Involvement of Ca2+-ATPase in suppressing the appearance of bovine helically motile spermatozoa with intense force prior to cryopreservation

In cattle, cryopreserved spermatozoa are generally used for artificial insemination (AI). Many of these specimens exhibit helical movement, although the molecular mechanisms underlying this phenomenon remain unclear. This study aimed to characterize helically motile spermatozoa, investigate the involvement of Ca²⁺-ATPase in suppressing the appearance of these spermatozoa prior to cryopreservation, and examine the potential of helical movement as an index of sperm quality. In the cryopreserved semen, approximately 50% of spermatozoa were helically motile, whereas approximately 25% were planarly motile. The helically motile samples swam significantly faster than those with planar movement, in both non-viscous medium and viscous medium containing polyvinylpyrrolidone. In contrast, in non-cryopreserved semen, planarly motile spermatozoa outnumbered those that were helically motile. Fluorescence microscopy with Fluo-3/AM and propidium iodide showed that flagellar [Ca²⁺]_i was significantly higher in cryopreserved live spermatozoa than in non-cryopreserved live ones. The percentage of non-cryopreserved helically motile spermatozoa was approximately 25% after washing, and this increased significantly to approximately 50% after treatment with an inhibitor of sarcoplasmic reticulum Ca²⁺-ATPases (SERCAs), “thapsigargin.” Immunostaining showed the presence of SERCAs in sperm necks. Additionally, the percentages of cryopreserved helically motile spermatozoa showed large inter-bull differences and a significantly positive correlation with post-AI conception rates, indicating that helical movement has the potential to serve as a predictor of the fertilizing ability of these spermatozoa. These results suggest that SERCAs in the neck suppress the cytoplasmic Ca²⁺-dependent appearance of helically motile spermatozoa with intense force in semen prior to cryopreservation.     

戊型肝炎病毒(HEV)猪间传播的动力学分析——对人类感染的潜在影响

【目的】戊型肝炎病毒(Hepatitis Evirus,HEV)感染是一种人畜共患病,而猪是HEV的病毒储库。在日本,HEV感染已成为猪的地方性猪病。阐明HEV在猪场中的详细传播机制,将有利于人们掌握特定年龄段猪群的感染状况,尤其是肉猪在屠宰前的感染状况。【结果】本文,作者重新分析了源于日本猪场的一个大型猪HEV血清阳性率数据集,以此评估HEV的感染力。结果表明,北海道、本州和九州三个地区猪场中的猪HEV感染力分别为3.45×10-2/d(95%的置信区间(下同):3.17～3.75×10-2/d)、2.68×10-2/d(2.28～3.14×10-2/d)和3.11×10-2/d(2.76～3.50×10-2/d)。利用作者建立的模型得到的估测结果表明,猪感染HEV的平均日龄在59.0～67.3d之间,其基本传染数R0按4.02～5.17的数量级递增。对特定年龄猪群在不同感染力下的敏感性分析显示,HEV感染力下降将提高猪感染HEV的年龄,而且会增加180日龄时散毒猪的数量。【结论】尽管作者的研究表明95%以上的猪是在150日龄前被感染的,但该模型显示感染力下降可以增加感染猪将HEV传播给人的风险。如果感染力开始下降,就有必要采取基本的应对策略(如在哺乳期末将未感染猪与感染猪分开)以最大限度地减少肥育阶段的HEV阳性猪数量。     

Strong antiviral activity of heated and hydrated dolomite--preliminary investigation

Heated and hydrated naturally occurring dolomite showed very strong antiviral activity. Infectivity of avian and human influenza, avian infectious bronchitis (coronavirus), Newcastle disease (paramyxovirus) and avian laryngotracheitis (herpesvirus) viruses dropped at least 1,000 fold following contact with the dolomite for five minutes at 4 degrees C. Dolomite is expected to be useful to inhibit the incidence of emerging and re-emerging infectious diseases.     

Effects of synchronization of donor cell cycle on embryonic development and DNA synthesis in porcine nuclear transfer embryos

The relationship between donor cell cycle and the developmental ability of somatic cell nuclear transfer (SCNT) embryos has not fully been elucidated. Donor cells that are usually prepared by serum starvation or confluent-cell culture for SCNT represent a heterogeneous population that includes mainly G0 phase cells, other cells in different phases of the cell cycle and apoptotic cells. In this study, we compared the developmental ability of porcine SCNT embryos reconstructed from G0 phase cells (G0-SCNT embryos) and strictly synchronized-G1 phase cells (G1-SCNT embryos), and examined the developmental rates and timing of first DNA synthesis. The G0 phase cells were synchronized by confluent culture, and the G1 phase cells were prepared from actively dividing M phase cells. The G1-SCNT embryos showed a significantly higher (P<0.05) developmental rate to the blastocyst stage per cleaved embryo (59%) than the G0-SCNT embryos (43%). Moreover, initiation of first DNA synthesis and cleavage occurred significantly earlier in the G1-SCNT embryos than in the G0-SCNT embryos. Delay of initiation of first DNA synthesis in the SCNT embryos by aphidicolin resulted in decreased developmental rates to the blastocyst stage without any effect on cleavage rates. Our data demonstrates that synchronized-G1 phase cells can be used as donor cells for SCNT embryos and that earlier initiation of first DNA synthesis may be important for subsequent development of SCNT embryos. The SCNT system using G1-synchronized cells, in terms of their highly uniform and viable cell states, can be useful for studying the reprogramming processes and embryonic development of SCNT embryos.     

