Preliminary study on alleviation of heat-induced intestinal inflammation through compensatory effects of glucose oxidase.

1. The influence of glucose oxidase (GOD) supplementation on growth, gut inflammation and its compensatory effects in broilers was investigated before and after heat stress.2. Before heat stress, one-day-old broilers were divided into two groups: the control (CON) and GOD (100 g/t complete feed) groups. On d 21, the CON group was equally divided into CON1 and CON2 groups, and heat stress (35°C) was applied to the CON2 and GOD groups for 8 h/day to the end of the study, d 27 of age. The chickens were either killed before heat stress and 2 d after heat stress for the determination of cytokines in the liver and ileum, serum antioxidant enzymes and ileal microbiota. Growth performance was determined before and 7 d after heat stress.3. The GOD decreased Clostridiales and Enterobacteriaceae families of bacteria and increased ileal nuclear factor-κB, interleukin-1ß, and interferon-γ (P < 0.05) before heat stress. The broilers exhibited compensatory effects, including increases in ileal sirtuin-1, heat shock protein 70 expression, liver nuclear factor erythroid 2-related factor 2 content, serum total antioxidant capacity and glutathione peroxidase level (P < 0.05). At 2 d after heat stress, inflammatory factors were increased in both the CON2 and GOD groups, but the levels were lower in the GOD than CON2 (P < 0.05). On d 7 after heat stress, GOS alleviated heat stress induced growth retardation (P < 0.05).4. These data suggested that GOD supplementation in broiler diets before heat stress stimulated intestinal oxidative stress and produced a compensatory response, which prevented a rapid increase in intestinal inflammatory factors and helped to maintain growth performance under heat stress.

Isolation and characterization of microsatellite loci in Quercus fabri (Fagaceae).

Quercus fabri is a pioneer species of secondary succession in evergreen broadleaved forests in China. In this study, we isolated and developed 12 polymorphic and 2 monomorphic microsatellite loci for Q. fabri using the biotin-streptavidin capture method. We characterized 12 polymorphic loci in 52 individuals from two populations. The number of alleles per locus ranged from 3 to 23. The observed and expected heterozygosities per locus were 0.033-0.773 and 0.138-0.924, respectively. These microsatellite loci will facilitate the studies on genetic variation, mating system, and gene flow of Q. fabri.

Sperm cryopreservation in different grouper subspecies and application in interspecific hybridization.

The present study established and confirmed an efficient technology for groupers: giant grouper Epinephelus lanceolatus, orange-spotted grouper E. coioides, seven-band grouper, E. septemfasciatus, and kelp grouper E. moara sperm cryopreservation and successfully applied the cryopreserved E. lanceolatus sperm into interspecific hybridization with E. coioides. For both E. lanceolatus and E. coioides, the best motility of postthaw sperm were achieved using 6% to 10% DMSO, 6% to 16% propylene glycol, and 6% ethylene glycol as cryoprotectants. Furthermore, we have successfully applied this method into the other two species of E. septemfasciatus (74.56 ± 5.45%) and E. moara (71.67 ± 5.10%) sperm cryopreservation and obtained high motility, respectively. Computer-assisted sperm motion analysis showed that the postthaw sperm of the four species of grouper could keep 30 to 35 minutes motile state in nature seawater. And the freezing-thawing process decreased the sperm motility, speed, and longevity but did not significantly change the sperm movement pattern, and the progressive linear motion still was the dominant movement pattern. For the four species of grouper, the ultrastructural analysis showed 70% to 80% of the spermatozoa had intact morphology with a little of swelling; 20% to 30% were damaged, such as swelling or rupture of head, midpiece, and tail region; and 10% to 20% were severely damaged. Whereas, by the microscopic observation, more than 90% of the postthaw sperm presented normal morphology. In the artificial insemination and hybridization experiment, high fertilization rates and hatching rates were achieved when using 10% DMSO (88.7 ± 5.3%, 85.3 ± 7.4%) and 10% propylene glycol (86.8 ± 3.3%, 83.1 ± 6.6%), with no significant difference in comparison with control (92.2 ± 1.4%, 87.9 ± 4.2%). In addition, we found the embryos from postthaw sperm of E. lanceolatus and E. coioides eggs developed and grew normally as reported in previous study on hybridization of groupers (E. coioides × E. lanceolatus) using cryopreserved sperm. The results of the present study further validated the safety of the cryopreserved sperm in breeding production by assessing the fertilization capacity, embryo development, and larval growth.

