Coculture with porcine luteal cells during in vitro porcine oocyte maturation affects lipid content, cortical reaction and zona pellucida ultrastructure.

CONTEXT: In pigs, in vitro fertilisation (IVF) is associated with high polyspermy rates, and for this reason, in vitro embryo production (IVP) is still an inefficient biotechnology. Coculture with somatic cells is an alternative to improve suboptimal in vitro maturation (IVM) conditions. AIM: This study was conducted to test a coculture system of porcine luteal cells (PLC) and cumulus-oocyte complexes (COC) to improve oocyte metabolism. METHODS: COC were matured in vitro with PLC. Oocyte lipid content, mitochondrial activity, zona pellucida (ZP) digestibility and pore size, cortical reaction and in vitro embryo development were assessed. KEY RESULTS: Coculture reduced cytoplasmic lipid content in the oocyte cytoplasm without increasing mitochondrial activity. Although ZP digestibility and ZP pore number were not different between culture systems, ZP pores were smaller in the coculture. Coculture impacted the distribution of cortical granules as they were found immediately under the oolemma, and more of them had released their content in the ZP. Coculture with porcine luteal cells during IVM increased monospermic penetration and embryo development after IVF. CONCLUSIONS: The coculture of COC with PLC affects the metabolism of the oocyte and benefits monospermic penetration and embryo development. IMPLICATIONS: The coculture system with PLC could be an alternative for the conventional maturation medium in pigs.

Coculture of porcine cumulus-oocyte complexes with porcine luteal cells during IVM: effect on oocyte maturation and embryo development.

Coculture with somatic cells is an alternative to improve suboptimal invitro culture conditions. In pigs, IVF is related to poor male pronuclear formation and high rates of polyspermy. The aim of this study was to assess the effect of a coculture system with porcine luteal cells (PLCs) on the IVM of porcine cumulus-oocyte complexes (COCs). Abattoir-derived ovaries were used to obtain PLCs and COCs. COCs were matured invitro in TCM-199 with or without the addition of human menopausal gonadotrophin (hMG; C+hMG and C-hMG respectively), in coculture with PLCs from passage 1 (PLC-1) and in PLC-1 conditioned medium (CM). In the coculture system, nuclear maturation rates were significantly higher than in the C-hMG and CM groups, but similar to rates in the C+hMG group. In cumulus cells, PLC-1 coculture decreased viability, early apoptosis and necrosis, and increased late apoptosis compared with C+hMG. PLC-1 coculture also decreased reactive oxygen species levels in cumulus cells. After IVF, monospermic penetration and IVF efficiency increased in the PLC-1 group compared with the C+hMG group. After invitro culture, higher blastocysts rates were observed in the PLC-1 group. This is the first report of a coculture system of COCs with PLCs. Our model could be an alternative for the conventional maturation medium plus gonadotrophins because of its lower rates of polyspermic penetration and higher blastocysts rates, key issues in porcine invitro embryo production.

The antioxidant dimethylthiourea improves IVF efficiency and decreases cumulus cell apoptosis in pigs.

Abattoir ovaries, which are the main source of oocytes for reproductive biotechnologies, arrive at the laboratory under ischaemic conditions. Reoxygenation generates reactive oxygen species (ROS) in ischaemic tissues, which could affect oocyte quality. The aim of this study was to evaluate the effect of supplementation of media with dimethylthiourea (DMTU) during the collection and washing of cumulus-oocyte complexes (COC) on ROS levels, COC apoptosis and oocyte nuclear and cytoplasmic maturation. Thus, the collection (TCM-199) and washing (TCM-199 with 10% porcine follicular fluid, sodium pyruvate and antibiotics) media were supplemented with 1 and 10mM DMTU. In the control group, the media were not supplemented with DMTU. Intracellular ROS levels decreased significantly in the DMTU-treated groups (P<0.05). Although no effects on rate of nuclear maturation were observed, DMTU significantly increased sperm penetration rates without increasing polyspermy (P<0.05). The addition of 10mM DMTU to the collection and washing media enhanced IVF efficiency. DMTU did not modify the early or late apoptosis of oocytes. Both concentrations of DMTU significantly increased viability and decreased the apoptosis of cumulus cells (P<0.05). These results suggest that the addition of 1 or 10mM of DMTU to the media during the collection and washing of porcine COCs is useful for decreasing cumulus apoptosis mediated by ROS and for optimising the IVF of porcine oocytes.

