Intracranial hemorrhage with direct oral anticoagulants in patients with brain tumors.

Essentials Intracranial hemorrhage (ICH) is common in patients with brain tumors. We compared rates of ICH with DOACs and low molecular weight heparin. DOACs were associated with a lower incidence of ICH in primary brain tumors. DOACs appear safe to administer to patients with brain tumors. SUMMARY: Background Direct oral anticoagulants (DOACs) are efficacious in the treatment of cancer-associated thrombosis but are associated with an increased risk of hemorrhage compared with low-molecular-weight heparin in certain malignancies. Whether the DOACs increase the incidence of intracranial hemorrhage (ICH) in patients with brain tumors is not established. Objectives To determine the cumulative incidence of ICH in DOACs compared with Low-molecular-weight heparin (LMWH) in patients with brain tumors and venous thromboembolism. Patients and methods A retrospective comparative cohort study was performed. Radiographic images for all ICH events were reviewed and the primary endpoint was cumulative incidence of ICH at 12 months following initiation of anticoagulation. Results and conclusions A total of 172 patients with brain tumors were evaluated (42 DOAC and 131 LMWH). In the primary brain tumor cohort (n = 67), the cumulative incidence of any ICH was 0% in patients receiving DOACs vs. 36.8% (95% confidence interval [CI], 22.3-51.3%) in those treated with LMWH, with a major ICH incidence of 18.2% (95% CI, 8.4-31.0). In the brain metastases cohort (n = 105), DOACs did not increase the risk of any ICH relative to enoxaparin, with an incidence of 27.8% (95% CI, 5.5-56.7%) compared with 52.9% (95% CI, 37.4-66.2%). Similarly, DOAC did not increase the incidence of major ICH in brain metastases, with a cumulative incidence 11.1% (95% CI, 0.5-40.6%) vs. 17.8% (95% CI, 10.2-27.2%). We conclude that DOACs are not associated with an increased incidence of ICH relative to LMWH in patients with brain metastases or primary brain tumors.

Effects of steroid treatment on growth, nutrient partitioning, and expression of genes related to growth and nutrient metabolism in adult triploid rainbow trout (Oncorhynchus mykiss).

The contribution of sex steroids to nutrient partitioning and energy balance during gonad development was studied in rainbow trout. Specifically, 19-mo old triploid (3N) female rainbow trout were fed treatment diets supplemented with estradiol-17ß (E2), testosterone (T), or dihydrotestosterone at 30-mg steroid/kg diet for a 1-mo period. Growth performance, nutrient partitioning, and expression of genes central to growth and nutrient metabolism were compared with 3N and age-matched diploid (2N) female fish consuming a control diet not supplemented with steroids. Only 2 N fish exhibited active gonad development, with gonad weights increasing from 3.7% to 5.5% of body weight throughout the study, whereas gonad weights in 3N fish remained at 0.03%. Triploid fish consuming dihydrotestosterone exhibited faster specific growth rates than 3N-controls (P < 0.05). Consumption of E2 in 3N fish reduced fillet growth and caused lower fillet yield compared with all other treatment groups (P < 0.05). In contrast, viscera fat gain was not affected by steroid consumption (P > 0.05). Gene transcripts associated with physiological pathways were identified in maturing 2N and E2-treated 3N fish that differed in abundance from 3N-control fish (P < 0.05). In liver these mechanisms included the growth hormone/insulin-like growth factor (IGF) axis (igf1, igf2), IGF binding proteins (igfbp1b1, igfbp2b1, igfbp5b1, igfbp6b1), and genes associated with lipid binding and transport (fabp3, fabp4, lpl, cd36), fatty acid oxidation (cpt1a), and the pparg transcription factor. In muscle, these mechanisms included reductions in myogenic gene expression (fst, myog) and the proteolysis-related gene, cathepsin-L, suggesting an E2-induced reduction in the capacity for muscle growth. These findings suggest that increased E2 signaling in the sexually maturing female rainbow trout alters physiological pathways in liver, particularly those related to IGF signaling and lipid metabolism, to partition nutrients away from muscle growth toward support of maturation-related processes. In contrast, the mobilization of viscera lipid stores appear to be mediated less by E2 and more by energy demands associated with gonad development. These findings improve the understanding of how steroids regulate nutrient metabolism to meet the high energy demands associated with gonad development during sexual maturation.

