Comparative stability and analytical performance of anti-miroestrol recombinant antibody in different cassettes.

Immunoassays are efficient for the phytochemical analysis of various matrices. However, producing an appropriate recombinant antibody for small molecules is challenging, resulting in costly analyses. In this study, we aimed to develop recombinant fragment antigen-binding (Fab) antibodies against miroestrol, a potent phytoestrogen marker of Pueraria candollei. Two expression cassettes of Fab were established for the production of active Fab antibodies using SHuffle® T7 Escherichia coli cells. The orientation of variable fragment heavy chain (VH) and variable fragment light chain (VL) in the expression vector constructs influences the reactivity, stability, and binding specificity of the resultant Fab. Stability testing of antibodies demonstrated that Fab is a more stable form of recombinant antibody than a single-chain variable fragment (ScFv) antibody in all conditions. Based on the obtained Fab, the ELISA specifically detected miroestrol in the range of 39.06-625.00 ng/mL. The intra- and inter-assay precisions were 0.74-2.98% and 6.57-9.76%, respectively. The recovery of authentic miroestrol spiked into samples was 106.70-110.14%, and the limit of detection was 11.07 ng/mL. The results for P. candollei roots and products determined using our developed ELISA with Fab antibody and an ELISA with anti-miroestrol monoclonal antibody (mAb) were consistent (R2 = 0.9758). The developed ELISA can be applied for the quality control of miroestrol derived from P. candollei. Therefore, the appropriate expression platform of Fab resulted in the stable binding specificity of the recombinant antibody and was applicable for immunoassays.Key points• ELISAs with Fab has higher sensitivity than that with ScFv.• Fab is more stable than ScFv.• Fab-based ELISA can be used for miroestrol determination of Pueraria candollei.

Mixtures of Mycotoxins, Phytoestrogens, and Other Secondary Metabolites in Whole-Plant Corn Silages and Total Mixed Rations of Dairy Farms in Central and Northern Mexico.

Mycotoxins and endocrine disruptors such as phytoestrogens can affect cattle health, reproduction, and productivity. Most studies of mycotoxins in dairy feeds in Mexico and worldwide have been focused on a few (regulated) mycotoxins. In contrast, less known fungal toxins, phytoestrogens, and other metabolites have been neglected and underestimated. This study analyzed a broad spectrum (>800) of mycotoxins, phytoestrogens, and fungal, plant, and unspecific secondary metabolites in whole-plant corn silages (WPCSs) and total mixed rations (TMRs) collected from 19 Mexican dairy farms. A validated multi-metabolite liquid chromatography/electrospray ionization-tandem mass spectrometric (LC/ESI-MS/MS) method was used. Our results revealed 125 of >800 tested (potentially toxic) secondary metabolites. WPCSs/TMRs in Mexico presented ubiquitous contamination with mycotoxins, phytoestrogens, and other metabolites. The average number of mycotoxins per TMR was 24, ranging from 9 to 31. Fusarium-derived secondary metabolites showed the highest frequencies, concentrations, and diversity among the detected fungal compounds. The most frequently detected mycotoxins in TMRs were zearalenone (ZEN) (100%), fumonisin B1 (FB1) (84%), and deoxynivalenol (84%). Aflatoxin B1 (AFB1) and ochratoxin A (OTA), previously reported in Mexico, were not detected. All TMR samples tested positive for phytoestrogens. Among the investigated dietary ingredients, corn stover, sorghum silage, and concentrate proportions were the most correlated with levels of total mycotoxins, fumonisins (Fs), and ergot alkaloids, respectively.

Advances in the Bioactivities of Phytochemical Saponins in the Prevention and Treatment of Atherosclerosis.

Atherosclerosis (AS) is a chronic inflammatory disease characterized by hardening and narrowing of arteries. AS leads to a number of arteriosclerotic vascular diseases including cardiovascular diseases, cerebrovascular disease and peripheral artery disease, which pose a big threat to human health. Phytochemicals are a variety of intermediate or terminal low molecular weight secondary metabolites produced during plant energy metabolism. Phytochemicals from plant foods (vegetables, fruits, whole grains) and traditional herb plants have been shown to exhibit multiple bioactivities which are beneficial for prevention and treatment against AS. Many types of phytochemicals including polyphenols, saponins, carotenoids, terpenoids, organic sulfur compounds, phytoestrogens, phytic acids and plant sterols have already been identified, among which saponins are a family of glycosidic compounds consisting of a hydrophobic aglycone (sapogenin) linked to hydrophilic sugar moieties. In recent years, studies have shown that saponins exhibit a number of biological activities such as anti-inflammation, anti-oxidation, cholesterol-lowering, immunomodulation, anti-platelet aggregation, etc., which are helpful in the prevention and treatment of AS. This review aims to summarize the recent advances in the anti-atherosclerotic bioactivities of saponins such as ginsenoside, soyasaponin, astra-galoside, glycyrrhizin, gypenoside, dioscin, saikosaponin, etc.

