The effects of two dietary synbiotics on growth performance, haematological parameters, and non-specific immune responses in Japanese eel (Anguilla japonica).

Feed additives have attracted increased attention in aquaculture due to their ability to modulate fish gut microbiota, resulting in improved fish growth and immunity. This study assessed two synbiotics' effects in Japanese eels: Bacillus subtilis with mannooligosaccharide (MOS) and Enterococcus faecium with fructooligosaccharide (FOS). Six diets, including a control (CON), oxytetracycline (OTC), and four synbiotic diets - B.subtilis at 1 × 106 and 107 CFU/g with 5 g/kg MOS (BS6MO and BS7MO) and E. faecium at 1 × 106 and 107 CFU/g with 5 g/kg FOS (EF6FO and EF7FO) - were fed to triplicate groups of 20 fish averaging 6.00 ± 0.07g for eight weeks. Fish fed the BSMOS diets showed significantly higher weight gain (WG), specific growth rate (SGR), and feed efficiency (FE) compared to the CON and OTC diets (P < 0.05), but not significantly different from those fed the EFFOS diets. Weight gain, SGR of fish fed EFFOS were not significantly different from those fed all other diets (P > 0.05). Fish fed the OTC diet showed a higher mean aspartate aminotransferase (AST) level, though the difference was not statistically significant. The myeloperoxidase (MPO) activity of fish fed the BS7MO diet was significantly higher than in all other diets, and the superoxide dismutase (SOD) activity of fish fed the BS7MO diet was also significantly higher than in the EF7FO diet. Overall, the BSMOS synbiotic diets were significantly more effective than the CON diet in enhancing fish survival against Vibrio anguillarum. Our findings suggest that synbiotics can be a preferable alternative to antibiotics in aquaculture.

Effects of streptozotocin and S-allyl-L-cysteine on motility, plasma membrane integrity, and mitochondrial activity of boar spermatozoa.

This study investigated the effects of streptozotocin (STZ) and S-allyl-L-cysteine (SAC) on motility, plasma membrane integrity, and mitochondrial activity of the boar sperm. STZ (0, 10, 50, and 100 µM) and SAC (0, 1, 5, 25, and 100 µM) were treated alone and co-treated in the fresh boar semen. The motility, plasma membrane integrity, and mitochondrial activity of sperm were analyzed at 3, 6, and 9 h after incubation. Boar semen was collected using the gloved-hand method from ten crossbred male pigs, and age of experimental ten male pigs is 24~27 months. The sperm plasma membrane integrity was analyzed using Live/Dead sperm kit. Mitochondrial activity was analyzed using rhodamine 123 and PI double-staining method. Additionally, sperm motility was evaluated according to standard method. Sperm motility, plasma membrane integrity, and mitochondrial activity were decreased in an STZ concentration-dependent manner (P < 0.05) and also were decreased by 10 µM STZ in all incubation times (P < 0.05). The motility, plasma membrane integrity, and mitochondrial activity of the sperm were increased at 5 µM SAC treatment, whereas it was decreased at 100 µM treatment. In addition, sperm motility, plasma membrane integrity, and mitochondrial activity were increased when co-treated with 50 µM STZ and 5 µM SAC group at 9 h after incubation (P < 0.05). Based on our results, STZ has a deleterious effect on sperm characteristics, and SAC can protect sperm motility, viability, and function of the sperm exposed to STZ.

Effects of food availability on growth performance and immune-related gene expression of juvenile olive flounder (Paralichthys olivaceus).

