protozoa from the kangaroo forestomach.

The methods used for culturing rumen protozoa were found to be unsatisfactory for growth of ciliate protozoa from the kangaroo forestomach. Based on published measurements of physical parameters in the marsupial forestomach, several modifications were incorporated into the procedure, i.e., an increase in % hydrogen in the gas phase, adjustment of initial pH of the medium to 6.9-7.0 range, feed only forage as a substrate and incubate at a lower temperature (33-36 °C). Only incubation at the lower temperature increased survival time of the kangaroo protozoa. Two species of Bitricha were still viable after 28 d in culture. Cultures had to be terminated at that time. One of the species differed considerably in size and shape from previously described species and based on 18S rRNA data, may represent a new species of Bitricha. The second species, present in low numbers was identified as Bitricha oblata. In a separate trial, Macropodinium yalanbense survived for 11 d, at which time these cultures also had to be terminated.

Protozoa and digestive tract parameters in blue wildebeest (Connochaetes taurinus) and black wildebeest (Connochaetes gnou), with description of Entodinium taurinus n. sp.

Rumen contents from four blue wildebeest (Connochaetes taurinus) and six black wildebeest (Connochaetes gnou) were collected from two locations during two winter culling seasons. A total of 16 species of protozoa were found in blue wildebeest with Dasytricha ruminantium, Opisthotrichum janus and Ostracodinium gracile occurring in all four animals. In black wildebeest, 23 species of protozoa were observed with only Diplodinium bubalidis (caudal spine morphotype) and Ostracodinium damaliscus being present in all animals. In the blue wildebeest, the total number of species in an individual animal varied from 9 to 11 and in the black wildebeest, the number ranged from 4 to 14. Concentrations of cells per ml ranged from 1110 to 5880 in the blue wildebeest and 3120 to 6240 in the black wildebeest. This study is the first report on protozoa species in the blue and black wildebeest. A new species of Entodinium is described, Entodinium taurinus n. sp., observed in the rumen contents of three blue wildebeest. Several physical parameters of the digestive tract were also measured, including distribution, pH and density of gastrointestinal contents in different sections of the total tract. In vitro gas production was estimated for rumen, cecum and colon contents.

Rumen protozoa in South African sheep with a summary of the worldwide distribution of sheep protozoa.

Protozoa species were identified in rumen contents of four domestic sheep (Ovis aries) from South Africa. All animals were fed a forage diet which consisted of 50% lucerne and 50% teff hay. Ten new host records were identified, bringing the total number of species and forms observed in sheep in South Africa to 30. The occurrence and geographic distribution of ciliate protozoa in both domestic and wild sheep from around the world are summarised. It was found that 15 genera and 131 species occur in domestic sheep globally.

Protozoa and digestive tract parameters of the impala.

Intestinal contents were collected from eight impala at three different localities during the winter hunting season (2005-2009), as well as from another 24 animals from a one-year trial at a game farm called Ditholo (2003-2004). Gas production, protozoa counts and several other physiological parameters were measured from both rumen and caecum or colon contents. Only higher ophryoscolecid and Isotrichidae species of protozoa were counted and identified. Ostracodinium gracile was present in all 32 impala. Eudiplodinium maggii was present in 31 animals and Eudiplodinium impalae and Epidinium (either ecaudatum or caudatum) in 30 animals. Dasytricha ruminantium was present in only 11 of the impala. Concentrations of protozoa were correlated with the season of sample collection and highly correlated with the animals living on the game farm. Gas production (mL/g of wet rumen ingesta) was weakly correlated with protozoa concentration but not with the season of collection.

Effect of external resistance on bacterial diversity and metabolism in cellulose-fed microbial fuel cells.

