In vivo characteristics of a transplantable Marek's disease lymphoblastoid cell line, MDCC-MSB1-41C

A Marek's disease (MD) lymphoblastoid cell line, MDCC-MSB1-41C, was highly transplantable and lethal for chickens. Autopsies showed extensive metastasis in various organs. The transplantabilities of the parent cell line, MDCC-MSB1, and another derivative line, MDCC-MSB1-33C, were transient. MD virus (MDV) could be isolated from the kidneys but not from the peripheral blood leukocytes of chickens inoculated with the MSB1-41C cell line. In addition, anti-MDV antibodies were produced both in chickens inoculated with this cell line and in controls raised with inoculated chickens, but several attempts to isolate MDV from this cell line in vitro failed.     

Sex differences in metabolic rate and swimming performance in pink salmon (Oncorhynchus gorbuscha): the effect of male secondary sexual traits

Do secondary sexual traits, such as large dorsal hump and hooked snout, decrease the swimming efficiency of male pink salmon during freshwater migration? This is the first study to address the effects of secondary sexual traits in pink salmon on oxygen uptake and swimming capacity. We conducted a laboratory experiment using a swimming respirometer and a field study using electromyogram (EMG) telemetry in the Shibetsu River, Hokkaido, Japan. We compared the relationship between MO₂ (mg O₂·kg^?1·h^?1) and swimming velocity U (m·s^?1) in male and female fish, and also investigated the effects of morphological traits (secondary sexual characters) on the relationship between MO₂ (mg O₂·kg^?1·h^?1) and swimming velocity U (m·s^?1). Additionally, we compared energy costs and swimming behaviour during upstream migration between male and female pink salmon. The laboratory experiment revealed that MO₂ exponentially increased with increasing U; this increase was described by MO₂ = 167.9e^1.23U for males and 144.9e^1.14U for females. Linear mixed models found that hump height and the upper jaw length in males significantly and positively affected the relationship between MO₂ and U; no effect was found in females. The field study found that swimming velocity for both sexes estimated from EMG calibration was lower than optimal swimming velocity (U_opt) calculated from the laboratory experiment. We suggest that pink salmon in the Shibetsu River do not swim at the optimal swimming velocity because of the short migration distance involved (20 km).     

Effects of ovary storage time and temperature on DNA fragmentation and development of porcine oocytes

The present study was conducted to investigate the effects of storage time and temperature of porcine ovaries on the quality and nuclear maturation in vitro of oocytes obtained from stored ovaries and their subsequent development after in vitro fertilization. The ovaries were stored in physiological saline for 0, 3, 6, 9 and 12 h at various temperatures (4, 15, 25 and 35 C). The pH of follicular fluid obtained from the ovaries, DNA fragmentation of the oocyte nucleus and meiotic competence of oocytes were examined. Some oocytes from ovaries stored at 15, 25 and 35 C for 6 h were fertilized in vitro, and then cultured for 7 days to examine the ability of embryos to develop to the blastocyst stage. When the ovaries were stored at 35 C, the pH of follicular fluid decreased and the proportions of oocytes with DNA fragmented nuclei increased as the storage time was prolonged, and the storage of ovaries for 6, 9 and 12 h resulted in lower maturation rates of oocytes. When the ovaries were stored at 4, 15, 25 and 35 C for 6 h, the storage at higher temperatures (> or =15 C) decreased the pH of follicular fluid and induced nucleic DNA fragmentation in higher proportions of oocytes. None of the oocytes from ovaries stored at 4 C reached metaphase II. The storage of ovaries at 15 C reduced the rates of in vitro fertilized oocytes and subsequent embryo development, but there were no significant differences in the rates of fertilization and blastocyst formation between oocytes from ovaries stored at 25 C and 35 C. Our findings indicate that the storage of ovaries at 25-35 C for 6 h is effective for maintaining the developmental competence of porcine oocytes even though the development rates were lower than those of ovaries stored at 35 C for 3 h.     