Space allowance influences individually housed Holstein male calves' age at feed consumption, standing behaviors, and measures of immune resilience before and after step-down weaning.

Many dairy calves in the southwestern regions of the United States are raised in wooden hutches with 1.23 m2 of free space that house 3 calves individually. Producers claim that calves in hutch systems are not ready to wean and be placed in groups at the country-average age of 6 wk. Calves may remain in this individual housing system until as late as 10 wk of age. The objective of this study was to modify space allowance of hutches and evaluate weaning readiness using age at solid feed consumption, standing behaviors, and measures of immunity. Calves were randomly assigned at 4 d of age to conventional (CONV; 1.23 m2 of space; n = 18), moderate (MOD; 1.85 m2 of space; n = 17), or maximized (MAX; 3.71 m2 of space; n = 19) space allowance in hutches. These modifications also changed the number of calves housed per hutch from 3 (CONV) to 2 (MOD) and 1 (MAX). Calves were fed milk replacer via bottle twice daily until weaning and offered ad libitum feed throughout the experiment. Step-down weaning was initiated (Wi) at age 53 or 54 d by withdrawal of the p.m. bottle and was completed (Wc) 11 d later by removal of the a.m. milk replacer. Accelerometer data for standing behaviors were collected relative to Wi (3 consecutive days to represent -4 wk, -3 d, 3 d, and 3 and 5 wk). Blood samples were collected in the a.m. just before Wi (d 0) and at d 3, 11, 14, and 18 after Wi. Calves provided with more space (MOD and MAX) compared with CONV calves consumed feed at an earlier age and had slightly healthier erythrocytes, greater circulating glucose, and fewer circulating eosinophils. The CONV calves had haptoglobin (Hp) responses to the stressors of both Wi and Wc and had more IFN-γ from whole blood stimulated with phytohemagglutinin-P. The MAX calves had the least active neutrophils (phagocytosis and oxidative burst), but MOD calves' leukocytes secreted the most TNF-α from whole blood stimulated with lipopolysaccharide. Just before and after weaning, MAX calves spent more time per day in the standing position than CONV and MOD calves and had an Hp response to Wc, but MOD calves did not have an Hp response to Wi or Wc. Based on these results, MOD calves were the most ready for weaning; therefore, they potentially can be moved to group housing at an earlier age than CONV calves, thus improving animal welfare concerns over space allowance and individual housing.

Effects of ractopamine hydrochloride on growth performance, carcass characteristics, and physiological response to different handling techniques.

Feedlot cattle ( = 128; BW = 549 ± 60 kg) were used to evaluate the effects of ractopamine hydrochloride (RAC) on growth performance, physiological response to handling, and mobility during shipment for slaughter in a study utilizing a split-plot design with a 2 × 2 factorial arrangement of treatments: 1) diet (CON [no ß-adrenergic agonist] vs. RAC [400 mg·animal·d ractopamine hydrochloride for 28 d]) and 2) handling intensity (HI; low-stress handling [LSH; cattle moved at a walking pace with no electric prod use] vs. high-stress handling [HSH; cattle moved at a minimum of a trot and an electric prod applied while in the alley for posthandling restraint and during loading for shipment to the abattoir]). Cattle fed RAC tended to have greater ADG and G:F ( = 0.06), and had greater HCW and LM area ( = 0.04). The HI treatments were applied on the day after the 28-d growth performance period. Blood samples were collected before HI treatment (baseline), after HI treatments (POSTHAND), after transport to the abattoir (POSTTRANS), and during exsanguination at slaughter. A diet × HI interaction ( = 0.01) was observed in the change in cortisol from baseline to POSTTRANS, and there tended ( ≤ 0.07) to be diet × HI interactions for the change in epinephrine from baseline to POSTHAND and for the change in creatine kinase (CK) from baseline to POSTTRANS. Feeding RAC and HSH both increased the change from baseline to POSTHAND in norepinephrine and pH ( ≤ 0.05). The HSH cattle also had greater changes from baseline to POSTHAND in blood HCO, base excess, partial pressure of CO, lactate, cortisol, and glucose ( ≤ 0.01). Ractopamine and HSH both produced greater increases in CK concentrations from baseline to slaughter ( < 0.01). Mobility was not affected by RAC at the feedlot or following an average 6-h lairage ( ≥ 0.43). This study confirms RAC improves growth performance and suggests metabolic acidosis, a precursor to fatigued cattle syndrome, develops in cattle allowed to trot without the use of a lead rider regardless of RAC administration. Cattle fed RAC displayed altered hormonal responses to handling and transport stress, and the overall proportion of cattle with compromised mobility appears to increase later in the marketing channel. These findings warrant additional research aimed at better understanding the physiological response to stress and protect the welfare of cattle during shipment for slaughter.