Simultaneous inclusion of sorghum and cottonseed meal or millet in broiler diets: effects on performance and nutrient digestibility.

Two experiments were conducted to investigate the use of sorghum, cottonseed meal and millet in broiler diets and their interaction when they are used simultaneously. In Experiment 1, a corn-soybean meal control diet was compared with eight experimental treatments based on low tannin sorghum (S30, S45 and S60), cottonseed meal (CM15, CM40) or both ingredients included in the same diet (S30/CM40, S45/CM25 and S60CM15). Results showed that BW gain was not affected by the inclusion of sorghum or cottonseed meal. However, feed intake tended to be affected by the cereal type with the highest values with sorghum-based diets. Feed conversion ratio increased (P<0.001) with sorghum-based diets compared with the control diet, whereas a combination of cottonseed meal and sorghum in the same diet did not affect the feed conversion ratio. Significant differences (P<0.001) were observed in apparent ileal digestibility (%) of protein and energy with the cottonseed meal and sorghum/cottonseed meal-based diets having lower protein and energy digestibility compared with corn-based diets. In Experiment 2, a control diet was compared with six diets in which corn was substituted at 60%, 80% or 100% by either sorghum or millet and other three diets with simultaneous inclusion of these two ingredients (S30/M30, S40/M40, S50/M50). Single or combined inclusion of sorghum and millet resulted in similar feed intake and growth performance as the control diet. Apparent ileal digestibility of protein and energy was higher with millet-based diets (P<0.001). Total tract digestibility of protein in sorghum and millet-based diets tended to decrease linearly with the increasing level of substitution. Sorghum-based diets resulted in lower total tract digestibility of fat compared with millet and sorghum/millet-based diets (P<0.001). Higher total tract digestibility of starch were obtained with the control diet and millet-based diets compared with the sorghum-based treatments. Results of the two experiments suggest that broiler growth performance was not affected by the dietary level of sorghum, millet or cottonseed meal. Nutrient digestion can, however, be affected by these feed ingredients.

Tolerance evaluation of overdosed dietary levels of 25-hydroxyvitamin D3 in growing piglets.

Forty-eight, cross-bred (GL × LW × P) piglets were used in a 42-day tolerance trial to assess the effects of feeding diets supplemented with vitamin D or increasing levels of 25-hydroxyvitamin D3 (25-OH-D3 ). Six-week-old piglets (24 castrate males, 24 females) were used. Two replicate groups of 6 piglets were randomized by weight and allocated to four dietary treatments. The control group (T1) was supplemented with 50 µg vitamin D3 /kg feed. The experimental groups received 25-OH-D3 at the recommended dose (T2: 50 µg/kg = 1x), at 250 µg/kg (T3: 5x) or at 500 µg/kg (T4: 10x) respectively. Feed intake and daily weight gain were measured weekly, and the animals were examined by a veterinarian daily. After 42 days, body mass, blood, urine, bone and tissue samples were analysed and a pathology examination conducted. Dietary treatments had no significant effect on final body mass or daily weight gain. The 25-OH-D3 plasma concentration in T1 was 17 ± 3 ng/ml (mean ± SD) while the respective values of the experimental groups were significantly increased in T2, T3 and T4. Tissue concentrations of 25-OH-D3 were higher in liver and muscle for T3 and T4 and in skin for T4 than in T1. However, neither gross pathology nor histology, nor blood and urine characteristics, nor bone parameters were affected by dietary treatments. Weight of organs as well as dry matter, ash and calcium content of kidneys remained unaffected by dietary 25-OH-D3 intake. Furthermore, no changes were observed for general indicators of health. The results of this study demonstrated that feeding piglets with 25-OH-D3 at 5 or 10 times the recommended level had no adverse effects on any of the biological parameters measured. It was concluded that 25-OH-D3 can be regarded as a supplement with a very high safety margin when used at the recommended level.