Mixtures of mycotoxins, phytoestrogens and pesticides co-occurring in wet spent brewery grains (BSG) intended for dairy cattle feeding in Austria.

Spent brewery grains (BSG) are the main by-product of beer production and are incorporated in rations of food-delivering animals, mainly dairy cows. Like other agricultural commodities, BSG can be contaminated by a broad spectrum of natural and synthetic undesirable substances, which can be hazardous to animal and human health as well as to the environment. The co-occurrence of mycotoxins, phytoestrogens, other fungal and plant secondary metabolites, along with pesticides, was investigated in 21 BSG samples collected in dairy farms in Austria. For this purpose, a validated multi-metabolite liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) was employed. Metabolites derived from Fusarium, Aspergillus, Alternaria and pesticide residues, were ubiquitous in the samples. Zearalenone (ZEN), T-2 and HT-2 toxins were the only regulated mycotoxin detected, albeit at concentrations below the European guidance values for animal feeds. Ergot alkaloids, Penicillium-derived metabolites, and phytoestrogens had occurrence rates of 90, 48 and 29%, respectively. Penicillium metabolites presented the highest levels among the fungal compounds, indicating contamination during storage. Aflatoxins (AFs), ochratoxins and deoxynivalenol (DON) were not detected. Out of the 16 detected pesticides, two fungicides, ametoctradin (9.5%) and mandipropamid (14.3%) revealed concentrations exceeding their respective maximum residue level (MRL) (0.01 mg kg-1) for barley in two samples. Although based on European guidance and MRL values the levels of the detected compounds probably do not pose acute risks for cattle, the impact of the long-time exposure to such mixtures of natural and synthetic toxicants on animal health and food safety are unknown and must be elucidated.

Cocktails of Mycotoxins, Phytoestrogens, and Other Secondary Metabolites in Diets of Dairy Cows in Austria: Inferences from Diet Composition and Geo-Climatic Factors.

Dairy production is a pivotal economic sector of Austrian and European agriculture. Dietary toxins and endocrine disruptors of natural origin such as mycotoxins and phytoestrogens can affect animal health, reproduction, and productivity. This study characterized the profile of a wide spectrum of fungal, plant, and unspecific secondary metabolites, including regulated, emerging, and modified mycotoxins, phytoestrogens, and cyanogenic glucosides, in complete diets of lactating cows from 100 Austrian dairy farms. To achieve this, a validated multi-metabolite liquid chromatography/electrospray ionization−tandem mass spectrometric (LC/ESI−MS/MS) method was employed, detecting 155 of >800 tested metabolites. Additionally, the most influential dietary and geo-climatic factors related to the dietary mycotoxin contamination of Austrian dairy cattle were recognized. We evidenced that the diets of Austrian dairy cows presented ubiquitous contamination with mixtures of mycotoxins and phytoestrogens. Metabolites derived from Fusarium spp. presented the highest concentrations, were the most recurrent, and had the highest diversity among the detected fungal compounds. Zearalenone, deoxynivalenol, and fumonisin B1 were the most frequently occurring mycotoxins considered in the EU legislation, with detection frequencies >70%. Among the investigated dietary factors, inclusion of maize silage (MS) and straw in the diets was the most influential factor in contamination with Fusarium-derived and other fungal toxins and metabolites, and temperature was the most influential among the geo-climatic factors.

Endocrine disruptors on and in fruits and vegetables: Estimation of the potential exposure of the French population.

Fruits and vegetables are considered to be healthy compared to fats, carbohydrates, and meats. However, their production involves plant protection products (PPPs) or they can contain phytoestrogens which may exhibit endocrine effects. Thus, the exposure to the main PPPs and to phytoestrogens known as endocrine disruptors (EDs) is estimated. PPPs include fungicides, growth substances, herbicides, and insecticides authorised in France. ED-PPPs exposure is estimated from the maximum residue limits (MRLs) of 70 potential ED-PPPs used in France on 64 fruits and vegetables. The estimated exposure to potential ED-PPPs is 509 µg/d and involves agonist and antagonist substances in complex mixtures. Anti-androgens are preeminent, at 353 µg/d. Exposure to genistein and daidzein is calculated from 140 measurements in 9 categories of food-items containing soy. The global exposure to isoflavones in France is evaluated at 6700 µg/d. Phytoestrogen exposure is much higher than that of ED-PPPs. Their endocrine effects should be considered.