Effect of handling intensity at the time of transport for slaughter on physiological response and carcass characteristics in beef cattle fed ractopamine hydrochloride.

The effects of handling intensity on the physiological response and carcass characteristics of feedlot cattle fed ractopamine hydrochloride were evaluated at the time of transport to slaughter. Eighty steers (BW = 668 ± 36 kg) representing 10 lots of similar breed, frame size, and degree of finish were blocked by lot, stratified by weight, and randomly assigned to 1 of 2 handling intensities (HI) over a 1,600 m dirt alley course: 1) low-stress handling (LSH) or 2) high-stress handling (HSH). For the LSH treatment, 4 penmates were kept at a walk with the use of a lead rider. For the HSH treatment, 4 penmates were kept at a minimum of a trot and received 2 applications of an electric prod (approximately 1 s per impulse) at 2 separate instances: first in the alley before post-handling sampling, and again during loading for transportation to the abattoir. Behavioral observations and physical indicators of stress were recorded a minimum of 1 h before handling (baseline), immediately after handling (POSTHAND), and while in lairage after a 200 km transport to the abattoir. Vital parameters were recorded at baseline and POSTHAND. Venous blood samples were collected via jugular venipuncture at baseline and POSTHAND, and mixed arterial and venous blood samples were collected during exsanguination at slaughter. Muscle tremors tended to be more prevalent in HSH cattle at POSTHAND ( = 0.10). The HSH cattle tended to have greater POSTHAND heart rate ( = 0.08); however, there was no effect of HI on POSTHAND respiration rate or rectal temperature ( 0.34). The HSH cattle had greater lactate, epinephrine, norepinephrine, cortisol, and glucose concentrations at POSTHAND ( ≤ 0.02). Additionally, HSH cattle had lower POSTHAND blood pH, bicarbonate, base excess, and partial pressure carbon dioxide ( < 0.0001). Bicarbonate concentrations were greater in HSH cattle at slaughter ( = 0.05); however, there were no differences between HI treatments for the remaining blood variables ( 0.11). Concentrations of stress hormones and CK were significantly greater at slaughter relative to baseline and POSTHAND for both LSH and HSH cattle ( < 0.001). These findings suggest cattle trotted without a lead rider develop metabolic acidosis, and illustrate the importance of low-stress handling at the time of transport for slaughter. Further research is warranted to develop strategies to mitigate stress at the time of transport and ensure the welfare of beef cattle presented to abattoirs.

Technical note: valuation of an enteric methane emissions measurement system for cattle.

Through the process of enteric fermentation, cattle produce methane (CH), a potent greenhouse gas (GHG). Growing public concern about climate change has increased the need for accurate methods of measuring CH emissions from cattle directly to improve emission inventories and evaluate emissions mitigation techniques. The present study evaluated a ventilated head box system capable of measuring CH and carbon dioxide (CO) emissions, and oxygen (O) consumption from cattle. Six Holstein heifers were used to measure CH and CO emissions and O consumption from 2 ad libitum intake measurement periods (ADAPT and ADLIB) and 1 measurement period (RESTRICT) with intake restricted to 2% of BW on a DM basis. Methane emissions during RESTRICT were significantly lower ( < 0.0001) than both ad libitum periods. Daily CH4 emission rates per animal were 235.0 ± 6.19 L/d, 228.3 ± 6.18 L/d, and 193.2 ± 8.88 L/d for the periods ADAPT, ADLIB, and RESTRICT, respectively. Carbon dioxide emission rates were 3627 ± 90.72 L/d, 3632 ± 90.47, and 3184 ± 104.79 L/d for the ADAPT, ADLIB, and RESTRICT periods, respectively. Oxygen consumption rates were 3391 ± 99.77 L/d, 3454 ± 99.57 L/d, and 3002 ± 111.36 L/d for the periods ADAPT, ADLIB, and RESTRICT, respectively. The head box system evaluated provides an accurate method of measuring emissions from cattle and can provide information about daily variations and peaks in emissions.

