Optimization of cryopreservation protocols for cooled-transported stallion semen.

Freezing cooled-transported semen allows veterinarians and breeders to collect and process the semen of stallions on farm, and then ship the semen to a semen freezing center. There, however, is a lack of standardization of shipping and freezing protocols. The objectives were to optimize and simplify protocols to freeze cooled-shipped semen. In Experiment 1, cooled-transported semen was centrifuged at room temperature or 5 °C before freezing. Sperm variables (motility, membrane integrity, acrosome integrity, membrane fluidity) were evaluated before and after freezing. Centrifugation temperature had no effect on post-thaw semen quality. In Experiment 2, cooled-transported semen was centrifuged at room temperature and cryopreserved in three semen freezing extenders. With use of the improved modified French formula, there was less post-thaw total and progressive motility compared with use of Botucrio or the improved lactose-EDTA formula (P<0.0001). Semen cryopreserved in the improved modified French formula also had a lesser percentage of sperm with intact membranes compared with lactose-EDTA, and a greater percentage of sperm with capacitation-like changes compared with Botucrio (P<0.0001). In Experiment 3, semen diluted in each extender was frozen conventionally or placed directly in a -80 °C ultra-freezer. Freezing in the ultra-freezer resulted in a lesser post-thaw sperm motility, but not membrane and acrosome integrity and capacitation-like changes. In conclusion, centrifugation and addition of freezing extender to cooled transported semen can be performed at room temperature or 5 °C. The Botucrio and lactose-EDTA formula are recommended for conventional cryopreservation of cooled-transported stallion semen as compared with the modified French formula.

Comparison of cushioned centrifugation and SpermFilter filtration on longevity and morphology of cooled-stored equine semen.

This study compares two methods for seminal plasma removal by evaluating sperm recovery rates, and motility and morphology of cooled-stored semen. Ejaculates were divided into three groups: control, filtration and cushioned centrifugation. Semen was extended to 25 million sperm/ml using a skim-milk-based extender and stored at 5°C for all groups. Sperm motility (total motility (%TM) and progressive motility (%PM)) was determined at 0, 24, 48 and 72 hours by a computer-assisted sperm analyser. Sperm morphology was assessed using differential interference microscopy. Overall, %TM of the centrifugation group was significantly higher than the filter group, but not significantly different than the control. No significant difference in %TM or %PM was detected for the control group and filter. Cushioned centrifugation was a superior method to obtain progressively motile sperm compared with control (P=0.03) and filter groups (P<0.001). No significant difference was found for the per cent of normal sperm cells and detached heads between the groups. This study demonstrated that cushioned centrifugation was a superior method to remove seminal plasma while preserving %TM and enhancing %PM for stallions under cooled storage over three days. However, as the differences appear to be negligible, the SpermFilter may represent an alternative for farms lacking a centrifuge.

Diagnosis and effects of urine contamination in cooled-extended stallion semen.

Urospermia is known to affect semen quality in many mammals, including stallions. Determinations of semen pH and creatinine and urea concentrations have been used to diagnose urine contamination in raw stallion semen. Unfortunately, practitioners suspecting urine contamination in cooled-shipped samples have no proven means to confirm the presence of urine. Therefore, the objectives of this study were (1) to assess the effects of urine contamination on sperm motility of extended fresh and cooled-stored stallion semen, (2) to evaluate the usefulness of semen color, odor, pH, and creatinine and urea concentrations for urospermia diagnosis, and (3) to evaluate the accuracy of a commercial blood urea nitrogen test strip in diagnosing urine contamination in extended-cooled stallion semen. Thirty-seven ejaculates were obtained from 11 stallions with no history of urospermia before division into 5 mL aliquots, and contamination with stallion urine. Each resulting sample was assessed for sperm motility, color, odor, pH, creatinine, and urea nitrogen concentration using both a semiquantitative test strip (Azostix), and a quantitative automated analyzer before and after cooling for 24 hour. Sperm motility parameters, pH, and creatinine and urea concentrations were analyzed using mixed models. Urine contamination decreased total and progressive motility in all samples before and after cooling (P < 0.05). Mean control total motility was 80% at 0 hour and 67% at 24 hours, whereas urine-contaminated samples ranged from 30% to 71% at 0 hour and 27% to 61% at 24 hours. Control mean urea (29 mg/dL) and creatinine (0.6 mg/dL) concentrations were significantly different (P < 0.05) from all urine-contaminated samples (158 mg/dL and 11.6 mg/dL, respectively) at 0 hour. Similarly, control mean urea (8 mg/dL) and creatinine (0.9 mg/dL) concentrations were significantly different than all urine-contaminated samples at 24 hours. Odor assessment presented moderate sensitivity (65%) and high specificity (100%), while color assessment presented low sensitivity (47%) and moderate specificity (79%) for urine in extended semen. Azostix strips were highly sensitive (95%) and specific (97%). Assessment of color, odor, and pH are not reliable methods to diagnose urine in experimentally contaminated cooled-stored stallion semen. Sperm motility parameters (in raw and cooled semen) are significantly reduced by the presence of urine in a concentration dependent. The results of the present study indicated that determination of urea and creatinine concentrations can be used to diagnose urospermia and that Azostix can be used as a point care method for diagnosing urine contamination in extended cooled stallion semen.