Inclusion of sorghum, millet and cottonseed meal in broiler diets: a meta-analysis of effects on performance.

A meta-analysis was conducted (i) to evaluate broiler response to partial or total substitution of corn by sorghum and millet and (ii) to determine the effect of soybean meal replacement by cottonseed meal in broiler diet. The database included 190 treatments from 29 experiments published from 1990 to 2013. Bird responses to an experimental diet were calculated relative to the control (Experimental-Control), and were submitted to mixed-effect models. Results showed that diets containing millet led to similar performance as the corn-based ones for all parameters, whereas sorghum-based diets decreased growth performance. No major effect of the level of substitution was observed with millet or cottonseed meal. No effect of the level of substitution of sorghum on feed intake was found; however, growth performance decreased when the level of substitution of corn by sorghum increased. Cottonseed meal was substituted to soybean meal up to 40% and found to increase feed intake while reducing growth performance. Young birds were not more sensitive to these ingredients than older birds since there was no negative effect of these ingredients on performance in the starter phase. Results obtained for sorghum pointed out the necessity to find technological improvements that will increase the utilization of these feedstuffs in broiler diet. An additional work is scheduled to validate these statistical results in vivo and to evaluate the interactions induced with the simultaneous inclusions of sorghum, millet and cottonseed meal in broiler feeding.

Urban vs. extra-urban environments: scales of variation of intertidal benthic assemblages in north Portugal.

Littoral areas are subject to severe and increasing pressures resulting from human activities occurring along or next to the coast. In this study, patterns of variability in the structure of rocky intertidal benthic assemblages and in the abundance of individual taxa were compared between locations close to the coastal cities of Porto and Vila Nova de Gaia (north Portugal) and reference locations far from it in much less urbanized conditions over a temporal scale of fourteen months and multiple spatial scales. Present findings indicated that assemblages were more heterogeneously distributed in the urban than in the extra-urban condition. The total number of taxa and several individual taxa displayed, in general, this same pattern of variability. This could be interpreted as the beginning of a habitat deterioration process with largely unpredictable consequences. The adopted sampling design supports the need for simultaneously including a range of temporal and spatial scales when evaluating responses of coastal marine biodiversity to anthropogenic disturbances.

Triennial Growth Symposium--Effects of dietary 25-hydroxycholecalciferol and cholecalciferol on blood vitamin D and mineral status, bone turnover, milk composition, and reproductive performance of sows.

To evaluate the role of vitamin D3 during gestation and lactation of sows, 2 independent experiments were performed with the aim of investigating sow reproductive performance, milk composition (study 1 only), and changes in blood status of 25-hydroxycholecalciferol (25-OH-D3), 1,25-dihydroxycholecalciferol (1,25-(OH)2-D3; study 2 only), minerals, and bone markers of sows during gestation and lactation. Study 1 comprised 39 primi- and multiparous crossbred sows fed 1 of 3 barley meal-based diets fortified with 200 IU/kg vitamin D3 (NRC, 1998; treatment DL), 2,000 IU/kg vitamin D3 (cholecalciferol; treatment DN), or 50 µg 25-OH-D3 (calcidiol; treatment HD)/kg feed. This study was conducted over a 4-parity period under controlled conditions. Study 2, running over 1 parity only, was performed in a commercial farm with 227 primi- and multiparous sows allocated to 2 dietary treatments: control (CON), receiving 2,000 IU vitamin D3/kg (equivalent to 50 µg/kg) feed (114 sows), and test (HYD), supplemented with 50 µg 25-OH-D3/kg feed (113 sows). Blood samples of sows were collected at 84 and 110d postcoitum and 1, 5, and 33 d postpartum (study 1) and at insemination and 28 and 80 d postinsemination as well as d 5 and 28 postpartum (study 2). Colostrum and milk samples in study 1 were obtained at 1, 9, and 33 d of lactation after oxytocin administration. Plasma 25-OH-D3 concentrations were increased (P < 0.05) in sows receiving 25-OH-D3 (HD and HYD) at any time of sampling whereas circulating plasma concentrations of 1,25-(OH)2-D3, Ca, and P were not affected by treatment. Milk concentrations of Ca and P were similar, but 25-OH-D3 content (except in colostrum) was clearly increased (P< 0.05) when 25-OH-D3 was fed. Most characteristics of sow reproductive performance responded similarly to the 2 sources and levels of vitamin D3, but weight gain of piglets between birth and weaning was decreased (P< 0.05) in offspring of DL and HD sows compared with animals of treatment DN (study 1). In study 2 total litter weight and birth weight per piglet were increased (P< 0.05) with 25-OH-D3 supplementation in comparison with the control (CON). Overall, feeding sows with 25-OH-D3 was considered to improve maternal supply with vitamin D3 and thereby maintain Ca homeostasis during gestation and lactation.

