Production of Diabetic Offspring Using Cryopreserved Epididymal Sperm by In Vitro Fertilization and Intrafallopian Insemination Techniques in Transgenic Pigs

Somatic cell nuclear transfer (SCNT) is a useful technique for creating pig strains that model human diseases. However, production of numerous cloned disease model pigs by SCNT for large-scale experiments is impractical due to its complexity and inefficiency. In the present study, we aimed to establish an efficient procedure for proliferating the diabetes model pig carrying the mutant human hepatocyte nuclear factor-1α gene. A founder diabetes transgenic cloned pig was generated by SCNT and treated with insulin to allow for normal growth to maturity, at which point epididymal sperm could be collected for cryopreservation. In vitro fertilization and intrafallopian insemination using the cryopreserved epididymal sperm resulted in diabetes model transgenic offspring. These results suggest that artificial reproductive technology using cryopreserved epididymal sperm could be a practical option for proliferation of genetically modified disease model pigs.     

Effect of conjugated linolenic acid (cis-9, trans-11, cis-13–18:3) on growth performance and lipid composition of fingerling rainbow trout Oncorhynchus mykiss

ABSTRACT:   This study investigated the effect of conjugated linolenic acid (CLNA) on the growth and lipid composition of fingerling rainbow trout Oncorhynchus mykiss . Thirty individuals in each tank with three dietary treatments and duplicate groups were subjected to three dietary levels (0:4, 2:2, 4:0) of CLNA and linolenic acid (LNA) at 16°C in the laboratory for a period of 9 weeks. Punicic acid ( cis -9, trans -11, cis -13–18:3) and linseed oil were used as sources of CLNA and LNA, respectively. The hepatosomatic index and body lipid content of the 2% CLNA dietary treatment increased significantly ( P  < 0.05) with respect to the control (LNA treatment). But the specific growth rate and percent gain did not improve significantly. After 6 weeks' observation, the 4% CLNA dietary treatment exhibited higher mortality and liver weight but lower weight gains (1.1–1.9 g). No significant differences were observed in the results of the feed efficiency and moisture as well as the protein content of the whole body among the treatments. Incorporation of CLNA into fish tissues was at a distinctly higher level in neutral lipids than in polar lipids. Increased levels of 22:6n-3 were found in the 4% CLNA treatment but decreased levels were found in the 2% CLNA treatment in both neutral and polar fractions. In the CLNA treatments, saturated fatty acid was relatively unaffected, the level of 18:1 was increased and the level of 18:2n-6 was decreased. These results indicate that the lower level (2%) of CLNA had no significant effect on the growth and the higher level (4%) of CLNA caused higher mortality as well as attenuating the growth of the fish. Dietary CLNA accumulated in a greater amount in the neutral lipids and modulated the fatty acid composition in the whole body of rainbow trout fingerlings.     

The pain-relieving effects of lactoferrin on oxaliplatin-induced neuropathic pain

Oxaliplatin (OXL) therapy often causes side effects including chronic peripheral neuropathy. We investigated the pain-relieving effects of recombinant human lactoferrin (rhLf) as well as a long-acting IgG-Fc fused rhLf (rhLf-Fc) on OXL-induced neuropathic pain. We used the hLf in this study, because the homology between mouse Lf and hLf is higher than that of bovine Lf. In addition, rhLf-Fc is expected to enhance the analgesic effect due to the life extension effect in the body. We administered OXL (2 mg/kg, i.v.) to mice twice weekly for 4 weeks. Phosphate buffered saline (PBS), rhLf (100 mg/kg, i.p.) or rhLf-Fc (100 mg/kg, i.p.) was administered once a week from day 15 to 32. We also assessed the continuous infusion of same drugs (10 mg/kg/day) into the external jugular vein by using an osmotic pump. Both of rhLf and rhLf-Fc significantly reduced the hypersensitivity to mechanical stimulation when they were administered intraperitoneally. The continuous infusion of rhLf resulted in a more pronounced effect. Histopathological analysis of sciatic nerve showed that both rhLf and rhLf-Fc tended to reduce nerve fiber damage, but no significant difference was observed in nerve fiber cross-sectional area. Therefore, it was suggested that rhLf or rhLf-Fc injection could be an option for controlling neuropathic pain, which are side effects of OXL.     

Application of microparticle diets for Japanese flounder Paralichthys olivaceus larvae

ABSTRACT:   A feeding trial was conducted with two newly developed microparticle diets (MD-Q and MD-V), which differed in the mixing ratios of two types of casein hydrolysates (C700 and C800) as the protein source. The ratio of C700 and C800 were adjusted to 7:3 in MD-Q and 6:4 in MD-V, respectively. Japanese flounder larvae were fed from 7 days after hatching (d.a.h.) to 32 d.a.h. on live food (LF), MD solely, MD + 1/3 LF (either of the MD and one-third quantity of the live food) and 1/3 LF (one-third quantity of live food alone), respectively. They were then switched to Kyowa diet B from 33 d.a.h. until 40 d.a.h. The larvae fed on MD-Q exclusively had a higher survival rate (36.4%) than those fed on MD-V (24.2%) by 22 d.a.h. Unfed larvae could survive up to only 12 d.a.h. Larvae fed on MD-Q + 1/3 LF also had a significantly higher survival rate (34.2%) than those on MD-V + 1/3 LF (16.3%) and 1/3 LF solely (15.4%) at 32 d.a.h. These results suggest the potential of MD-Q as a part replacement for LF from the early developmental stage in the seed production of Japanese flounder.     