Protective Effect of Antiapoptosis Potency of Prolonged Preservation by Desiccation Using High-Pressure Carbon Monoxide on Isolated Rabbit Hearts.

Heart transplantation has been widely accepted as a therapy for end-stage heart failure. Mitigation of ischemia-reperfusion injury by inhibiting the apoptotic process plays an important role in organ transplantation. Desiccation using high-pressure carbon monoxide (CO) is a new method of preserving donor hearts; however, its mechanism of antiapoptosis remains unclear. This study was intended to elucidate the efficacy and mechanism of preservation by desiccation for 18 hours using high-pressure CO on myocardial apoptosis. Rabbit heterotopic abdominal cardiac transplantation models were established. New Zealand rabbits were divided randomly into 3 groups: naive group (n = 16), HTK group (n = 16), and desiccation using high-pressure CO group (n = 16). The donor hearts of the naive group were transplanted immediately after being extracted. In the HTK group, the donor hearts were extracted and steeped in 4°C HTK cardioplegic solution for 18 hours and then transplanted; in the desiccation using high-pressure CO group, the donor hearts were extracted and exposed to a gas mixture (Po2 = 3200 hPa, Pco = 800 hPa) in the chamber before being preserved in a refrigerator at 4°C for 18 hours and then transplanted. Apoptotic cardiomyocytes were detected using TUNEL technique and histopathology was performed by hematoxylin-eosin staining. The expression of the ratio of Bcl-2/Bax and caspase-3 proteins was detected using the Western blot method. These findings suggest that compared with traditional HTK preservation, preservation by desiccation using high-pressure CO could alleviate rabbits' myocardial histopathology and apoptosis induced by ischemia-reperfusion injury through adjusting the ratio of Bcl-2/Bax protein expression, thus resulting in the reduction of expression of caspase-3.

Summer flounder (Paralichthys dentatus) sperm cryopreservation and application in interspecific hybridization with olive flounder (P olivaceus).

The present study established an efficient technology for summer flounder (Paralichthys dentatus) sperm cryopreservation, and successfully applied the cryopreserved sperm into interspecific hybridization with olive flounder (P olivaceus). The best motility of postthaw sperm (78.00 ± 4.70% and 76.60 ± 7.90%), fertilization rates (95.70 ± 3.60% and 79.40 ± 5.20%), and hatching rates (93.10 ± 4.00% and 77.20 ± 2.90%) were achieved when using propylene glycol or DMSO as cryoprotectant. Furthermore, we have successfully improved the cryopreservation method from using 2-mL cryotube to 5-mL cryotube, and the dilution ratio has been increased to 4:1. By this method, the cryopreservation efficiency has been improved to 30 times of that with routine method. Computer-assisted sperm motion analysis showed the freezing-thawing process decreased the sperm speed but did not significantly change the sperm movement pattern, and the progressive linear motion still was the dominant movement pattern. The ultrastructural analysis showed 50% to 60% of the spermatozoa had normal morphology, 20% to 30% were slightly damaged, such as swelling or rupture of head, midpiece, and tail region, and 10% to 20% were severely damaged. In the artificial hybridization experiment, high fertilization rates and hatching rates were achieved when using 15% DMSO (95.7 ± 3.6% and 79.4 ± 5.2%, respectively) and 15% propylene glycol (93.1 ± 4.0% and 77.2 ± 2.9%, respectively), with no significant difference in comparison with control (94.7 ± 2.6% and 72.5 ± 6.5%, respectively). In addition, we found the embryos and larvae from postthaw sperm of P dentatus developed and grew normally. The results of the present study further validated the safety of the cryopreserved sperm in breeding production by assessing the fertilization capacity, embryo development, and larval growth.

Germline-specific and sexually dimorphic expression of a dead end gene homologue in turbot (Scophthalmus maximus).

Germ cells are indispensable for gonadal development and fertility. However, the physiological mechanisms regulating germ cell development in marine fish are poorly understood due to a lack of germ cell markers. The dead end (dnd) gene is a vertebrate-specific component of germplasm crucial for primordial germ cells (PGCs) migration and development in teleosts. In this study, we identified a dnd homologue (Smdnd) in turbot (Scophthalmus maximus) and investigated its expression pattern during embryogenesis and gonadal development. The deduced amino acid sequence of Smdnd shared several conserved motifs of Dnd homologues as well as high identity to other Dnd proteins. Phylogenetic analysis revealed that the SmDnd was closely related to its teleost counterparts. Reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization revealed that Smdnd transcripts could be exclusively detected in germ cells, including presumptive PGC and adult male and female germ cells. In addition, an interesting sexually dimorphic expression of Smdnd during gonadal development was observed by real-time PCR. Female turbot showed greater (P < 0.05) Smdnd expression than male before sex maturation. This difference reduced gradually due to the upregulation of Smdnd in the male during the period corresponding to spermatogonia proliferation and meiosis. These results indicate that Smdnd can be used as a germ cell marker in turbot. In addition, the temporal and sex differences in Smdnd expression indicate that this gene may play different roles in gonadal development in both sexes.

