Localization of putative pituitary stem/progenitor cells in female dairy cattle

Research on sex-determining region Y-box 2 (SOX2)-positive pituitary stem/progenitor cells, as a source of hormone-producing cells, is progressing rapidly in rodents. However, the stem/progenitor cells supplying hormone-producing cells that are essential for growth, reproduction, and lactation in bovines have not yet been identified. In this study, we characterized SOX2-positive cells in the pituitary gland of dairy cattle (Holstein heifers) after sexual maturity. Immunofluorescence analysis revealed that the localization pattern of SOX2-positive cells in the dairy cattle pituitary gland was similar to that observed in the rodent pituitary gland; the marginal cell layer (MCL), dense cell clusters, and single cells scattered in the parenchyma of the anterior lobe. Furthermore, most of the SOX2-positive cells were positive for the pituitary stem/progenitor cell niche markers E-cadherin and cytokeratin 8+18, which have been reported in rodents. In addition, in the MCL of the anterior lobe, there was a subpopulation of SOX2-positive cells positive for paired-related homeobox 1 and 2, whereas negative for S100β. Moreover, in the parenchyma of the anterior lobe, co-localization of SOX2 and pituitary hormones was infrequent. In summary, this study reveals the localization of putative pituitary stem/progenitor cells positive for SOX2 in dairy cattle. These results provide valuable information to support further investigation of cell supply in the dairy cattle pituitary gland.     

Sarcoplasmic Reticulum Ca2+-ATPase Activity and Glycogen Content in Various Fiber Types after Intensive Exercise in Thoroughbred Horses

To find a new parameter indicating muscle fitness in Thoroughbred horses, we examined time-dependent recovery of glycogen content and sarcoplasmic reticulum (SR) Ca²⁺-ATPase activity of skeletal muscle after intensive treadmill running. Two repeated 50-sec running sessions (13 m/sec) were performed on a flat treadmill (approximately 90%VO₂max). Muscle samples of the middle gluteal muscle were taken before exercise (pre) and 1 min, 20 min, 60 min, and 24 hr after exercise. Muscle fiber type composition was determined in the pre muscle samples by immunohistochemical staining with monoclonal antibody to myosin heavy chain. SR Ca²⁺-ATPase activity of the muscle and glycogen content of each muscle fiber type were determined with biochemical analysis and quantitative histochemical staining, respectively. As compared to the pre value, the glycogen content of each muscle fiber type was reduced by 15–27% at 1 min, 20 min, and 60 min after the exercise and recovered to the pre value at 24 hr after exercise test. These results indicate that 24 hr is enough time to recover glycogen content after short-term intensive exercise. The mean value of the SR Ca²⁺-ATPase activity showed a slight decrease (not significant) immediately after exercise, and complete recovery at 60 min after exercise. There were no significant relationship between the changes in glycogen content of each muscle fiber type and SR Ca²⁺-ATPase. Although further studies are needed, SR Ca²⁺-ATPase is not a useful parameter to detect muscle fitness, at least in Thoroughbred horses.     

Differences in Muscle Fiber Recruitment Patterns between Continuous and Interval Exercises

We evaluated differences in muscle fiber recruitment patterns between continuous and interval training to develop an optimal training program for Thoroughbred horses. Five well trained female thoroughbred horses (3–4 years old) were used. The horses performed two different exercises on a 10% inclined treadmill: 90%VO₂ max for 4 min (continuous) and 90% VO₂ max for 2 min × 2 times with 10-min interval (interval). Muscle samples were obtained from the middle gluteal muscle before and immediately after the exercises. Four muscle fiber types (type I, IIA, IIA/X, and IIX) were immunohistochemically identified, and the optical density of periodic acid Schiff staining (OD-PAS) in each fiber type and glycogen content of the muscle sample were determined by quantitative histochemical and biochemical procedures, respectively. No significant differences were found in the OD-PASs and glycogen contents between the continuous and interval exercises, but the decreases in OD-PAS of fast-twitch muscle fibers were obvious after interval as compared to continuous exercise. Interval exercise may be a more effective training stimulus for the glycolytic capacity of fast-twitch muscle fiber. The data about muscle fiber recruitment can provide significant insights into the optimal training program not only for thoroughbred horses, but also for human athletes.     