External resistance affects the performance of microbial fuel cells (MFCs) by controlling the flow of electrons from the anode to the cathode. The purpose of this study was to determine the effect of external resistance on bacterial diversity and metabolism in MFCs. Four external resistances (20, 249, 480, and 1000 Ω) were tested by operating parallel MFCs independently at constant circuit loads for 10 weeks. A maximum power density of 66 mW m(-2) was achieved by the 20 Ω MFCs, while the MFCs with 249, 480, and 1000 Ω external resistances produced 57.5, 27, and 47 mW m(-2), respectively. Denaturing gradient gel electrophoresis analysis of partial 16S rRNA genes showed clear differences between the planktonic and anode-attached populations at various external resistances. Concentrations of short chain fatty acids were higher in MFCs with larger circuit loads, suggesting that fermentative metabolism dominated over anaerobic respiration using the anode as the final electron acceptor.

Effects of diet and weight gain on circulating tumour necrosis factor-α concentrations in Thoroughbred geldings.

Low-grade inflammation precedes the development of obesity-related metabolic disorders in humans, but whether the same is true in the horse is not known. The objective of this study was to examine the effects of weight gain and diet on the inflammatory state of horses as determined by serum concentrations of tumour necrosis factor-α (TNF), an inflammatory cytokine. Fifteen mature Thoroughbred geldings with an initial body weight (BW) of 519±12 kg and body condition score (BCS) of 4.3±0.1 were fed a diet of hay plus a concentrate that was either high in non-structural carbohydrates (NSC) (i.e. starch and sugar), similar to those commercially available (CON) or one that had the energy source replaced with fat and fibre (FAT) for 32 weeks. Weight gain was achieved by feeding an additional 20 Mcal/day in excess of digestible energy maintenance requirements and resulted in a final BW of 608±12 kg and BCS of 6.9±0.1. Horses were exercised twice daily at a walk during the weight gain period. Horses were assessed bi-weekly for BW and BCS. Serum TNF was analysed from blood samples collected at 4-week intervals. Although treatment groups began the study with similar mean serum TNF concentrations, 12 weeks of FAT feeding promoted a decrease in circulating TNF that was maintained throughout the study with the exception of weeks 20 and 32. For either diet, there were no linear correlations between serum TNF concentration and BCS when horses increased in BCS from four to seven. The higher level of TNF observed in horses fed the CON diet indicates an increase in some level of systemic inflammation that was independent of their weight gain from a moderately thin to fleshy condition. The influence of diet on serum TNF concentrations should be investigated in horses fed to maintain body condition.

Nutrient intake during an elite level three-day event competition is correlated to inflammatory markers and antioxidant status.

REASONS FOR PERFORMING STUDY: Dietary intake and feeding management practices could affect the degree of physiological stress and subsequent performance of high level 3-day event horses. OBJECTIVES: To assess whether a relationship exists between dietary intake levels of selected nutrients and the inflammatory and antioxidant status in horses competing in a high level 3-day event. MATERIALS AND METHODS: Riders competing in a CCI2*/CCI3* 3-day event (n = 35) answered a nutritional management survey conducted by the investigators at the competition. Actual and recommended intakes of vitamin E, potassium (K), calcium (Ca), phosphorus (P) and magnesium (Mg) were calculated using the manufacturer or NRC values. Blood samples, bodyweight and body condition score of horses were taken precompetition, 30 min and 18-24 h after cross-country, but before stadium jumping. Data were analysed using a mixed model ANOVA with repeated measures and Pearson's product moment correlation. RESULTS: Estimated daily intakes of vitamin E, K, Ca, P and Mg for horses were higher than daily recommended levels (P < 0.05). In response to competition, tumour necrosis factor-α (TNFα; P = 0.0002), nitric oxide (NO; P = 0.013) and ß-carotene (BC; P < 0.0001) decreased, creatine kinase (P < 0.0001) and aspartate aminotransferase (P = 0.001) increased, and α-tocopherol and retinol did not change. Intake of vitamin E, K, Ca, P and bodyweight were negatively correlated with TNFα (P < 0.05). Vitamin E and bodyweight were also negatively correlated with NO (P < 0.05). Pasture intake and BC were positively correlated (P < 0.0001). CONCLUSIONS: The decline in systemic inflammatory markers is probably due to increased utilisation or excretion and decreased production related to the increased oxidative stress experienced by horses during competition. High bodyweights could also predispose horses to a higher level of inflammation during 3-day event competition.