Unfavorable environmental conditions and inappropriate culture practices have increased the vulnerability of cultured fish to disease infection. Up to date many studies have aimed to determine a feeding regimen to maximize productivity; however, very little information on immune responses of cultured fish in response to underfeeding or overfeeding is available. Therefore, a preliminary study was conducted to evaluate effects of graded feeding levels (i.e., food availability) on growth performance and immune-related gene expression of juvenile olive flounder (Paralichthys olivaceus). Six different feeding rates including 1, 4, 7, 10, 13, and 16% body weight per day (BW/d) were randomly assigned to three replicate tanks stocking 150 fish (average initial body weight: 0.27 ±â€¯0.02 g; mean ±â€¯SD) per tank. A feeding trial lasted for two weeks. Based on the results of the weight gain, nutrient gain, and whole-body compositions and energy content, the feeding rate of 10%, 13%, and 16% BW/d resulted in high nutritional status, whereas the feeding rate of 1% and 4% BW/d resulted in low nutritional status. Intermediate nutritional status was observed at the feeding rate of 7% BW/d. In the given rearing conditions the optimum feeding rate resulting in the maximum growth was estimated to be 11.9% BW/d based on the quadratic broken-line regression model, chosen as the best-fit model among the tested models. Expression of immune-related genes including IL-8 and IgM was significantly down-regulated in the flounder fed at 1% BW/d in comparison to those fed at 7% BW/d. Interestingly, expression of these genes in the flounder fed at 10%, 13%, and 16% BW/d was relatively down-regulated in comparison to that of the flounder fed at 7% BW/d. Although no statistical difference was detected, overall response patterns of other immune-related genes, including TLR3, polymeric Ig receptor, lysozyme C-type, GPx, SOD, and Trx followed what IL-8 and IgM exhibited in response to the various feeding rates. Given the current challenges in aquaculture of the flounder our findings suggest to prohibit underfeeding or overfeeding (i.e., ad-libitum feeding) when culturing the young flounder.

Effects of feed restriction on the upper temperature tolerance and heat shock response in juvenile green and white sturgeon.

The objective of the current study was to investigate the effects of feed restriction on whole-organism upper thermal tolerance and the heat shock response of green and white sturgeon to determine how changes in food amount might influence physiological performance of each species when faced with temperature stress. Two parallel feed restriction trials were carried out for juvenile green (202g; 222-day post hatch: dph) and white sturgeon (205g; 197-dph) to manipulate nutritional status at 12.5%, 25%, 50%, or 100% of optimum feeding rate (100% OFR were 1.6% and 1.8% body weight/day, respectively) for four weeks. Following the trials, the critical thermal maximum (CTMax, 0.3°C/min) of sturgeon (N=12/treatment/species) was assessed as an indicator of whole-organism upper thermal tolerance. To assess temperature sensitivity, sturgeon (N=9/treatment/species) were acutely transferred to two temperature treatments (28°C and 18°C as a handling control) for 2h followed by 2h of recovery at 18°C before being sacrificed, and gill, brain, and mucus sampled for measurements of 70-kDa heat shock protein levels (Hsc/Hsp70). Feeding rate had species-specific effects on CTMax in green and white sturgeon such that CTMax of green sturgeon decreased as the magnitude of feed restriction increased; whereas, CTMax of white sturgeon did not change with feed restriction. Elevated temperature (28°C) and feed restriction increased Hsc/Hsp70 levels in the gill tissue of green sturgeon, while heat shock increased Hsc/Hsp70 levels in the mucus of white sturgeon. Our results suggest that green sturgeon may be more susceptible to temperature stress under food-limited conditions.

RLIP76 regulates Arf6-dependent cell spreading and migration by linking ARNO with activated R-Ras at recycling endosomes.

R-Ras small GTPase enhances cell spreading and motility via RalBP1/RLIP76, an R-Ras effector that links GTP-R-Ras to activation of Arf6 and Rac1 GTPases. Here, we report that RLIP76 performs these functions by binding cytohesin-2/ARNO, an Arf GTPase guanine exchange factor, and connecting it to R-Ras at recycling endosomes. RLIP76 formed a complex with R-Ras and ARNO by binding ARNO via its N-terminus (residues 1-180) and R-Ras via residues 180-192. This complex was present in Rab11-positive recycling endosomes and the presence of ARNO in recycling endosomes required RLIP76, and was not supported by RLIP76(Δ1-180) or RLIP76(Δ180-192). Spreading and migration required RLIP76(1-180), and RLIP76(Δ1-180) blocked ARNO recruitment to recycling endosomes, and spreading. Arf6 activation with an ArfGAP inhibitor overcame the spreading defects in RLIP76-depleted cells or cells expressing RLIP76(Δ1-180). Similarly, RLIP76(Δ1-180) or RLIP76(Δ180-192) suppressed Arf6 activation. Together these results demonstrate that RLIP76 acts as a scaffold at recycling endosomes by binding activated R-Ras, recruiting ARNO to activate Arf6, thereby contributing to cell spreading and migration.