Mycotoxins, Phytoestrogens and Other Secondary Metabolites in Austrian Pastures: Occurrences, Contamination Levels and Implications of Geo-Climatic Factors.

Pastures are key feed sources for dairy production and can be contaminated with several secondary metabolites from fungi and plants with toxic or endocrine-disrupting activities, which possess a risk for the health, reproduction and performance of cattle. This exploratory study aimed to determine the co-occurrences and concentrations of a wide range of mycotoxins, phytoestrogens and other secondary metabolites in grazing pastures. Representative samples of pastures were collected from 18 Austrian dairy farms (one sample per farm) between April to October 2019. After sample preparation (drying and milling) the pastures were subjected to multi-metabolite analysis using LC-MS/MS. In total, 68 metabolites were detected, including regulated zearalenone and deoxynivalenol (range: 2.16-138 and 107-505 µg/kg on a dry matter (DM) basis, respectively), modified (3-deoxynivalenol-glucoside, HT-2-glucoside) and emerging Fusarium mycotoxins (e.g., enniatins), ergot alkaloids and Alternaria metabolites along with phytoestrogens and other metabolites. Aflatoxins, fumonisins, T-2 toxin, HT-2 toxin and ochratoxins were not detected. Of the geo-climatic factors and botanical diversity investigated, the environment temperature (average of 2 pre-sampling months and the sampling month) was the most influential factor. The number of fungal metabolites linearly increased with increasing temperatures and temperatures exceeding 15 °C triggered an exponential increment in the concentrations of Fusarium and Alternaria metabolites and ergot alkaloids. In conclusion, even though the levels of regulated mycotoxins detected were below the EU guidance levels, the long-term exposure along with co-occurrence with modified and emerging mycotoxins might be an underestimated risk for grazing and forage-fed livestock. The one-year preliminary data points out a dominant effect of environmental temperature in the diversity and contamination level of fungal metabolites in pastures.

High resolution mass spectrometry workflow for the analysis of food contaminants: Application to plant toxins, mycotoxins and phytoestrogens in plant-based ingredients.

An analytical workflow including mass spectral library, generic sample preparation, chromatographic separation, and analysis by high-resolution mass spectrometry (HRMS) was developed to gain insight into the occurrence of plant toxins, mycotoxins and phytoestrogens in plant-based food. This workflow was applied to 156 compounds including 90 plant toxins (pyrrolizidine alkaloids, tropane alkaloids, glycoalkaloids, isoquinoline alkaloids and aristolochic acids), 54 mycotoxins (including ergot alkaloids and Alternaria toxins) and 12 phytoestrogens (including isoflavones, lignans and coumestan) in plant-based protein ingredients, cereal and pseudo-cereal products. A mass spectral library was built based on fragmentation spectra collected at 10 different collision energies in both positive and negative ionisation modes for each toxin. Emphasis was put on a generic QuEChERS-like sample preparation followed by ultra-high-pressure liquid chromatography using alkaline mobile phase allowing the separation of more than 50 toxic pyrrolizidine alkaloids. HRMS acquisition comprised a full-scan event for toxins detection followed by data-dependent MS2 for toxin identification against mass spectrum. Method performance was evaluated using fortified samples in terms of sensitivity, repeatability, reproducibility and recovery. All toxins were positively identified at levels ranging from 1 µg kg-1 to 100 µg kg-1. Quantitative results obtained by a standard addition approach met SANTE/12682/2019 criteria for 132 out of 156 toxins. Such a workflow using generic, sensitive and selective multi-residue method allows a better insight into the occurrence of regulated and non-regulated toxins in plant-based foods and to conduct safety evaluation and risk assessments when needed.

Miroestrol Quantification in Pueraria mirifica Crude Drugs and Products by Single-Reference UPLC/PDA/MS Using Relative Molar Sensitivities to Kwakhurin.