Germ cells and fertilization differences among Japanese flounder Paralichthys olivaceus, summer flounder Paralichthys dentatus and their first and second generations.

The morphology of gametes and the fertilization biology of Japanese flounder Paralichthys olivaceus (Po), summer flounder Paralichthys dentatus (Pd) and their hybrids were examined. Multiple generations (two hybrids: Poâ™€× Pd♂ (F1) and Pdâ™€× Po♂; two backcrosses: F1â™€× Po♂ and F1â™€× Pd♂) were obtained by artificial insemination. Comparison of egg ultrastructure among Po, Pd and F1 showed the morphology of micropyle region and the distribution density of pores were species specific. There were c. 100-200 accessory openings around the micropyle in Po, but not in Pd and F1. The zona radiata thickness and number of parallel bands were similar between F1 and Po, which were different from Pd. Comparison of spermatozoa ultrastructure revealed a close relationship between Po and Pd. Cytologically, the six crosses obeyed normal fertilization and cleavage processes, and only one male pronucleus was observed in a fertilized egg, indicating a monospermic fertilization pattern. Analysis of the time distribution from fertilization to first cleavage revealed an obvious delay at pronucleus fusion in the Pd × Po cross. The delay might indicate some cytoplasmic-nuclear incompatibility during the process of fertilization.

Skeletal development and abnormalities of the vertebral column and of the fins in hatchery-reared turbot Scophthalmus maximus.

To describe the skeletal development and abnormalities in turbot Scophthalmus maximus, samples were collected every day from hatching to 60 days after hatching (DAH). A whole-mount cartilage and bone-staining technique was used. Vertebral ontogeny started with the formation of anterior haemal arches at 5·1 mm standard length (L(S) ) c. 11 DAH, and was completed by the full attainment of parapophyses at 16·9 mm L(S) c. 31 DAH. Vertebral centra started to develop at 6·3 mm L(S) c. 16 DAH and ossification in all centra was visible at 11·0 mm L(S) c. 25 DAH. The caudal fin appeared at 5·1 mm L(S) c. 11 DAH and ossification was visible at 20·6 mm L(S) c. 37 DAH. The onset of dorsal and anal fin elements appeared at 5·8 mm L(S) c. 15 DAH and 6·3 mm L(S) c. 16 DAH, respectively. Ossifications of both dorsal fin and anal fin were visible at 20·6 mm L(S) c. 37 DAH. The pectorals were the only fins present before first feeding, their ossifications were completed at 23·5 mm L(S) c. 48 DAH. Pelvic fins began forming at 7·2 mm L(S) c. 19 DAH and calcification of the whole structure was visible at 19·8 mm L(S) c. 36 DAH. In the present study, 24 types of skeletal abnormalities were observed. About 51% of individuals presented skeletal abnormalities, and the highest occurrence was found in the haemal region of the vertebral column. As for each developmental stage, the most common abnormalities were in the dorsal fin during early metamorphic period (stage 2), vertebral fusion during climax metamorphosis (stage 3) and caudal fin abnormality during both late-metamorphic period (stage 4) and post-metamorphic period (stage 5). Such research will be useful for early detection of skeletal malformations during different growth periods of reared S. maximus.

Digestive enzyme activities of turbot (Scophthalmus maximus L.) during early developmental stages under culture condition.

Digestive enzyme activities were analysed in turbot (Scophthalmus maximus) from hatching until 60 days after hatching (DAH). Trypsin sharply increased to the climax at 17 DAH and decreased until 31 DAH followed by a stable level thereafter. Amylase was determined at 4 DAH, reached the maximum value at 19 DAH and declined sharply to 39 DAH and remained at a low level thereafter, suggesting the carbohydrate component should remain at a low level in formulated diets. Pepsin was detected at 9 DAH and increased to 34 DAH and then remained at a stable level. The above results revealed pancreatic enzymes are no longer main enzymes for food digestion after the formation of functional stomach. Leucine-alanine peptidase (Leu-ala) and alkaline phosphatase (AP) and leucine aminopeptidase N (LAP) were found in newly hatched larvae. Both AP and LAP activities markedly increased to 23 DAH, decreased abruptly to 50 DAH and increased gradually to 60 DAH. Leu-ala reached the plateau from 23 to 39 DAH, followed by a decline to 46 DAH and an increase until 60 DAH. The brush border membrane (BBM)-bound enzyme activities increased from 30% at 31 DAH to 81% at 38 DAH of the total activities, indicating the maturation of intestinal tract.