RLIP76 regulates HIF-1 activity, VEGF expression and secretion in tumor cells, and secretome transactivation of endothelial cells.

This study was undertaken to reveal the mechanisms by which RLIP76 regulates endothelial cell angiogenic responses. RLIP76 is an effector of the angiogenic modulator, R-Ras. RLIP76 is overexpressed in many tumors, required for tumor angiogenesis, and blockade of RLIP76 results in tumor regression in multiple models. We report here that RLIP76 was required for expression and secretion of vascular endothelial growth factor (VEGF) in carcinoma and melanoma cells. Conditioned medium derived from RLIP76-depleted tumor cells, but not control knockdown cells, could not stimulate proliferation, migration, or Matrigel cord formation in endothelial cell cultures, which indicates that RLIP76 regulates angiogenic components of the tumor cell secretome. Recombinant VEGF added to conditioned medium from RLIP76-knockdown tumor cells restored these endothelial cell functions. Transcriptional activity of hypoxia-inducible factor 1 (HIF-1), which drives VEGF expression, was blocked in RLIP76-depleted tumor cells. RLIP76 was required for PI3-kinase activation, known to regulate HIF-1, in these cells. However, HIF-1α expression and nuclear localization were unaffected by RLIP76 knockdown, which suggests that RLIP76 regulates HIF-1 at the functional level. Thus, RLIP76 regulates tumor cell transactivation of endothelial cells via control of VEGF expression and secretion, providing a new important link in the mechanism of tumor cell induction of angiogenesis.

Effects of feed restriction on salinity tolerance in white sturgeon (Acipenser transmontanus).

A multistressor study was conducted to investigate interactive effects of nutritional status and salinity on osmoregulation of juvenile white sturgeon. Our hypothesis was that lower nutritional status would decrease the salinity tolerance of juvenile white sturgeon. A four-week feed restriction (12.5%, 25%, 50%, 100% of optimum feeding rate: OFR defined as the rate (% body weight per day) at which growth is maximal) trial was performed, and relevant indices of nutritional status were measured. Following the trial, sturgeon were acutely exposed to various salinities (0, 8, 16, 24 ppt) for 120 h, and relevant osmoregulatory measurements were made at 12, 72, and 120 h post-salinity exposures. The feed-restriction trial resulted in a graded nutritional response with the most feed-restricted group (12.5% OFR) showing the lowest nutritional status. The salinity exposure trial showed clear evidence that lower nutritional status decreased the salinity tolerance of juvenile white sturgeon. Increasing salinities resulted in significant alterations in osmoregulatory indices of all feeding groups; however, a significantly slower acclimatory response to 24 ppt was detected in the most feed-restricted group compared to the non-feed-restricted group (100% OFR). Furthermore, evaluation of the effect of nutritional status on the relationship between osmoregulatory measurements and body size showed that there was a significant negative relationship between osmoregulatory performance and body size within the most feed-restricted group. This suggests that there is a certain body size range (200-300 g based on our finding) where juvenile white sturgeon can maximize osmoregulatory capacity at a salinity of 24 ppt.

The RLIP76 N-terminus binds ARNO to regulate PI 3-kinase, Arf6 and Rac signaling, cell spreading and migration.

