Bulls fed a high-gain diet decrease blastocyst formation after in vitro fertilization.

In brief: Paternal high-gain diet reduces blastocyst development following in vitro fertilization and embryo culture but does not affect gene expression or cellular allocation of resultant blastocysts. Abstract: Bulls used in cattle production are often overfed to induce rapid growth, early puberty, and increase sale price. While the negative consequences of undernutrition on bull sperm quality are known, it is unclear how a high-gain diet influences embryo development. We hypothesized that semen collected from bulls fed a high-gain diet would have a reduced capacity to produce blastocysts following in vitro fertilization. Eight mature bulls were stratified by body weight and fed the same diet for 67 days at either a maintenance level (0.5% body weight per day; n = 4) or a high-gain rate (1.25% body weight per day; n = 4). Semen was collected by electroejaculation at the end of the feeding regimen and subjected to sperm analysis, frozen, and used for in vitro fertilization. The high-gain diet increased body weight, average daily gain, and subcutaneous fat thickness compared to the maintenance diet. Sperm of high-gain bulls tended to have increased early necrosis and had increased post-thaw acrosome damage compared with maintenance bulls, but diet did not affect sperm motility or morphology. Semen of high-gain bulls reduced the percentage of cleaved oocytes that developed to blastocyst stage embryos. Paternal diet had no effect on the number of total or CDX2-positive cells of blastocysts, or blastocysts gene expression for markers associated with developmental capacity. Feeding bulls a high-gain diet did not affect sperm morphology or motility, but increased adiposity and reduced the ability of sperm to generate blastocyst-stage embryos.

Seminal plasma modulates expression of endometrial inflammatory meditators in the bovine†.

Seminal plasma has conventionally been viewed as a transport and survival medium for mammalian sperm; however, its role now extends beyond this process to actively targeting female tissues. Studies in rodents, swine, and humans demonstrate that seminal plasma induces molecular and cellular changes within the endometrium or cervix following insemination. Seminal-plasma-induced alterations to the maternal environment have been theorized to facilitate embryo development, modulate maternal immunity toward the conceptus, and potentially improve pregnancy success. It is unknown if bovine seminal plasma modulates the uterine environment following insemination in the cow, where routine use of artificial insemination reduces maternal exposure to seminal plasma. We hypothesize that seminal plasma modulates the expression of inflammatory mediators in the endometrium, altering the maternal environment of early pregnancy. In vitro, seminal plasma altered intact endometrial explant expression of CSF2, IL1B, IL6, IL17A, TGFB1, IFNE, PTGS2, and AKR1C4. Furthermore, endometrial epithelial cell CSF2, CXCL8, TGFB1, PTGS2, and AKR1C4 expression were increased after seminal plasma exposure, while endometrial stromal cell CSF2, IL1B, IL6, CXCL8, IL17A, TGFB1, PTGS2, and AKR1C4 expression were increased following seminal plasma exposure. Endometrial expression of IL1B was increased in the cow 24 h after uterine infusion of seminal plasma, while other evaluated inflammatory mediators remained unchanged. These data indicate that seminal plasma may induce changes in the bovine endometrium in a temporal manner. Understanding the role of seminal plasma in modulating the maternal environment may aid in improving pregnancy success in cattle.

S-nitrosylation and S-glutathionylation of Cys134 on troponin I have opposing competitive actions on Ca2+ sensitivity in rat fast-twitch muscle fibers.

Nitric oxide is generated in skeletal muscle with activity and decreases Ca2+ sensitivity of the contractile apparatus, putatively by S-nitrosylation of an unidentified protein. We investigated the mechanistic basis of this effect and its relationship to the oxidation-induced increase in Ca2+ sensitivity in mammalian fast-twitch (FT) fibers mediated by S-glutathionylation of Cys134 on fast troponin I (TnIf). Force-[Ca2+] characteristics of the contractile apparatus in mechanically skinned fibers were assessed by direct activation with heavily Ca2+-buffered solutions. Treatment with S-nitrosylating agents, S-nitrosoglutathione (GSNO) or S-nitroso-N-acetyl-penicillamine (SNAP), decreased pCa50 ( = -log10 [Ca2+] at half-maximal activation) by ~-0.07 pCa units in rat and human FT fibers without affecting maximum force, but had no effect on rat and human slow-twitch fibers or toad or chicken FT fibers, which all lack Cys134. The Ca2+ sensitivity decrease was 1) fully reversed with dithiothreitol or reduced glutathione, 2) at least partially reversed with ascorbate, indicative of involvement of S-nitrosylation, and 3) irreversibly blocked by low concentration of the alkylating agent, N-ethylmaleimide (NEM). The biotin-switch assay showed that both GSNO and SNAP treatments caused S-nitrosylation of TnIfS-glutathionylation pretreatment blocked the effects of S-nitrosylation on Ca2+ sensitivity, and vice-versa. S-nitrosylation pretreatment prevented NEM from irreversibly blocking S-glutathionylation of TnIf and its effects on Ca2+ sensitivity, and likewise S-glutathionylation pretreatment prevented NEM block of S-nitrosylation. Following substitution of TnIf into rat slow-twitch fibers, S-nitrosylation treatment caused decreased Ca2+ sensitivity. These findings demonstrate that S-nitrosylation and S-glutathionylation exert opposing effects on Ca2+ sensitivity in mammalian FT muscle fibers, mediated by competitive actions on Cys134 of TnIf.

