Improved survival of vitrified in vivo-derived porcine embryos

An efficient cryopreservation protocol for porcine morulae was investigated with three types of vitrification having different cooling rates (Exp. 1). Survival of embryos vitrified after removal of cytoplasmic lipid droplets was also examined by means of the minimum volume cooling (MVC) method (Exp. 2). In Exp. 1, the morula stage embryos were vitrified with a 0.25 ml plastic straw (ST-method), gel loading tip (GLT-method) and the MVC-method, respectively, and stored in liquid nitrogen after which they were warmed in sucrose solutions with cryoprotectants being subsequently removed in a stepwise manner. In Exp. 2, morulae were centrifuged with 7.5 microg/ml cytocharasin B at 12000 x g for 20 min to polarize the cytoplasmic lipid droplets that were then removed from the embryos by micromanipulation (delipation). Both those delipated at the morula stage and the intact embryos at the morula to blastocyst stages were vitrified by the MVC-method. In vitro survival of the vitrified embryos was assessed in both experiments by culturing in NCSU-23 + 10% FCS for 48 h. In vitro developments of vitrified embryos after warming to blastocysts were 20% (6/30) for the ST-method, 39% (18/46) for the GLT-method, and 60% (26/43) for the MVC-method. Embryo survival was further improved by vitrification after delipation (95%, 35/37) compared to intact vitrified morulae (24/42, 57%, P<0.001) and blastocysts (23/31, 74%, P<0.05). Moreover, the number of cells in blastocysts (92 +/- 25) derived from the delipated-vitrified morulae was comparable to those derived from intact control non-vitrified embryos (103 +/- 31). Our results demonstrate that vitrified porcine morulae have the highest survival when using the MVC-method in conjunction with delipation.     

Contribution of insulin to the ascorbate recycling system in the chicken liver

The effect of insulin on the ascorbate recycling system in the chicken liver was examined. First, insulin was injected subcutaneously into the chicken, and liver glutathione‐dependent dehydroascorbate reductase (GSH‐DHAR) activity was determined. Insulin increased liver GSH‐DHAR activity, but did not affect plasma and liver ascorbate concentration. Dehydroascorbate increased plasma and liver ascorbate levels, and liver GSH‐DHAR activity. However, distinct changes in plasma insulin level were not observed by dehydroascorbate injection. In addition, reduction of external dehydroascorbate in cultured chicken hepatocytes could not be observed in an insulin‐deprived culture, although the cells reduced external dehydroascorbate in a serum‐free culture with insulin. We concluded that insulin affects the ascorbate recycling system as an essential factor in the chicken liver.     

Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen

To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA.     

Immunostimulating and growth-promoting effects of sugar cane extract (SCE) in chickens

Polymorphonuclear cells of the peripheral blood in the chicken significantly increased their phagocytosis when cultured with sugar cane extract (SCE; 250-1,000 microg/ml) for 24 hr. Chickens orally administered SCE (500 mg/kg/day) for 3 or 6 consecutive days at 1 week of age showed significantly higher body weight and gain in body weight/day and a lower food conversion ratio within the growing period of 6 weeks than physiological saline-administered control chickens. Furthermore, oral administration of SCE also resulted in significantly higher immune responses against sheep red blood cells and Brucella abortus. These results suggest that SCE has immunostimulating and growth promoting effects in chickens.     

Sugar expressions on the vaginal epithelium in pregnant mice

Sugar expressions were examined on the epithelium of both the middle portion of the vagina and the vaginal portion of the cervical canal (CC) in pregnant mice to understand the pathogenesis of bacterial infection in the female reproductive organ by using a panel of lectins. As a result, N-acetylglucosamine was positive before pregnant day (P) 7 but negative after P10 and at diestrus on both the vagina and the CC. In addition, some differences in sugar expressions were seen between them. These results suggest that sugar expressions on the mucosal surface would change not only site-specifically but also time-dependently, and these sugar differences indicate the possibility of the alteration of the settled bacterial species on the vaginal mucosa in pregnancy.     