RLIP76 is a multifunctional protein involved in tumor growth and angiogenesis, and a promising therapeutic target in many cancers. RLIP76 harbors docking sites for many proteins, and we have found that it interacts with ARNO, a guanine nucleotide exchange factor for Arf6, and that RLIP76 regulates activation of Rac1 via Arf6, and regulates cell spreading and migration in an ARNO and Arf6-dependent manner. Here we show that ARNO interacts with the RLIP76 N-terminal domain, and this domain was required for RLIP76-dependent cell spreading and migration. We identified two sites in the RLIP76 N-terminus with differential effects on ARNO binding and downstream signaling: Ser29/Ser30 and Ser62. Ser29/30 mutation to Alanine inhibited ARNO interaction and was sufficient to block RLIP76-dependent cell spreading and migration, as well as RLIP76-dependent Arf6 activation. In contrast, RLIP76(S62A) interacted with ARNO and supported Arf6 activation. However, both sets of mutations blocked Rac1 activation. RLIP76-mediated Rac and Arf6 activation required PI3K activity. S29/30A mutations inhibited RLIP76-dependent PI3K activation, but S62A mutation did not. Together these results show that ARNO interaction with the RLIP76 N-terminus regulates cell spreading and motility via PI3K and Arf6, independent of RLIP76 control of Rac.

Three-dimensional reconstruction of neovasculature in solid tumors and basement membrane matrix using ex vivo X-ray microcomputed tomography.

OBJECTIVE: To create accurate, high-resolution 3D reconstructions of neovasculature structures in xenografted tumors and Matrigel plugs for quantitative analyses in angiogenesis studies in animal models. METHODS: The competent neovasculature within xenografted solid tumors or Matrigel plugs in mice was perfused with Microfil, a radioopaque, hydrophilic polymerizing contrast agent, by systemic perfusion of the blood circulation via the heart. The perfused tumors and plugs were resected and scanned by X-ray micro-CT to generate stacks of 2D images showing the radioopaque material. A nonbiased, precise postprocessing scheme was employed to eliminate background X-ray absorbance from the extravascular tissue. The revised binary image stacks were compiled to reveal the Microfil-casted neovasculature as 3D reconstructions. Vascular structural parameters were calculated from the refined 3D reconstructions using the scanner software. RESULTS: Clarified 3D reconstructions were sufficiently precise to allow measurements of vascular architecture to a diametric limit of resolution of 3 µm in tumors and plugs. CONCLUSIONS: Ex vivo micro-CT can be used for 3D reconstruction and quantitative analysis of neovasculature including microcirculation in solid tumors and Matrigel plugs. This method can be generally applied for reconstructing and measuring vascular structures in three dimensions.

Effects of Different Dietary Cadmium Levels on Growth and Tissue Cadmium Content in Juvenile Parrotfish, Oplegnathus fasciatus.

This feeding trial was carried out to evaluate the effects of different dietary cadmium levels on growth and tissue cadmium content in juvenile parrotfish, Oplegnathus fasciatus, using cadmium chloride (CdCl2) as the cadmium source. Fifteen fish averaging 5.5±0.06 g (mean±SD) were randomly distributed into each of twenty one rectangular fiber tanks of 30 L capacity. Each tank was then randomly assigned to one of three replicates of seven diets containing 0.30 (C0), 21.0 (C21), 40.7 (C41), 83.5 (C83), 162 (C162), 1,387 (C1,387) and 2,743 (C2,743) mg cadmium/kg diet. At the end of sixteen weeks of feeding trial, weight gain (WG), specific growth rate (SGR) and feed efficiency (FE) of fish fed C21 were significantly higher than those of fish fed C83, C162, C1,387 and C2,743 (p<0.05). Weight gain, SGR and FE of fish fed C0, C21 and C41 were significantly higher than those of fish fed C162, C1,387 and C2,743. Protein efficiency ratio of fish fed C0, C21 and C41 were significantly higher than those of fish fed C1,387 and C2,743. Average survival of fish fed C0, C21, C41 and C162 were significantly higher than that of fish fed C2,743. Tissue cadmium concentrations increased with cadmium content of diets. Cadmium accumulated the most in liver, followed by gill and then muscle. Muscle, gill and liver cadmium concentrations of fish fed C0, C21, C41 and C83 were significantly lower than those of fish fed C162, C1,387 and C2,743. Based on the ANOVA results of growth performance and tissue cadmium concentrations the safe dietary cadmium level could be lower than 40.7 mg Cd/kg diet while the toxic level could be higher than 162 mg Cd/kg diet.