Concordance between Results of Medium-term Liver Carcinogenesis Bioassays and Long-term Findings for Carcinogenic 2-Nitropropane and Non-carcinogenic1-Nitropropane in F344 Rats

This study was conducted to determine the concordance of results for a pair of structural isomers, 2-nitropropane (2-NP) and 1-nitropropane (1-NP), using the rat medium-term liver carcinogenesis bioassay (Ito test) and previously published long-term carcinogenicity tests. Male F344 rats were given a single intraperitoneal injection of DEN (200 mg/kg b.w.) to initiate hepatocarcinogenesis. After 2 weeks, they received per os 0, 0.8, 4 or 20 mg/kg/day of 2-NP or 1-NP six times a week and were subjected to two-thirds partial hepatectomy at week 3. Non-initiated groups receiving 0 or 20 mg/kg/day were also included. The animals were sacrificed for quantitative analysis of GST-P-positive foci at week 8. With the highest dose of 2-NP, significantly increased numbers and areas of GST-P-positive foci were demonstrated as compared with the respective control but were not noted with 1-NP. In the non-DEN-initiated groups, many small GST-P-positive foci of less than 0.2 mm in diameter were also induced in the rats treated with 2-NP at 20 mg/kg/day but were lacking with 1-NP. These results strongly support that 2-NP is a complete hepatocarcinogen with a potent initiation activity, whereas 1-NP is not.     

Measurement of telomerase activity in bovine leukaemia virus infected cows

Telomerase adds new telomeric sequences to the end of chromosomal DNA in order to overcome the end-replication problem. The upregulation of telomerase activity in tumours has been reported in humans and some mammals and is considered to be a tumour marker; however, such activity has not been investigated in cows. Therefore, we investigated telomerase activity in bovine leukaemia, the most common tumour in cows and its relationship with the bovine leukaemia virus (BLV) infection, which is the major cause of leukaemia. Telomerase activity was detected in 25 of 29 bovine leukaemia tissue samples. In peripheral blood lymphocytes (PBL) from BLV-infected cases that did not develop the tumour, telomerase activity was detected in 11 of 71 cases (15.5%). When these cases were classified based on serological tests and the peripheral blood lymphocyte count, the telomerase activity was observed to be the highest in the seropositive, non-lymphoproliferative (PBL<8000 microl(-1)) cases (three of seven cases, 42.9%), and not observed in the lymphoproliferative cases (PBL<16,000 microl(-1)) except in one case. Although the precise pathogenesis of BLV-related diseases remains obscure, persistent lymphocytosis is considered as a pre-neoplastic state. In contrast, our results suggested that given the fact that telomerase activity indicates tumour development, the aleukaemic stage could be defined as the 'pre-neoplastic state'. In conclusion, similar to many tumours in humans, telomerase activity was detected in bovine leukaemia; further, this activity can be a potentially useful prediction marker for tumour development and/or a good therapeutic target.     

Impact of land use on nitrogen concentration in groundwater and river water

Abstract

The aim of this study was to evaluate the impact of land use on nitrate nitrogen (NO₃-N) in shallow groundwater (G-N) and total nitrogen (N) in river water (R-N). The study area consisted of 26 watersheds (1342 km²) covering 72% of Kagawa Prefecture in Japan. We estimated G-N specific concentrations, which showed the magnitude of the upland fields, paddy fields, forests and urban land-use contributions to watershed-mean G-N. G-N specific concentrations were gained as partial regression coefficients using a multiple regression analysis of the watershed-mean G-N concentrations and the land-use ratios in each of the 26 watersheds. The results showed that the G-N specific concentration, which was gained as the partial regression coefficient for the multiple regression analysis, was 15.2 mg L^?1, 10.3 mg L^?1, 2.3 mg L^?1 and 2.5 mg L^?1 for the upland fields, paddy fields, forests and urban land-use types, respectively. R-N pollution load runoff to the river mouth was calculated by multiplying R-N specific concentration (previously reported) by river flow at the river mouth. Similarly, G-N pollution load arrival to groundwater was calculated by multiplying G-N specific concentration by the groundwater flow. The R-N pollution load runoff was 19.3 kg ha^?1 y^?1, 7.7 kg ha^?1 y^?1, 1.7 kg ha^?1 y^?1 and 7.6 kg ha^?1 y^?1, while the G-N pollution load arrival was 7.3 kg ha^?1 y^?1, 5.0 kg ha^?1 y^?1, 1.1 kg ha^?1 y^?1 and 1.2 kg ha^?1 y^?1, for upland fields, paddy fields, forests and urban areas, respectively. These results showed that the N in river water and groundwater was derived mainly from runoff and leaching from croplands. Therefore, the relationships between watershed-mean non-absorbed, applied nitrogen (NAA-N: nitrogen applied to cropland via fertilizer and manure without being absorbed by crops), R-N concentration and watershed-mean G-N concentration were investigated. A curvilinear correlation was observed between NAA-N and R-N concentrations (r² = 0.68) except for one small, high-density, urban watershed, and a weak linear correlation was observed between NAA-N and G-N concentrations (r² = 0.42).     