Owing to occasional health damages caused by health food products derived from Pueraria mirifica (PM), the Japanese government has designated PM as an "ingredient calling for special attention." Miroestrol is a specific isoflavone isolated from PM and possesses very strong estrogenic activity enough to induce side effects in small amount. Therefore, routine analyses for miroestrol quantification is recommended to control the safety and quality of PM products. However, miroestrol content in PM is quite low, and commercial reagent for its detection is rarely available. In this study, we developed a quantitative analysis method for miroestrol in PM without using its analytical standard by using the relative molar sensitivity (RMS) of miroestrol to kwakhurin, another PM-specific isoflavone, as a reference standard. The RMS value was obtained by an offline combination of 1H-quantitative NMR spectroscopy and a LC/photo diode array (PDA) and miroestrol content was determined by single-reference LC/PDA using RMS. Furthermore, we investigated miroestrol content in commercially available PM crude drugs and products, and the RMS method was compared with the conventional calibration curve method in terms of performance. The rate of concordance of miroestrol contents determined by two method was 89-101%. The results revealed that our developed LC/PDA/MS method with RMS using kwakhurin as a reference standard was accurate for routine monitoring of miroestrol content in PM crude drugs and products to control their quality.

Implications of a Soy-Based Diet for Animal Models.

The use of animal models in fundamental or pre-clinical research remains an absolute requirement for understanding human pathologies and developing new drugs. In order to transpose these results into clinical practice, many parameters must be taken into account to limit bias. Attention has recently been focused on the sex, age or even strain of each animal, but the impact of diet has been largely neglected. Soy, which is commonly used in the diet in varying quantities can affect their physiology. In order to assess whether the presence of soy can impact the obtained results, we studied the impact of a soy-based diet versus a soy-free diet, on diastolic function in a rat model based on transgenic overexpression of the ß3-adrenergic receptors in the endothelium and characterized by the appearance of diastolic dysfunction with age. Our results show that the onset of diastolic dysfunction is only observed in transgenic male rats fed with a soy-free diet in the long term. Our study highlights the importance of the diet's choice in the study design process, especially regarding the proportion of soy, to correctly interpret the outcome as low-cost diets are more likely to be highly concentrated in soy.

Is There Such a Thing as "Anti-Nutrients"? A Narrative Review of Perceived Problematic Plant Compounds.

Plant-based diets are associated with reduced risk of lifestyle-induced chronic diseases. The thousands of phytochemicals they contain are implicated in cellular-based mechanisms to promote antioxidant defense and reduce inflammation. While recommendations encourage the intake of fruits and vegetables, most people fall short of their target daily intake. Despite the need to increase plant-food consumption, there have been some concerns raised about whether they are beneficial because of the various 'anti-nutrient' compounds they contain. Some of these anti-nutrients that have been called into question included lectins, oxalates, goitrogens, phytoestrogens, phytates, and tannins. As a result, there may be select individuals with specific health conditions who elect to decrease their plant food intake despite potential benefits. The purpose of this narrative review is to examine the science of these 'anti-nutrients' and weigh the evidence of whether these compounds pose an actual health threat.

Molecularly imprinted polymers and capillary electrophoresis for sensing phytoestrogens in milk.

Dairy cow feed contains, among other ingredients, soybeans, legumes, and clover, plants that are rich in phytoestrogens. Several publications have reported a positive influence of phytoestrogens on human health; however, several unfavorable effects have also been reported. In this work, a simple, selective, and eco-friendly method of phytoestrogen isolation based on the technique of noncovalent molecular imprinting was developed. Genistein was used as a template, and dopamine was chosen as a functional monomer. A layer of molecularly imprinted polymers was created in a microtitration well plate. The binding capability and selective properties of obtained molecularly imprinted polymers were investigated. The imprinted polymers exhibited higher binding affinity toward chosen phytoestrogen than did the nonimprinted polymers. A selectivity factor of 6.94 was calculated, confirming satisfactory selectivity of the polymeric layer. The applicability of the proposed sensing method was tested by isolation of genistein from a real sample of bovine milk and combined with micellar electrokinetic capillary chromatography with UV-visible detection.

Evaluation of white Kwao Krua (Pueraria candollei Grah. ex Benth.) products sold in Thailand by molecular, chemical, and microscopic analyses.