Fish Welfare-Related Issues and Their Relevance in Land-Based Olive Flounder (Paralichthys olivaceus) Farms in Korea.

Korean aquaculture has expanded considerably in recent decades; however, this growth has often prioritized quantity over fish welfare. Therefore, we analyzed the aquaculture practices of olive flounder, the predominant species in Korean consumption and production, within the framework of fish welfare. We conducted extensive interviews and surveys across olive flounder farms in Jeju-do and Wando to examine prevalent issues impacting fish welfare in aquaculture. These issues include stressors, mass mortality events, and disease outbreaks, all of which strain the welfare of farmed fish. Moreover, our survey revealed farmers' varying perceptions of fish welfare, highlighting the necessity for a cohesive approach. Accordingly, we propose recommendations to enhance fish welfare and establish a more sustainable aquaculture model in Korea. Ensuring fish welfare in aquaculture operations requires a comprehensive approach that considers the physiological and behavioral needs of fish throughout the farming lifecycle. By prioritizing fish welfare, Korean aquaculture can strengthen its growth while maintaining ethical standards and ensuring the well-being of farmed fish. This welfare-centric approach is crucial for the long-term sustainability and resilience of the Korean aquaculture industry. By addressing welfare concerns and promoting responsible practices, Korean aquaculture can foster an ethically sound and sustainable future.

Angiogenesis and Apoptosis: Data Comparison of Similar Microenvironments in the Corpus Luteum and Tumors.

The corpus luteum is a temporary endocrine gland formed in the ovary after ovulation, and it plays a critical role in animal reproductive processes. Tumors rely on the development of an adequate blood supply to ensure the delivery of nutrients and oxygen and the removal of waste products. While angiogenesis occurs in various physiological and pathological contexts, the corpus luteum and tumors share similarities in terms of the signaling pathways that promote angiogenesis. In the corpus luteum and tumors, apoptosis plays a crucial role in controlling cell numbers and ensuring proper tissue development and function. Interestingly, there are similarities between the apoptotic-regulated signaling pathways involved in apoptosis in the corpus luteum and tumors. However, the regulation of apoptosis in both can differ due to their distinct physiological and pathological characteristics. Thus, we reviewed the biological events of the corpus luteum and tumors in similar microenvironments of angiogenesis and apoptosis.

Effects of Melatonin and Silymarin on Reactive Oxygen Species, Nitric Oxide Production, and Sperm Viability and Motility during Sperm Freezing in Pigs.

Sperm during the freezing and thawing process is damaged by oxidative stress. Thus, its antioxidant scavenger is essential for sperm survival and death in frozen-thawed semen. We used melatonin and silymarin in experiments after the dose-dependent experiment. Our study aimed to identify the effect of melatonin and silymarin on the motility and viability of sperm, reactive oxygen species (ROS), and nitric oxide (NO) production in frozen-thawed boar semen. Melatonin and silymarin were treated alone and cotreated in the fresh boar semen. Boar semen was collected using the gloved-hand method from ten crossbred pigs, and samples were used in the experiments. We evaluated sperm viability using SYBR-14 and PI kit, and ROS and NO production were detected by DCF-DA and DAF-2, respectively. The sperm motility was not significantly different between non-treatment and treatment. ROS and NO production in frozen-thawed sperm were decreased by melatonin and silymarin. Moreover, silymarin significantly reduced NO production more than melatonin. Melatonin and silymarin enhanced the viability of sperm. We suggest that melatonin and silymarin are essential antioxidants in semen cryopreservation for protecting sperm damage and maintaining sperm viability. Melatonin and silymarin may be useful antioxidants in freezing boar sperm.

Effect of Magnetized Freezing Extender on Membrane Damages, Motility, and Fertility of Boar Sperm Following Cryopreservation.

