Forages and pastures symposium: forage biodegradation: advances in ruminal microbial ecology.

The rumen microbial ecosystem provides ruminants a selective advantage, the ability to utilize forages, allowing them to flourish worldwide in various environments. For many years, our understanding of the ruminal microbial ecosystem was limited to understanding the microbes (usually only laboratory-amenable bacteria) grown in pure culture, meaning that much of our understanding of ruminal function remained a "black box." However, the ruminal degradation of plant cell walls is performed by a consortium of bacteria, archaea, protozoa, and fungi that produces a wide variety of carbohydrate-active enzymes (CAZymes) that are responsible for the catabolism of cellulose, hemicellulose, and pectin. The past 15 years have seen the development and implementation of numerous next-generation sequencing (NGS) approaches (e.g., pyrosequencing, Illumina, and shotgun sequencing), which have contributed significantly to a greater level of insight regarding the microbial ecology of ruminants fed a variety of forages. There has also been an increase in the utilization of liquid chromatography and mass spectrometry that revolutionized transcriptomic approaches, and further improvements in the measurement of fermentation intermediates and end products have advanced with metabolomics. These advanced NGS techniques along with other analytic approaches, such as metaproteomics, have been utilized to elucidate the specific role of microbial CAZymes in forage degradation. Other methods have provided new insights into dynamic changes in the ruminal microbial population fed different diets and how these changes impact the assortment of products presented to the host animal. As more omics-based data has accumulated on forage-fed ruminants, the sequence of events that occur during fiber colonization by the microbial consortium has become more apparent, with fungal populations and fibrolytic bacterial populations working in conjunction, as well as expanding understanding of the individual microbial contributions to degradation of plant cell walls and polysaccharide components. In the future, the ability to predict microbial population and enzymatic activity and end products will be able to support the development of dynamic predictive models of rumen forage degradation and fermentation. Consequently, it is imperative to understand the rumen's microbial population better to improve fiber degradation in ruminants and, thus, stimulate more sustainable production systems.

Forage degradation in the rumen is critical to producing ruminant animals. For many years, scientists were limited to biochemical techniques to understand how ruminal microbes degraded forage, impairing our understanding of which microbes were involved with degrading which forage components. However, we have understood that as the ruminant opened up plant cells to microbial activity, a succession of microbes was involved in colonizing and breaking fiber into increasingly smaller pieces. The recent development of sequencing techniques has allowed a more detailed understanding of changes in the microbial population of the rumen during forage degradation and the types of degradative enzymes produced by this complex microbial ecosystem. We described the enzymes involved in the degradation of specific forage components, how their end products impact the microbial population through cross-feeding interactions, and how fermentation products can impact food animal production.

Utilization of canola and sunflower meals as replacements for soybean meal in a corn silage-based stocker system.

Two experiments were conducted to evaluate 3 silage-based stocker diets. In Exp. 1, diets were fed to a total of 276 animals over a period of 3 yr and performance data was collected. In Exp. 2, the same diets were subjected to in vitro digestion for 5 time periods: 0, 6, 12, 24, and 48 h, to evaluate IVDMD, production of fermentation end products, and efficiency of transformation of energy. The experimental diets were similar, except for their protein supplements. They were composed of: 1) 74% corn silage, 15.2% ground ear corn, and 10.8% soybean meal (SBM); 2) 74.4% corn silage, 9.8% ground ear corn, and 15.8% canola meal (CAN); 3) 74.5% corn silage, 9.8% ground ear corn, and 15.7% sunflower meal (SUN). Results from Exp. 1 showed that DMI was similar across all treatments (P = 0.167), but ADG was greater (P = 0.007) for animals fed either SBM or CAN than for animals fed SUN (1.29, 1.28, and 1.20 kg/d, respectively). Both CAN and SUN significantly reduced (P < 0.001) daily feeding cost per animal in comparison to SBM. Exp. 2 revealed that total VFA production was similar for all treatments (P = 0.185), and greatest molar proportions of propionate were observed for SBM and CAN (P = 0.02). Additionally, IVDMD was highest for SBM (P < 0.001). Regression analysis showed that most of the evaluated traits followed a quadratic trend for incubation times (P ≤ 0.02). On average, the in vitro technique used in this study was able to account for 97.03% of the caloric transformations suffered by DE throughout the different incubation times. Overall, our findings revealed that although animals receiving SUN had the cheapest daily feeding cost, important traits like ADG and feed conversion rate were negatively affected by this treatment. In contrast, data showed that CAN was an effective replacement for SBM for it maintained similar animal performance while decreasing feed costs. Therefore, from a producer standpoint, CAN is a viable alternative to replace the more costly SBM diet in silage-based stocker operations.