α-TEA cooperates with MEK or mTOR inhibitors to induce apoptosis via targeting IRS/PI3K pathways.

BACKGROUND: α-Tocopherol ether-linked acetic acid (α-TEA) is a promising agent for cancer prevention/therapy based on its antitumour actions in a variety of cancers. METHODS: Human breast cancer cells, MCF-7 and HCC-1954, were used to study the effect of α-TEA using Annexin V/PI staining, western blot analyses, and siRNA knockdown techniques. RESULTS: α-Tocopherol ether-linked acetic acid suppressed constitutively active basal levels of pAKT, pERK, pmTOR, and their downstream targets, as well as induced both cell types to undergo apoptosis. Phosphoinositide 3-kinase (PI3K) inhibitor wortmannin suppressed pAKT, pERK, pmTOR, and their downstream targets, indicating PI3K to be a common upstream mediator. In addition, α-TEA induced increased levels of pIRS-1 (Ser-307), a phosphorylation site correlated with insulin receptor substrate-1 (IRS-1) inactivation, and decreased levels of total IRS-1. Small interfering RNA (siRNA) knockdown of JNK blocked the impact of α-TEA on pIRS-1 and total IRS-1 and impeded its ability to downregulate the phosphorylated status of AKT, ERK, and mTOR. Combinations of α-TEA+MEK or mTOR inhibitor acted cooperatively to induce apoptosis and reduce basal levels of pERK and pmTOR. Importantly, inhibition of MEK and mTOR resulted in increased levels of pAKT and IRS-1, and α-TEA blocked them. CONCLUSIONS: Downregulation of IRS-1/PI3K pathways via JNK are critical for α-TEA and α-TEA+MEK or mTOR inhibitor-induced apoptosis in human MCF-7 and HCC-1954 breast cancer cells.

Validation and comparison of two methods of measuring lactate in equine plasma.

REASONS FOR PERFORMING STUDY: Some methods of lactate (LA) measurement have not been validated appropriately for use in horses. OBJECTIVES: To validate 2 LA analysers (YSI 2300 Stat Plus and TDx Lactic Acid Assay) for use with equine plasma and to compare plasma [LA] determined by the 2 methods. METHODS: Both instruments were evaluated for linearity, parallelism, recovery and precision using serial dilutions of standard LA solutions and equine plasma and then comparing results with linear regression or paired t tests. Plasma [LA] results were compared in 275 blood samples collected from horses exercising at various intensities using Bland-Altman analysis. Level of significance was P < 0.05. RESULTS: YSI exhibited good linearity for both LA standards and equine plasma (P < 0.05) at 0-30 mmol/l. TDx had good linearity at 0-12 mmol/l (P < 0.05); with LA standard solutions >12 mmol/l and with equine plasma, linearity was decreased. YSI exhibited good parallelism between LA standards and equine plasma LA measurements throughout the 0-30 mmol/l range (P > 0.05). Parallelism was poor with TDx (P < 0.05). Mean ± s.d. % recovery was 101.7 ± 3.4% for YSI (acceptable) and 110.6 ± 8.4% for TDx (unacceptable). Within-run and mean between-run coefficients of variation (CV) for plasma samples tested from 3.3-29.5 mmol/l were 0.4-3.0% for YSI. CVs for samples tested from 2.8-8.0 mmol/l were 17.4-24.1% for TDx. In 275 plasma samples, [LA] ranged from 0.1-42.7 mmol/l and 0.3-50.6 mmol/l for the YSI and TDx methods, respectively. The difference in plasma [LA] determined by the 2 methods was -1.0 ± 3.2 mmol/l, documenting that the TDx overestimated the YSI results by a mean value of 1 mmol/l. CONCLUSIONS: It was concluded that the YSI method was a reliable method for measuring equine plasma [LA] from 0-30 mmol/l. The TDx method was found not to be suitable for use with equine plasma due to greater variability in measurements (high CV).