Magnetized water is defined as the amount of water that has passed through a magnet. The magnetic field weakens the hydrogen bonds between the water molecules, leading to the magnetized liquid acquiring special characteristics such as easy supercooling and forming smaller ice crystals. We researched the influences of a magnetized freezing extender on cell membrane damage and in vitro fertilization of boar sperm during cryopreservation. The freezing extenders were passed through 0, 2000, 4000, and 6000 gausses (G) of magnetic devices using a liquid cycling pump system and then used for the sperm freezing process. The damage to plasma, acrosomal, and mitochondrial membranes in frozen-thawed spermatozoa was investigated by flow cytometry, and motility was assessed using the CASA system. The fertility of frozen-thawed sperm was estimated using in vitro fertilization. The damage to the membranes was significantly decreased in the magnetized freezing extender by the 6000 G magnetic field compared to that of the control in frozen-thawed sperm, and motility was increased in the 6000 G group. Although there were no significant differences in the cleavage rates of in vitro fertilized oocytes among the treatment groups, the ratio of blastocyst formation increased in the magnetized freezing extender groups compared with that in the control group. The number of blastocysts was significantly higher in the 4000 G group than in the 0 G group. In conclusion, these results suggest that a magnetized freezing extender could improve the freezability of sperm and the development of oocytes fertilized in vitro with frozen-thawed sperm.

Effect of Four Functional Feed Additives on Growth, Serum Biochemistry, Antioxidant Capacity, Gene Expressions, Histomorphology, Digestive Enzyme Activities and Disease Resistance in Juvenile Olive Flounder, Paralichthys olivaceus.

An 8-week feeding trial was executed to evaluate the efficacy of four functional feed additives in replacing antibiotics in juvenile olive flounder, Paralichthys olivaceus, fed with a low-fish-meal diet. A basal diet without feed additives was used as a control (CON); other diets were formulated by supplementing 0.50% taurine (TW), 0.30% peptide (PT), 0.23% mineral water (MW), 0.35% yeast-extracted nucleotides (GRO), 0.35% GRO + 0.50% taurine (GROTW), 0.35% GRO + 0.30% peptide (GROPT) and 0.35% GRO + 0.23% mineral water (GROMW) into the basal diet; in addition, one diet was supplemented with oxytetracycline (OTC) at 0.5% as a positive control. Triplicate groups of 25 fish with an average weight of 5.15 ± 0.06 g (mean ± SD) were fed one of the nine experimental diets. At the end of the feeding trial, the weight gain, specific growth rate and protein efficiency ratio of fish fed the GRO, GROMW, GROPT and GROTW diets were significantly higher than those of fish fed the CON diet (p < 0.05). The feed efficiency of fish fed the GRO, GROMW, GROPT and GROTW diets was significantly higher than that of fish fed the TW and OTC diets. However, the survival, hepatosomatic index, viscerosomatic index and condition factor of fish, as well as their whole-body proximate composition, were not significantly affected by the experimental diets (p > 0.05). The serum glutamic pyruvic transaminase of fish fed the GROPT diet was significantly lower than that of fish fed the CON diet. However, glutamic oxaloacetic transaminase, glucose and total protein were not significantly affected by the experimental diets (p > 0.05). The serum superoxide dismutase activity of fish fed the PT, TW, GRO, GROMW, GROPT and GROTW diets was significantly higher than that of fish fed the CON diet. The lysozyme activity of fish fed the PT, GRO, GROMW, GROPT and GROTW diets was significantly higher than that of fish fed the CON and OTC diets. The myeloperoxidase activity of fish fed the TW, GRO, GROMW, GROPT and GROTW diets was significantly higher than that of fish fed the CON, PT and MW diets (p < 0.05). The flounder growth hormone gene expression of fish fed the TW, GRO, GROMW, GROPT, GROTW and OTC diets was significantly higher than that of fish fed the CON, PT and MW diets (p < 0.05). The interleukin 1ß and interleukin 10 gene expressions of fish fed the GRO, GROMW, GROPT and GROTW diets were significantly higher than those of fish fed the CON, PT, TW and MW diets (p < 0.05). Intestinal histology showed a significantly higher villi length for fish fed the GRO, GROMW, GROPT and GROTW diets compared to that of fish fed the CON diet (p < 0.05). Digestive enzyme activities such as trypsin activity were significantly higher in fish fed the GROMW, GROPT and GROTW diets than those in the rest of the diet groups (p < 0.05). Amylase activity in fish fed the MW, GRO, GROMW, GROPT, GROTW and OTC diets was significantly higher than that of fish fed the PT, TW and CON diets (p < 0.05). On the other hand, the lipase activity of fish fed the TW, GRO, GROMW, GROPT and GROTW diets was significantly higher than that of fish fed the CON, PT, MW and OTC diets (p < 0.05). The cumulative survival rate of fish fed the PT, GROTW, GROPT and GROMW diets was significantly higher than that of fish fed the CON, TW and MW diets after thirteen days of the challenge testing. Overall, the results demonstrate that the GRO, GROMW, GROPT and GROTW diets could be beneficial feed additives to replace antibiotics in juvenile olive flounder fed low-fish-meal diets.

