With recent poaching of southern white rhinoceros (SWR [Ceratotherium simum simum]) reaching record levels, the need for a robust assurance population is urgent. However, the global captive SWR population is not currently self-sustaining due to the reproductive failure of captive-born females. Dietary phytoestrogens have been proposed to play a role in this phenomenon, and recent work has demonstrated a negative relationship between diet estrogenicity and fertility of captive-born female SWR. To further examine this relationship, we compared gut microbial communities, fecal phytoestrogens, and fertility of SWR to those of another rhinoceros species—the greater one-horned rhinoceros (GOHR [Rhinoceros unicornis]), which consumes a similar diet but exhibits high levels of fertility in captivity. Using 16S rRNA amplicon sequencing and mass spectrometry, we identified a species-specific fecal microbiota and three dominant fecal phytoestrogen profiles. These profiles exhibited various levels of estrogenicity when tested in an in vitro estrogen receptor activation assay for both rhinoceros species, with profiles dominated by the microbial metabolite equol stimulating the highest levels of receptor activation. Finally, we found that SWR fertility varies significantly not only with respect to phytoestrogen profile, but also with respect to the abundance of several bacterial taxa and microbially derived phytoestrogen metabolites. Taken together, these data suggest that in addition to species differences in estrogen receptor sensitivity to phytoestrogens, reproductive outcomes may be driven by the gut microbiota’s transformation of dietary phytoestrogens in captive SWR females. IMPORTANCE Southern white rhinoceros (SWR) poaching has reached record levels, and captive infertility has rendered SWR assurance populations no longer self-sustaining. Previous work has identified dietary phytoestrogens as a likely cause of this problem. Here, we investigate the role of gut microbiota in this phenomenon by comparing two rhinoceros species to provide the first characterizations of gut microbiomes for any rhinoceros species. To our knowledge, our approach, combining parallel sequencing, mass spectrometry, and estrogen receptor activation assays, provides insight into the relationship between microbially mediated phytoestrogen metabolism and fertility that is novel for any vertebrate species. With this information, we plan to direct future work aimed at developing strategies to improve captive reproduction in the hope of alleviating their threat of extinction.

Greenhouse studies were planted at the R.R. Foil Plant Science Research Center in Starkville, MS. In the efficacy trial, pots were seeded with barnyardgrass (Echinochloa crus-galli), broadleaf signalgrass (Urochloa platyphylla), and giant foxtail (Setaria faberi). In the lab detection trial, only barnyardgrass was seeded. Both studies consisted of 16 treatments with four replications per treatment. The treatments consisted of clethodim, glyphosate, dicamba, and 2,4-D applied singularly and in combination with each other. Each herbicide combination was applied with three application methods: tank mixture, sequential applications where the synthetic auxin was applied first (auxin applied first), and sequential applications where glyphosate or clethodim was applied first (auxin applied second). The auxin applied second method had higher visual estimations of control ratings and lower biomass weights compared to the other two methods. The auxin applied second method had more glyphosate and clethodim detected with the use of liquid chromatography tandem mass spectrometry.

29With recent poaching of southern white rhinoceros (Ceratotherium simum simum; SWR) 30 reaching record levels, the need for a robust assurance population is urgent. However, the global 31 captive SWR population is not currently self-sustaining due to the reproductive failure of 32 captive-born females. Dietary phytoestrogens have been proposed to play a role in this 33 phenomenon, and recent work has demonstrated a negative relationship between diet 34 estrogenicity and fertility of captive-born female SWR. To further examine this relationship, we 35 compared gut microbial communities, fecal phytoestrogens, and fertility of SWR to another 36 rhinoceros species-the greater one-horned rhinoceros (Rhinoceros unicornis; GOHR), which 37 consumes a similar diet but exhibits high levels of fertility in captivity. Using 16S rRNA 38 amplicon sequencing and mass spectrometry, we identified a species-specific fecal microbiota 39 and three dominant fecal phytoestrogen profiles. These profiles exhibited varying levels of 40 estrogenicity when tested in an in vitro estrogen receptor activation assay for both rhinoceros 41 species, with profiles dominated by the microbial metabolite, equol, stimulating the highest 42 levels of receptor activation. Finally, we found that SWR fertility varies significantly with 43 respect to phytoestrogen profile, but also with the abundance of several bacterial taxa and 44 microbially-derived phytoestrogen metabolites. Taken together, these data suggest that in 45 addition to species differences in estrogen receptor sensitivity to phytoestrogens, reproductive 46 outcomes may be driven by gut microbiota's transformation of dietary phytoestrogens in captive 47