RRR-alpha-tocopheryl succinate induces MDA-MB-435 and MCF-7 human breast cancer cells to undergo differentiation.

RRR-alpha-Tocopheryl succinate (vitamin E succinate, VES) is a potent antitumor agent, inducing DNA synthesis arrest, differentiation, and apoptosis. Because little is known about VES-induced differentiation, studies reported here characterize VES effects on the differentiation status of human breast cancer cell lines and investigate possible molecular mechanisms involved. VES-induced differentiation of human MCF-7 and MDA-MB-435 breast cancer cells was characterized by morphological changes, induction of lipid droplets, induction of beta-casein mRNA expression, and down-regulation of Her2/neu protein. In contrast, VES treatment of normal human mammary epithelial cells, MCF-10A cells, and T-47D cells did not induce differentiation. Studies addressing mechanisms showed that neither antibody neutralization of the transforming growth factor-beta signaling pathway nor expression of a dominant-negative mutant of c-Jun N-terminal kinase blocked the ability of VES to induce differentiation; however, treatment of cells with PD 98059, a chemical inhibitor of mitogen-activated protein kinase kinase (MEK1/2), blocked the ability of VES to induce differentiation.

Activation of extracellular signal-regulated kinase and c-Jun-NH(2)-terminal kinase but not p38 mitogen-activated protein kinases is required for RRR-alpha-tocopheryl succinate-induced apoptosis of human breast cancer cells.

RRR-alpha-tocopherol succinate (vitamin E succinate, VES) is a potent, selective apoptotic agent for cancer cells but not normal cells. VES has been shown to inhibit the growth of a wide variety of tumor cells in cell culture and animal models. Studies addressing mechanisms of action of VES-induced apoptosis have identified transforming growth factor-beta, Fas/CD95-APO-1, and mitogen-activated protein kinase (MAPK) signaling pathway involvement. Here we show that MAPKs, the extracellular signal-regulated kinases (ERK), and the stress-activated protein kinases, c-Jun NH2-terminal kinases (JNK), but not p38, are critical mediators in VES-induced apoptosis of human breast cancer MDA-MB-435 cells. VES activates ERK1/2 and JNK both in level and duration of kinase activity. Expression of dominant negative mutants of ERK1, MAPK/ERK activator-1, or JNK1 but not p38 blocked phosphorylation of the substrate glutathione S-transferase-c-Jun and inhibited VES-induced apoptosis. Increased phosphorylation and transactivation activity of nuclear transcription factors c-Jun, ATF-2, and Elk-1 are observed after VES treatments; however, only c-Jun and ATF-2 appear to be involved in VES-induced apoptosis based on antisense blockage experiments. Collectively, these results imply a critical role for ERK1 and JNK1 but not p38 in VES-induced apoptosis of human MDA-MB-435 breast cancer cells.

Technical note: using calcium carbonate as an osmolar control treatment for acid-base studies in horses.

The efficacy of using calcium carbonate as an osmolar control treatment for acid-base studies in horses receiving alkalizing compounds was evaluated. Six mares were nasogastrically intubated with isomolar quantities of sodium or calcium as sodium bicarbonate or calcium carbonate or with water during three treatment periods. Doses of the carbonic acid salts were 500 mg/kg sodium bicarbonate mixed with 4 L of distilled water (positive control) and 595 mg/kg calcium carbonate mixed with 2 L of distilled water to yield isoosmolar treatments. Four liters of distilled water served as the negative control. Jugular venous blood samples were drawn before intubation and at hourly intervals for 6 h after intubation. The serum electrolytes Na+ and K+, blood pH, and HCO3- were determined. The sodium bicarbonate treatment increased blood pH and HCO3- (P < 0.01) above both the water and CaCO3 treatments. No differences (P > 0.05) were found between the water and CaCO3 treatments. These data indicate that calcium carbonate may serve as a suitable osmolar control treatment for studying the effects of treatments that affect acid-base status of horses.

