Intratesticular expression of mRNAs of both interferon γ and tumor necrosis factor α is significantly increased in experimental autoimmune orchitis in mice

Experimental autoimmune orchitis (EAO) is one of the models of immunological male infertility. Murine EAO is CD4+T cell-dependent and classically induced by immunization with a testicular homogenate and adjuvants. We previously established that immunization with viable syngeneic testicular germ cells (TGC) can also induce murine EAO with no use of any adjuvant. Analyses of this EAO model have already revealed that cultured spleen cells of immunized mice secreted interferon (IFN)-γ and that treatment of the immunized mice with anti-IFN-γ monoclonal antibodies significantly suppressed the EAO. It is known that both IFN-γ and tumor necrosis factor (TNF)-α are representative cytokines of Th1 cells and exhibit local toxicity toward the seminiferous epithelium in vivo. However, changes in these two cytokines in EAO-affected testes have not yet been investigated. Therefore, in the present study, we investigated the expression of intratesticular IFN-γ and TNF- α mRNAs in TGC-induced EAO using real-time RT-PCR. The results demonstrated that the intratesticular mRNAs for both IFN-γ and TNF-α significantly increased, while other cytokines such as IL-1α, IL-1β, IL-6 and TGF-β did not show dramatic changes in the immunized mice. These results suggest that secretion of significant amounts of IFN-γ and TNF-α in situ contributes to the spermatogenic disturbance in EAO.     

Analysis of radiosensitivity of cancer stem‐like cells derived from canine cancer cell lines

Cancer stem‐like cells (CSCs) are self‐renewing cells comprising a small subpopulation in tumours, and generate differentiated progeny through asymmetric division. It has been shown that CSCs are resistant to ionizing radiation, and this feature could be one of the mechanisms of tumour recurrence after radiation therapy. Much attention has been focused on to target CSCs; however, difficult of isolating CSCs and lack of knowledge on their radiosensitivity have limited this kind of research in veterinary medicine. In the present study, sphere‐forming cells (SC), cultured using sphere formation method, were isolated from four type of canine tumour cell lines and evaluated if they have CSCs‐like properties by expression of CSCs markers (real‐time polymerase chain reaction) and capacity of tumorigenesis (xenograft transplantation in nude mice), and were assessed radiosensitivity (clonogenic survival assay) and DNA repair kinetics (immunofluorescence staining for p53‐binding protein 1) after X‐ray irradiation in comparison with the corresponding normal adherent culture cells (AC). All SCs were isolated using sphere formation and showed high gene expression of CD133 and tumorigenic ability as compared with AC. All SCs were significantly resistant against X‐ray irradiation as compared with AC. In addition, the amount of DNA double‐strand breaks after X‐ray irradiation were significantly lower in SC compared with the corresponding AC. These results indicate that SC isolated through sphere formation possess CSCs‐like characteristics and CSCs are important factor that affect radiosensitivity in canine tumours. In addition, radioresistance of CSCs may depend on reaction of DNA double‐strand break after X‐ray exposure.     

Phytotoxin Production and Aerial Mycelium Formation by Streptomyces scabies and S. acidiscabies in Vitro

Culture conditions (pH, Ca²⁺, phosphate, and temperature) on phytotoxin production (thaxtomin A and concanamycins A and B) and aerial mycelium formation were examined for two strains each of Streptomyces scabies and S. acidiscabies. Thaxtomin production decreased at pH 7.5 and increased at 15°C in S. scabies and increased at 30 mM phosphate in S. acidiscabies. Concanamycin production was stimulated by the addition of CaCl₂ and was correlated with formation of aerial mycelium. Aerial mycelium formation in S. scabies was greater the higher the pH, Ca²⁺ concentration and temperature, whereas that in S. acidiscabies was unaffected. Received 2 November 2000/ Accepted in revised form 27 April 2001     

Potential of anaerobic digestate of dairy manure in suppressing soil‐borne plant disease

Frequent use of pesticides to control soil‐borne plant disease leads to environmental pollution and the development of pesticide resistance in phytopathogens. Soil amendment is considered to have the potential of suppressing plant disease because of its biological properties. However, information on anaerobic digestate is limited. In this study, potential of antagonistic activities of anaerobic digestate against phytopathogens were investigated by detecting the amounts of antagonistic bacteria (Bacillus and Pseudomonas) in anaerobic digestates of dairy manure. The results showed that anaerobic digestion increased the total amounts of Bacillus and Pseudomonas in digestate. Bacillus suppressed growth of phytopathogens, while Pseudomonas did not show any antagonistic activities. These results indicated that Bacillus was an effective antagonistic bacterium in digestate against phytopathogens. Furthermore, two selected isolates, B11 (Bacillus subtilis) and B59 (Bacillus licheniformis), were applied in field experiments and showed significant reduction in percent infection of potato late blight (Phytophthora infestans). These results demonstrate the benefits of digestate in suppressing soil‐borne plant diseases caused by antagonistic bacteria.     

