Objectives: Effluents contain a diverse abundance of antibiotic resistance genes that augment the resistome of receiving aquatic environments. However, uncertainty remains regarding their temporal persistence, transcription and response to anthropogenic factors, such as antibiotic usage. We present a spatiotemporal study within a river catchment (River Cam, UK) that aims to determine the contribution of antibiotic resistance gene-containing effluents originating from sites of varying antibiotic usage to the receiving environment. Methods: Gene abundance in effluents (municipal hospital and dairy farm) was compared against background samples of the receiving aquatic environment (i.e. the catchment source) to determine the resistome contribution of effluents. We used metagenomics and metatranscriptomics to correlate DNA and RNA abundance and identified differentially regulated gene transcripts. Results: We found that mean antibiotic resistance gene and transcript abundances were correlated for both hospital (ρ = 0.9, two-tailed P <0.0001) and farm (ρ = 0.5, two-tailed P  <0.0001) effluents and that two β-lactam resistance genes (bla_GES and bla_OXA) were overexpressed in all hospital effluent samples. High β-lactam resistance gene transcript abundance was related to hospital antibiotic usage over time and hospital effluents contained antibiotic residues. Conclusions: We conclude that effluents contribute high levels of antibiotic resistance genes to the aquatic environment; these genes are expressed at significant levels and are possibly related to the level of antibiotic usage at the effluent source.

Summary. In Exp. 1, 4 groups of 50 recently weaned ewes were exposed to various degrees of contact with rams for 65 days, followed by exposure to novel rams for 4 days. Ovarian activity in the ewes was determined by laparoscopy on Days 29, 65 and 69 of treatment. There were no treatment differences in the percentage of ewes ovulating on Day 4 whereas by Day 29 more ewes in clear fenceline and full ram contact were ovulating compared to controls (P < 0·05, P < 0·001). After 65 days ovarian activity was significant only in those ewes in full contact with rams (P < 0·001). Between 89 and 95% of ewes remaining anovulatory after 65 days ovulated after 4 days of full contact with novel rams. In Exp. 2, 4 groups of about 30 anovulatory ewes were exposed to various degrees of contact with rams for 5 days. Ovarian activity was assessed before and after treatment by laparoscopy. After 5 days, more ewes were ovulating in response to full ram contact than in any other treatment (P < 0·05) and more ewes in fenceline contact with rams or with rams plus ewes were ovulating than in the isolated control treatment (P < 0·01). In Exp. 3, 6 groups of about 40 anovulatory ewes were exposed to face masks with and without rams' wool and/or various degrees of contact with rams for 5 days. More ewes were ovulating after 5 days in the group in full physical contact with rams than in any other group (P < 0·01). Ram contact through a clear fence either with or without masks stimulated more ewes to ovulate than masks alone or isolation from rams (P < 0·01). The additional exposure to rams' wool did not increase the percentage of ewes ovulating in response to fenceline ram contact or masks alone but exposure to masks themselves with or without rams' wool did stimulate ovulation compared to isolated controls (P < 0·05, P < 0·01 respectively). These results indicate that fenceline ram contact was effective in inducing ovulation in a high proportion of seasonally anovulatory ewes but less effective in lactationally anovulatory ewes. Furthermore, maximum stimulation of ovulation required full physical contact with rams in all cases. Visual and tactile/behavioural stimuli from the ram therefore appear most important in mediating the ram effect whereas olfactory cues from Merino rams' wool were ineffective either alone or in addition to fenceline ram exposure. Pre-isolation of ewes from rams was not required when novel rams were used to induce subsequent ovulation. Keywords: ram stimuli; induced ovulation; ewes