Nutrition-associated problems facing elite level three-day eventing horses.

REASONS FOR PERFORMING STUDY: The main goal of feeding elite 3-day event horses is to deliver nutrients in optimal amounts to allow the horse to maximize its health and performance. However, improper nutritional management and/or physiological stressors related to intense training and competition may increase the risk of nutrition-associated disorders in these horses. An understanding of the nutrition-associated problems contributing to poor performance is critical to the health and welfare of the horse. OBJECTIVES: To characterize the nutrition-associated problems affecting top level 3-day event horses during 2008. METHODS: Contact information for riders competing in the 2 highest levels of 3-day eventing in 2008 was obtained from the United States Eventing Association. A survey containing 10 questions pertaining to participant demographics and nutrition-associated problems experienced by their horses was mailed and e-mailed to the 81 individuals fitting our criteria of living in USA and Canada. Data was collected in April and May 2009. RESULTS: Twenty-nine of 81 riders completed the survey (35.8%). Respondents rode a total of 45 horses in top level 3-day events in 2008. The top 5 nutrition-associated problems that horses faced at a significantly higher level than the other problems (P < 0.0001) were gastric ulcers (42.2%), joint problems (37.7%), decreased appetite (31.1%), weight loss (31.1%) and hyperexcitability (22.2%). There was no significant difference in frequency of problems between home and competition (P = 0.22). CONCLUSIONS: Horses competing at a high level of 3-day eventing in 2008 were at risk of reduced performance given the significant rate of gastric ulcers, decreased appetite and weight loss. Research addressing specific causes of and/or feeding management changes that would reduce the incidence of these problems in these horses is needed to ensure optimal health and performance.

Physiological characteristics of several rumen protozoa grown in vitro with observations on within and among species variation.

When fed equal amounts of substrate, two Epidinium caudatum clone cultures of markedly different size produced similar volumes of microbial protoplasm. Addition of up to 50% volume of 72h culture medium had no inhibitory effects on growth of Epidinium. Two clone cultures of Epidinium caudatum from Australia had longer generation times and showed less substrate attachment when compared to Ohio clones of this same species. Substitution of alfalfa for orchardgrass in the normal substrate increased Epidinium concentrations, while feeding only ground orchardgrass or alfalfa resulted in a marked decrease or disappearance of the protozoa. Eudiplodinium impalae, isolated from rumen contents of a steer in Australia, was successfully cultured, with generation times for this species averaging 11.3h. Reducing particle size of the substrates by ball-milling was detrimental for growth of Entodinium and Epidinium; however, Eudiplodinium increased in concentration. Significant concentration differences were observed among six clone cultures of Epidinium obtained from Europe. A generation time of 18.7h was measured for Enoploplastron triloricatum when the culture was transferred every 12h. Lowering the incubation temperature to 34°C completely inhibited protozoal growth of Epidinium and Entodinium exiguum after 12 days, but not for Entodinium caudatum.

Voluntary intake and digestibility of teff hay fed to horses.