Influence of all-trans retinoic acid on pigmentation and skeletal formation in larval Japanese flounder

ABSTRACT: The effect of all- trans retinoic acid (atRA) on pigmentation and skeletal formation of Japanese flounder Paralichthys olivaceus was investigated. Five groups of flounder larvae were fed live food enriched with 0.5 mL docosahexaenoic acid (DHA) 38G oil containing 10 mg of atRA/10 L of culture medium during different developmental stages; that is, A–B, C–D, E–F, G–H, and I. The control group was fed live food enriched with only DHA 38G oil. Flounder that were fed live food enriched with atRA during the A–B stages showed albinism, and mandible and severe caudal defects (albinism 75.7%, lower jaw defects 75%, caudal defects 100%). Occurrence of these abnormalities in other groups was 0%, 1–4%, and 4.5–10.7%, respectively. Administration of atRA during the A–B stages also caused a high number of vertebrae mainly in the caudal area. Moreover, additional abdominal vertebrae had formed in 25% of fish that were fed live food enriched with atRA in the A–B stages. These results indicate that the effect of atRA is dependent on the developmental stage of flounder larvae and they also suggest that morphological and color abnormalities in flounder were induced by atRA accumulated in live food (rotifers 13 mg/g; Artemia 1.6 mg/g), especially during the A–B stages.     

Effect of different photoperiod cycles on metabolic rate and energy loss of both fed and unfed young tilapia Oreochromis niloticus: Part I

ABSTRACT: The influence of different photoperiod cycles (3L : 3D, 6L : 6D, 12L : 12D, and 24L : 24D) on the metabolic rate and energy loss of either fed or unfed young tilapia Oreochromis niloticus (bodyweight 8.6–9.5 g) was investigated at 28°C. A computer-operated respirometer with a closed tank was used to prevent water from evaporating into the air or condensing from the air. The photoperiods acted as strong Zeitgeber (i.e., cue or synchronizer) during the experiments with either fed or unfed fish. A photoperiod-mediated metabolic cycle was demonstrated in fed and unfed fish in which oxygen consumption was higher during the light phase compared with during the dark phase for all photoperiods. Mean oxygen consumption during the 3L : 3D, 6L : 6D, 12L : 12D, and 24L : 24D periods for fed and unfed fish were 685.06 mg/kg per h and 299.33 mg/kg per h; 658.52 mg/kg per h and 284.80 mg/kg per h; 591.09 mg/kg per h and 249.62 mg/kg per h; and 500.64 mg/kg per h and 239.14 mg/kg per h, respectively. The highest post-prandial increase in energy loss was recorded during the 3L : 3D period (145.88 kJ/kg per day), followed by 141.19 kJ/kg per day during the 6L : 6D, 128.70 kJ/kg per day during the 12L : 12D, and 99.92 kJ/kg per day during the 24L : 24D periods. The results suggest that higher energy conservation would be achieved if fish are exposed to longer rather than shorter photoperiod cycles.     

Effect of lactoferrin on murine sperm apoptosis induced by intraperitoneal injection of lipopolysaccharide

Genital bacterial infection is one of the most important causes of infertility, however, bacteria frequently exist in seminal fluid. Sperm express Toll-like receptors (TLRs) on their cell surfaces and bacterial recognition by TLRs induces sperm apoptosis. In this study, we examined the lactoferrin (LF) potentiality on sperm apoptosis induced by bacterial lipopolysaccharide (LPS). The TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay indicated that TUNEL-positive sperm cells were scarce in the group treated with LF and LPS (LF/LPS group) compared to the group treated with LPS only (LPS group). In addition, real-time RT-PCR detected lower mRNA expression levels of apoptosis-associated genes in the LF/LPS group compared to the LPS group. These results indicate that LF treatment of semen might decrease LPS-induced apoptosis of sperm.     

Novel polymorphism of the canine dopamine receptor D4 gene intron II region

Various dog breeds are remarkably different from each other not only in their sizes and shapes but also in behavioral traits, suggesting that some of these characteristics are under genetic control. However, little is known about genes related to behavioral traits in canine species. In humans, it has been reported that the dopamine receptor D4 gene (DRD4) includes polymorphism at several regions that relate to personality or psychiatric disorders. In an earlier study by the authors of the present study, the polymorphisms in canine DRD4 exon III and exon I regions were reported. In the present study, a novel polymorphism in canine DRD4 intron II was found based on a 17 base pair insertion/deletion, and the two alleles detected were named P (shorter allele) and Q (longer allele). The allelic distribution in 28 breeds of dog, including a total of 1114 unrelated individuals, were then investigated. Both P and Q alleles were detected in most of the breeds investigated; however, the frequencies of P and Q differed greatly between breeds. With respect to classification based on breed origin, P and Q alleles were frequent in Occidental and Oriental breeds, respectively. Furthermore, two subspecies of wolves, the ancestors of dogs, were analyzed for the comparison of allele frequencies with dogs, and the P allele was predominant in both European and Chinese wolves.