Changes in vaginal microbiology associated with the use of progesterone-releasing intravaginal devices

PROGESTERONE-releasing devices are commonly used in the control of bovine reproduction (Peters and Ball 1995). However, because the devices provide a surface for bacterial growth away from endometrial defences and change the vaginal environment, their use stimulates the proliferation of vaginal bacteria, resulting in a profuse vaginal discharge (Bulman and others 1978). However, this discharge resolves spontaneously after removal of the device from most animals (Bulman and others 1978, Arbeiter and Pohl 1985). The effect on the vaginal flora, particularly at the time of insemination, is less clear. Bulman and others (1978) found no significant effect of a progesterone-releasing intravaginal device (PRID; Sanofi Animal Health) on the vaginal flora, seven and 14 days after the device was removed, but they did not investigate the vaginal flora at insemination. Thus, it has not been demonstrated that intravaginal devices do not result in the proliferation of potentially harmful bacteria. This is particularly important when such devices are used soon after parturition in order to stimulate the early return of ovarian cyclicity. At this stage, a prolonged period of progesterone dominance may predispose to ascending infection from the vagina (Noakes 1996). As part of a study designated to investigate the use of progesterone-releasing devices in the establishment of early cyclicity, the changes in the bacterial population in the anterior vagina resulting from a seven-day treatment period with one of two progesterone-releasing intravaginal devices (PRID; Sanofi Animal Health, and Eazi-Breed CIDR; Animal Reproduction Technologies) were determined. As an early onset of cyclicity was the aim of the treatment, the devices were inserted 14 days after parturition, rather than the data sheet recommendation of at least 21 days. Thirty-four cows were selected from the ADAS Bridgets winter calving herd. Each cow was allocated at random to one of the two treatments, with the device being inserted 14 days after parturition. Cows in both treatment groups were injected with 25 mg of dinoprost (Enzaprost; Sanofi Animal Health) two days before the device was inserted, as part of the regimen for stimulating early cyclicity. The anterior vagina of each cow was swabbed on three occasions; immediately before the device was inserted, immediately after the device was removed, and 56 hours after the device was removed (corresponding to the usual time of insemination). The devices were removed by gently pulling the exposed string. Two protected swabs, taken via a speculum, were taken from each cow on each occasion, one for aerobic culture (Venturi Transport Swab; Bibby Sterilin) and one for anaerobic culture (Anaerobic Culturette; Becton Dickinson). After collection, the swabs were placed in a refrigerator for storage at approximately 4°C for a maximum of 48 hours, before being transferred to the Veterinary Laboratories Agency, Winchester, for culture. For the majority of samples, culture began within 12 hours. All swabs submitted were allocated a unique reference number for the day they arrived. This number then generated a worksheet for each day’s swabs which contained all the details of the work carried out, including the date and the time of arrival of the samples at the laboratory, and when the plates were inoculated with the swabs. It could therefore be ascertained which swabs were delayed for 48 hours, thereby allowing a comparison to be made with other results and any anomalies to be detected. Any delay in inoculation would have been due to swabs being taken/arriving at a weekend, which was minimal. The company supplying the anaerobic culture quotes: ‘providing manufacturers’ instructions are followed, the Anaerobic Culturette dual ampoule system provides the additional advantage of a reduced oxygen environment within 10 minutes for up to 48 hours after activation in its unique, self-contained, gas-generating envelope. ’ Viability up to 48 hours has been established for organisms including Bacteroides species and Peptostreptococcus species and data remain on file at Becton Dickinson Microbiology Systems. Charcoal swabs were inoculated onto blood agar (CM271; Oxoid) with 5 per cent sterile defibrinated sheep’s blood and MacConkey’s agar (CM7; Oxoid) and then incubated aerobically for up to 48 hours at 37°C. Colonies were identified using standard bacteriological techniques (Carter and Cole 1990, Cowan and Steel 1993). The swabs for anaerobic culture were inoculated on to fastidious anaerobic blood agar (90; LAB M) containing 5 per cent sterile sheep’s blood (FABA), FABA plus nalidixic acid/vancomycin (X090; LAB M) for the selection of Gram-negative organisms, and FABA plus neomycin (X015; LAB M) for the selection of Gram-positive organisms. The plates were incubated for 48 hours at 37°C in an anaerobic cabinet (Don Whitley). All organisms grown were subcultured aerobically and anaerobically to detect ‘true anaerobes’. The resulting anaerobes were then identified using standard bacteriological techniques (Carter and Cole 1990, Cowan and Steel 1993). For statistical analysis, the bacteria identified were placed in one of the following three categories: very important (category 1); important (category 2); and other (category 3). The categorisation was based on potential pathogenicity in the environment of the bovine reproductive tract (Paisley and others 1986, Bonnett and others 1991, Bekana and others 1994, Cohen and others 1996, Noakes 1996). Forty-nine different bacteria were identified from analysis of the swabs. The categorisation for the first two categories is shown in Table 1. The other 33 bacteria cultured from the swabs were categorised as category 3 and were considered to be sample contaminants or unimportant in the context of this study. For statistical analysis, at each swabbing occasion the most serious category of bacteria cultured from either the aerobic or anaerobic swab was determined. For this analysis, category 3 also included those cows from which no bacteria were isolated. Analysis of these data using Fisher’s exact test demonVeterinary Record (2000) 146, 760-762