Contribution of mutation I142M in fusion protein and Q44R in matrix protein of Newcastle disease virus to virulence in ducks

Although verogenic Newcastle disease viruses (NDVs) generally cause subclinical infection in waterfowls such as ducks, NDVs with high virulence in waterfowl have been sporadically reported. We previously reported that the NDV d5a20b strain, which is obtained by serial passaging of the velogenic 9a5b strain in domestic ducks, showed increased virulence in ducks (Hidaka et al., 2021). The d5a20b strain had 11 amino acid substitutions in its P/V, M, F, HN, and L proteins as compared to 9a5b. In the present study, we generated a series of recombinant (r) NDVs with these amino acid substitutions to identify the molecular basis of virulence of NDV in ducks, and evaluated their influences on virulence and in vitro viral properties. Each of the single amino acid substitutions in either the F protein I142M or the M protein Q44R contributed to the enhancement of intracerebral and intranasal pathogenicity in domestic ducks. The cell-cell fusion activity of the virus with F I142M was five times higher than that of the parental r9a5b. The virus with M Q44R rapidly replicated in duck embryo fibroblasts. Additionally, the rM+F+HN strain, which has the same amino acid sequences as d5a20b in M, F, and HN proteins, showed the highest level of virulence and replication efficiency among the generated recombinant viruses, nearly comparable to rd5a20b. These results suggest that multiple factors are involved in the high growth ability of NDV in duck cells, leading to increased virulence in vivo.     

GM1 gangliosidosis in a Japanese domestic cat: a new variant identified in Hokkaido,Japan

A male Japanese domestic cat with retarded growth in Hokkaido, Japan, showed progressive motor dysfunction, such as ataxia starting at 3 months of age and tremors, visual disorder and seizure after 4 months of age. Finally, the cat died of neurological deterioration at 9 months of age. Approximately half of the peripheral blood lymphocytes had multiple abnormal vacuoles. Magnetic resonance imaging showed bisymmetrical hyperintensity in the white matter of the parietal and occipital lobes in the forebrain on T2-weighted and fluid-attenuated inversion recovery images, and mild encephalatrophy of the olfactory bulbs and temporal lobes. The activity of lysosomal acid β-galactosidase in leukocytes was negligible, resulting in the biochemical diagnosis of GM1 gangliosidosis. Histologically, swollen neurons characterized by accumulation of pale, slightly granular cytoplasmic materials were observed throughout the central nervous system. Dysmyelination or demyelination and gemistocytic astrocytosis were observed in the white matter. Ultrastructually, membranous cytoplasmic bodies were detected in the lysosomes of neurons. However, genetic analysis did not identify the c.1448G>C mutation, which is the single known mutation of feline GM1 gangliosidosis, suggesting that the cat was affected with a new variant of the feline disease.     

Ability of salmon growth hormone to accelerate somatic growth of juvenil abalone Haliotis discus hannai

ABSTRACT:   The ability of salmon growth hormone (sGH) to accelerate the somatic growth of juvenile abalone Haliotis discus hannai by immersion was examined. After immersion for one hour into a sGH-rich solution at a concentration of 30 mg/L, the sGH immunoreactivity in the body fluid of abalone was maximal after one day, and levels were still detectable at two days. No immunoreactivity was observed in the control group. Following immersion for one hour at one or two-week intervals for 120 weeks into the sGH-rich solution at 30 mg/L, the sGH-immersed abalone exhibited a significant increase in shell length and body weight. On the other hand, abalone immersed into a salmon prolactin (sPRL)-rich solution at 30 mg/L for one hour showed a lower increase in both shell length and body weight than the sGH-immersed abalone. No increases in shell length and body weight were observed in either the bovine serum albumin-immersed abalone or untreated controls. These results provide evidence that sGH can be transported from ambient water into the circulatory system of abalone, and can subsequently improve the somatic growth of juveniles.     

Identification of reaction products of acylated anthocyanins from red radish with peroxyl radicals

Red radish anthocyanin extract, which consists of 12 known acylated anthocyanins, was reacted with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) to generate peroxyl radicals under acidic pH conditions at 37 degrees C. The reaction products were isolated using preparative HPLC, and their chemical structures were determined to be p-hydroxybenzoic acid (1), 6-O-(E)-p-coumaroyl-2-O-beta-d- glucopyranosyl-alpha-d-glucopyranoside (3), p-coumaric acid (4), 6-O-(E)-feruloyl-2-O-beta-d-glucopyranosyl-alpha-d-glucopyranoside (5), and ferulic acid (6). Some products were not identified. HPLC analyses of the mixture of acylated pelargonidin isolated from red radish and AAPH revealed that the acylated pelargonidins possess the radical scavenging ability on some common sites even if the characteristics of the intramolecular acyl units are different. Degradation rates of acylated pelargonidins and the formation rates of the resulting reaction products were found to be quite different.