Immunohistochemical study on the delayed progression of epithelial apoptosis in follicle-associated epithelium of rat Peyer's patch

It is well known that some caspases in apoptosis is involved in determinant of terminal differentiation and maturation of various cells. Our previous study ultrastructurally clarified the differentiation into M cells from immature microvillous epithelial cells and the redifferentiation from M cells to microvillous epithelial cells in the follicle-associated epithelium (FAE) of rat Peyer's patch. In this study, the difference of epithelial apoptosis between the FAE of Peyer's patch and intestinal villi was immunohistochemically investigated in rat jejunoileum. As a result, cleaved caspase-3 was limited to several epithelial cells at the tip of FAE, whereas almost all of the epithelial cells were cleaved caspase-3 positive in intestinal villi. Cleaved caspase-9 was detected only in a few exfoliating or exfoliated epithelial cells of both FAE and intestinal villi. Nuclear DNA-fragmentation was detected only in several epithelial cells of the tip of FAE, while it was expressed from the middle regions in the intestinal villi. The DNase I expression of the epithelial cytoplasm was much weaker in FAE than in intestinal villi. Bcl-x expression was restricted in the apical cytoplasms of epithelial cells in the FAE, whereas it was restricted in whole cytoplasms in villous epithelial cells. These findings suggest that the progression of the apoptotic process in the epithelial cells of FAE is later than in the intestinal villi, so that the possibility of epithelial differentiation might be remained in the FAE, unlike in the intestinal villi.     

Effect of infection timing on fusarium head blight and mycotoxin accumulation in open- and closed-flowering barley

ABSTRACT Barley has two flowering types, chasmogamous (open-flowering) and cleistogamous (closed-flowering). We examined the effect of the timing of Fusarium graminearum infection on Fusarium head blight (FHB) and mycotoxin accumulation in barley cultivars with different flowering types using greenhouse experiments. In the first experiment, 13 cultivars were spray inoculated at two different developmental stages, and the severity of FHB was evaluated. The effect of the timing of infection differed among cultivars. Cleistogamous cultivars were resistant at anthesis but susceptible at 10 days after anthesis, whereas chasmogamous cultivars were already susceptible at anthesis. In the second experiment, five cultivars were inoculated at three different developmental stages and the concentrations of deoxynivalenol (DON) and nivalenol (NIV) in mature grain were analyzed. Cleistogamous cultivars accumulated more mycotoxins (DON and NIV) when inoculated 10 or 20 days after anthesis than when inoculated at anthesis, whereas chasmogamous cultivars accumulated more mycotoxins when inoculated at anthesis. Thus, the most critical time for F. graminearum infection and mycotoxin accumulation in barley differs with cultivar, and likely is associated with the flowering type. Late infection, even without accompanied FHB symptoms, was also significant in terms of the risk of mycotoxin contamination.     

Phospholipase A<Subscript>1</Subscript> activity of crude enzyme extracted from the ovaries of skipjack tuna

The phospholipid class composition, fatty acid composition and phospholipase A₁ (PLA₁) activity from the ovaries of skipjack tuna were compared with those of six other species of marine fish. In the skipjack ovaries, the lysophosphatidylcholine (LPC) proportion for the phospholipid, the docosahexaenoic acid (DHA) percentage for the total fatty acids of the phospholipids and the PLA₁ activity of the crude enzyme were the highest among those of the seven species. The optimum pH and temperature for the PLA₁ activity of the crude enzyme from the skipjack ovaries were in the range of pH 6–7 and 20–30°C, respectively, and calcium ions were not required. As a substrate, phosphatidylcholine was more easily hydrolyzed than phosphatidylethanolamine by this enzyme, and the plasmalogen-type phospholipid was much lower than the acyl-type phospholipid. After a 6-h hydrolysis reaction of the purified phospholipid extracted from the mixed ovaries of skipjack and yellowfin tuna by this enzyme, the LPC ratio of the phospholipid increased from 20 to 72.6% and the percentage of DHA for the total fatty acids of the phospholipid also increased. Thus, skipjack ovaries might possibly be used as a source of PLA₁.     

The agronomic benefit of phosphate rock application with elemental sulfur depends on the reactivity and fertilizer placement

Abstract

A possible way to improve phosphate rock (PR) agronomic performance is through the addition of elemental sulfur (S⁰). We used ³²P isotope dilution method to assess the P taken up by crops treated with PR. Two experiments, one with common bean and other with upland rice, were carried out, to evaluate the effect of S⁰ on the agronomic performance of two contrasting PR, applied in different methods. Gafsa (GPR) and Patos de Minas (PPR) were used as the high and low reactivity PR, respectively. The experiments were arranged in completely randomized design on factorial structure 2?×?2 × 2?+?2; which means 2 PR (GPR and PPR) versus 2?S⁰ condition (with or without-S⁰) versus 2 application methods (band and broadcast) plus 2 additional treatments (control and triple superphosphate). In band application the S⁰ increased the amount of P uptake by plants from fertilizer of GPR from 2.57 to 9.86?mg pot^?1 for common bean and of 2.26 to 7.05?mg pot^?1 to rice. Regardless the management adopted, less than 2% of P applied as PPR has been taken up by crops. The addition of S⁰ as a strategy to increase the agronomic performance of PR is PR characteristics dependent and fertilizers placement.