Long-term use of corn coproducts as a source of protein in beef finishing diets and the effects on carcass characteristics and round muscle quality.

A finishing trial was conducted during the late spring and summer of 2 consecutive years to evaluate long-term feeding of corn gluten feed and dried distillers' grains with solubles in finishing rations in the southeastern United States on feedlot performance, carcass characteristics, and meat quality attributes. Each year, 36 steers (yr 1 BW = 396 ± 18 kg; yr 2 BW = 436 ± 23 kg) were assigned to 1 of 3 finishing diets that contained 1) 25% dried corn gluten feed (CGF), 2) 25% dried distillers' grains plus solubles (DDGS), or 3) 10% soybean meal and 15% ground corn (SBM) and evaluated over a 100-d feedlot period. All steers were previously fed their respective diets at 25% of DM in a corn silage-based stockering system for 84 d. During the 100-d feedlot trial, weights were recorded and carcass traits were estimated via ultrasound on d -0, 50, and 100. All steers were subsequently harvested under federal inspection and had carcass data collected for quality and yield traits. At 48 h postmortem, the rectus femoris (RF), vastus lateralis (VL), and semimembranosus (SM) were collected for proximate analysis and aged for 7, 14, and 21 d for Warner-Bratzler shear and sensory analysis. Diet did not affect ( ≥ 0.14) BW, DMI, or ultrasound composition traits; however, DDGS steers had greater ADG ( = 0.05) than SBM steers and had greater ( = 0.04) G:F than CGF or SBM steers. There were no differences in carcass characteristics due to diet except the CGF carcasses had greater LM area and marbling scores ( ≤ 0.05). Protein source did not affect proximate composition, but the RF had greater percent moisture and lower percent protein compared with the VL and SM and greater percent lipid than the SM ( ≤ 0.01). Shear force analysis revealed a diet × aging period interaction ( = 0.04) where DDGS steaks were similar across all aging periods; however, steaks from SBM and CGF carcasses became more tender after 14 and 21 d of aging, respectively. Sensory panel results indicate that DDGS steaks were more tender than CGF and SBM steaks ( = 0.02) and steak tenderness increased, as expected, with aging ( < 0.01). The RF was rated as being more tender ( < 0.01) than the VL and SM, which were similar ( > 0.05). These data show that long-term use of CGF or DDGS at 25% DM will have a minimal impact on animal performance, carcass characteristics, or sensory traits of selected round cuts.

Nutritional value of ensiled grocery food waste for cattle.

Assessment of nutrient variability, feed value, ensiling capability, intake, and digestibility of grocery food waste recycled from large retail stores was conducted in 3 experiments. In Exp. 1, 115 proximate nutrient analyses of grocery byproduct feed (GBP) from stores in the southern United States from April 8, 2011, to November 18, 2012, were evaluated for variation in nutrient concentration. Grocery byproduct feed was characterized as being a readily fermentable, high-moisture energy feed with an average DM content of 17.5 ± 3.7% and TDN of 89.8 ± 7.1%. In Exp. 2 and 3, grocery food waste consisting of fruit, vegetables, and bakery items from large retail stores in the Atlanta, GA, area was used for ensiling and feeding studies. The GBP material for Exp. 2 was processed on farm into homogenous slurry and treated to reduce its moisture content and preserved in experimental silos. Drying treatments included 3 levels of citrus pulp substitution (8, 16, and 24% as-fed basis), or passively removing liquid as seepage after stacking for 24 h, or oven drying (24 h at 80°C). All GBP mixtures effectively ensiled after 28 d, as determined by changes in pH, soluble carbohydrates, and fermentation acids. Ensiled GBP was moderately stable during 72-h aerobic exposure. In Exp. 3, a feeding/digestibility trial, 8 yearling Holstein steers were used in a replicated 4 × 4 Latin Square and fed 4 incremental levels of ensiled GBP in total mixed rations (TMR). Steers were fed 0, 18, 36, and 54% ensiled GBP as part of a TMR containing 68% wheat silage and 32% concentrate on a DM basis. The rations averaged 35.9, 30.7, 26.8, and 23.8% DM with incremental levels of GBP. Steers increased DM intake and digestibility when fed increasing GBP (P < 0.5). Digestible energy and TDN were linearly related to the level of GBP fed (P < 0.01). The TDN content of GBP was 82.7% (DM basis) and similar to predicted TDN values from commercial feed analyses of GBP. The feeding and nutritive value of ensiled GBP indicates it can be priced to be used effectively as an energy supplement in TMR for cattle.