The objective of this study was to evaluate nutrient composition, voluntary DMI, and apparent DM digestibility of teff hay cut at 3 different stages of maturity to evaluate its potential as a preserved forage for horses. Six mature Quarter Horse mares (12 +/- 3 yr; 553 +/- 39 kg of BW) were used in a replicated balanced Latin square design with 3 periods and 3 maturities of teff hay. Eragrostis tef ('Tiffany' teff) was planted in May and harvested at the boot, early-heading, or late-heading stage of maturity through the summer. Horses were acclimated to a mixture of maturities of teff hay for 8 d before the beginning of the study. After this acclimation period, each period consisted of a 9-d voluntary DMI phase, followed by a 3-d DM digestibility phase. The percentages of nonstructural carbohydrates (NSC) increased from 5.4% in the boot stage to 8.4% in the late-heading stage, whereas concentrations of CP, K, Fe, and Mn decreased. The Ca:P ratio was 2.0 ± 0.3 for all maturities. Horses had less DMI of late-heading teff hay (1.5% BW) than teff hay of other maturities (1.8% BW; P < 0.05), indicating a preference for the earlier maturities. The intake and nutrient composition of the boot and early-heading maturities was sufficient to meet 90 to 97% of the average DE of the horses and most other nutrient requirements. Digestibility decreased from boot to late-heading teff hay for DM, CP, ADF, and NDF (P < 0.05). Digestibility increased from boot to early-heading to late-heading hay for nonfiber carbohydrates and water-soluble carbohydrates (P < 0.05). For all maturities of teff hay, the NSC intake was below 10% of the total intake. In conclusion, the low NSC and DE of teff hay grown in central Pennsylvania under the conditions in this study make it an appropriate forage source for obese horses and those at risk for laminitis or other metabolic disorders.

Rumen protozoal diversity in the Spanish ibex (Capra pyrenaica hispanica) as compared with domestic goats (Capra hircus).

Rumen protozoal diversity in the Spanish ibex (SI) was studied in males (n=4), females (n=7) and young (n=4) from the Maestrazgo (Spain) and contrasted with domestic goats (n=3; DG) of the same region. There were no differences among SI types in protozoal concentration or in the number of protozoal species. Only protozoa from the genus Entodinium were observed in SI (seven species), the highest numbers corresponding to E. damae, E. ovibos and E. parvum. DG harboured threefold more species than SI. Nine to 10 Entodinium spp. were observed, but E. ovibos was absent from the rumen of DG, and E. damae was in only one animal. E. caudatum (caudatum, dubardi and lobospinosum morphotypes) occurred in the highest percentage, and E. dubardi, E. exiguum and E. parvum were quite abundant. Four genera of the subfamily Diplodiniinae and the genera Isotricha and Dasytricha from the family Isotrichidae were detected in DG. Epidinium (two DG) and Ophryoscolex (one DG) were also observed. Denaturing gradient gel electrophoresis analysis agreed with microscopic classification, showing up to 8 and 16 bands in SI and DG samples, respectively. The three DG clustered together (similarity index over 0.84), and separately from SI (similarity index over 0.86), with only 0.58 similarity between host species.

Effects of direct-fed microbial supplementation on digestibility and fermentation end-products in horses fed low- and high-starch concentrates.