Automatic joint alignment measurements in pre- and post-operative long leg standing radiographs.

OBJECTIVES: For diagnosis or treatment assessment of knee joint osteoarthritis it is required to measure bone morphometry from radiographic images. We propose a method for automatic measurement of joint alignment from pre-operative as well as post-operative radiographs. METHODS: In a two step approach we first detect and segment any implants or other artificial objects within the image. We exploit physical characteristics and avoid prior shape information to cope with the vast amount of implant types. Subsequently, we exploit the implant delineations to adapt the initialization and adaptation phase of a dedicated bone segmentation scheme using deformable template models. Implant and bone contours are fused to derive the final joint segmentation and thus the alignment measurements. RESULTS: We evaluated our method on clinical long leg radiographs and compared both the initialization rate, corresponding to the number of images successfully processed by the proposed algorithm, and the accuracy of the alignment measurement. Ground truth has been generated by an experienced orthopedic surgeon. For comparison a second reader reevaluated the measurements. Experiments on two sets of 70 and 120 digital radiographs show that 92% of the joints could be processed automatically and the derived measurements of the automatic method are comparable to a human reader for pre-operative as well as post-operative images with a typical error of 0.7° and correlations of r = 0.82 to r = 0.99 with the ground truth. CONCLUSIONS: The proposed method allows deriving objective measures of joint alignment from clinical radiographs. Its accuracy and precision are on par with a human reader for all evaluated measurements.

Effects of a blend of essential oil compounds and benzoic acid on performance of broiler chickens as revealed by a meta-analysis of 4 growth trials in various locations.

A series of growth trials with broiler chicks was conducted in various geographical locations to evaluate the efficacy of a novel eubiotic feed additive (EFA) at various dietary inclusion levels on performance of growing chicks. The EFA product consisted of a blend of essential oil compounds (thymol, eugenol, piperine) with benzoic acid, all belonging to the group of flavoring substances. Although variable in responses, the overall results indicated that 300 mg/kg of this EFA represented an optimum supplementation dose for generation of beneficial performance effects in broilers. A meta-analysis with all data from the 300 mg/kg EFA-supplemented treatments in comparison with the non-supplemented controls revealed that the eubiotic product significantly improved BW on d 21 (+2.0%; P = 0.0021) and on d 42 (+1.4%; P = 0.0151). Furthermore, the birds on the EFA 300 mg/kg treatment expressed a higher average daily gain in the starter phase (d 1-21; +2.1%; P = 0.0023) and over the entire experimental period (d 1-42; +1.5%; P = 0.0154). Feed conversion ratio was more favorable with dietary EFA supplementation (-0.6%; P = 0.0414), when compared with the control birds. Mortality was considered normal and was not affected by the dietary treatment (control = 3.09%; EFA 300 mg/kg = 3.26%). In conclusion, 300 mg/kg of this new eubiotic product demonstrated to effectively improve performance of broiler chicks under various husbandry conditions.

Effect of source and quantity of dietary vitamin D in maternal and creep diets on bone metabolism and growth in piglets.