Study on permeability of DMSO in embryos of red seabream (Pagrus major) by capillary electrophoresis.

The objectives were to investigate the permeability of DMSO to red seabream (Pagrus major) embryos by capillary electrophoresis and the effects of DMSO concentrations (5 to 40 percent, volume basis) and immersion times (10, 30 and 60 min) on hatching rate and morphology. The results suggested the internal DMSO concentrations were positively related with the external concentrations and exposure times, while the hatching rate was negatively related. The hatching rate decreased drastically (less than 50 percent) after exposure in 35 percent, 20 percent and 15 percent DMSO for over 10, 30 and 60 min, respectively. In all groups, when hatching rate was greater 50 percent, the internal DMSO concentration was less than 2 percent, which was still insufficient for successful cryopreservation. Morphological changes indicated the chorion was permeable to the cryoprotectant. A sign of dehydration in yolk were observed, for a significant decrease in the maximal yolk sac diameter. However, further research was needed to investigate whether the DMSO permeated into the yolk.

Changes in RNA, DNA, protein contents and growth of turbot Scophthalmus maximus larvae and juveniles.

The growth potential of turbot Scophthalmus maximus larvae and juveniles was studied using nucleic acid-based indices and protein variables. The experiment was carried out from 4 to 60 days post hatching (dph). A significant increase in instantaneous growth rate during metamorphosis and retarded growth rate during post-metamorphic phase were observed. Ontogenetic patterns of DNA, RNA and protein all showed developmental stage-specific traits. The RNA:DNA ratio decreased up to 12 dph, then increased rapidly till 19 dph and fluctuated until 35 dph followed by a decline to the end. The RNA:DNA ratio was positively correlated with growth rate of juveniles during the post-metamorphic phase, whereas this ratio was not a sensitive indicator of growth during the pre-metamorphic phase and metamorphosis. The protein:DNA ratio showed a similar tendency to the RNA:DNA ratio. Changes of DNA content and protein:DNA ratio revealed that growth of S. maximus performed mainly by hyperplasia from 4 to 12 dph and hypertrophy until 21 dph during the pre-metamorphic larval phase. Growth was dominantly hypertrophical from the early- to mid-metamorphosing phase and hyperplastic thereafter. The results show that the DNA content and protein:DNA ratio can evaluate growth rates of larval and juvenile S. maximus on a cellular level.

Extra- and intra-cellular ice formation of red seabream (Pagrus major) embryos at different cooling rates.

The ice crystal formation is assumed as the most lethal factor for the failure of fish embryo cryopreservation and intracellular ice formation (IIF) plays a central role in cell injury during cooling. The objectives were to observe the morphological changes of red seabream (Pagrus major) embryo during the cooling-thawing process, and to examine the effect of cryoprotectant and cooling rate on the temperatures of oil globule ice formation (T(OIF)), extra-cellular ice formation (T(EIF)) and intracellular ice formation (T(IIF)) using cryomicroscope. After thawing, the morphological changes of embryos were observed and recorded by the video attachment and monitor under the microscope. During the cooling process, three representative phenomena were observed: oil globule gradually turned bright firstly, then the whole field of view flashed and the embryo blackened. Cooling rate affect the temperature of both extra- and intra-cellular ice formations. T(EIF) and T(IIF) at high cooling rate were much lower than that at low cooling rate. And the value of T(EIF)-T(IIF) increased from 0.45 to 11.11 degrees C with the increase of cooling rate from 3 to130 degrees C/min. Taken together, our results suggested that high cooling rate with proper cryoprotectant would be a good option for red seabream embryo cryopreservation.

Effect of cryoprotectants on hatching rate of red seabream (Pagrus major) embryos.