The mitotic-inhibiting herbicide pronamide controls susceptible annual bluegrass (Poa annua L.) pre- and post-emergence, but in some resistant populations, post-emergence activity is compromised, hypothetically due to a target-site mutation, lack of root uptake, or an unknown resistance mechanism. Three suspected pronamide-resistant (LH-R, SC-R, and SL-R) and two pronamide-susceptible (BS-S and HH-S) populations were collected from Mississippi golf courses. Dose-response experiments were conducted to confirm and quantify pronamide resistance, as well as resistance to flazasulfuron and simazine. Target-sites known to confer resistance to mitotic-inhibiting herbicides were sequenced, as were target-sites for herbicides inhibiting acetolactate synthase (ALS) and photosystem II (PSII). Pronamide absorption, and translocation was investigated following foliar and soil applications. Dose-response experiments confirmed pronamide resistance of LH-R, SC-R, and SL-R populations, as well as instances of multiple resistance to ALS and PSII inhibiting herbicides. Sequencing of the α-tubulin gene confirmed the presence of a mutation that substituted isoleucine for threonine at position 239 (Thr239-Ile) in LH-R, SC-R, SL-R, and BS-S populations. Foliar application experiments failed to identify differences in pronamide absorption and translocation between the five populations, regardless of harvest time. All populations had limited basipetal translocation—only 3–13% of the absorbed pronamide—across harvest times. Soil application experiments revealed that pronamide translocation was similar between SC-R, SL-R, and both susceptible populations across harvest times. The LH-R population translocated less soil-applied pronamide than susceptible populations, 24, 72, and 168 HAT, suggesting that reduced acropetal translocation may contribute to pronamide resistance. This study reports three new pronamide-resistant populations, two of which are resistant to two modes-of-action (MOA), and one of which is resistant to three MOA. Results suggest that both target-site- and translocation-based mechanisms may be associated with pronamide resistance. Further research is needed to confirm the link between pronamide resistance and the Thr239-Ile mutation of the α-tubulin gene.

Objective To determine synovial butorphanol concentrations and mechanical nociceptive threshold (MNT) changes after butorphanol intravenous regional limb perfusion (IVRLP). Study design Experimental Animals Six adult horses. Methods Cephalic IVRLP was performed with 10 mg butorphanol in sedated horses with a wide rubber tourniquet and a total volume of 30 mL. Radiocarpal synovial fluid and serum concentrations along with MNT were evaluated prior to and 0.5, 1, 2, 4, and 6 hours after IVRLP. Butorphanol concentrations were determined with liquid chromatography coupled to tandem mass spectrometry positive electrospray ionization. Results Butorphanol concentrations reached mean (SD) peak concentrations of 9.47 ng/mL (±12.00) in synovial fluid and 3.89 ng/mL (3.29) in serum 30 minutes after IVRLP. Concentrations remained above baseline for 4 hours in synovial fluid (P ≤ .017) and for 2 hours in serum (P ≤ .016). The only difference in MNT was detected 1 hour after IVRLP, when MNT were higher in controls than in treated horses (P = .047). Conclusion Butorphanol IVRLP seemed well tolerated and resulted in measurable levels of butorphanol in the radiocarpal synovial fluid of five of six horses. Clinical significance Intravenous regional limb perfusion appears to be a viable alternative to administer butorphanol, but additional investigation is required to evaluate the dose and local concentrations required for analgesia.