Space allowance influences individually housed Holstein bull calf innate immune measures and standing behaviors after castration at 3 weeks of age.

Dairy calves in the Southwest regions of the United States are typically raised individually in wooden hutches with 1.23 m2 of space. The objective of the study was to determine if increased space allowance in wooden hutches influences measures of innate immunity and behaviors of Holstein bull calves pre- and postcastration. Calves were randomly assigned at 4 d of age to conventional (CONV; 1.23 m2 of space; n = 18), moderate (MOD; 1.85 m2 space; n = 17), or maximized space allowance (MAX; 3.71 m2 space; n = 19) in hutches. Calves were surgically castrated at 24 d of age. Peripheral whole blood samples were collected at -1, +1, +5, and +12 d of castration. Accelerometer loggers (n = 16 calves per treatment) were used from -3 to +5 d of castration to assess standing behaviors. All calves decreased total standing duration the day of castration versus precastration. Overall, MAX spent the most time in the stand position postcastration versus CONV and MOD. Within treatments, MOD and MAX had increased plasma cortisol 1 d postcastration versus precastration. A treatment × time tendency was observed for cortisol at 12 d postcastration; MAX had the least circulating cortisol. A treatment × time tendency for circulating haptoglobin (Hp) was observed and Hp was greatest among CONV 1 d pre- and 12 d postcastration. Compared with precastration, CONV had increased Hp at 1, 5, and 12 d, whereas MOD had increased Hp at 5 d, and Hp remained similar within MAX. A treatment × time tendency for tumor necrosis factor-α (TNF-α) from lipopolysaccharide-stimulated whole blood was observed; at 1 d postcastration, MOD had the most TNF-α, whereas MAX had the least. Within MAX, calves had increased TNF-α from precastration to 5 d postcastration. A treatment × time interaction was observed for whole blood bactericidal activity against Escherichia coli (WB anti-E). The CONV tended to have the greatest WB anti-E at d -1, but at d 1 and 5 postcastration, CONV had the least WB anti-E. Overall, MAX had less intensity of neutrophil oxidative burst versus CONV and MOD. The lower response of neutrophil oxidative burst and slower Hp secretion after castration is indicative that the wound site likely had less microbial exposure. The findings of this study suggest that calves housed with more space are potentially at less risk of too much inflammation after castration, which may likely be due to the effects of increased space on hide cleanliness and increased standing time.

Review: Effects of Ractopamine Hydrochloride (Paylean) on welfare indicators for market weight pigs.

This review summarizes the effects of ractopamine hydrochloride (RAC) dose (5, 7.5, 10, and 20 mg/kg) on market weight pig welfare indicators. Ractopamine hydrochloride (trade name Paylean) is a ß-adrenergic agonist that was initially approved in the U.S. in 1999 at doses of 5 to 20 mg/kg to improve feed efficiency and carcass leanness. However, anecdotal reports suggested that RAC increased the rate of non-ambulatory (fatigued and injured) pigs at U.S. packing plants. This led to the addition of a caution statement to the Paylean label, and a series of research studies investigating the effects of RAC on pig welfare. Early research indicated that: (1) regardless of RAC administration, fatigued (non-ambulatory, non-injured) pigs are in a state of metabolic acidosis; (2) aggressive handling increases stress responsiveness at 20 mg/kg RAC, while 5 mg/kg reduces stress responsiveness to aggressive handling. Given this information, dosage range for Paylean was changed in 2006 to 5 to 10 mg/kg in market weight pigs. Subsequent research on RAC demonstrated that: (1) RAC has minimal effects on mortality, lameness, and home pen behavior; (2) RAC fed pigs demonstrated inconsistent prevalence and intensity of aggressive behaviors; (3) RAC fed pigs may be more difficult to handle at doses above 5 mg/kg; and (4) RAC fed pigs may have increased stress responsiveness and higher rates of non-ambulatory pigs when subjected to aggressive handling, especially when 20 mg/kg of RAC is fed.