Ca(2+) leakage out of the sarcoplasmic reticulum is increased in type I skeletal muscle fibres in aged humans.

KEY POINTS: The amount of Ca(2+) stored in the sarcoplasmic reticulum (SR) of muscle fibres is decreased in aged individuals, and an important question is whether this results from increased Ca(2+) leakage out through the Ca(2+) release channels (ryanodine receptors; RyRs). The present study examined the effects of blocking the RyRs with Mg(2+), or applying a strong reducing treatment, on net Ca(2+) accumulation by the SR in skinned muscle fibres from Old (∼70 years) and Young (∼24 years) adults. Raising cytoplasmic [Mg(2+)] and reducing treatment increased net SR Ca(2+) accumulation in type I fibres of Old subjects relative to that in Young. The densities of RyRs and dihydropyridine receptors were not significantly changed in the muscle of Old subjects. These findings indicate that oxidative modification of the RyRs causes increased Ca(2+) leakage from the SR in muscle fibres in Old subjects, which probably deleteriously affects normal muscle function both directly and indirectly. ABSTRACT: The present study examined whether the lower Ca(2+) storage levels in the sarcoplasmic reticulum (SR) in vastus lateralis muscle fibres in Old (70 ± 4 years) relative to Young (24 ± 4 years) human subjects is the result of increased leakage of Ca(2+) out of the SR through the Ca(2+) release channels/ryanodine receptors (RyRs) and due to oxidative modification of the RyRs. SR Ca(2+) accumulation in mechanically skinned muscle fibres was examined in the presence of 1, 3 or 10 mm cytoplasmic Mg(2+) because raising [Mg(2+)] strongly inhibits Ca(2+) efflux through the RyRs. In type I fibres of Old subjects, SR Ca(2+) accumulation in the presence of 1 mm Mg(2+) approached saturation at shorter loading times than in Young subjects, consistent with Ca(2+) leakage limiting net uptake, and raising [Mg(2+)] to 10 mm in such fibres increased maximal SR Ca(2+) accumulation. No significant differences were seen in type II fibres. Treatment with dithiothreitol (10 mm for 5 min), a strong reducing agent, also increased maximal SR Ca(2+) accumulation at 1 mm Mg(2+) in type I fibres of Old subjects but not in other fibres. The densities of dihydropyridine receptors and RyRs were not significantly different in muscles of Old relative to Young subjects. These findings indicate that Ca(2+) leakage from the SR is increased in type I fibres in Old subjects by reversible oxidative modification of the RyRs; this increased SR Ca(2+) leak is expected to have both direct and indirect deleterious effects on Ca(2+) movements and muscle function.

Contrasting effects of progesterone on fertility of dairy and beef cows.

The role of progesterone in maintaining pregnancy is well known in the bovine. Subtle differences exist between dairy and beef cows because of differing concentrations of progesterone during recrudescence of postpartum estrous cycles, rate of follicular growth and maturation, proportions of 2- and 3-follicular wave cycles, and other effects on pregnancy outcomes per artificial insemination (P/AI). Because proportions of anovulatory cows before the onset of the artificial insemination (AI) period are greater and more variable in beef (usually ranging from 30 to 70%) than dairy (25%) cows, AI programs were developed to accommodate anovulatory and cycling beef cows enrolled therein. Incorporating a progestin as part of an AI program in beef cows improved P/AI by reducing the proportion of cows having premature luteal regression and short post-AI luteal phases. In both genotypes, prolonged dominant follicle growth in a reduced progesterone milieu resulted in increased (1) LH pulses, (2) preovulatory follicle diameter, and (3) concentrations of estradiol and a subsequently larger corpora lutea (CL). In contrast, the progesterone milieu during growth of the ovulatory follicle in an ovulation control program does not seem to affect subsequent P/AI in beef cows, whereas in dairy cows follicle development in an elevated compared with a low progesterone environment increases P/AI. Progesterone status in beef cows at the onset of ovulation synchronization is not related to P/AI in multiparous cows, whereas P/AI was suppressed in primiparous cows that began a timed AI program in a low-progesterone environment. In timed AI programs, elevated concentrations of progesterone just before PGF2α and reduced concentrations at AI are critical to maximizing subsequent P/AI in dairy cows, but seemingly much less important in beef cows. By inducing ancillary CL and increasing concentrations of progesterone, human chorionic gonadotropin may increase P/AI when administered to beef cows 7d after AI or at embryo transfer, and its success seems to depend on induction of ancillary CL, whereas in dairy cows increased fertility was detected in cows with multiple CL, human chorionic gonadotropin-enhanced progesterone from original CL, or both. Pregnancy losses after AI are less frequent in beef cows and are not associated with pre-AI progesterone or cycling status, whereas losses in dairy cows are inversely related to progesterone and adversely affected in anovular dairy cows. Genotype and nutritional management likely influence several physiological differences including circulating concentrations of progesterone and responses to supplemental progesterone.