Dietary protein restriction and fat supplementation diminish the acidogenic effect of exercise during repeated sprints in horses.

A restricted protein diet supplemented with amino acids and fat may reduce the acidogenic effects of exercise. Twelve Arabian horses were assigned to a 2 x 2 factorial experiment: two fat levels: 0 or 10 g/100 g added corn oil and two crude protein levels: 7.5 g/100 g (supplemented with 0.5% L-lysine and 0.3% L-threonine) or 14.5 g/100 g. The experiment began with a 4-wk diet accommodation period followed by a standard exercise test consisting of six 1-minute sprints at 7 m/s. Horses were interval trained for 11 wk followed by another exercise test with sprints at 10 m/s. Blood samples were taken at rest and during the exercise tests. Plasma was analyzed for PCO(2), PO(2), Na(+), K(+), Cl(-), lactate, pH and total protein. Bicarbonate, strong ion difference and total weak acids were calculated. Data were analyzed using repeated-measures analysis of variance. Venous pH was higher in the low protein group during the first test (P = 0.0056) and strong ion difference became higher (P = 0.022) during sprints in the low protein group. During the second test, venous pH and bicarbonate were higher for the low protein high fat group (P = 0.022 and P = 0.043, respectively) and strong ion difference became higher (P = 0.038) at the end of exercise in the low protein groups. These results show that restriction of dietary protein diminishes the acidogenic effect of exercise, especially in combination with fat adaptation.

Muscular dystrophy in female dogs.

The most common form of muscular dystrophy in dogs and humans is caused by mutations in the dystrophin gene. The dystrophin gene is located on the X chromosome, and, therefore, disease-causing mutations in dystrophin occur most often in males. Therefore, females with dystrophin deficiency or other forms of muscular dystrophy may be undiagnosed or misdiagnosed. Immunohistochemistry was used to analyze dystrophin and a number of other muscle proteins associated with muscular dystrophy in humans, including sarcoglycans and laminin alpha2, in muscle biopsy specimens from 5 female dogs with pathologic changes consistent with muscular dystrophy. The female dogs were presented with a variety of clinical signs including generalized weakness, muscle wasting, tremors, exercise intolerance, gait abnormalities, and limb deformity. Serum creatine kinase activity was variably high. One dog had no detectable dystrophin in the muscle; another was mosaic, with some fibers normal and others partly dystrophin-deficient. A 3rd dog had normal dystrophin but no detectable laminin alpha2. Two dogs could not be classified. This study demonstrates the occurrence of dystrophin- and laminin alpha2-associated muscular dystrophy and the difficulty in clinical diagnosis of these disorders in female dogs.

Dietary fat supplementation effects on in vitro nutrient disappearance and in vivo nutrient intake and total tract digestibility by horses.