Circulating Fibroblast Growth Factor 21 (Fgf21) as Diagnostic and Prognostic Biomarker in Renal Cancer.

BACKGROUND: The finding of new biomarkers is needed to have a better sub-classification of primary renal tumors (RCC) as well as more reliable predictors of outcome and therapy response. In this study, we evaluated the role of circulating FGF21, an endocrine factor, as a diagnostic and prognostic biomarker for ccRCC. MATERIALS AND METHODS: Serum samples from healthy controls (HC), clear cell and chromophobe RCC cancer patients were obtained from the serum biobank "Biobanco Público de Muestras Séricas Oncológicas" (BPMSO) of the "Instituto de Oncología "Ángel H. Roffo". Serum FGF21 and leptin were measured by ELISA while other metabolic markers were measured following routinely clinical procedures. RESULTS: One of our major findings was that FGF21 levels were significantly increased in ccRCC patients compared with HC. Moreover, we showed an association between the increased serum FGF21 levels and the shorter disease free survival in a cohort of 98 ccRCC patients, after adjustment for other predictors of outcome. CONCLUSION: Our results suggest that higher FGF21 serum level is an independent prognostic biomarker, associated with worse free-disease survival.

Vesicles Cytoplasmic Injection: An Efficient Technique to Produce Porcine Transgene-Expressing Embryos.

The use of vesicles co-incubated with plasmids showed to improve the efficiency of cytoplasmic injection of transgenes in cattle. Here, this technique was tested as a simplified alternative for transgenes delivery in porcine zygotes. To this aim, cytoplasmic injection of the plasmid alone was compared to the injection with plasmids co-incubated with vesicles both in diploid parthenogenic and IVF zygotes. The plasmid pcx-egfp was injected circular (CP) at 3, 30 and 300 ng/µl and linear (LP) at 30 ng/µl. The experimental groups using parthenogenetic zygotes were as follows: CP naked at 3 ng/µl (N = 105), 30 ng/µl (N = 95) and 300 ng/µl (N = 65); Sham (N = 105); control not injected (N = 223); LP naked at 30 ng/µl (N = 78); LP vesicles (N = 115) and Sham vesicles (N = 59). For IVF zygotes: LP naked (N = 44) LP vesicles (N = 94), Sham (N = 59) and control (N = 79). Cleavage, blastocyst and GFP+ rates were analysed by Fisher's test (p < 0.05). The parthenogenic CP naked group showed lower cleavage respect to control (p < 0.05). The highest concentration of plasmids to allow development to blastocyst stage was 30 ng/µl. There were no differences in DNA fragmentation between groups. The parthenogenic LP naked group resulted in high GFP rates (46%) and also allowed the production of GFP blastocysts (33%). The cytoplasmic injection with LP vesicles into parthenogenic zygotes allowed 100% GFP blastocysts. Injected IVF showed higher cleavage rates than control (p < 0.05). In IVF zygotes, only the use of vesicles produced GFP blastocysts. The use of vesicles co-incubated with plasmids improves the transgene expression efficiency for cytoplasmic injection in porcine zygotes and constitutes a simple technique for easy delivery of plasmids.

Wooden hutch space allowance influences male Holstein calf health, performance, daily lying time, and respiratory immunity.