Piglets are born with reduced plasma concentrations of 25-hydroxycholecalciferol (25-OH-D(3)) and are thus highly predisposed to vitamin D deficiency. Furthermore, sow milk contains little vitamin D, and the slow intestinal vitamin D absorption of sows limits the efficacy of dietary vitamin D supplementation. Hence, the neonate depends, to a large extent, on the vitamin D stores built up in fetal tissues from maternal sources. The current study was undertaken to evaluate whether the source and quantity of dietary vitamin D provided to the gestating and lactating sow, and also directly in the form of creep feed to the piglet, would influence the vitamin D status, growth performance, and skeletal development of piglets. A total of 39 primiparous and multiparous sows were randomly assigned to 1 of 3 dietary treatments (13 in each treatment), supplemented with either 5 or 50 µg of the commonly used cholecalciferol (vitamin D(3)) or 50 µg of 25-OH-D(3) per kilogram of feed. By wk 3 of lactation, piglets were offered a creep diet with vitamin D supplementation according to the treatment of the dam, and they were offered the same creep diets after weaning at d 35 of age until they reached a BW of approximately 20 kg. When dietary 25-OH-D(3) was provided, circulating concentrations of 25-OH-D(3) in piglet serum increased (P < 0.05) as early as d 21 and later at d 33 and 77, indicating greater body stores in those animals. Bone-breaking strength and cortical bone mineral content and density at the tibial midshaft of piglets were reduced (P < 0.05) when vitamin D(3) was supplemented at 5 µg/kg compared with the bone traits of other groups, but no differences (P > 0.05) were observed between the 2 other groups. After weaning, ADFI was greater (P < 0.05) and growth performance tended (P = 0.08) to improve when doses of 50 µg/kg were administered, regardless of the vitamin D source. In conclusion, supplementation of the diet with 50 µg/kg of either source of vitamin D was proved to be adequate in meeting the needs of gestating sows and in permitting the accumulation of vitamin D in fetal tissues, as well as for normal skeletal mineralization and growth in the offspring. Furthermore, the markedly improved vitamin D status of piglets whose mothers received 25-OH-D(3) possibly resulted from greater tissue reserves present at birth and a greater availability of vitamin D when released from those stores.

Response to selection for bacterial cold water disease resistance in rainbow trout.

A family-based selection program was initiated at the National Center for Cool and Cold Water Aquaculture in 2005 to improve resistance to bacterial cold water disease (BCWD) in rainbow trout. The objective of this study was to estimate response to 2 generations of selection. A total of 14,841 juvenile fish (BW = 3.1 g; SD = 1.1 g) from 230 full-sib families and 3 randomly mated control lines were challenged intraperitoneally with Flavobacterium psychrophilum, the bacterium that causes BCWD, and mortalities were observed for 21 d. Selection was applied to family EBV derived from a proportional-hazards frailty (animal) model while constraining rate of inbreeding to

Evidence of major genes affecting stress response in rainbow trout using Bayesian methods of complex segregation analysis.

As a first step toward the genetic mapping of QTL affecting stress response variation in rainbow trout, we performed complex segregation analyses (CSA) fitting mixed inheritance models of plasma cortisol by using Bayesian methods in large full-sib families of rainbow trout. To date, no studies have been conducted to determine the mode of inheritance of stress response as measured by plasma cortisol response when using a crowding stress paradigm and CSA in rainbow trout. The main objective of this study was to determine the mode of inheritance of plasma cortisol after a crowding stress. The results from fitting mixed inheritance models with Bayesian CSA suggest that 1 or more major genes with dominant cortisol-decreasing alleles and small additive genetic effects of a large number of independent genes likely underlie the genetic variation of plasma cortisol in the rainbow trout families evaluated. Plasma cortisol is genetically determined, with heritabilities of 0.22 to 0.39. Furthermore, a major gene with an additive effect of -42 ng/mL (approximately 1.0 genetic SD) is segregating in this rainbow trout broodstock population. These findings provide a basis for designing and executing genome-wide linkage studies to identify QTL for stress response in rainbow trout broodstock and markers for selective breeding.

Involvement of phospholipase C and intracellular calcium signaling in the gonadotropin-releasing hormone regulation of prolactin release from lactotrophs of tilapia (Oreochromis mossambicus).