Addition of fat to the diet of the equine is a popular method of increasing energy density of the diet while reducing feed intake. Reducing feed intake is of interest to race horse trainers because additional feed is seen as additional weight and, therefore, a hindrance to performance. Limited information is available regarding the interactions of fat with other dietary components, particularly fiber, in the equine digestive system. The effect of dietary fat on in vitro nutrient disappearance in equine cecal fluid was studied in Exp. 1 using a split-plot design within a 2 x 2 Latin square. Two ponies were fed alfalfa (ALF) alone or alfalfa plus 100 g/d corn oil. Five substrates were used to determine in vitro DM disappearance, OM disappearance, NDF disappearance, and total dietary fiber (TDF) disappearance. The substrates included: ALF, tall fescue (TF), red clover (RC), soybean hulls (SBH), and rolled oats (RO). Fat supplementation did not affect in vitro DM, OM, or NDF disappearance. Addition of fat to the diet increased (P < 0.05) the disappearance of NDF in RO. Among substrates, in vitro DM and OM disappearance were highest (P < 0.05) for RO, followed by SBH, ALF, RC, and TF. In vitro NDF and TDF disappearance were highest (P < 0.05) for SBH, followed by RO, ALF, RC, and TF. In Exp. 2, the effects of varying levels of fat on nutrient intake and total tract digestibility were examined using a 4 x 4 Latin square design. Four mature mares were fed a 60% forage-40% concentrate diet containing different concentrations of fat: 0% supplemental fat control (C); 5% supplemental corn oil (5% CO); 10% supplemental corn oil (10% CO); or 15% supplemental corn oil (15% CO). Treatment did not affect intake of the concentrate portion of the diet or CP, gross energy, or NDF intake. Mares consuming the C diet had the highest (P < 0.05) intake of alfalfa cubes, DM, and OM, followed by those on the 10, 5, and 15% CO treatments, respectively. Treatment did not affect nutrient digestibility. Mares consuming the 15% CO diet had the highest (P < 0.05) fat digestibility, whereas those consuming C had the lowest fat digestibility. Fat in the form of CO generally had little effect on in vitro and in vivo nutrient digestibilities in horses.

Psychophysiological correlates of individual differences in patterns of hemodynamic reactivity.

The present study delineates a method for the quantification of six hemodynamic reactivity patterns, in response to a laboratory stressor, and examines the psychophysiological correlates of individual differences in these patterns. One hundred and ninety-four young adult men and women participated in rest periods and two laboratory stressors, mental arithmetic and an anger recall interview. Measures were taken of blood pressure, heart rate, and cardiac output, from which total peripheral resistance was derived, as well as state reports of feelings during the tasks. Six hemodynamic reactor patterns were identified: Non-reactors, Mild Myocardials, Mild Vasculars, Myocardials, Vasculars, and Dual Reactors, each associated with a unique profile of cardiac output and total peripheral resistance change. Myocardial reactors to the interview had the highest resting levels of blood pressure and total peripheral resistance. Dual reactors had the largest increases in diastolic reactivity; Dual and Myocardial reactors had the largest increases in systolic reactivity. The extreme reactor groups (Dual, Myocardial, Vascular) all reported greater task invigoration than the Non-reactors, who reported greater efforts to relax. Reactor groups were similar on anger-related trait affect. Based on both resting blood pressure and magnitude of task-induced reactivity, Myocardial and Dual reactors may be at the greatest risk for subsequent hypertension.

From "reducing" to "coping with" uncertainty: reconceptualizing the central challenge in breast self-exams.

An ideology of uncertainty reduction pervades scholarly and popular discourse on breast self-exams (BSE). Women are encouraged to understand BSE as an activity that reduces uncertainty about their health. Moreover, uncertainties about the procedure itself are conceived as barriers to BSE. In turn, reducing these uncertainties is seen as the key to promoting BSE. We argue that the ideology of uncertainty reduction is both descriptively and prescriptively inadequate and potentially a threat to women's health. We further contend the ideology should be replaced by a framework that illuminates processes of coping with uncertainty. Several major characteristics of such a framework, as well as implications for medical practice, are discussed and illustrated within the context of BSE.

Breast self-examination pamphlets: a content analysis grounded in fear appeal research.

In this study, we used the topic of breast self-examination (BSE) to illustrate how content analysis of promotional texts (already in existence, in the process of being created, or both) can provide supplementary data to that derived from audience analysis. Specifically, we used content analysis to isolate messages in BSE pamphlets that are consistent with the variables of severity, susceptibility, response efficacy, and self-efficacy, identified by existing fear appeal research and supported by other persuasion research as critical to the construction of effective health promotion messages. We then used statistical analyses to describe the relation among these 4 message variables. Our findings suggested that BSE pamphlets contain an unbalanced proportion of threat to efficacy arguments. Additionally, the efficacy messages were substantively weak. We contrasted these messages against the relatively strong mammography arguments contained in these pamphlets. We then provided recommendations for formulating stronger persuasive arguments in BSE promotional materials.

An alternative approach for achieving cardiovascular baseline: viewing an aquatic video.