Dairy calves in the western United States are commonly raised individually in wooden hutches with a space allowance of 1.23m(2)/calf. Recent legislative initiatives in California and across the United States were passed regarding concern over space allowance for farm animals. The objective of this study was to determine if rearing male Holstein calves in wooden hutches modified to increase space allowance would influence measures of performance, lying time per day, health, and respiratory immunocompetence. At 4d of age, 60 calves were randomly assigned to 1 of 3housing treatments: (1) conventional housing (CONV; 1.23m(2)/calf), (2) 1.5 × CONV (MOD; 1.85m(2)/calf), or (3) 3 × CONV (MAX; 3.71m(2)/calf). Intakes of milk and solid feed were recorded daily and body weight was measured at 0, 3, 6, 10, and 12 wk of age. For the first 3 wk of the trial, calves were scored daily for fecal consistency, hydration, and hide cleanliness. In addition, calves were scored for respiratory health (i.e., nasal and eye discharge, ear position) until 7 wk of age. The total lying duration per day was recorded using data loggers at 3, 6, and 10 wk of age. Eight clinically healthy calves from each treatment were sensitized with subcutaneous ovalbumin (OVA) and then challenged with aerosolized OVA to assess calf respiratory immunity at 11 wk of age. Bronchoalveolar lavage fluid (BALF) was collected 4d after the OVA challenge and analyzed for leukocyte differentials and OVA-specific IgG, IgG1, IgA, and IgE. Calf average daily gain and body weight were positively associated with space allowance at approximately 3 wk before weaning and throughout postweaning, respectively. A greater space allowance decreased lying time after 46d. Space allowance did not influence fecal consistency, but there was a tendency for MAX calves to take 1d longer to recover from loose feces than MOD calves. The MAX calves had the fewest (%) observations with feces on their body compared with CONV or MOD. At 3 wk of age, peripheral eosinophil concentrations decreased with increased space allowance. However, observations (%) of eye discharge increased with greater space allowance. Among calves challenged with OVA, MOD calves had the least BALF OVA-IgE, and the percent of BALF eosinophils decreased with increased space allowance. Increased space allowance for calves raised in wooden hutches may improve some measures of calf performance, health, and respiratory immunocompetence.

The effects of technology use in feedlot production systems on feedlot performance and carcass characteristics.

The objectives of this study were to examine the effects of feedlot production systems with and without the use of a ß-adrenergic agonist compared to an all-natural production program on feedlot performance and carcass characteristics. Crossbred beef steers ( = 336; initial BW = 379 ± 8 kg) were randomized to 1 of 3 treatments in a randomized complete block design (RCBD; 14 steers/pen; 8 pens/treatment). Treatments consisted of an all-natural treatment (NAT), a conventional treatment (CONV), and a conventional treatment with a ß-agonist (CONV-Z). All treatments were fed the same basal diet with NAT cattle receiving no growth promoting technologies. The CONV and CONV-Z cattle were implanted with 40 mg of estradiol and 200 mg of trenbolone acetate (TBA) on d 0 and were fed 33 and 9 mg/kg of monensin and tylosin daily, respectively. The CONV-Z cattle were fed zilpaterol hydrochloride (ZH) at 6.76 mg/kg (90% DM basis) for the last 20 days on feed (DOF) There was no effect of treatment on DMI ( = 0.83); however, CONV-Z steers gained 3.8% faster (1.64 vs. 1.58 kg/d; < 0.01) and were 5.3% more efficient (0.160 vs. 0.152; < 0.01) than CONV steers, and CONV steers gained 32.8% faster (1.58 vs. 1.19 kg/d; < 0.01) and were 26.7% more efficient (0.152 vs. 0.120; < 0.01) than NAT steers. There was a 35.7% improvement in estimated carcass gain (1.29 vs. 0.95 kg/d; < 0.01) and a 32.6% improvement in carcass efficiency (0.126 vs. 0.095; < 0.01) for CONV-Z steers compared to NAT steers. Hot carcass weight was increased by 8 kg for CONV-Z steers compared to CONV steers (394 vs. 386 kg; = 0.05) and 46 kg compared to NAT steers (394 vs. 348 kg; < 0.01). Longissimus muscle area was increased by 3.6 cm for CONV-Z steers compared to CONV steers (92.29 vs. 88.67 cm; = 0.02) and 12.1 cm for CONV-Z steers compared to NAT steers (92.29 vs. 80.16 cm; < 0.01), resulting in a 9.6% unit increase in USDA yield grade (YG) 1 (15.14 vs. 5.52%; < 0.05) and a 21.6% unit reduction in USDA YG 3 for CONV-Z steers compared to CONV steers (30.70 vs. 52.32%; < 0.05). The CONV-Z steers had a lower marbling score compared to the other treatments (432; 0.01), resulting in an 11.7% unit increase (20.70 vs. 9.03%; < 0.05) in USDA Select carcasses compared to CONV steers. The results of this experiment show that CONV-Z and CONV production results in a significant improvement in feedlot performance and USDA YG compared to NAT.