Use of corn gluten feed and dried distillers grains plus solubles as a replacement for soybean meal and corn for supplementation in a corn silage-based stocker system.

Corn gluten feed and dried distillers grains plus solubles (DDGS) were evaluated as replacements for soybean meal and ground ear corn when supplemented with corn silage during 2 yr of a beef cattle stockering program. Experiment 1: In YR 1, 104 steers (initial BW = 305 ± 30 kg), and in YR 2, 56 steers and 38 heifers (initial BW = 301 ± 32 kg) were stratified by weight and assigned to 1 of 9 groups. Each group was randomly assigned to 1 of 3 corn silage-based (75% of DM) diets supplemented with: i) corn gluten feed (CGF), ii) DDGS, or iii) soybean meal and ground ear corn (CSBM) at 25% of DM. On d 0, 28, 56, and 84, BW and BCS were recorded. Additionally, ribeye area, 12th rib fat thickness, intramuscular fat, and rump fat thickness were assessed via ultrasound on 9 (YR1) and 4 (YR 2) steers per pen that were randomly assigned as observational units. Average daily gain was greater (P < 0.05) for steers fed DDGS and CSBM compared with CGF (1.08, 1.08, and 0.94 kg/d, respectively). Average DMI (P < 0.05) was less for DDGS compared with CSBM with CGF intermediate (18.1, 18.8, 20.2 g/kg BW, respectively), and the resulting G:F was greatest for DDGS (P = 0.01). Cost per kilogram of BW gain was least for DDGS (P > 0.05). Ultrasound data indicated no differences (P ≥ 0.13) in predicted carcass traits among treatments. Experiment 2: Diets from Exp. 1 were subjected to in vitro digestion for incubation times of 0, 2, 4, 8, 16, 24, 48, and 72 h to estimate DM degradation, gas production kinetics, and CP fractions. The potentially degradable DM fraction was greater (P = 0.01) for CSBM compared with CGF and DDG. Total gas production and rate of gas production was not different among treatments (P > 0.42). Rumen degradable protein was greatest for CSBM and least for DDG (P = 0.001). These data indicate that DDGS can be used to replace soybean meal and corn in silage-based stocker systems to decrease feed costs without compromising animal performance and CGF may decrease animal performance.

Effect of long-term corn by-product feeding on beef quality, strip loin fatty acid profiles, and shelf life.