Gonadotropin-releasing hormone (GnRH) is a potent stimulator of prolactin (PRL) secretion in various vertebrates including the tilapia, Oreochromis mossambicus. The mechanism by which GnRH regulates lactotroph cell function is poorly understood. Using the advantageous characteristics of the teleost pituitary gland from which a nearly pure population of PRL cells can be isolated, we examined whether GnRH might stimulate PRL release through an increase in phospholipase C (PLC), inositol triphosphate (IP3), and intracellular calcium (Ca(i)2+) signaling. Using Ca(i)2+ imaging and the calcium-sensitive dye fura-2, we found that chicken GnRH-II (cGnRH-II) induced a rapid dose-dependent increase in Ca(i)2+ in dispersed tilapia lactotrophs. The Ca(i)2+ signal was abolished by U-73122, an inhibitor of PLC-dependent phosphoinositide hydrolysis. Correspondingly, cGnRH-II-induced tPRL188 secretion was inhibited by U-73122, suggesting that activation of PLC mediates cGnRH-II's stimulatory effect on PRL secretion. Pretreatment with 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an inhibitor of Ca2+ release from intracellular stores, impeded the effect of cGnRH-II on Ca(i)2+. To further address the possible involvement of intracellular Ca2+ stores, IP3 concentrations in the tilapia rostral pars distalis (RPD containing 95-99% PRL cells) was determined by a radioreceptor assay. We found that GnRH-II induces a rapid (<5min) and sustained increase in IP3 concentration in the RPD. Secretion of tPRL(188) in response to cGnRH-II was suppressed by Ca2+ antagonists (TMB-8 and nifedipine). These data, along with our previous findings that show PRL release increases with a rise in Ca(i)2+, suggest that GnRH may elicit its PRL releasing effect by increasing Ca(i)2+. Furthermore, the rise in Ca(i)2+ may be derived from PLC/IP3-induced mobilization of Ca2+ from intracellular stores along with influx through L-type voltage-gated Ca2+ channels.

Argus--a new database system for Web-based analysis of multiple microarray data sets.

The ongoing revolution in microarray technology allows biologists studying gene expression to routinely collect >10(5) data points in a given experiment. Widely accessible and versatile database software is required to process this large amount of raw data into a format that facilitates the development of new biological insights. Here, we present a novel microarray database software system, named Argus, designed to process, analyze, manage, and publish microarray data. Argus imports the intensities and images of externally quantified microarray spots, performs normalization, and calculates ratios of gene expression between conditions. The database can be queried locally or over the Web, providing a convenient format for Web-publishing entire microarray data sets. Searches for regulated genes can be conducted across multiple experiments, and the integrated results incorporate images of the actual hybridization spots for artifact screening. Query results are presented in a clone- or gene-oriented fashion to rapidly identify highly regulated genes, and scatterplots of expression ratios allow an individual ratio to be interpreted in the context of all data points in the experiment. Algorithms were developed to optimize response times for queries of regulated genes. Supporting databases are updated easily to maintain current gene identity information, and hyperlinks to the Web provide access to descriptions of gene function. Query results also can be exported for higher-order analyses of expression patterns. This combination of features currently is not available in similar software. Argus is available at http://vessels.bwh.harvard.edu/software/Argus.

In vitro hormone induction of final oocyte maturation in striped bass (Morone saxatilis) follicles is inhibited by blockers of phosphatidylinositol 3-kinase activity.

Oocyte germinal vesicle breakdown (GVBD) was induced in striped bass ovarian fragments when tissues were incubated with 100-nM recombinant human insulin-like growth factor-I (rhIGF-I), 25-IU human chorionic gonadotropin (hCG) ml(-1), or 290 nM of the maturation-inducing steroid (MIS), 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S). Inhibitors of phosphatidylinositol 3-kinase (PI 3-K), wortmannin (100 nM) and LY 294002 (50 microM), inhibited GVBD induced by these hormones. Furthermore, the inhibitors attenuated hCG-induced steroid hormone synthesis. Previous studies report that gap junction uncouplers inhibit GVBD induced by hCG, but not by rhIGF-I, in striped bass. We show that 20beta-S-induced GVBD is also attenuated by 1 mM 1-heptanol or 1-octanol without being affected by incubation with 3 mM ethanol. Thus, the effects of inhibiting PI 3-K activity on GtH and MIS actions are similar to effects of uncoupling gap junctions. These data suggest that PI 3-K activity is required for GtH- MIS- and IGF-I induction of GVBD in striped bass. Our data are also consistent with the notion that a ligand that regulates PI 3-K activity, possibly an IGF, participates in maintenance of gap junctional communication required for maximal GtH and MIS action.