Bill E. Kunkle Interdisciplinary Beef Symposium: Practical developments in managing animal welfare in beef cattle: what does the future hold?

Interest in the welfare of cattle in the beef industry has intensified over time because of ethical concerns and varying societal perceptions that exist about the treatment and living conditions of farm animals. The definition of welfare will vary according to an individual's philosophies (how one defines and prioritizes what is "good"), experiences (societal and cultural influences of animal roles and relationships), and involvement in the livestock industry (knowledge of how livestock operations work and why). Many welfare concerns in the beef industry could be mitigated by enhancing traditional husbandry practices that utilize practical improvements to alleviate or eliminate heat stress, pain from routine husbandry procedures, negative cattle handling, and the transitional effects of weaning, dry feeding, transportation, and comingling of calves. Recent concerns about the potential welfare effects of feeding technologies such as ß-adrenergic agonists (BAA) have emerged and led to industry-wide effects, including the removal of a single BAA product from the market and the development of BAA-specific welfare audits. Altogether, the beef industry continues to be challenged by welfare issues that question a large range of practices, from traditional husbandry to newer technological advancements. As welfare awareness increases, efforts to improve livestock care and management must focus on scientific investigations, practical solutions, consumer perceptions, and educational tools that advance knowledge and training in livestock welfare. Furthermore, the future of beef cattle welfare must align welfare concerns with other aspects of sustainable beef production such as environmental quality, profitability, food safety, and nutritional quality.

Cross-talk between tumor cells and the microenvironment at the metastatic niche.

This review presents recent information about the cross-talk between the tumor cells and the microenvironment in the target organ of metastasis at the premetastatic and metastatic stage. The development of metastatic foci is driven not only by the tumor cells intrinsic properties, but also by the interplay with resident and foreign cells located at particular niches in the target organ. The primary tumor modulates the metastatic target through the production of soluble factors that mobilize cells from distant organs like the bone marrow, which in turn localize in the metastatic niche. There is also strong evidence indicating that some primary tumors induce a fertile ground for the tumor cell at the target organ even before the arrival of the disseminated tumor cell (premetastatic niche). The relationship between the players of the metastatic setting is dynamic and shows a high degree of plasticity. Tumor cells change through the acquisition of genetic and/or epigenetic alterations that provide adaptive advantages and the metastatic niche is remodeled by incoming cell types or newly secreted soluble mediators, as a result a reciprocal dialogue is established that invokes new levels of molecular and cellular complexity. Unraveling the mechanisms that sustain the metastatic niche will allow a better understanding of the biology of the disseminated tumor cell, the design of new therapeutic approaches and, hopefully, the improvement of cancer patients' survival.

The copper-chelating agent, trientine, suppresses tumor development and angiogenesis in the murine hepatocellular carcinoma cells.

Angiogenesis is now recognized as a crucial process in tumor development, including hepatocellular carcinoma (HCC). Since HCC is known as a hypervascular tumor, anti-angiogenesis is a promising approach to inhibit the HCC development. Trientine dihydrochloride (trientine) is used in clinical practice as an alternative copper (Cu)-chelating agent for patients with Wilson's disease of penicillamine intolerance. In our study, we examined the effect of Cu-chelating agents on tumor development and angiogenesis in the murine HCC xenograft model. Although both trientine and penicillamine in the drinking water suppressed the tumor development, trientine exerted a more potent inhibitory effect than penicillamine. In combination with a Cu-deficient diet, both trientine and penicillamine almost abolished the HCC development. Trientine treatment resulted in a marked suppression of neovascularization and increase of apoptosis in the tumor, whereas tumor cell proliferation itself was not altered. In vitro studies also exhibited that trientine is not cytotoxic for the tumor cells. On the other hand, it significantly suppressed the endothelial cell proliferation. These results suggested that Cu plays a pivotal role in tumor development and angiogenesis in the murine HCC cells, and Cu-chelators, especially trientine, could inhibit angiogenesis and enhance apoptosis in the tumor with consequent suppression of the tumor growth in vivo. Since trientine is already used in clinical practice without any serious side effects as compared to penicillamine, it may be an effective new strategy for future HCC therapy.