Effects of voltage strength during electroporation on the development and quality of in vitro‐produced porcine embryos

This study was conducted to determine suitable conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro‐produced porcine zygotes by electroporation. In the first experiment, when putative zygotes derived from in vitro fertilization (IVF) were electroporated by either unipolar or bipolar pulses, keeping the voltage, pulse duration and pulse number fixed at 30 V/mm, 1 msec and five repeats, respectively, the rate of blastocyst formation from zygotes electroporated by bipolar pulses decreased compared to zygotes electroporated by unipolar pulses. In the second experiment, the putative zygotes were electroporated by electroporation voltages ranging from 20 V/mm–40 V/mm with five 1‐msec unipolar pulses. The rate of cleavage and blastocyst formation of zygotes electroporated at 40 V/mm was significantly lower (p < .05) than that of zygotes electroporated at less than 30 V/mm. Moreover, the apoptotic nuclei indices of blastocysts derived from zygotes electroporated by voltages greater than 30 V/mm significantly increased compared with those from zygotes electroporated by voltages less than 25 V/mm (p < .05). When zygotes were electroporated with Cas9 mRNA and single‐guide RNA (sgRNA) targeting site in the FGF10 exon 3, the proportions of blastocysts with targeted genomic sequences were 7.7% (2/26) and 3.6% (1/28) in the embryos derived from zygotes electroporated at 25 V/mm and 30 V/mm, respectively. Our results indicate that electroporation at 25 V/mm may be an acceptable condition for introducing Cas9 mRNA and sgRNA into pig IVF zygotes under which the viability of the embryos is not significantly affected.     

Changes in plasma concentrations of S100A7 and S100A8 in dairy cows during pregnancy

This study was carried out to examine the changes in plasma concentrations of the Ca‐binding antimicrobial proteins S100A7 and S100A8 during pregnancy in dairy cows. Holstein Friesian cows (n = 19) were inseminated with Holstein Friesian semen. Blood was collected at days 30, 60, 90, 120, 150, 180, 210, 240 and 270 after insemination. Plasma was used for measuring the concentrations of S100A7 and S100A8. Both S100A7 and S100A8 concentrations showed similar patterns during gestation; they increased during the midgestation, between days 90 and 180, and then declined before calving. The findings indicated that plasma concentrations of S100A7 and S100A8 did not change significantly during pregnancy in cows. Further studies are required to determine the roles of S100A7 and S100A8 in physiological function during pregnancy in dairy cows.     

Effects of Plant Species on CH4 and N2O Fluxes from a Volcanic Grassland Soil in Nasu, Japan

To investigate the effects of plant species in grassland on methane (CH₄) and nitrous oxide (N₂O) fluxes from soil, fluxes from an orchardgrass ( Dactylis glomerata L.) grassland, white clover ( Trifolium repens L.) grassland and orchardgrass/white clover mixed grassland were measured weekly from April 2001 to March 2002 using a vented closed chamber method. Related environmental parameters (soil inorganic N content, soil pH (H₂O) value, soil moisture content, soil temperature, grass yield, and the number of soil microorganisms) were also regularly monitored. On an annual basis, CH₄ consumption in the soil of the orchardgrass grassland, white clover grassland and orchardgrass/white clover mixed grassland was 1.8, 2.4, and 1.8 kg C ha⁻¹ year⁻¹, respectively. The soil bulk density of the white clover grassland was lower than that of the other grasslands. Fluxes of CH₄ were positively correlated with the soil moisture content. White clover increased the CH₄ consumption by improving soil aeration. Nitrogen supply to the soil by white clover did not decrease the CH₄ consumption in the soil of our grasslands. On the other hand, annual N₂O emissions from the orchardgrass grassland, white clover grassland, and orchardgrass/white clover mixed grassland were 0.39, 1.59, and 0.67 kg N ha⁻¹ year⁻¹, respectively. Fluxes of N₂O were correlated with the NO₃⁻ content in soil and soil temperature. White clover increased the N₂O emissions by increasing the inorganic N content derived from degrading white clover in soil in summer.     

Seasonal variations in the concentration of antimicrobial components in milk of dairy cows

The incidence of bovine mastitis and the bulk milk somatic cell count (BMSCC) are influenced by season, which may be associated with innate immune functions, including antimicrobial components in mammary glands. Therefore, the present study was conducted to examine the effect of season on antimicrobial components in milk. Rectal temperature and plasma cortisol, thyroxine, and derivatives of reactive oxygen metabolites (d‐ROMs) were measured as stress parameters. Concentrations of lactoferrin (LF), lingual antimicrobial peptide (LAP), psoriasin (S100A7), and Immunoglobulin A (IgA) in milk were measured as indicators of innate immune function. LF and LAP concentrations were significantly lower in summer than in winter and spring, respectively, whereas the concentration of S100A7 was significantly lower in winter than in spring and autumn. The rectal temperature was significantly higher in summer than in other seasons, whereas plasma cortisol, thyroxine, and d‐ROMs did not exhibit any seasonal variation. In conclusion, even though stress parameters were not changed, the concentration of antimicrobial components, such as LF and LAP, decreased in summer, which may explain the frequent occurrence of mastitis during this season.