Fertilization Ability of Porcine Oocytes Reconstructed from Ooplasmic Fragments Produced and Characterized after Serial Centrifugations

Mitochondria are reported to be critical in in vitro maturation of oocytes and subsequent embryo development after fertilization, but their contribution for fertilization has not been investigated in detail. In the present study, we investigate the contribution of mitochondria to fertilization using reconstructed porcine oocytes by fusion of ooplasmic fragments produced by serial centrifugations (centri-fusion). Firstly, we evaluated the characteristics of ooplasmic fragments. Three types of fragments were obtained by centrifugation of porcine oocytes matured in vitro for 46 h: brownish (B), transparent (T) and large (L) fragments containing both B and T parts in a fragment. The production efficiencies of these types of fragments were 71.7, 91.0 and 17.8 fragments/100 oocytes, respectively. In experiments, L fragments were excluded because they contained both brownish and transparent components that were apparently intermediate between B and T fragments. Observations by confocal microscopy after staining with MitoTracker Red CMXRos^® and transmission electron microscopy revealed highly condensed active mitochondria in B fragments in contrast to T fragments that contained only sparse organelles. We reconstructed oocytes by fusion of a karyoplast and two cytoplasts from B and T fragments (B and T oocytes, respectively). The B oocytes showed higher sperm penetration (95.8%) and male pronuclear formation rates (94.2%) by in vitro fertilization than T oocytes (66.7% and 50.0%, respectively). These results suggest that the active mitochondria in oocytes may be related to their ability for fertilization.     

Prevention of thaw-rigor during frozen storage of bigeye tuna Thunnus obesus and meat quality evaluation

Thaw-rigor is often found in frozen meat of bigeye tuna Thunnus obesus. Excessive amounts of drip loss and stiffness greatly lower the commercial value of tuna meat. In order to prevent thaw-rigor in meat stored at −60°C post-capture, we adapted a temperature shift technique that stores the meat at −7°C for 1 day or −10°C for 7 days before thawing. Biochemical changes in muscle of bigeye tuna before and after the temperature shift to −7 or −10°C were characterized. Contents of ATP, NAD⁺, glycogen, and creatine phosphate decreased after the temperature shift. NAD⁺ levels decreased faster than ATP levels and were highly correlated with the rigor index. Thaw-rigor occurred in muscle containing NAD⁺ at 1 μmol/g and ATP at 7 μmol/g. On the other hand, the meat color of tuna during frozen storage changed to brown depending on the storage temperature and reflected the rate of metmyoglobin (met-Mb) formation. Met-Mb formation increase was dependent on the decrease in NADH levels during the frozen storage. A temperature shift technique with storage at −7°C for 1 day or −10°C for 7 days before thawing prevented thaw-rigor and met-Mb formation.     

Association between energy status early postpartum and subsequent embryonic mortality in high‐yielding recipient cows

High‐yielding Holstein‐Friesian recipients (n = 43) were used in order to investigate the relation between energy balance status during the early postpartum period and subsequent embryonic mortality after transferring good‐quality frozen embryos. Blood samples were collected during the second, third, fourth, fifth, sixth and seventh weeks postpartum in order to measure energy status indicators. These indicators include β hydroxyl butyric acid (BHBA), non‐esterified fatty acid (NEFA), total cholesterol (T‐chol), glucose and blood urea nitrogen (BUN). Moreover, body condition scores (BCS) were assessed during the same period. Pregnancy diagnosis by ultrasonography at the 28th day postestrus and embryo viability was ascertained until 45 days postestrus in order to detect late embryonic mortality (LEM). The pregnancy rate on day 28 was 44.2% (19/43); however, five cows (11.6%) experienced LEM by day 45. Based on the non‐return rate at day 24, non‐pregnant animals, as diagnosed by ultrasonography, were allocated into animals with longer estrus intervals than 24 days (32.5%; mid‐embryonic mortality (MEM) group) and animals returning to estrus by day 24 postestrus (23.5%; early embryonic mortality (EEM) group). At week 5 postpartum, BCS was significantly (P < 0.05) lower in the LEM group than that of pregnant (PREG), EEM and MEM groups. NEFA was significantly higher in animals that experienced LEM (LEM group) at week 7 postpartum (289.6 ± 47.0 µEq/L; P < 0.01) than that of PREG (196.8 ± 16.0 µEq/L), EEM (157.2 ± 18.6 µEq/L) and MEM groups (191.5 ± 14.4 µEq/L). In conclusion, lower BCS at week 5 postpartum and higher NEFAs at week 7 postpartum may be associated with subsequent LEM in high‐yielding recipient cows.     