Roles of prostaglandin F2alpha and hydrogen peroxide in the regulation of Copper/Zinc superoxide dismutase in bovine corpus luteum and luteal endothelial cells.

BACKGROUND: Prostaglandin F2alpha (PGF) induces luteolysis in cow by inducing a rapid reduction in progesterone production (functional luteolysis) followed by tissue degeneration (structural luteolysis). However the mechanisms of action of PGF remain unclear. Reactive oxygen species (ROS) play important roles in regulating the luteolytic action of PGF. The local concentration of ROS is controlled by superoxide dismutase (SOD), the main enzyme involved in the control of intraluteal ROS. Thus SOD seems to be involved in luteolysis process induced by PGF in cow. METHODS: To determine the dynamic relationship between PGF and ROS in bovine corpus luteum (CL) during luteolysis, we determined the time-dependent change of Copper/Zinc SOD (SOD1) in CL tissues after PGF treatment in vivo. We also investigated whether PGF and hydrogen peroxide (H2O2) modulates SOD1 expression and SOD activity in cultured bovine luteal endothelial cells (LECs) in vitro. RESULTS: Following administration of a luteolytic dose of PGF analogue (0 h) to cows at the mid-luteal stage, the expression of SOD1 mRNA and protein, and total SOD activity in CL tissues increased between 0.5 and 2 h, but fell below the initial (0 h) level at 24 h post-treatment. In cultured LECs, the expression of SOD1 mRNA was stimulated by PGF (1-10 microM) and H2O2 (10-100 microM) at 2 h (P<0.05). PGF and H2O2 increased SOD1 protein expression and total SOD activity at 2 h (P<0.05), whereas PGF and H2O2 inhibited SOD1 protein expressions and total SOD activity at 24 h (P<0.05). In addition, H2O2 stimulated PGF biosynthesis at 2 and 24 h in bovine LECs. Overall results indicate that, SOD is regulated by PGF and ROS in bovine LECs. SOD may play a role in controlling intraluteal PGF and ROS action during functional and structural luteolysis in cows.

Dietary Supplementation with γ-Aminobutyric Acid Improves Growth, Digestive Enzyme Activity, Non-Specific Immunity and Disease Resistance against Streptococcus iniae in Juvenile Olive Flounder, Paralichthys olivaceus.

Recent research is increasingly shedding light on the important role that microbial metabolites such as γ-aminobutyric acid (GABA) play in the context of nutrition, cognition, immune function, and the modulation of the gut microbiome. Yet, very few trials were conducted to assess the effects of its supplementation on biomarkers of fish health. Therefore, an eight-week feeding trial was devised to evaluate GABA supplementation in juvenile olive flounder, (Paralichthys olivaceus). A total of 630 fish with an average weight of 4.90 ± 0.10 g (±SD) were randomly assigned to one of seven triplicate groups and fed a non-GABA supplemented diet (CON, with 92 mg/kg GABA content), a positive control with 4 g/kg oxytetracycline (OTC), and five other diets supplemented with 50, 100, 150, 200 and 250 mg/kg GABA (corresponding to a total GABA content of 154, 229, 282, 327 and 352 mg/kg, respectively). Growth, blood chemistry, nonspecific immunity, digestive enzyme activity and disease resistance were assessed. The results showed that 100 and 150 mg/kg GABA supplementation consistently yielded significant improvements (p < 0.05) in growth, intestinal amylase, serum lysozyme, and survival against infection with Streptococcus iniae. Based on polynomial analysis, the optimal supplementation level was determined to be 237 mg/kg. These results support GABA as an important functional feed additive in juvenile olive flounder.

