16S rRNA gene profiling of bacterial communities mediating production of tsetse attractive phenols in mammalian urine.

Several types of odours are involved in the location of host animals by tsetse (Diptera: Glossinidae), a vector of animal African trypanosomiasis. Host animals' ageing urine has been shown to be the source of a phenolic blend attractive to the tsetse. Nevertheless, limited research has been performed on the microbial communities' role in the production of phenols. This study aimed at profiling bacterial communities mediating the production of tsetse attractive phenols in mammalian urine. Urine samples were collected from African buffalo (Syncerus caffer), cattle (Bos taurus) and eland (Taurotragus oryx) at Kongoni Game Valley Ranch and Kenyatta University in Kenya. Urine samples, of each animal species, were pooled and left open to age in ambient conditions. Bacteriological and phenols analyses were then carried out, at 4 days ageing intervals, for 24 days. Phenols analysis revealed nine volatile phenols: 4-cresol, ortho-cresol, 3-cresol, phenol, 3-ethylphenol, 3-propylphenol, 2-methyloxyphenol, 4-ethylphenol and 4-propylphenol. Eight out of 19 bacterial isolates from the ageing urine revealed the potential to mediate production of phenols. 16S rRNA gene characterisation of the isolates closely resembled Enterococcus faecalis KUB3006, Psychrobacter alimentarius PAMC 27887, Streptococcus agalactiae 2603V, Morganella morganii sub.sp. morganii KT, Micrococcus luteus NCTC2665, Planococcus massiliensis strain ES2, Ochrobactrum pituitosum AA2 and Enterococcus faecalis OGIRF. This study established that some of the phenols emitted from mammalian urine, which influence the tsetse's host-seeking behaviour, are well characterised by certain bacteria. These results may allow the development of biotechnological models in vector control that combines the use of these bacteria in the controlled release of semiochemicals.

Detection of estrus in Indian blackbuck: behavioural, hormonal and urinary volatiles evaluation.

The determination of the reproductive status is one of the most important factors for effective wild life conservation and management, and effective use of assisted reproductive techniques like artificial insemination, in vitro fertilization/embryo transfer depends on the knowledge of the basic reproductive physiology. In this context the reproductive status of female blackbucks (Antelope cervicapra L.) was assessed by behaviour and determination, sex steroid hormones in faeces and urinary volatile compounds. The male and female blackbucks exhibited as many as 31 different reproductive/courtship behaviour patterns. Particularly, the males showed a more extensive repertoire: i.e. 23 behavioural patterns by territorial males, 11 by bachelor males and 4 by females. The behaviours such as, mounting, Flehmen, clockwise and anticlockwise movements were significantly higher in male blackbuck when exposed to estrus. By contrast, such courtship behaviours were completely absent in male when exposed to diestrus. It clearly indicates that, the estrus female produces specific chemical cues (pheromone) through urine, which would involve in attracting the conspecifics. In addition, the average faecal oestrogen concentration was significantly higher (p<0.05) during the estrus faecal than the proestrus and diestrus periods. In contrast, the faecal progesterone concentration was significantly higher (p<0.05) during the diestrus faecal sample than that of proestrus and estrus faecal sample. Twenty-eight volatiles are identified, across the three reproductive phases (i.e. proestrus, estrus and diestrus) of sexually mature and prepubertal females. Amongst, the compounds 2-methyl-3-butyn-2-ol, 3,7-dimethylnonane, 3-phenyl-2-propen-1-ol and 2-hydroxybenzoic acid occurred only during estrus which may be considered as marker for detection of estrus which would ultimately help for artificial insemination in captive condition. The findings of the present study suggest that the non-invasive approaches like reproductive behaviours, faecal steroids and estrus-specific urinary volatiles could serve as good indicators for detection of estrus for blackbuck.

Assessment of dominance hierarchy through urine scent marking and its chemical constituents in male blackbuck Antelope cervicapra, a critically endangered species.

In ungulates the process of chemical communication by urinary scent marking has been directly related to reproductive dominance, territorial defense and proximity to resources. The differences in the frequency of urine marking and chemical composition of urine of males Antelope cervicapra before, during and after the dominance hierarchy period were assessed. The variations in the urine marking and its chemical profiles of dominant males (n=9), bachelors (n=5) and sub-adult males (n=5) were compared to find out how the dominance hierarchy influences the confined blackbuck herd under semi-natural captive conditions. The frequency of urine marking is significantly higher (p<0.001) in dominant males. Twenty-eight major constituents were identified in the urine of dominant males (before, during and after the dominance hierarchy period), bachelor and sub-adult males. Among these, three specific compounds namely, 3-hexanone (I), 6-methyl-5-hepten-2-one (II) and 4-methyl-3-heptanone (III) were seen only in dominant males urine during the dominance hierarchy period. Based on the behavioural observation and the unique chemical constituents in the urine, it is concluded that the dominant male scent odor suppresses aggression, scent marking, scent production and territorial patrolling activities of subordinate males, through which the dominant male establish their hierarchy and attains success in reproduction.

Analysis of urinary progesterone metabolites with behavioral correlation in Guenther's dik-dik (Madoqua guentheri).

Urine samples and behavioral data were collected from nine adult female Guenther's dik-dik (Madoqua guentheri) from September 1989-November 1992. The durations of predefined individual behaviors were recorded during focal sampling periods and ad libitum. Immunoreactive pregnanediol-3-glucuronide (PdG) concentration in dik-dik urine was determined by radioimmunoassay. The immunoreactive PdG radioimmunoassay was validated for use with dik-dik by determining parallelism [F(alpha=0.05,1,4) = 0.04) between a serially diluted PdG standard and serially diluted dik-dik urine. Behavioral estrus was manifested by lordosis. Eighty-nine percent of the estrous behavior occurred during or within 2 days of the interluteal phase period, as delineated by immunoreactive PdG concentrations; thus, a high degree of correlation between hormone concentrations and behavioral data was observed. The mean (+/-SD) luteal phase, interluteal phase, and estrous cycle lengths were 14.4+/-5.5 days (range, 6-29 days; n = 50), 6.6+/-5.1 days (range, 2-33 days; n = 50), and 20.2+/-6.6 days (range, 10-43 days; n = 48), respectively. During the collection period, one animal conceived, with a gestation period of 170 days. Estrous cyclicity occurred throughout the year, with no evidence of seasonality. Cochromatography of pooled urine samples with radiolabeled PdG indicated that the major progesterone metabolite detected by the immunoreactive PdG antibody during luteal phase and pregnancy was not PdG. This is the first detailed description of female dik-dik reproductive endocrine activity.