Contractile properties and sarcoplasmic reticulum calcium content in type I and type II skeletal muscle fibres in active aged humans.

KEY POINTS: Muscle weakness in old age is due in large part to an overall loss of skeletal muscle tissue, but it remains uncertain how much also stems from alterations in the properties of the individual muscle fibres. This study examined the contractile properties and amount of stored intracellular calcium in single muscle fibres of Old (70 ± 4 years) and Young (22 ± 3 years) adults. The maximum level of force production (per unit cross-sectional area) in fast twitch fibres in Old subjects was lower than in Young subjects, and the fibres were also less sensitive to activation by calcium. The amount of calcium stored inside muscle fibres and available to trigger contraction was also lower in both fast- and slow-twitch muscle fibres in the Old subjects. These findings indicate that muscle weakness in old age stems in part from an impaired capacity for force production in the individual muscle fibres. ABSTRACT: This study examined the contractile properties and sarcoplasmic reticulum (SR) Ca(2+) content in mechanically skinned vastus lateralis muscle fibres of Old (70 ± 4 years) and Young (22 ± 3 years) humans to investigate whether changes in muscle fibre properties contribute to muscle weakness in old age. In type II fibres of Old subjects, specific force was reduced by ∼17% and Ca(2+) sensitivity was also reduced (pCa50 decreased ∼0.05 pCa units) relative to that in Young. S-Glutathionylation of fast troponin I (TnIf ) markedly increased Ca(2+) sensitivity in type II fibres, but the increase was significantly smaller in Old versus Young (+0.136 and +0.164 pCa unit increases, respectively). Endogenous and maximal SR Ca(2+) content were significantly smaller in both type I and type II fibres in Old subjects. In fibres of Young, the SR could be nearly fully depleted of Ca(2+) by a combined caffeine and low Mg(2+) stimulus, whereas in fibres of Old the amount of non-releasable Ca(2+) was significantly increased (by > 12% of endogenous Ca(2+) content). Western blotting showed an increased proportion of type I fibres in Old subjects, and increased amounts of calsequestrin-2 and calsequestrin-like protein. The findings suggest that muscle weakness in old age is probably attributable in part to (i) an increased proportion of type I fibres, (ii) a reduction in both maximum specific force and Ca(2+) sensitivity in type II fibres, and also a decreased ability of S-glutathionylation of TnIf to counter the fatiguing effects of metabolites on Ca(2+) sensitivity, and (iii) a reduction in the amount of releasable SR Ca(2+) in both fibre types.

Identification of seven novel HLA class I alleles in New Zealand.

Seven new HLA class I alleles have been identified in the New Zealand population in the process of routine HLA typing and they are described here. Unusual bead positivity in Luminex typing identified potential new alleles in a bone marrow registry donor (B*40:285) and two HIV patients prior to abacavir prescription (B*14:02:09, B*41:29). In addition, four new class I alleles were identified through class I sequencing-based typing (SBT) outside of exons 2 and 3. One mutation was identified in exon 4 (new allele C*12:125) and three have been found in exon 5, an exon rarely sequenced. Two stem cell transplant recipients (B*07:02:45, C*03:279) had novel mutations in exon 5 and one was found in exon 5 of a potentially matched unrelated donor from DKMS, previously thought to be B*40:02:01 (B*40:303).

Sarcoplasmic reticulum Ca2+ uptake and leak properties, and SERCA isoform expression, in type I and type II fibres of human skeletal muscle.

The Ca(2+) uptake properties of the sarcoplasmic reticulum (SR) were compared between type I and type II fibres of vastus lateralis muscle of young healthy adults. Individual mechanically skinned muscle fibres were exposed to solutions with the free [Ca(2+)] heavily buffered in the pCa range (-log10[Ca(2+)]) 7.3-6.0 for set times and the amount of net SR Ca(2+) accumulation determined from the force response elicited upon emptying the SR of all Ca(2+). Western blotting was used to determine fibre type and the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoform present in every fibre examined. Type I fibres contained only SERCA2 and displayed half-maximal Ca(2+) uptake rate at ∼pCa 6.8, whereas type II fibres contained only SERCA1 and displayed half-maximal Ca(2+) uptake rate at ∼pCa 6.6. Maximal Ca(2+) uptake rate was ∼0.18 and ∼0.21 mmol Ca(2+) (l fibre)(-1) s(-1) in type I and type II fibres, respectively, in good accord with previously measured SR ATPase activity. Increasing free [Mg(2+)] from 1 to 3 mM had no significant effect on the net Ca(2+) uptake rate at pCa 6.0, indicating that there was little or no calcium-induced calcium release occurring through the Ca(2+) release channels during uptake in either fibre type. Ca(2+) leakage from the SR at pCa 8.5, which is thought to occur at least in part through the SERCA, was ∼2-fold lower in type II fibres than in type I fibres, and was little affected by the presence of ADP, in marked contrast to the larger SR Ca(2+) leak observed in rat muscle fibres under the same conditions. The higher affinity of Ca(2+) uptake in the type I human fibres can account for the higher relative level of SR Ca(2+) loading observed in type I compared to type II fibres, and the SR Ca(2+) leakage characteristics of the human fibres suggest that the SERCAs are regulated differently from those in rat and contribute comparatively less to resting metabolic rate.