Sequence conservation among fish myostatin orthologues and the characterization of two additional cDNA clones from Morone saxatilis and Morone americana.

Myostatin (MSTN) negatively regulates mammalian skeletal muscle growth and development by inhibiting myoblast proliferation. Mice and cattle possessing mutant MSTN alleles display a 'double muscling' phenotype characterized by extreme skeletal muscle hypertrophy and/or hyperplasia. MSTN orthologues have been previously characterized in 12 vertebrate species, including the white bass Morone chrysops. Presented herein is the identification and characterization of novel cDNA clones from two additional Morone species: saxatilis (striped bass) and americana (white perch), which were obtained by PCR amplification and subsequent TA-cloning. The predicted amino acid sequence of each cDNA clone contains a putative signal sequence, conserved cysteine residues and a RXXR proteolytic processing site. The different Morone proteins were 97-99% identical to each other and approximately 91, 81, 68 and 67% identical to the tilapia, zebrafish, mammalian and avian proteins, respectively. However, the bioactive domains, which lie downstream of each processing site, were considerably more conserved. They were 99-100% identical within the genus and were approximately 99, 95, 88 and 88% identical to the tilapia, zebrafish, mammalian and avian domains, respectively. This high level of sequence conservation among all known MSTN orthologues suggests that the structure/function relationship of each is equally well conserved among vertebrates.

Isolation and characterization of myostatin complementary deoxyribonucleic acid clones from two commercially important fish: Oreochromis mossambicus and Morone chrysops.

In mammals, skeletal muscle mass is negatively regulated by a muscle-derived growth/differentiating factor named myostatin (MSTN) that belongs to the transforming growth factor-beta superfamily. Although putative MSTN homologs have been identified from several vertebrates, nonmammalian orthologs remained poorly defined. Thus, we isolated and characterized MSTN complementary DNA clones from the skeletal muscle of the tilapia Oreochromis mossambicus and the white bass Morone chrysops. The nucleic and amino acid sequences from both fish species are highly homologous to the previously identified mammalian and avian orthologs, and both possess conserved cysteine residues and putative RXXR proteolytic processing sites that are common to all transforming growth factor-beta family members. Western blotting of conditioned medium from human embryonal kidney (HEK293) cells overexpressing a His-tagged tilapia MSTN indicates that the secreted fish protein is processed in a manner similar to mouse MSTN. However, in contrast to mice, MSTN expression in tilapia is not limited to skeletal muscle as it occurs in many tissues. Furthermore, the timing of MSTN expression in developing tilapia larvae coincides with myogenesis. These results suggest that the biological actions of MSTN in the tilapia and possibly in other fishes may not be limited to myocyte growth repression, but may additionally influence different cell types and organ systems.

Effects of insulin-like growth factor-I on in vitro final oocyte maturation and ovarian steroidogenesis in striped bass, Morone saxatilis.

Recombinant human (rh) insulin-like growth factor-I (IGF-I) was more potent than rhIGF-II at inducing in vitro germinal vesicle breakdown (GVBD), a marker for resumption of meiosis, in oocytes of striped bass. Treatment of ovarian fragments containing oocytes in intact follicles with rhIGF-I increased concentrations of estradiol-17beta and maturation-inducing steroid (MIS) 17,20beta, 21-trihydoxy-4-pregnen-3-one (20beta-S) in the culture medium and decreased testosterone levels. The follicles were too immature for oocytes to complete GVBD in response to 20beta-S (MIS incompetent) or hCG. Addition of 20beta-S to cultures did not increase the percentage of oocytes completing GVBD in response to rhIGF-I or rhIGF-II. Bovine insulin was without effect on GVBD or steroid production. Incubation of MIS-competent follicles with actinomycin D, cyanoketone, trilostane, 1-heptanol, or 1-octanol had no effect on rhIGF-I-induced GVBD, but attenuated hCG-induced GVBD and 20beta-S production. Cycloheximide inhibited rhIGF-I-induced GVBD. Collectively, these observations indicate that IGF-I can induce GVBD via MIS- and transcription-independent pathways without coupled gap junctions between oocytes and granulosa cells or among granulosa cells, but requires protein synthesis to do so. An rhIGF-I analogue that does not bind IGF-binding proteins, des(1,3)IGF-I, was more potent than rhIGF-I in inducing GVBD, suggesting ovarian IGF-binding proteins may inhibit IGF-I action.