The objective of this study was to evaluate the meat quality and shelf life of steaks from steers fed dried distillers grains with solubles (DDGS) or dried corn gluten feed (CGF) compared with soybean meal with corn (SBM) as a protein supplement from weaning to slaughter. Angus cross steers (n = 81; BW = 306 ± 26.1 kg) were randomly assigned to pens (n = 9) and fed a stocker diet of corn silage (75% of DM) with DDGS, CGF, or SBM and ground ear corn. After 84 d of stockering, 12 steers (BW = 397 ± 15.3 kg) were randomly selected from each treatment and finished using the same protein supplement at 25% of DM for 100 d. Carcass data were collected (24 h) and the longissimus lumborum was fabricated into steaks at 48 h postmortem. Steaks were assigned to proximate analysis, Warner-Bratzler shear force (7-, 14-, or 21-d aging), and retail display (1, 3, 6, or 9 d). Protein source did not affect carcass yield, quality, or longissimus lumborum composition (P > 0.05). After 7 d of aging, DDGS and CGF steaks were more tender (P < 0.01) than SBM, but were similar (P = 0.30) after 14 and 21 d of aging. Feeding corn by-products did not influence subjective overall color acceptance (P = 0.17) in this study, but acceptance declined over time (P < 0.01). Subjective redness was similar (P > 0.05) among diets except SBM steaks were more red (P < 0.01) than DDGS after 9 d. On d 3 and 6 of retail display, CGF steaks exhibited more discoloration (P < 0.04) than SBM or DDGS steaks. However, after 9 d DDGS steaks were more discolored (P < 0.01) than CGF or SBM. Objective L* was lighter for CGF (P < 0.04) over 9 d of display, and all treatments became darker (P < 0.01) as time increased. Redness (a*) declined (P < 0.01) over time with SBM steaks maintaining more color in the red spectrum than CGF and DDGS after 6 d of display. Protein source did not affect (P > 0.05) the rate of lipid oxidation. Total SFA concentrations were similar (P > 0.05) among treatments; however, total MUFA were less (P < 0.05) and total PUFA concentrations were greater (P < 0.05) in DDGS steaks compared with SBM or CGF steaks. These data show that DDGS or CGF can be fed as a protein supplement at 25% DM from weaning until slaughter while maintaining meat quality when compared with steers fed soybean meal as a protein supplement.

Immuno-analysis of phospho-Akt in primary human breast cancers.

The aim of this study was to investigate the expression of the activated (phosphorylated) form of Akt (Ser473) in primary breast cancer and to correlate the results with clinicopathological and prognostic variables for clinically relevant associations. Phospho-Akt expression was studied using immunoblot analysis in 49 invasive breast carcinomas (median follow-up time 55 months, range 7-74 months). We assessed the level of phospho-Akt in different types of primary breast cancers and compared the use of autoradiograph X-ray film with a PVDF-DAB-staining system. Twelve percent of the tumours had no phospho-Akt protein, 25% had low phospho-Akt expression, 51% had intermediate expression and 12% had high phospho-Akt expression. No relationship was observed between phospho-Akt and tumour grade, tumour size or nodal status. A significant relationship was demonstrated between phospho-Akt score and oestrogen receptor status (P=0.014). Univariate analysis demonstrated that intermediate levels of phospho-Akt in breast tumour tissue are associated with a lower probability of developing recurrences (P=0.035), while in multivariate analyses, none of the phospho-Akt levels appeared to be independent predictors of disease recurrence or death. In addition, it has been clearly established that a suitable composition of reagents and components such as PVDF membranes treated with DAB substrate will enable the performing of sensitive immuno-analyses.

Upregulation of CD44 expression by interleukins 1, 4, and 13, transforming growth factor-beta1, estrogen, and progestogen in human cervical adenocarcinoma cell lines.

Although cervical adenocarcinoma constitutes approximately 10-20% of primary malignant tumors of the uterine cervix, its pathogenesis is less well understood than that of the corresponding squamous cancer. CD44 is a cell surface glycoprotein postulated to play a role in many biologic processes including tumor growth and metastasis. We have previously reported from immunohistochemical studies that a particular CD44 variant (CD44v5) is consistently overexpressed in endocervical neoplasia. It thus has potential as a diagnostic marker and even as a target for therapeutic approaches directed against specific epitopes. The aim of this study was to investigate which cytokines and hormones are capable of modulating CD44v5 expression, using a cell culture model. The effects of interleukin (IL)-1alpha, IL-1beta, IL-4, IL-13, transforming growth factor (TGF)-beta1, estrogen, and progestogen on CD44v5 expression were examined in cultures of three human cervical adenocarcinoma cell lines (HeLa, HeLa229, and HS588T). Expression was assessed using dual fluorescence-labeled flow cytometry and western blotting techniques. It was found that incubation of cultures for 72 h with IL-1alpha, IL-1beta, IL-4, IL-13, TGF-beta1 (all at 0.1-10 ng/mL), estrogen (5-10 ng/mL), or progestogen (5-20 ng/mL) induced significant upregulation of CD44v5. These factors are likely to exert a similar stimulatory influence in vivo and may contribute to the process of carcinogenesis.