Impacts of reproductive technologies on beef production in the United States.

Estimations of world population growth indicate that by the year 2050 we will reach nine billion habitants on earth. These estimates impose a tremendous challenge in the current agricultural systems as food supply will need to increase by 100 % in the next 40 years (Food and Agriculture Organization of the United Nations 2009). Beef will be a primary protein source that will assist in meeting the requirements for a portion of the protein in diets of this expanding global populace. Beef is a high-quality protein that contains all essential amino acids for the human body and also contains additional essential nutrients such as iron, zinc, B vitamins, riboflavin, selenium, choline, and conjugated linoleic acid (CLA). Adopting reproductive technologies at greater rates than currently used is a viable method to dramatically enhance production efficiency of beef cattle enterprises.Artificial insemination (AI), estrous synchronization and fixed-time AI (TAI), semen and embryo cryopreservation, multiple ovulation and embryo transfer (MOET), in vitro fertilization, sex determination of sperm or embryos, and nuclear transfer are technologies that are used to enhance the production efficiency of beef operations. In many cases, the development of these technologies is responsible for significant changes to traditional livestock production practices. However, adoption of these technologies appears to has not grown at the same rate in the United States as other formidable beef producing nations. For example, sales of beef semen for AI increased from 3.3 to 11.9 million units between 1993 and 2011 in Brazil, whereas that in the United States has increased from 2.9 to 3.8 million units during the same period. The significant increases in adoption of reproductive technologies in developing countries is likely as a result of the development of practical estrous synchronization and TAI systems that have allowed beef producers the opportunity to eliminate detection of estrus in their AI programs with a high degree of success. In the United States, slow adoption rates of these technologies may result in a future loss of international market share of beef products as other nations take advantage not only of the additional kilogram of beef that can be produced but also the improved quality of beef that can be realized through incorporation of reproductive technologies and resultant genetic improvement. However, current difficulties the US producers have with the incorporation of applied reproductive technologies, such as TAI, MOET, and sex semen, must not be the reason to overlook and incorporate more traditional reproductive technologies such as castration, breeding season management, or weaning. In many cases, beef producers in the United States fail to incorporate these more traditional technologies, which results in a reduction in production efficiency of the US beef industry. This chapter will focus on both traditional and more developed reproductive technologies that will play a role in enhancing future production efficiencies of the US beef cattle production system.

Endogenous and maximal sarcoplasmic reticulum calcium content and calsequestrin expression in type I and type II human skeletal muscle fibres.

The relationship between sarcoplasmic reticulum (SR) Ca(2+) content and calsequestrin (CSQ) isoforms was investigated in human skeletal muscle. A fibre-lysing assay was used to quantify the endogenous Ca(2+) content and maximal Ca(2+) capacity of the SR in skinned segments of type I and type II fibres from vastus lateralis muscles of young healthy adults. Western blotting of individual fibres showed the great majority contained either all fast or all slow isoforms of myosin heavy chain (MHC), troponins C and I, tropomyosin and SERCA, and that the strontium sensitivity of the force response was closely indicative of the troponin C isoform present. The endogenous SR Ca(2+) content was slightly lower in type I compared to type II fibres (0.76 ± 0.03 and 0.85 ± 0.02 mmol Ca(2+) per litre of fibre, respectively), with virtually all of this Ca(2+) evidently being in the SR, as it could be rapidly released with a caffeine-low [Mg(2+)] solution (only 0.08 ± 0.01 and <0.07 mmol l(-1), respectively, remaining). The maximal Ca(2+) content that could be reached with SR Ca(2+) loading was 1.45 ± 0.04 and 1.79 ± 0.03 mmol l(-1) in type I and type II fibres, respectively (P < 0.05). In non-lysed skinned fibres, where the SR remained functional, repeated cycles of caffeine-induced Ca(2+) release and subsequent Ca(2+) reloading similarly indicated that (i) maximal SR Ca(2+) content was lower in type I fibres than in type II fibres (P < 0.05), and (ii) the endogenous Ca(2+) content represented a greater percentage of maximal content in type I fibres compared to type II fibres (∼59% and 41%, respectively, P < 0.05). Type II fibres were found on average to contain ∼3-fold more CSQ1 and ∼5-fold less CSQ2 than type I fibres (P < 0.001). The findings are consistent with the SR Ca(2+) content characteristics in human type II fibres being primarily determined by the CSQ1 abundance, and in type I fibres by the combined amounts of both CSQ1 and CSQ2.