A study was conducted to determine whether direct-fed microbials (DFM) could be used to increase digestibility and minimize the risk of acidosis associated with feeding an increase in the amount of starch fed to horses. Fifteen mature Thoroughbred geldings were randomly assigned to 1 of 3 treatments in a 3 x 3 Latin square design balanced for carryover effects. Within each 26-d period, horses were offered grass hay + low-starch concentrate (LS; 1.2 g of starch x kg of BW(-1) x meal(-1)) from d 1 to 13 and then were abruptly changed to hay + high-starch concentrate (HS; 2.4 g of starch.kg of BW(-1)x meal(-1)) on d 14 continuing through d 26. The DFM treatments were offered in concentrate pellets at a target dosage of 10(8) cfu/(50 kg of BW x d) as follows: no DFM (CON; control), Lactobacillus acidophilus (LAC1; single-species DFM), or a mixture of L. acidophilus, Lactobacillus casei, Bifidobacterium bifidum, and Enterococcus faecium (LAC4; multiple-species DFM). Total feces were collected over 72 h from d 11 to 13 (LS; low dietary starch collection), from d 15 to 17 (AC; abrupt change in dietary starch collection), and at the end of each experimental period, from d 24 to 26 (HS; high dietary starch collection). Data collected consisted of total DM intake and fecal output, fecal pH, fecal acetate and propionate concentrations, and viable numbers of DFM in the feed. With the exception of Fe digestibility, there were no starch x DFM interactions. There was an effect of starch level (P 0.10). Horses supplemented with LAC4 had increased ether extract (P < 0.05) and a tendency for decreased Na (P < 0.10) digestibilities compared with CON horses. All DFM-supplemented horses had increased Cu (P < 0.05) and Fe and numerically increased Zn digestibilities compared with CON horses. Fecal pH decreased (P < 0.05), and fecal propionate concentration increased (P < 0.05) as dietary starch content changed from LS to HS. There was a tendency for elevated fecal pH (P < 0.10) in LAC1 horses compared with CON horses. These results confirm that increasing starch in the equine diet can enhance nutrient digestibility of the diet. Supplementing equine diets with either a single or mixed strain direct-fed lactic acid bacteria had limited effects on nutrient digestibility or on reducing the risk of acidosis associated with feeding high-starch concentrates to horses. The potential response of DFM supplementation should be evaluated when a more acute acidotic state is induced in horses than in the current study.

An investigation of the relationship between fecal and rumen bacterial concentrations in sheep.

With no acceptable method for collecting fresh rumen fluid from zoo ruminants, it was proposed that fecal bacterial concentrations may be correlated with rumen bacteria. If so, fecal bacterial concentrations could be used to study both the effects of diet on rumen bacteria as well as rumen abnormalities. Total and cellulolytic bacterial concentrations were determined in whole rumen contents and feces of sheep using a most-probable-number (MPN) assay. In a Latin square design, four crossbred ewes were fed diets of 100% long or chopped orchardgrass hay (OH) and 60% ground or whole shelled corn plus 40% chopped OH. In a second trial, the sheep were fed a pelleted complete feed at varying levels of intake i.e., control at 2.0% of body weight and at 1.8, 1.6, and 1.2% of body weight. Higher total rumen bacterial concentrations (P<0.01) were found on the high concentrate diets as compared with the high forage diets. Grinding the corn also increased total bacterial concentrations (P<0.05). Fecal concentrations of total bacteria were higher (P<0.01) with the high concentrate diets. Chopping the forage decreased the concentration of fecal cellulolytic bacteria (P<0.05) but had no effect on their concentration in the rumen. An inverse linear relationship (P<0.01) was observed between total bacterial concentrations in the feces and diet intake. Although relationships were observed between the rumen and feces for total and cellulolytic bacterial concentrations, they were dependent on diet, particle size, and level of intake. Thus, fecal bacterial concentrations cannot be used to reliably predict rumen bacterial concentrations. Zoo Biol 27:100-108, 2008. (c) 2008 Wiley-Liss, Inc.

Electricity generation from cellulose by rumen microorganisms in microbial fuel cells.

In microbial fuel cells (MFCs) bacteria generate electricity by mediating the oxidation of organic compounds and transferring the resulting electrons to an anode electrode. The objective of this study was to test the possibility of generating electricity with rumen microorganisms as biocatalysts and cellulose as the electron donor in two-compartment MFCs. The anode and cathode chambers were separated by a proton exchange membrane and graphite plates were used as electrodes. The medium in the anode chamber was inoculated with rumen microorganisms, and the catholyte in the cathode compartment was ferricyanide solution. Maximum power density reached 55 mW/m(2) (1.5 mA, 313 mV) with cellulose as the electron donor. Cellulose hydrolysis and electrode reduction were shown to support the production of current. The electrical current was sustained for over 2 months with periodic cellulose addition. Clarified rumen fluid and a soluble carbohydrate mixture, serving as the electron donors, could also sustain power output. Denaturing gradient gel electrophoresis (DGGE) of PCR amplified 16S rRNA genes revealed that the microbial communities differed when different substrates were used in the MFCs. The anode-attached and the suspended consortia were shown to be different within the same MFC. Cloning and sequencing analysis of 16S rRNA genes indicated that the most predominant bacteria in the anode-attached consortia were related to Clostridium spp., while Comamonas spp. abounded in the suspended consortia. The results demonstrated that electricity can be generated from cellulose by exploiting rumen microorganisms as biocatalysts, but both technical and biological optimization is needed to maximize power output.

