Prevention of estrus in the queen with chlormadinone acetate administered orally.

The possibility of estrus prevention in the queen by the oral administration of chlormadinone acetate was examined. The animals used were 29 mature and 15 immature queens. For 16 mature females, 4-12.5 mg was given daily by mouth for 7 days every 3 months. Ten of the 16 queens given this treatment came into estrus within 4 months of the first treatment. For 28 females including the immature, 2-12.5 mg was given once a week throughout the experiment. This treatment prevented estrous activity for at least 1 year. In the queens in this study, the side effects were not observed excepting an increase in body weight during treatment. Our results showed that oral administration of this drug weekly is safe and reliable for long-range prevention of estrus in queens.     

Histological and immunohistochemical evaluation of granulosa cells during different stages of folliculogenesis in bovine ovaries

Bovine granulosa cells (GC) vary in their morphological aspect during different stages of folliculogenesis. In this study, 10 morphologically normal bovine ovaries were collected to study the structural aspects of different stages of GC using intermediate filament protein antibodies including cytokeratin AE1/AE3 (AE1/AE3), vimentin, nectin‐4 and desmin. Hormonal immunolocalization was assessed using the immunomarkers anti‐Müllerian hormone (AMH) and inhibin alpha. In addition, tumour markers and proliferation markers using c‐erbB‐2 oncoprotein and proliferating cell nuclear antigen, respectively, were investigated. The immunolabelling of AE1/AE3 in GC was strongest in the early follicle stage and gradually decreased when reaching the Graafian follicle stage. Its immunolabelling increased again as the stage progressed from stage I to stage III. The immunolabelling of inhibin alpha was inversely proportional to that of AE1/AE3 in the developing ovarian follicles as their immunolabelling is opposite to each other during folliculogenesis. AMH was immunopositive in almost all GC stages in different intensities and percentages, except for some negative staining in the atretic IV follicles. The atretic IV follicle is a unique type of atretic follicle that shows Call‐Exner body formation, which was mainly found in older cows in this study. The distinct patterns of immunoreactivity for various types of immunomarkers in the different GC stages will play an important role in diagnostic assistance of various follicle conditions, including cystic ovaries and GC tumours.     

Discrimination of “grazing milk” using milk fatty acid profile in the grassland dairy area in Hokkaido

Milk produced by the grazing system, referred to as “grazing milk” contains many components required for human health. The milk fatty acid (FA) profile is strongly associated with the diet on the farms. In the present study, based on the FA profile of farmer's bulk milk, we determined how to discriminate between milk produced on grazing and on a confinement system. A field survey was conducted four times (grazing and confinement season) in the Konsen (29 farms) and Okhotsk (25 farms) area in Hokkaido. Farmer's bulk milk samples and details of feeding management were collected and the FA profile of milk was measured. Milk produced during the grazing season contained less C16:0 and cis‐9 C16:0, and more C18:0, cis‐9 C18:1, trans‐11 C18:1, cis‐9,12 C18:2, cis‐9,trans‐11 C18:2 and cis‐9,12,15 C18:3 than milk produced during the confinement season. Discrimination analysis using 16 FA revealed that almost all milk samples were discriminated correctly (confinement season: 90% correct and 10% borderline, grazing season: 88% correct, 9% borderline and 3% incorrect). For farmers that were categorized incorrectly and were considered borderline in the grazing season, the dependency on pasture was low compared with that for farmers correctly discriminated. Therefore, to claim “grazing milk”, a high dependency on pasture is required for grazing dairy farmers.     

Relationship of rumen fill and fermentation to diurnal and seasonal variation of herbage intake in dairy cows grazed on perennial ryegrass pasture

To clarify the effect of digesta weight in the reticulorumen on diurnal and seasonal fluctuations in herbage intake, six ruminally cannulated, non‐lactating dairy cows were grazed on perennial ryegrass/white clover pasture during morning and evening sessions in spring and autumn. The digesta weight of fresh matter, dry matter (DM) and fiber in the reticulorumen at the beginning and the end of each grazing session was lower in spring than in autumn (P < 0.01). Although the digesta weight was similar between the sessions at the beginning of grazing, it was greater for the evening than for the morning at the end of grazing (P < 0.01). The large particles proportion in the digesta was lower for the morning than the evening (P < 0.01), and it was lower in spring than in autumn (P < 0.01). The concentrations of volatile fatty acids in rumen fluid were generally higher in spring compared with autumn. The herbage DM intake during the evening was greater compared with the morning in both seasons (P < 0.01). However, there was no difference in herbage DM intake between seasons. The results showed that the rumen digesta fill was not the sole factor explaining diurnal and seasonal variation of herbage intake in grazing dairy cows.     