Ca2+-dependent proteolysis of junctophilin-1 and junctophilin-2 in skeletal and cardiac muscle.

Excessive increases in intracellular [Ca(2+)] in skeletal muscle fibres cause failure of excitation-contraction coupling by disrupting communication between the dihydropyridine receptors in the transverse tubular system and the Ca(2+) release channels (RyRs) in the sarcoplasmic reticulum (SR), but the exact mechanism is unknown. Previous work suggested a possible role of Ca(2+)-dependent proteolysis in this uncoupling process but found no proteolysis of the dihydropyridine receptors, RyRs or triadin. Junctophilin-1 (JP1; ∼90 kDa) stabilizes close apposition of the transverse tubular system and SR membranes in adult skeletal muscle; its C-terminal end is embedded in the SR and its N-terminal associates with the transverse tubular system membrane. Exposure of skeletal muscle homogenates to precisely set [Ca(2+)] revealed that JP1 undergoes Ca(2+)-dependent proteolysis over the physiological [Ca(2+)] range in tandem with autolytic activation of endogenous µ-calpain. Cleavage of JP1 occurs close to the C-terminal, yielding a ∼75 kDa diffusible fragment and a fixed ∼15 kDa fragment. Depolarization-induced force responses in rat skinned fibres were abolished following 1 min exposure to 40 µm Ca(2+), with accompanying loss of full-length JP1. Supraphysiological stimulation of rat skeletal muscle in vitro by repeated tetanic stimulation in 30 mm caffeine also produced marked proteolysis of JP1 (and not RyR1). In dystrophic mdx mice, JP1 proteolysis is seen in limb muscles at 4 and not at 10 weeks of age. Junctophilin-2 in cardiac and skeletal muscle also undergoes Ca(2+)-dependent proteolysis, and junctophilin-2 levels are reduced following cardiac ischaemia-reperfusion. Junctophilin proteolysis may contribute to skeletal muscle weakness and cardiac dysfunction in a range of circumstances.

Impacts of learning experiences within an online extension initiative on application of research-based principles by beef stakeholders.

The objective of this study was to evaluate, characterize and quantify the learning experiences and subsequent application of research-based technologies by beef producers upon conclusion of an online extension certification program (44 Farms International Beef Cattle Academy, IBCA). Upon conclusion of the program, paricipants were invited to complete a structured interview. Interview transcripts (n = 19) were coded, categorized, and merged into four overarching themes: Strengths, Struggles, Courses, and Geographical origin. Within Strengths, the most frequent codes were Connections, Application, and Instructor Experience, with 61, 53, and 50 coded segments respectively. Within Struggles, the most frequent codes were Time Management, Level of Knowledge, and Language issues, with 27, 18, and 15 coded segments, respectively. For Courses in the program, the most frequently mentioned were Nutrition, Reproduction, and Genetics, with 35, 28, and 24 coded segments respectively. Correlation between codes was evaluated using Pearson and only statistically significant (P ≤ 0.05) correlations were included in the matrices for network analysis. Interpretation of the generated network analysis map (P ≤ 0.05; Q = 0.468) including all four categories of codes revealed close relationships between Application and the Strengths of Time management, Instructor Experience, and Connections. Application was also directly related to the Courses of Reproduction and Genetics, and the Struggle of Student Engagement and Guidance. Geographical origin was an important factor mediating different correlations. Developing countries (Brazil, Panama, Dominican Republic, and South Africa) were more closely related to the Struggle of Tuition cost, which, in turn was related to the perceived Prestige of the program. In Europe (Romania, Germany, and Kazakhstan), a stronger correlation to the Struggles of Material Relevance and Language Issues was described. Collectively, these results support the positive impact of a comprehensive and interactive extension initiative to leverage application of research-based principles by beef stakeholders around the world. Further, these outcomes indicate that the most valued aspects of the program regarding application are related to interpersonal experience with faculty and peers of the industry (Instructor Experience and Connections) and that perception of struggles and strengths is greatly influenced by socio-cultural aspects of the learning community.

S-glutathionylation of troponin I (fast) increases contractile apparatus Ca2+ sensitivity in fast-twitch muscle fibres of rats and humans.