Hypothermic storage of porcine zygotes in serum supplemented with chlorogenic acid

The current study was conducted to investigate the effects of 100% foetal bovine serum (FBS) and 100% porcine follicular fluid (pFF) as a storage medium on the developmental competence of porcine zygotes stored at 25°C for 24 hr. Moreover, we evaluated the additive effects of chlorogenic acid (CGA) in the storage medium. When in vitro‐produced zygotes were stored at 25°C for 24 hr in tubes containing either tissue culture medium (TCM) 199 supplemented with 1 mg/ml bovine serum albumin (BSA), 100% of FBS or 100% of pFF, the rate of blastocyst formation was significantly higher in 100% of FBS than in BSA‐containing TCM 199. When the effects of CGA supplementation in 100% of FBS on the development of zygotes stored at 25°C for 24 hr was evaluated, more zygotes stored with 50 µM CGA developed to blastocysts compared with the other concentrations of CGA. When the formation date and quality of blastocysts derived from zygotes stored in 100% of FBS supplemented with 50 µM CGA were investigated, the highest ratio of blastocysts formation in the storage group appeared 1 day later than in the non‐stored control group. However, a higher proportion of blastocysts with apoptotic nuclei was observed in the stored group as compared to the non‐stored group. In conclusion, 100% of FBS is available for a short storage medium of porcine zygotes. The supplementation of 50 µM CGA into the storage medium improves the rates of blastocyst formation of zygotes after storage, but the quality of embryos from the stored zygotes remains to be improved.     

Evaluation criterion for response to selection with constraint

The aims of the present study are to represent the concept of restricted breeding values algebraically and to propose a criterion for evaluating the genetic responses achieved by using a restricted selection procedure. An additive genetic mixed model characterized by multiple traits with constraints was assumed. If the random errors approach zero and the fixed effects can be completely estimated correctly in the model, the restricted best linear unbiased predictor of breeding values ( u _R) is equal to , where G ₀, C ₀, and u are the additive genetic variance‐covariance matrix for the q traits, the matrix for restriction, and the vector of breeding values, respectively. Therefore, if we want to evaluate the response to restricted selection, such as by a stochastic computer simulation study with known breeding values, we can use u _R as only one criterion.     

Genetic parameter estimation for number born alive at different parities in Landrace and Large White pigs

We estimated genetic parameters for number born alive (NBA) from the first to the seventh parities in Landrace and Large White pigs using three models. Analyzing 55,160 farrowing records for 12,677 Landrace dams and 43,839 for 10,405 Large White dams, we used a single‐trait animal model to estimate the heritability of NBA at each parity and a two‐trait animal model and a single‐trait random regression model to estimate the genetic correlations between parities. Heritability estimates of NBA at each parity ranged from 0.08 to 0.13 for Landrace and from 0.05 to 0.16 for Large White. Estimated genetic correlations between parities in all cases were positive. Genetic correlations between the first and second parities were slightly lower than those between other neighboring parities. Genetic correlations between more distant parities tended to be lower, in some cases <0.8. The results indicate the necessity to investigate the applicability of evaluating NBA at different parities as different traits (e.g., the first and later parities), although a repeatability model might still be reasonable.     

Correlations between mitochondrial respiration activity and residual feed intake after divergent genetic selection for high‐ and low‐ oxygen consumption in mice

The aims of the present study were to identify the differences between two mouse lines (high (H)‐ and low (L)‐oxygen consumption) in terms of mitochondrial respiratory activity when GMP (glutamate, malate, and pyruvate) and succinic acid are used as substrates and to examine the relationship between mitochondrial respiration activity and feed efficiency in both lines. The average daily feed intake, feed conversion ratio (FCR), and residual feed intake (RFI) were significantly higher in the H than the L line. The correlation between FCR and RFI was significant (r = 0.60, p < 0.05). RFI was effective as an indicator of feed efficiency. When succinic acid was used as a substrate, mitochondrial respiration states 2–4, ACR, and proton leak were significantly higher in the H than the L line. When GMP was used as a substrate, respiration states 3 and 4 in the H line were significantly higher than those in the L line, and there were significant positive correlations between FCR and RFI and mitochondrial respiration states 2–4. The results indicated that selection for high or low OC changed the basal metabolic rates estimated from liver mitochondrial respiration activity and feed efficiency.     

Comparison of three mobilization protocols for peripheral blood stem cell apheresis with Spectra Optia continuous mononuclear cell protocol in healthy dogs

Peripheral blood stem cell (PBSC) transplantation following consolidation therapy is a feasible treatment option for canine haematological malignancies. In veterinary medicine, haematopoietic stem cells are generally mobilized into peripheral circulation using a granulocyte colony‐stimulating factor (G‐CSF). This pilot study aimed to evaluate the haematopoietic stem cell mobilization effect of three different regimens for PBSC apheresis with Spectra Optia continuous mononuclear cell (CMNC) protocol in healthy dogs. Stem cell mobilization was performed using high‐dose plerixafor (CXCR‐4 inhibitor) alone, a G‐CSF alone, or a combination of the low‐dose plerixafor and G‐CSF. Three dogs were assigned to each mobilization protocol. Regardless of the mobilization protocol, the total blood volume processed was uniformly set as 270 mL/kg and many PBSCs, defined as CD34+/CD45^dim cells, within the apheresis product were compared. Changes in complete blood count, PBSC counts, and blood chemistry analysis were monitored before, during, and after apheresis. All dogs tolerated the apheresis procedure using the Spectra Optia system with minimal adverse effects. The mean PBSC counts of the apheresis products for plerixafor, G‐CSF, and the combination groups were 1.3 ± 0.24, 4.2 ± 0.47, and 6.4 ± 0.9 × 10⁶ cells/kg, respectively. The apheresis procedure using Spectra Optia CMNC protocol in dogs is safe and feasible. Furthermore, PBSC mobilization with a combination of G‐CSF and plerixafor appeared more effective than either compound alone in mobilizing PBSC to the peripheral blood in dogs.