Increase of ruminal fiber digestion by cellobiose and a twin strain of Saccharomyces cerevisiae live cells in vitro

This experiment was designed to investigate the effects of different concentrations of cellobiose (CB) or a twin strain of Saccharomyces cerevisiae live cells (YST) (20, 40 and 60 mg/60 mL), and CB + YST (60 + 20, 60 + 40, 60 + 60 mg/60 mL) on mixed ruminal microorganism fermentation in vitro. Ruminal fluid was collected from a cow, mixed with phosphate buffer (1:2) and incubated (60 mL) anaerobically at 38°C for 24 h with or without supplement plus 400 mg (dry matter [DM] basis) substrate (hay plus concentrate, 1.5:1). The medium pH numerically decreased with CB and CB + YST, but was unchanged with YST. The total volatile fatty acid and proportion of propionate increased (P < 0.05) in all cases. The proportion of acetate decreased (P < 0.05) with CB and CB + YST, but increased (P < 0.05) with YST and that of butyrate increased (P < 0.05) with CB and CB + YST, but decreased (P < 0.05) with YST. Ammonia‐N decreased (P < 0.05) with CB and CB + YST, but was unchanged with YST. The number of protozoa was unchanged, and that of cellulolytic bacteria increased (P < 0.05) in all cases. Total gas production increased (P < 0.05) in all cases. Methane decreased, hydrogen was unchanged by YST and both gases were unchanged by CB and CB + YST. The in vitro disappearance of DM and neutral detergent fiber increased (P < 0.05) by 11.2% and 8.9%, 9% and 8.5%, and 12.1% and 10.2% in the case of CB, YST and CB + YST, respectively. Therefore, the dietary supplementation of CB and/or YST may improve ruminal fermentation and digestibility.     

Sulfated glycofuranans as inhibitors of melanoma lung metastasis

Chemically synthesized (1 5)--d-glucofuranan, (1 5)--d-galactofuranan, (1 5)--d-xylofuranan, (1 5)--L-arabinofuranan, natural xylan, and curdlan were sulfated to investigate their inhibitory activities on B16-BL6 lung metastasis and anticoagulant activities. (1 5)--d-Glucofuranan sulfate, (1 5)--d-galactofuranan sulfate, xylan sulfate, and curdlan sulfate had binding abilities with B16-BL6 melanoma lysate. The inhibitory activities of sulfated polysaccharides on B16-BL6 lung metastasis selected by heparin binding assay were in the order (1 5)--d-galactofuranan sulfate > (1 5)---d-glucofuranan sulfate > xylan sulfate curdlan sulfate. Furthermore, (1 5)--d-galactofuranan sulfate, (1 5)--d-glucofuranan sulfate, and xylan sulfate had not only high inhibitory activity on B16-BL6 lung metastasis but also low anticoagulant activity. The correlation between chemical structure and biological activity is discussed.Part of this paper was presented at the 10th International Synposium on Wood and Pulping Chemistry, Yokohama, Japan, June 1999     

Sapling architecture and growth in the co-occurring species Castanopsis cuspidata and Quercus glauca in a secondary forest in western Japan

This study quantitatively compared the sapling (height 62–289cm) architecture and growth of Castanopsis cuspidata and Quercus glauca, both of which are major components in the temperate zone of western Japan, under shaded light conditions in secondary forest. When the sapling architectures were compared at the same support mass (trunk + branch mass), C. cuspidata had a larger crown area but a smaller height gain than did Q. glauca owing to the allocation of more biomass to lateral branches in C. cuspidata. The above-ground relative growth rate (RGR) of C. cuspidata (0.442gg^–1 year^–1) was nearly twice that of Q. glauca (0.256gg^–1year^–1), primarily as a result of a greater total leaf area per above-ground biomass (LAR) in C. cuspidata (56cm²g^–1) as compared to Q. glauca (33cm²g^–1). Because it has a disadvantage in height gain, related to its allocation pattern of biomass, C. cuspidata realized the same height growth (RGR_H) as Q. glauca, despite the large biomass production. The great potential for photosynthesis in C. cuspidata, which results from its vigorous lateral spreading, is presumed to give it a long-term advantage over Q. glauca in the shaded forest understory. Q. glauca invests preferentially in trunk biomass, possibly giving it an advantage in sunny sites as opposed to a shaded forest understory.