Feeding Strategies for Adapting Lake Sturgeon (Acipenser fulvescens) Larvae to Formulated Diets at Early Life Stages.

Cost-effective feeding management is required to support conservation hatcheries for lake sturgeon (Acipenser fulvescens), an ecologically important species in the Great Lakes region. This study investigated an approach to transition lake sturgeon larvae from live feed (Artemia) to formulated feed and its effect on growth performance, survival, and response to acute hypoxia stress. The first experiment showed that sturgeon had similar (p > 0.05) growth and survival when fed Artemia or the combined feeding of Artemia with the commercial diet (crude protein, 551 g/kg diet). Feeding solely on the commercial or lab-made (crude protein, 491 g/kg diet) diet significantly reduced growth and survival (p < 0.05). In the second experiment, the growth performance of sturgeon (14 days post-hatch, DPH) fed with either Artemia only or combined feeding different feeding durations of two, three, and four weeks followed by a complete transition to the commercial diet. At the end of six weeks, the 3- and 4-week combined feeding periods resulted in significantly higher body weight and survival compared to the 2-week combined and the Artemia only feeding treatments. In the last experiment, sturgeons (27 DPH) were fed only with Artemia or combined feeding of Artemia with the commercial diet for four weeks followed by the complete transition to the commercial diet for two weeks. Eighteen fish from each treatment were investigated the response to acute hypoxic conditions (gradual decrease in dissolved oxygen level from 8 to 2.3 mg/L at the rate of 1 mg/L per hour). When the dissolved oxygen was between 3 and 4 mg/L, the mortality rate of the combination-fed sturgeon (11.7%) was significantly lower than those fed only Artemia (83.3%). These results clearly demonstrate that a commercial diet can partially replace Artemia at early life stages to improve growth, survival, and hypoxia tolerance and thus its co-feeding with Artemia is recommended.

The Role of the Guanosine Nucleotide-Binding Protein in the Corpus Luteum.

The corpus luteum is a temporary endocrine gland in the ovary. In the ovarian cycle, repeated patterns of specific cellular proliferation, differentiation, and transformation occur that accompany the formation and regression of the corpus luteum. Molecular mechanism events in the ovarian microenvironment, such as angiogenesis and apoptosis, are complex. Recently, we focused on the role of RAS protein in the ovarian corpus luteum. RAS protein plays a vital role in the modulation of cell survival, proliferation, and differentiation by molecular pathway signaling. Additionally, reproductive hormones regulate RAS activity in the cellular physiological function of ovarian follicles during pre-ovulatory maturation and ovulation. Thus, we have reviewed the role of RAS protein related to the biological events of the corpus luteum in the ovary.

Prostaglandin F2 Alpha Triggers the Disruption of Cell Adhesion with Cytokeratin and Vimentin in Bovine Luteal Theca Cells.

Intermediate filaments (IFs) maintain cell-cell adhesions and are involved in diverse cellular processes such as cytokinesis, cell migration and the maintenance of cell structure. In this study, we investigated the influence of prostaglandin F2 alpha (PGF2α) on cytokeratin and vimentin IFs, Rho-associated protein kinase (ROCK), and cell-cell adhesion in bovine luteal theca cells (LTCs). The luteal cells were isolated from bovine corpus luteum (CL), and the LTCs were treated with 0, 0.01, 0.1 and 1.0 mM PGF2α. Cytokeratin, vimentin and desmoplakin proteins were disrupted and the ROCK protein was significantly increased in PGF2α-treated LTCs. In addition, cell-cell adhesion was significantly (p < 0.05) decreased in the PGF2α-induced LTCs compared to control group (0 mM PGF2α). In conclusion, PGF2α affected the adhesion of cell to cell via disruption of desmoplakin, cytokeratin and vimentin, additionally increasing ROCK in bovine LTCs. These results may provide a better understanding of the mechanism of bovine CL regression.