Herbage intake and ruminal digestion of dairy cows grazed on perennial ryegrass pasture either in the morning or evening

This study aimed to clarify diurnal fluctuations of herbage intake, ruminal fermentation of herbage carbohydrates and proteins, and digesta particulate weight in the rumen of grazing dairy cows. Six ruminally cannulated, non‐lactating dairy cows were grazed on perennial ryegrass/white clover pasture either in the morning (04.00 to 08.00 hours) or the evening (16.00 to 20.00 hours). Cows grazed in the evening spent more time (P < 0.01) and consumed more herbage (P < 0.01) compared with cows grazed in the morning. Higher (P < 0.05) daily mean concentrations of total volatile fatty acid, propionate and n‐butyrate in rumen fluid were observed for cows grazed in the evening compared with cows grazed in the morning. Although cows grazed in the evening ingested more crude protein compared with cows grazed in the morning, no significant difference in NH₃‐N concentration in rumen fluid was observed between them. The ratio of purine‐derivative concentration to creatinine concentrations was higher (P < 0.01) in the urine of cows grazed in the evening than in cows grazed in the morning. These results clearly indicated that evening grazing was advantageous for dairy cows compared with morning grazing, in terms of ruminal fermentable energy intake and nitrogen utilization efficiency.     

Detection of Mycobacterium avium subsp. paratuberculosis in ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture

The aims of this study were to develop a new real-time quantitative PCR (QPCR) assay based on IS900 for detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP) DNA in faeces, and to use this to detect infected sheep. Both the C and S strains of MAP were detected by the QPCR assay, and no cross reactions were detected with 51 other species of mycobacteria including 10 which contained IS900-like sequences. One copy of IS900 fragment cloned into plasmid pCR2.1 and 1 fg of MAP genomic DNA were consistently detected, while in spiked faecal samples the detection limit was 10 viable MAP per gram of ovine faeces. A total of 506 individual ovine faecal samples and 27 pooled ovine faecal samples with known culture results were tested. The QPCR assay detected 68 of 69 BACTEC culture positive individual faeces and there was a strong relation between time to detection in culture and DNA quantity measured by QPCR (r= -0.70). In pooled faecal samples, QPCR also agreed with culture (kappa=0.59). MAP DNA was detected from some culture negative faecal samples from sheep exposed to MAP, suggesting that the QPCR has very high analytical sensitivity for MAP in faecal samples and detects non-viable MAP in ovine faeces. None of the faecal samples from 176 sheep that were not exposed to MAP were positive in QPCR. This is the first report of a direct faecal QPCR assay that has similar sensitivity to a gold standard radiometric culture assay.     

Androgen induces production of male effect pheromone in female goats

Previously we showed that the primer pheromone responsible for the "male effect" was produced in specific skin regions of castrated male goats by androgen treatments. In the present study, we examined whether androgen can also induce production of the male effect pheromone in female goats. Capsules containing dihydrotestosterone (DHT) or testosterone (T) were subcutaneously implanted into six ovariectomized (OVX) goats for 28 days. Small skin samples were collected from the head and rump regions, and the pheromone activity of their ether extracts was examined using a bioassay that monitors the electrophysiological manifestation of the hypothalamic gonadotropin-releasing hormone pulse generator as multiple-unit activity. Behaviors of OVX goats towards ovary-intact estrous goats were also examined before and at the end of DHT or T treatment. Before androgen treatment, neither the head nor rump skin samples in OVX goats showed pheromone activity. DHT treatment induced pheromone activity in the head skin sample of six OVX goats and in the rump skin sample of two OVX goats. Similar results were obtained by T treatment. In addition, OVX goats treated with T showed masculine-type sexual behaviors such as courtship and mounting behaviors towards the estrous goats. These results demonstrate that androgen is capable of inducing primer pheromone activity in the female and suggest that the synthesis pathway of the male effect pheromone exists in both sexes in the goat.     

Plant species identification using fecal DNAs from red-eared slider and Reeves’ pond turtle in agricultural canals for rural ecosystem conservation

Fecal DNA samples from the red-eared slider and Reeves’ pond turtle, suspected pests of lotus root paddies, were used to identify the plant species eaten by these turtles in order to develop a strategy for rural ecosystem conservation. The fecal samples were obtained from young and adult individuals (mostly female) of both species living in agricultural canals surrounding lotus root paddies in Tokushima Prefecture, Japan. The samples were screened for the presence or absence of DNA from nine plant species using PCR and plant species-specific primers for the rbcL gene of chloroplast DNA. In the red-eared slider, our analysis identified seven plant species in the fecal DNA samples of adults and three plant species in those of young individuals. In Reeves’ pond turtle, our analysis identified two plant species from adult fecal samples and one species from those of young individuals. Thus, adult red-eared sliders consume a greater range of plants than young red-eared sliders or Reeves’ pond turtles. Both turtle species, independently of age, consumed lotus plants and were likely to cause feeding damage to lotus roots. Considering the plant species detected in adult red-eared sliders and these plant habitats, we suggest that this adult turtle is likely to travel between the agricultural canals and the lotus root paddies. These findings will help the development of strategies for preventing damage to lotus roots by these turtles; furthermore, they indicate that fecal DNA analysis will be applicable to investigation of the feeding habits of other animal species.