Oxidation can decrease or increase the Ca2+ sensitivity of the contractile apparatus in rodent fast-twitch (type II) skeletal muscle fibres, but the reactions and molecular targets involved are unknown. This study examined whether increased Ca2+ sensitivity is due to S-glutathionylation of particular cysteine residues. Skinned muscle fibres were directly activated in heavily buffered Ca2+ solutions to assess contractile apparatus Ca2+ sensitivity. Rat type II fibres were subjected to S-glutathionylation by successive treatments with 2,2'-dithiodipyridine (DTDP) and glutathione (GSH), and displayed a maximal increase in pCa50 (−log10 [Ca2+] at half-maximal force) of ∼0.24 pCa units, with little or no effect on maximum force or Hill coefficient. Partial similar effect was produced by exposure to oxidized gluthathione (GSSG, 10 mM) for 10 min at pH 7.1, and near-maximal effect by GSSG treatment at pH 8.5. None of these treatments significantly altered Ca2+ sensitivity in rat type I fibres. Western blotting showed that both the DTDP­GSH and GSSG­pH 8.5 treatments caused marked S-glutathionylation of the fast troponin I isoform (TnI(f)) present in type II fibres, but not of troponin C (TnC) or myosin light chain 2. Both the increased Ca2+ sensitivity and glutathionylation of TnI(f) were blocked by N-ethylmaleimide (NEM). S-nitrosoglutathione (GSNO) also increased Ca2+ sensitivity, but only in conditions where it caused S-glutathionylation of TnI(f). In human type II fibres from vastus lateralis muscle, DTDP­GSH treatment also caused similar increased Ca2+ sensitivity and S-glutathionylation of TnI(f). When the slow isoform of TnI in type I fibres of rat was partially substituted (∼30%) with TnI(f), DTDP­GSH treatment caused a significant increase in Ca2+ sensitivity (∼0.08 pCa units). TnIf in type II fibres from toad and chicken muscle lack Cys133 present in mammalian TnIf, and such fibres showed no change in Ca2+ sensitivity with DTDP­GSH nor any S-glutathionylation of TnI(f) (latter examined only in toad). Following 40 min of cycling exercise in human subjects (at ∼60% peak oxygen consumption), TnI(f) in vastus lateralis muscle displayed a marked increase in S-glutathionylation (∼4-fold). These findings show that S-glutathionylation of TnI(f), most probably at Cys133, increases the Ca2+ sensitivity of the contractile apparatus, and that this occurs in exercising humans, with likely beneficial effects on performance.

Clinical utility of the Glasgow Prognostic Score in patients undergoing curative nephrectomy for renal clear cell cancer: basis of new prognostic scoring systems.

BACKGROUND: Measurement of the systemic inflammatory response in malignancy has been recently refined using a selective combination of C-reactive protein and albumin (modified Glasgow Prognostic Score, mGPS). This has prognostic value in patients with metastatic kidney cancer. This study examines the prognostic value of the mGPS in patients undergoing curative nephrectomy for clear cell cancer. METHODS: Patients with localised renal cell carcinoma undergoing potentially curative resection between March 1997 and July 2007 in a single institution were prospectively studied. The mGPS, University of California Los Angeles Integrated Staging System (UISS), 'Stage Size Grade Necrosis' (SSIGN), Kattan and Leibovich scores were constructed. RESULTS: A total of 169 patients were studied. The minimum follow-up was 49 months; the median follow-up of the survivors was 98 months. During this period, 35 patients died of their cancer; a further 24 patients died of intercurrent disease. On univariate survival analysis of the scoring systems, Kattan (P<0.05), UISS (P<0.001), SSIGN (P<0.001) and Leibovich (P<0.001) were significantly associated with cancer-specific survival. Using cancer-specific mortality at 4 years as an endpoint, the area under the receiver operator curve was 0.726 (95% CI 0.629-0.822; P=0.001) for Kattan, 0.776 (95% CI 0.671-0.880; P<0.001) for UISS, 0.812 (95% CI 0.733-0.892; P<0.001) for SSIGN, 0.778 (95% CI 0.666-0.889; P<0.001) for Leibovich and 0.800 (95% CI 0.687-0.912; P<0.001) for the mGPS scoring system. On multivariate analysis of significant independent scoring systems and mGPS, UISS (HR 3.08, 95% CI 1.54-6.19, P=0.002) and mGPS (HR 5.13, 95% CI 2.89-9.11, P<0.001) were significant independent predictors of cancer-specific survival. CONCLUSIONS: The present prospective study shows that the mGPS, an inflammation-based prognostic score, is at least equivalent to and independent of other current validated prognostic scoring systems for patients undergoing curative nephrectomy for renal clear cell cancer. The mGPS is simple, measured preoperatively, based on well-standardised, widely available protein assays, and therefore provides an objective and rational basis before treatment for future staging systems in patients with operable renal cancer.

Expression and prognostic significance of Src family members in renal clear cell carcinoma.