Phylogenetic analysis of hindgut microbiota in Hokkaido native horses compared to light horses

The analysis of 16S rDNA sequence of bacteria in feces of Hokkaido native horses and light horses were performed to compare the hindgut microbiota between the two breeds. One hundred and four bacterial 16S rDNA clones (57 clones from four native horses and 47 clones from two light horses) were obtained. Only four sequences (3.8% of total sequences) showed 97% or more similarity to known species. The sequences were mainly affiliated with Cytophaga–Flavobacter–Bacteroides and low GC Gram‐positive bacteria (LGCGP). Proportion of LGCGP was higher in light horses. Other phyla including Verrucomicrobia, Spirochaetes and Archaea were detected only for native horses, suggesting high diversity of microbiota in native horses. In LGCGP, clusters related to known cellulolytic species were found only for native horses, while a cluster related to soluble sugar‐utilizing species was detected only for light horses. The library composition‐comparing software LIBSHUFF showed significant (P < 0.05) difference of fecal microbiota between the horse breeds. The number of Fibrobacter succinogenes‐related sequence and the frequency of detection of novel groups were found to be higher in native horses by selective amplification analysis. The results suggest that genetic diversity and population size of the F. succinogenes group are higher in the hindgut of native horses.     

Reproductive and growth performance in Jin Hua pigs cloned from somatic cell nuclei and the meat quality of their offspring

Somatic cell cloning is expected to be a valuable method for conserving genetic resources in pigs. In this study, we compared the reproductive and growth performance of Jin Hua cloned pigs with that of naturally bred Jin Hua pigs. In addition, we generated offspring from the cloned sows and examined the productivity and quality of meat in the progeny. The birth weights and growth rates of somatic cell-cloned pigs were similar to those of Jin Hua pigs. The cloned pigs reached puberty very early, and this is typical of the Jin Hua breed. Furthermore, reproductive performance, in terms of traits such as gestation period, litter size, and raising rate in the cloned pigs were similar to Jin Hua pigs. Although the offspring of the cloned (OC) pigs had lower birth weights than the Jin Hua breed, the daily weight gain of the OC pigs was significantly higher, especially at the finishing stage. The carcass quality of the OC pigs had similar characteristics to the Jin Hua breed, namely thick back fat and a small loin area. Furthermore, the meat qualities of the OC pigs were similar to those of Jin Hua pigs in terms of intramuscular fat content and tenderness. These results demonstrate that cloned pigs and their offspring were similar to the Jin Hua breed in most of the growth, reproductive, and meat productive performances. This strongly suggests that pigs cloned from somatic cell nuclei have the potential to be a valuable genetic resource for breeding.     

Lack of Hepatocarcinogenicity of Combinations of Low Doses of 2-amino-3, 8-dimethylimidazo[4,5- f ]quinoxaline and Diethylnitrosamine in Rats: Indication for the Existence of a Threshold for Genotoxic Carcinogens

The purposes of the present study were to evaluate the hepatocarcinogenicity of concurrent treatment of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and diethylnitrosamine (DEN) in rats and to determine whether no effect levels of combinations of these two different structural categories of genotoxic hepatocarcinogens exist. Two 16-week rat hepatocarcinogenesis assays were performed using a total of 790 male F344 rats. In experiment 1, we evaluated the effects of concurrent treatment of a subcarcinogenic dose of DEN on rat hepatocarcinogenesis induced by various doses of MeIQx. In experiment 2, we determined hepatocarcinogenicities of combinations of MeIQx and DEN at subcarcinogenic doses, low carcinogenic doses and high carcinogenic doses. Quantitative analyses of glutathione S-transferase placental form (GST-P)-positive foci, a preneoplastic lesion of the liver in rats, revealed that concurrent treatment with subcarcinogenic doses of DEN did not enhance MeIQx-induced rat hepatocarcinogenicity. We also found that concurrent treatment with combinations of subcarcinogenic doses of DEN and MeIQx was not hepatocarcinogenic, indicating that the combined effects of subcarcinogenic doses of DEN and MeIQx were neither additive nor synergistic. Moreover, concurrent treatment with low carcinogenic doses of these 2 carcinogens did not show additive or synergistic effects. Synergetic effects were observed only in rats coadministered high carcinogenic doses of the 2 carcinogens. These results demonstrate the existence of no effect levels of combinations of these 2 genotoxic hepatocarcinogens, and provide new evidence supporting our idea that there is a threshold, at least a practical threshold, that should be considered when evaluating the risk of genotoxic carcinogens.