Due to the importance of baseline and recovery levels in the computation of reactivity, two studies were conducted to determine an alternative method to traditional rest for achieving baseline and recovery levels of cardiovascular measurements. Watching a relaxing, aquatic video was compared with a traditional resting baseline to determine the better method for achieving low baseline levels. In addition, watching the video was compared with traditional rest during 5-min post-task recovery periods. Systolic (SBP) and diastolic blood pressure (DBP) decreased more during the baseline period when subjects viewed the video than when subjects rested quietly. Similarly, subjects displayed greater recovery following the mental tasks when they watched a video than when they merely sat quietly. It is recommended that researchers standardize baseline procedures by showing a relaxing video before administering tasks for the assessment of cardiovascular reactivity.

Vitamin E succinate induces apoptosis in human prostate cancer cells: role for Fas in vitamin E succinate-triggered apoptosis.

The apoptosis-triggering properties of vitamin E succinate (VES, RRR-alpha-tocopheryl succinate) for human LNCaP and PC-3 prostate carcinoma cells and normal PrEC human prostate epithelial cells were investigated. LNCaP and PC-3 cells were sensitive to VES-induced apoptosis, with 100% and 60% of cells undergoing apoptosis after three days of treatment with 10 micrograms of VES/ml, respectively. PrEC cells were resistant to VES-induced apoptosis. Treatment of prostate cells with agonistic anti-Fas antibody triggered apoptosis in approximately 50% of PC-3 cells within 48 hours, whereas LNCaP and PrEC cells were resistant. Prostate cells simultaneously treated with VES and agonistic anti-Fas antibodies revealed 1) no effect on PrEC cells, 2) an additive effect on Fas-sensitive PC-3 cells, and 3) a synergistic effect on LNCaP cells. VES treatment of LNCaP cells caused depletion of cytosolic 43-kDa Fas, enhanced membrane levels of 43-kDa Fas, and induced Fas sensitivity. PC-3 cells expressed high levels of membrane 43-kDa Fas that were enhanced by VES treatments. Fas ligand expression by LNCaP cells was enhanced by VES treatments. In summary, VES triggers apoptosis in human prostate carcinoma cells but not normal prostate cells in vitro, and VES modulates Fas signaling.

Stable expression of a new chimeric fluorescent reporter in the human malaria parasite Plasmodium falciparum.

Stable transfection of a new, chimeric reporter in the human malaria parasite Plasmodium falciparum confers green fluorescence and methotrexate resistance that can be quantitated by Western blotting and flow cytometry. This provides a sensitive, live reporter for exploitation of genomic and high-throughput assays for the identification of new pathogenic determinants.

N-(4-hydroxyphenyl)retinamide activation of transforming growth factor-beta and induction of apoptosis in human breast cancer cells.

N-(4 hydroxyphenyl)retinamide (4-HPR), a synthetic derivative of all-trans-retinoic acid, induces DNA synthesis arrest and apoptosis in human breast cancer cells in a dose- and time-dependent manner. MDA-MB-435 cells treated with 3 microM 4-HPR exhibited 58% and 75% DNA synthesis arrest after 1 and 2 days of treatment and 31%, 39%, 48%, and 56% apoptosis after 3, 4, 5, and 6 days of treatment, respectively. Conditioned media from 4-HPR-treated MDA-MB-435 cells contained 63 and 57 pg of active transforming growth factor-beta (TGF-beta) per 10(6) cells after 1 and 2 days of treatment, whereas conditioned media from control cells contained only 9 pg/10(6) cells. TGF-beta involvement in 4-HPR-induced apoptosis, but not DNA synthesis arrest, in MDA-MB-435 cells was demonstrated by 1) blockage of 4-HPR-induced apoptosis by 66-75% after treatment of cells with neutralizing antibodies to TGF-beta s, 2) blockage of 4-HPR-induced apoptosis by 64-67% after transient transfection of cells with antisense oligomers to TGF-beta 1 or TGF-beta type II receptor, 3) blockage of 4-HPR-induced apoptosis by approximately 50% after inhibition of latent TGF-beta activation, and 4) demonstration that human breast cancer cells (T47D) defective in TGF-beta signaling were refractive to 4-HPR-induced apoptosis. These data indicate that 4-HPR is a potent activator of TGF-beta and that TGF-beta participates in 4-HPR-induced apoptosis of human breast cancer cells.