BACKGROUND: The aim of this study was to determine whether Src family kinases (SFK) are expressed in renal cell cancer and to assess their prognostic significance. METHODS: mRNA expression levels were investigated for the 8 SFK members by quantitative real-time PCR in 19 clear cell cancer tissue samples. Immunohistochemical staining was utilised to assess expression of Src kinase, dephosphorylated Src kinase at Y(530) (SrcY(530)), phosphorylated Src at Y(419) (SrcY(419)) and the downstream focal adhesion kinase (FAK) marker at the Y(861) site (FAK Y(861)) in a cohort of 57 clear cell renal cancer specimens. Expression was assessed using the weighted histoscore method. RESULTS: Src, Lyn, Hck, Fgr and Fyn were the most highly expressed in renal cancer. All members were more highly expressed in T2 disease, and furthermore expression levels between T2 and T3 disease showed a significant decrease for Lck, Lyn, Fyn, Blk and Yes (P=0.032). Assessment of membrane, cytoplasm and nuclear expression of Src kinase, SrcY(530) and SrcY(419) were not significantly associated with cancer-specific survival. High expression of cytoplasmic FAK Y(861) was associated with decreased cancer-specific survival (P=0.001). On multivariate analysis, cytoplasmic FAK Y(861) was independently associated with cancer-specific survival (hazard ratio 3.35, 95% CI 1.40-7.98, P=0.006). CONCLUSION: We have reported that all SFK members are expressed in renal cell carcinoma. The SFK members had their highest levels of expression before the disease no longer being organ confined. We hypothesise that these SFK members are upregulated before the cancer spreading out-with the organ and given that Src itself is not associated with cancer-specific survival but the presence of FAK Y(861), a downstream marker for SFK member activity is associated with decreased cancer-specific survival, we hypothesise that another SFK member is associated with decreased cancer-specific survival in renal cell cancer.

Prospective study of the role of inflammation in renal cancer.

BACKGROUND: The local and systemic inflammatory responses provide prognostic information in cancer. The modified Glasgow Prognostic Score (mGPS) provides additional prognostic information than C-reactive protein (CRP) alone when assessing the systemic inflammation in cancer. The aim of this study was to determine the role of local and systemic inflammation in renal cancer. METHODS: The cohort consisted of 79 patients who had undergone potential curative resection. Systemic inflammation, mGPS, was constructed by measuring preoperative CRP and albumin concentrations and the Klintrup-Makinen score was evaluated histologically for the local inflammatory response. Pathological parameters such as T stage, grade and tumour necrosis were also assessed. The local inflammatory response was assessed by examining all inflammatory cells at the tumour edge on diagnostic haematoxylin and eosin slides. RESULTS: On univariate analysis, T stage (p < 0.001), grade (p = 0.044) and mGPS (p < 0.001) were significant predictors of cancer-specific survival. On multivariate analysis, mGPS (hazard ratio 8.64, 95% confidence interval 3.5-21.29, p < 0.001) was the only significant independent predictor of cancer-specific survival. CONCLUSION: A preoperative systemic inflammatory response as measured by the mGPS is an independent predictor of poor cancer-specific survival in renal cancer in patients undergoing potential curative resection.

Intake, ruminal fermentation parameters, and apparent total-tract digestibility by beef steers consuming Pensacola bahiagrass hay treated with calcium oxide.

To determine the effect of CaO-treated Pensacola bahiagrass (Paspalum notatum) hay on intake, ruminal fermentation parameters, and apparent total-tract digestibility of nutrients, nine ruminally cannulated Angus-crossbred steers were used in a triplicated 3 × 3 Latin square design. Steers had ad libitum access to either 1) untreated dry hay (DH; n = 8); 2) hay at 50% DM treated with 8.9% CaCO3 (dry matter [DM] basis; CC; n = 9); or 3) hay at 50% DM treated with 5% CaO (DM basis; CO; n = 8). Water was added to reach 50% DM in the CC and CO diets. Ruminal fluid and blood samples were collected every 3 h for 24 h. Ruminal fluid was analyzed for pH, volatile fatty acids (VFA), and ammonia-nitrogen (NH3-N). Blood was analyzed for plasma urea nitrogen (PUN). Hay and fecal samples were collected for 4 d, four times daily for hay and twice daily for feces, to determine apparent total-tract digestibility of nutrients. The hay provided to steers during the digestibility period was analyzed for in vitro organic matter digestibility (IVOMD) for 48 h. Data were analyzed as repeated measures for blood and ruminal fermentation parameters. Total DM intake was not affected (P ≥ 0.674) by treatment. A treatment effect (P < 0.001) was observed for average ruminal pH, where steers consuming CO had the greatest pH (P < 0.001). Ruminal concentration of NH3-N tended (P = 0.059) to be reduced in steers consuming CO. There was a treatment × time interaction (P = 0.023) on concentrations of PUN, where at 3 h DH and CO were lesser than CC (P ≤ 0.050) and at 21 h DH was lesser than CC (P = 0.020). Total VFA, acetate, propionate, butyrate, branched-chain VFA, and valerate concentrations were affected by treatment (P ≤ 0.035), where a reduction (P ≤ 0.034) occurred in steers consuming CO. No treatment differences were observed for total-tract digestibility of DM (P = 0.186), organic matter (P = 0.169), or crude protein (P = 0.152); however, steers consuming DH had greater neutral detergent fiber (P = 0.038) than CC and tended to be greater than CO (P = 0.082). The CO hay had greater (P = 0.005) IVOMD compared with DH and tended (P = 0.100) to be greater than CC. Bahiagrass hay treated with CaO may reduce ruminal fermentation, as indicated by decreased total VFA concentration without altering DM intake. The addition of CaO did not improve the digestibility of bahiagrass hay in vivo; however, in vitro results are contradictory and warrant further elucidation.