Changes in serum concentrations and pituitary content of the two prolactins and growth hormone during the reproductive cycle in female tilapia, Oreochromis mossambicus, compared with changes during fasting.

Patterns of change in serum concentrations and pituitary content of GH and two tilapia prolactins (PRL177 and PRL188) were examined during the reproductive cycle of female tilapia, Oreochromis mossambicus, adapted to fresh water and to seawater. Changes in these hormones during fasting were examined to elucidate whether changes observed during brooding could be attributed to a reduction in feeding during brooding. Serum concentrations of GH increased prior to pituitary content during the brooding phase of the reproductive cycle. In contrast, pituitary content of GH increased prior to serum concentrations during fasting. There was no consistent pattern of change in serum or pituitary PRL levels during the reproductive cycle, among experiments. Serum concentrations of PRL177 were elevated in all fasted fish, whereas PRL188 was elevated during fasting in males but not females. The increases in the serum concentration of PRLs and GH, and in the pituitary content of GH in response to fasting support the notion that these hormones are involved in the regulation of the use of metabolic substrates in tilapia. We conclude that reduced food intake during brooding may contribute to changes in serum and pituitary levels of the PRLs and GH observed during the reproductive cycle. Nevertheless, differences between changes in serum and pituitary GH during brooding and fasting suggest GH has actions in reproduction, and changes in GH during brooding are not only in response to fasting.

3,5,3'-Triiodothyronine (T3) clearance and T3-glucuronide (T3G) appearance kinetics in plasma of freshwater-reared male tilapia, Oreochromis mossambicus.

Distribution and metabolism of the thyroid hormone 3,5, 3'-l-triiodothyronine (T3) were studied in several ways to gain insights into these processes in the warm water fish tilapia Oreochromis mossambicus. Trace doses of 125I-labeled T3 (T*3)1 were injected intraarterially, extraarterially, or intraperitoneally in freshwater-reared male tilapia to explore plasma clearance kinetic responses to these different input modalities. Multicompartmental analysis of the plasma clearance data indicated a kinetic distribution of T*3 much like that reported for the rat and human, with about 2% of total body T*3 in plasma, 5% in rapidly exchanging tissues such as kidney and liver, and 93% in slowly exchanging tissues such as muscle. However, plasma clearance rates (PCR, 5.37 mL/h . 100 g body wt) and plasma appearance rates (PAR3 = PCR x [T3] plasma = 36.3 ng/h . 100 g body wt) were quite different than these indices in rat and human and 5 to 50 times larger than values reported for rainbow trout. On a whole-body basis, normalized for body weight, the tilapia we studied produced and accumulated much more T3 than rat, human, or rainbow trout. Enzymatic and chromatographic analyses of the plasma clearance data samples indicated substantial production of labeled glucuronide, but not sulfate, conjugates of iodothyronines (TiG) of unknown origin appearing in plasma. The TiG appeared beginning a few hours postinjection, peaked at 6 hours, and yielded a predicted steady-state TiG level of 8.3% of the T3 level in plasma. In contrast, in published studies, no conjugates were detected in rainbow trout plasma from 2 to 24 h after iv injection of T*3, T*4, or reverse-T*3, although conjugates of all were present in bile. To our knowledge, although T3 and T4 sulfate conjugates are present in the sera of several mammals, this is the first quantification of iodothyronine glucuronides reported in blood of any species under normal conditions. This might have physiological significance for the tilapia, with T3G providing a reversible storage form of T3 in blood, as has been suggested for sulfate conjugates of T3 and T4 in blood of several mammals.