Production and localization of activins and activin type IIA and IIB receptors by the human endosalpinx.

Fallopian tubes from ten premenopausal women were collected and examined for the presence of inhibin, activin and its type IIA and IIB receptors (ActRIIA and ActRIIB) in the endosalpinx. Immunocytochemistry demonstrated clear staining for the betaA, betaB subunits and ActRIIA and ActRIIB that increased in intensity from the isthmus to the ampulla. No staining for the alpha subunit was observed. Whilst the staining of the betaA subunit and ActRIIA was seen in almost every epithelial cell, staining for the betaB subunit and ActRIIB was more variable. In situ hybridization and RT-PCR confirmed the presence of mRNA for the betaA, betaB subunits and ActRIIA and ActRIIB. These results indicated that the epithelium of the uterine tube is able to synthesize activin but not inhibin and has receptors for activin. Activins may thus act as paracrine regulators of tubal epithelial cell function, and embryonic activity may also bind to epithelial receptor and initiate intracellular processes that alter epithelial cell secretions.

Production of activins by the human endosalpinx.

Uterine tubes from 11 premenopausal and 6 postmenopausal women were collected and examined for the presence of inhibin, activin, and follistatin in the endosalpinx. Immunocytochemistry of tissue from both the isthmic and ampullary regions demonstrated clear staining for the beta(A)- and beta(B)-subunits that increased in intensity from the isthmus to the ampulla. Staining for follistatin showed a similar pattern, but no staining for the alpha-subunit was observed. Although staining for the beta(A)-subunit was seen in almost every epithelial cell, staining for the beta(B)-subunit was more variable. Western blotting showed a band with an apparent molecular mass of 28 kDa (corresponding to the activin dimer) and a band of approximately 60 kDa (corresponding to the pro-protein of activin). In situ hybridization confirmed the presence of mRNA for the beta(A)- and beta(B)-subunits in the endosalpinx. These results indicate that the endosalpinx is able to synthesize activin, not inhibin, suggesting that in premenopausal women they may have an important role in the biology of the developing embryo. The role in postmenopausal women is less certain, but could lead to the stimulation of FSH secretion by the pituitary gland or other autocrine/paracrine function within the uterine tube.

Inverse relationship between the expression of the human papillomavirus type 16 transcription factor E2 and virus DNA copy number during the progression of cervical intraepithelial neoplasia.

The human papillomavirus type 16 (HPV-16) status of 43 cervical biopsies, which had been characterized histologically as normal, various grades of cervical intraepithelial neoplasia (CIN) and invasive squamous cell carcinoma, was examined by using (i) a novel antibody against the HPV-16 E2 protein, (ii) sensitive HPV-16 DNA in situ hybridization and (iii) microdissection/PCR for the E2 ORF. The data indicate that E2 protein expression is highest in koilocytes in lower-grade CIN (I), but decreases with increasing grade, whereas the detection of HPV DNA is delayed until CIN I/II, rising to the highest levels in carcinoma cells. Co-localization of E2 with HPV-16 DNA-positive cells was most commonly observed in koilocytes in CIN II lesions. PCR analyses of microdissected epithelium from the same or serial sections indicated that E2 ORFs were retained in an intact form in a number of higher-grade CIN lesions and invasive carcinomas.

p53 expression, p21 expression and the apoptotic index in endometrioid endometrial adenocarcinoma.