The objectives were to investigate the effect of cryoprotectants on the hatching rate of red seabream embryos. Heart-beat embryos were immersed in: five permeable cryoprotectants, dimethyl sulfoxide (DMSO), glycerol (Gly), methanol (MeOH), 1,2-propylene glycol (PG), and ethylene glycol (EG), in concentrations of 5-30% for 10, 30, or 60 min; and two non-permeable cryoprotectants: polyvinylpyrrolidone (PVP), and sucrose (in concentrations of 5-20% for 10 or 30 min). The embryos were then washed and incubated in filtered seawater until hatching occurred. The hatching rate of the embryos treated with permeable cryoprotectants decreased (P<0.05) with increased concentration and duration of exposure. In addition, PG was the least toxic permeable cryoprotectant, followed by DMSO and EG, whereas Gly and MeOH were the most toxic. At a concentration of 15% and 30 min exposure, the hatching rate of the embryos immersed in PG was 93.3+/-7.0% (mean+/-S.D.), however, in DMSO, EG, Gly, and MeOH, it was 82.7+/-10.4, 22.0+/-5.7, 0.0+/-0.0, and 0.0+/-0.0%, respectively. Hatching rate of embryos treated with PVP decreased (P<0.05) with the increase of concentration and exposure time, whereas for embryos treated with sucrose, there was no significant decrease in comparison with the control at the concentrations used.

Preliminary studies on the vitrification of red sea bream (Pagrus major) embryos.

The objective was to identify an appropriate cryoprotectant and protocol for vitrification of red sea bream (Pagrus major) embryos. The toxicity of five single-agent cryoprotectants, dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycerol (GLY), and methyl alcohol (MeOH), as well as nine cryoprotectant mixtures, were investigated by comparing post-thaw hatching rates. Two vitrifying protocols, a straw method and a solid surface vitrification method (copper floating over liquid nitrogen), were evaluated on the basis of post-thaw embryo morphology. Exposure to single-agent cryoprotectants (10% concentration for 15 min) was not toxic to embryos, whereas for higher concentrations (20 and 30%) and a longer duration of exposure (30 min), DMSO and PG were better tolerated than the other cryoprotectants. Among nine cryoprotectant mixtures, the combination of 20% DMSO+10% PG+10% MeOH had the lowest toxicity after exposure for 10 min or 15 min. High percentages of morphologically intact embryos, 50.6+/-16.7% (mean+/-S.D.) and 77.8+/-15.5%, were achieved by the straw vitrifying method (20.5% DMSO+15.5% acetamide+10% PG, thawing at 43 degrees C and washing in 0.5M sucrose solution for 5 min) and by the solid surface vitrification method (40% GLY, thawing at 22 degrees C and washing in 0.5M sucrose solution for 5 min). After thawing, morphological changes in the degenerated embryos included shrunken yolks and ruptured chorions. Furthermore, thawed embryos that were morphologically intact did not consistently survive incubation.

Flow cytometry and ultrastructure of cryopreserved red seabream (Pagrus major) sperm.

The objectives were to assess motility, fertilizing capacity, structural integrity, and mitochondrial function in fresh versus frozen-thawed (15% DMSO was used as a cryoprotectant) sperm from red seabream (Pagrus major). Mean (+/-S.D.) rates of motility, fertilization and hatching of frozen-thawed sperm were 81.0+/-5.4, 92.8+/-1.9, and 91.8+/-5.2%, respectively; for fresh sperm, they were 87.5+/-7.7, 95.8+/-2.4, and 93.8+/-4.2%. Although motility was lower in frozen-thawed versus fresh sperm (P<0.05), there was no effect (P>0.05) of cryopreservation on fertilization or hatching. Based on scanning and transmission electron microscopy, 77.8+/-5.6% of fresh sperm had normal morphology, whereas for frozen-thawed sperm, 63.0+/-7.2% had normal morphology, 20.6+/-3.1% were slightly damaged (e.g. swelling or rupture of head, mid-piece and tail region as well as mitochondria), and 16.4+/-4.2% were severely damaged. Sperm were stained with propidium iodide and Rhodamine 123 to assess plasma membrane integrity and mitochondrial function, respectively, and examined with flow cytometry. For fresh sperm, 83.9% had an intact membrane and functional mitochondria, whereas for frozen-thawed sperm, 74.8% had an intact membrane and functional mitochondria, 12.7% had a damaged membrane, 9.9% had nonfunctional mitochondria, and 2.6% had both a damaged membrane and nonfunctional mitochondria. In conclusion, ultrastructure and flow cytometry were valuable for assessment of frozen-thawed sperm quality; cryopreservation damaged the sperm but fertilizing ability was not significantly decreased.