Voluntary intake and digestibility of reed canarygrass and timothy hay fed to horses.

Thousands of hectares of timothy (Phleum pretense L.) grown in the Mid-Atlantic region are infected by cereal rust mite (Abacarus hysterix) that causes discoloration and curling of leaves, decreased nutritional quality, and substantial decreases in yield. A decline in production of timothy hay can lower income for hay producers and cause horse owners to search for alternative hays. Low alkaloid reed canarygrass (Phalaris arundinacea L.) hay has potential as an alternative to timothy hay because it grows well in the Mid-Atlantic region, is believed to have a similar nutrient quality to timothy, and is not as susceptible to cereal rust mite. Eleven mature, stalled Thoroughbred geldings (549 +/- 12.1 kg) that were exercised daily were used to compare voluntary DMI and apparent nutrient DM digestibility of timothy and low-alkaloid Chiefton variety reed canarygrass hay. Horses were paired by age and BW and randomly assigned to timothy or reed canarygrass hay during a 14-d period to measure voluntary DMI followed by a 4-d period to measure apparent DM digestibility. Both hays met the minimum requirements for DE, CP, Ca, P, K, Fe, and Mn, but they did not meet the minimum requirements for Cu, Zn, and Na for horses at maintenance and averaging 550 kg of BW. Timothy hay seemed to have a lower CP concentration (14.4%) compared with reed canarygrass hay (17.1%) and a more desirable Ca:P ratio at 1.6:1 compared with 0.8:1 for reed canarygrass hay. Horses fed timothy consumed more hay (P <0.001) during the voluntary DMI period compared with horses fed reed canarygrass. Greater voluntary DMI of timothy occurred on d 1, 3, and 5 (P <0.05), but DMI was similar for other days. Apparent DM digestibility was greater in horses fed timothy hay by 9.6% compared with horses fed reed canarygrass hay (P <0.05). Horses fed timothy had greater DM digestibility of ADF (P = 0.001), NDF (P = 0.001), sugar (P = 0.05), and Ca (P = 0.001) but lower apparent DM digestibility of CP (P = 0.012) and crude fat (P = 0.004). Timothy hay was superior in voluntary DMI and apparent DM digestibility compared with low-alkaloid reed canarygrass hay fed to horses.

Effect of pH on viability of Entodinium caudatum, Entodinium exiguum, Epidinium caudatum, and Ophryoscolex purkynjei in vitro.

Cultures of Entodinium caudatum, Entodinium exiguum, Epidinium caudatum, and Ophryoscolex purkynjei were grown and transferred in poorly buffered media prepared using different concentrations of sodium bicarbonate and a nitrogen gas phase. By transferring every 12 or 24 h, culture pH was gradually decreased until the protozoa disappeared. The cultures were transferred by placing half of the culture into an equal volume of fresh medium, resulting in pH fluctuations similar to those in the rumen, resulting from fermentation, eating, and saliva production. All four species appeared to maintain their concentrations around pH 5.8, but numbers decreased as pH values fell below 5.6. The four species were similar in that they all survived above pH 5.3. These results differ from previous reports in which Entodinium species appeared to be more tolerant to low pH than all other species of rumen ciliates. No adaptation to low pH was observed in Epidinium caudatum cultures after recovery from pH 5.4 medium containing only one or two viable cells.