AIMS: Although several genetic abnormalities are known to occur in endometrial cancer, including tp53 gene mutation, the pathogenesis of this common malignancy remains poorly defined. We investigated the relationship between overexpression of p53 protein, p21 protein expression and apoptosis in endometrial carcinoma. METHODS AND RESULTS: Sixteen cases of endometrial carcinoma in which polymerase chain reaction analysis had demonstrated the absence of a tp53 gene mutation were selected on the basis of p53 protein expression; p21 protein expression and the apoptotic index were then determined for each case. The proportion of cells in each case expressing p53 and p21 protein immunoreactivity was compared with the apoptotic index. Overall, no significant correlation was demonstrated between p53 and p21 immunoreactivity, or between either p53 or p21 and the apoptotic index. CONCLUSIONS: Factors other than p53 are involved in the regulation of p21 expression and apoptosis in endometrioid endometrial adenocarcinomas without p53 mutations. Despite the small numbers used in this study, the data suggest a correlation between low levels of p53 immunoreactivity and apoptosis. We postulate that high levels of p53 immunoreactivity may be due to abnormal stabilization of the p53 protein. Follow-up studies are needed with a larger data set.

Direct sequencing of the p53 gene shows absence of mutations in endometrioid endometrial adenocarcinomas expressing p53 protein.

AIMS: To investigate the mutational status of the p53 gene (Tp53) in endometrial carcinomas expressing p53 protein by direct gene sequencing. METHODS AND RESULTS: Eighteen cases of endometrial carcinoma, selected on the basis of p53 protein expression as detected immunohistochemically by the monoclonal antibody DO7, were microdissected in the p53 positive areas. DNA was extracted and subjected to polymerase chain reaction (PCR) for exons 5-8 which code for conserved areas containing mutational hot-spots. The PCR amplified material was then sequenced using an ABI automated sequencer and analysed using BLAST software at the NCBI web site. All sequences analysed were wild-type. CONCLUSION: The results confirm that expression of p53 protein may occur in endometrial adenocarcinoma without mutation and may be due to stabilization of the protein during its normal function, possibly by an mdm2 mediated process.

Evaluation of three commercial tick removal tools.

OBJECTIVE: To evaluate three commercially available tick removal tools against medium-tipped nontissue tweezers. METHODS: We evaluated three commercially available tick removal tools against medium-tipped tweezers. Three inexperienced users randomly removed attached American dog ticks (Dermacentor variabilis Say) and lone star ticks (Amblyomma americanum L.) from laboratory rabbits in a university animal facility using all tools during one removal session. RESULTS: Tick damage occurring from removal and quantity of attachment cement were compared. No tool removed nymphs without damage and all tools removed adults of both species successfully. American dog ticks proved easier to remove than lone star ticks, whose mouthparts often remained in the skin. CONCLUSIONS: Nymphal ticks were consistently removed more successfully with commercial tools when compared with tweezers but with more difficulty than adults were removed. The commercial tick removal tools tested are functional for removal of nymphs and adults and should be considered as viable alternatives to medium-tipped tweezers.

Organizational effectiveness: toward an integrated model for schools of nursing.

Assessing the quality of academic institutions involves much more than the opinions of peers or experts. Examination of the organizational effectiveness of schools of nursing has been neglected. Current emphasis on assessing educational outcomes has diverted attention from the construct, organizational effectiveness, and more comprehensive theory-driven approaches to evaluation. This review of the organizational effectiveness literature focuses on the major assessment models: goal attainment, human relations, open systems, internal processes, culture, and life cycle. Attention is given to the influence of organizational maturation on an integrated model of organizational effectiveness. Selected macrolevel studies of schools of nursing are examined, and an agenda for nursing research is proposed.

Toxoplasma gondii antibodies in woodchucks (Marmota monax) from Pennsylvania.

Serum samples from 545 woodchucks Marmota monax from 22 counties in Pennsylvania were examined for antibodies to Toxoplasma gondii by the modified direct agglutination test. Fifty-one woodchucks (9.4%) had antibodies to T. gondii, with 10% at dilutions of 1:25, 2% at dilutions of 1:50, and 4% at dilutions of 1:500. This is the first report of T. gondii antibodies in woodchucks.

Prevalence of Toxoplasma gondii antibodies in sera of hunter-killed white-tailed deer in Pennsylvania.

Serum samples from 593 white-tailed deer in Pennsylvania that were killed by hunters in 1991 were examined for Toxoplasma gondii antibodies, by use of the modified agglutination test. Sixty percent (357/593) of the deer had T gondii antibodies; 10% had titer of 25, 23% had titer of 50, and 27% had titer > or = 500. Sex-specific differences in prevalence were not detected.