With the ever-growing desire to increase efficiency in beef cattle production, researchers have developed strategies such as treating poor-quality forages with chemicals to increase the digestibility of fiber fractions, consequently increasing their energy value for cattle feeding. Calcium oxide has been proposed as a replacement to more caustic chemicals used in the past (e.g., NaOH) and data indicate that it can promote similar and effective outcomes. The current study evaluated the effects of bahiagrass hay treated with calcium oxide on ruminal fermentation parameters, apparent total-tract digestibility of nutrients, and intake by beef steers consuming hay ad libitum as the sole ingredient in their diet. Additionally, in vitro organic matter digestibility was evaluated on the hay provided to steers to assess treatment effectiveness. Results indicated that steers consuming bahiagrass hay treated with calcium oxide had 1) increased pH and reduced volatile fatty acids concentrations in the rumen; 2) reduced or tendency for reduction on total-tract digestibility of fiber fractions; and 3) no effect on intake, all when compared with steers consuming untreated hay. In contrast, in vitro results indicated that organic matter digestibility was increased when the forage was treated with calcium oxide.

Modulation of contractile apparatus Ca2+ sensitivity and disruption of excitation-contraction coupling by S-nitrosoglutathione in rat muscle fibres.

S-Nitrosoglutathione (GSNO) is generated in muscle and may S-glutathionylate and/or S-nitrosylate various proteins involved in excitation­contraction (EC) coupling, such as Na+-K+-ATPases, voltage-sensors (VSs) and Ca2+ release channels (ryanodine receptors,RyRs), possibly changing their properties. Using mechanically skinned fibres from rat extensor digitorum longus muscle, we sought to identify which EC coupling processes are most susceptible to GSNO-modulated changes and whether these changes could be important in muscle function and fatigue. For comparison, we examined the effect of other oxidation, nitrosylation, or glutathionylation treatments (S-nitroso-N-acetyl-penicillamine (SNAP), hydrogen peroxide,2,2-dithiodipyridine and reduced glutathione) on twitch and tetanic force, action potential (AP) repriming, sarcoplasmic reticulum (SR) Ca2+ loading and leakage, and contractile apparatus properties. None of the treatments detectably altered AP repriming, indicating that t-system excitability was relatively insensitive to such oxidative modification. Importantly, the overall effect on twitch and tetanic force of a given treatment was determined primarily by its action on Ca2+ sensitivity of the contractile apparatus. For example, S-nitrosylation with the NO• donor,SNAP, caused matching decreases in the contractile Ca2+ sensitivity and twitch response, and GSNO applied ∼10 min after preparation had very similar effects. The only exception was when GSNO was applied immediately after preparation, which resulted in irreversible decreases in twitch and tetanic responses even though it concomitantly increased Ca2+ sensitivity by∼0.1 pCaunits, the latter evidently due to S-glutathionylation of the contractile apparatus. This decrease in AP-mediated force responses was due to impaired VS­RyR coupling and was accompanied by increased Ca2+ leakage through RyRs. Such oxidation-related impairment of coupling could be responsible for prolonged low frequency fatigue in certain circumstances.

Effects of supplemental progesterone on the development, metabolism and blastocyst cell number of bovine embryos produced in vitro.

The objectives of the present experiment were to determine whether supplementation with progesterone (LO, 1 ng mL(-1) or HI, 100 ng mL(-1)) during either the first (Culture-1, Day 1 to 3) or second (Culture-2, Day 4 to 7) phase of culture of in vitro-produced embryos alters embryo development, embryo metabolism or blastocyst cell number. The percentage of oocytes that cleaved, the percentage of cleaved embryos that developed to the morula stage or greater, the blastocyst stage or greater or the hatched blastocyst stage were similar among treatments. Quantities of glucose metabolised per blastocyst per hour were similar, but when metabolic data was normalised for numbers of cells in each blastocyst, the LO treatment during Culture-2 metabolised more glucose (P=0.03) compared with all other treatments. Embryos receiving LO progesterone tended to have greater (P=0.085) metabolism of glucose compared with embryos receiving HI progesterone. Quantities of pyruvate oxidised per blastocyst per hour, and per cell, were similar among treatments. The number of cells per blastocyst in the control group was increased (P=0.039) compared with cells in progesterone-treated groups. In conclusion, supplementation with progesterone during the culture of in vitro-produced embryos does not appear to improve embryo characteristics.