In vitro activity of a Solanum tuberosum extract against mammary carcinoma cells.

We investigated the antitumor properties of a Solanum tuberosum extract (STE) on F3II mouse mammary carcinoma cells. STE significantly inhibited adhesion on fibronectin-coated surfaces and blocked migration of tumor cells in vitro. A major gelatinolytic activity (gelatinase) of 82 kD was identified in STE by zymographic analysis and characterized by exposure to different experimental conditions. Proteolytic activity of STE may be responsible, at least in part, for the in vitro effects on mammary carcinoma cells.

Nafoxidine modulates the expression of matrix-metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in endothelial cells.

During angiogenesis, proteases and their inhibitors interact in the remodelling of the basement membrane. It has been demonstrated that nafoxidine has antiangiogenic activity in the chick egg chorioallantoic membrane assay, but the precise mechanism of action is unknown. We have analyzed the effect of the partial estrogen antagonist nafoxidine on human umbilical vein endothelial cells (HUVEC). Our data indicated that in nafoxidine-treated endothelial cells MMP-2 was activated. Nafoxidine upregulated, in a dose-dependent manner, the secretion of a 66 kDa TIMP-1 dimer, that lacks anti-MMP activity and inhibited angiogenesis in the endothelial cord formation assay. We can postulate that nafoxidine induces an increase in TIMP-1, which has antiangiogenic activity in the late stages of tube formation, independent of its capacity to inhibit MMPs.

Expression of metalloproteinases (MMP-1, MMP-2, and MMP-9) and their inhibitors (TIMP-1 and TIMP-2) in schistosomal portal fibrosis.

Focal extracellular matrix degradation morphologically identified in human portal pipestem fibrosis due to Schistosoma mansoni did not express immunohistochemical reactivity for metalloproteinases (MMP-1, MMP-2, and MMP-9) and their inhibitors (TIMP-1 and TIMP-2). However, when active schistosomal periovular granulomas were present, a strong reactivity for MMP-1, MMP-2, TIMP-1, and TIMP-2 was observed. No reactivity was ever observed for MMP-9. However, the positive pattern of immunohistochemical expression was not seen in old fibrotic periovular granulomas, which were sometimes situated in other areas of the same microscopic section. Positive staining for MMPs and TIMPs was observed at the same time in hepatocytes and within the apical portion of bile duct epithelium. These findings are consistent with the concept that matrix degradation in recent and old fibroses, in addition to differing at the ultrastructural level, also differs in immunohistochemical expression of metalloproteinases and their inhibitors.

Soluble factors from the target organ enhance tumor cell angiogenesis: role of laminin SIKVAV sequence.

The ability of tumor cells to respond to microenvironmental factors present in the target organ determines in part the successful development of a metastasis. In a previous work it was demonstrated that the conditioned medium (CM) from lungs of normal mice stimulates in vitro migration, proliferation and uPA activity of cells from a murine mammary adenocarcinoma moderately metastatic to lung. This CM also enhanced local and metastatic tumor growth. Here, we show that lung CM enhanced neovascularization when inoculated together with LM3 tumor cells into the skin of syngeneic mice. A similar tumor-induced angiogenesis response was obtained when lung CM was injected systemically. Western blot analysis of lung CM revealed the presence of some laminin fragments containing the sequence SIKVAV. To determine whether those molecules were responsible for the observed angiogenic effects, the CM was depleted of the peptides containing the SIKVAV sequence. We observed that the SIKVAV-depleted lung CM lost its ability to induce an enhancement of the tumor neovascular response. Our results suggest a role for the target organ in facilitating the neovascularization of tumor cells, probably through the participation of active peptides derived from the proteolytic degradation of the basement membrane component laminin.