The tuberous roots of Pueraria candollei Grah. ex Benth. (Fabaceae), commonly known as white Kwao Krua, are used to relieve menopausal symptoms in Thai traditional medicine because they contain phytoestrogens. Black and red Kwao Krua crude drugs exist as well, but they have different botanical origins and pharmacological activities. There is a high demand for white Kwao Krua products, but because of the limited availability of the plant material, it is suspected that the adulteration and misidentification of white Kwao Krua crude drugs and products occur. In this study, we authenticated white Kwao Krua products collected from Thai herbal markets by molecular, chemical, and microscopic analyses. The nucleotide sequences in the internal transcribed spacer (ITS) and trnH-psbA regions of 23 samples of authentic P. candollei were analyzed, and both regions were found to have intraspecific DNA polymorphisms. Based on the single nucleotide polymorphisms in the ITS1 region, species-specific primer sets of P. candollei were designed to authenticate white Kwao Krua and differentiate it from red and black Kwao Krua. Only the PCR products of KWP02 were not amplified by the primer sets. Isoflavonoid contents and microscopic features were used to support the results of molecular analysis to clarify the botanical origin of white Kwao Krua. Molecular, chemical and microscopic methods confirmed that all the Thai Kwao Krua products examined in this study contained authentic "white Kwao Krua" as claimed on their labels.

The relationship of obesity with lifestyle and dietary exposure to endocrine-disrupting chemicals.

Phytoestrogens are natural components of plants, including numerous foods that form part of the habitual diet of humans and animals. They have similar estrogenic effects to those of synthetic endocrine disrupters such as monomers of plastic materials, e.g., polycarbonates and epoxy resins. The most frequently used monomer is bisphenol A (BPA), which has been found to migrate from drink and food packaging, plastic baby bottles, and the coating of cans. Numerous studies have associated exposure to endocrine disrupters with obesity, classifying them as obesogens. The objectives of this study were to estimate the dietary exposure to phytoestrogens and BPA in a group of Spanish schoolchildren and to estimate their potential obesogenic effects. The diet of this population of healthy Spanish children was estimated to have a mean total estrogenic capacity of 5.10-12 M eq.E2 (5 pmol/day). The effects of this additional estrogenic burden are highly controversial, and no definitive conclusion has been reached. Thus, some authors consider exposure to these substances with estrogenic activity to be positive at certain stages of life, whereas others regard it as posing a risk at any age. In the present population of children, the likelihood of normal weight versus obesity was significantly related to the total proliferative effect (OR = 0.51, p = 0.026) as well as to the energy expenditure on physical activity, with lesser activity implying a greater risk of obesity (OR = 13.54, p = 0.001). Further research is warranted on the obesogenic effects of exposure to endocrine disruptors present in foods.

New Evaluation of Isoflavone Exposure in the French Population.

The study relates the present evaluation of exposure to estrogenic isoflavones of French consumers through two approaches: (1) identification of the isoflavone sources in the French food offering, (2) a consumption-survey on premenopausal women. For the foodstuff approach 150 food-items were analysed for genistein and daidzein. Additionally, 12,707 labels of processed-foods from French supermarket websites and a restaurant-supplier website were screened, and 1616 foodstuffs of interest were retained. The sources of phytoestrogens considered were soy, pea, broad bean and lupine. A price analysis was performed. A total of 270 premenopausal women from the French metropolitan territory were interviewed for their global diet habits and soy consumption and perception. In supermarkets, there were significantly less selected foodstuffs containing soy than in restaurant (11.76% vs. 25.71%, p < 0.01). There was significantly more soy in low price-foodstuff in supermarket (p < 0.01). Isoflavone levels ranged from 81 to 123,871 µg per portion of the analyzed soy containing foodstuff. Among the women inquired 46.3% claimed to have soy regularly. Isoflavone intake >45 mg/day is associated to vegan-diet (p < 0.01). In total, 11.9% of soy-consumers had a calculated isoflavone intake >50 mg/day. This dose can lengthen the menstrual cycles. The actual exposure to phytoestrogen is likely to have an effect in a part of the French population.

Phytoestrogens, phytosteroids and saponins in vegetables: Biosynthesis, functions, health effects and practical applications.

Phytoestrogens are non-steroidal secondary metabolites with similarities in structure and biological activities with human estrogens divided into various classes of compounds, including lignans, isoflavones, ellagitannins, coumestans and stilbenes. Similarly, phytosteroids are steroidal compounds of plant origin which have estrogenic effects and can act as agonists, antagonists, or have a mixed agonistic/antagonistic activity to animal steroid receptors. On the other hand, saponins are widely distributed plant glucosides divided into triterpenoid and steroidal saponins that contribute to plant defense mechanism against herbivores. They present a great variation from a structural point of view, including compounds from different classes. In this chapter, the main vegetable sources of these compounds will be presented, while details regarding their biosynthesis and plant functions will be also discussed. Moreover, considering the significant bioactive properties that these compounds exhibit, special focus will be given on their health effects, either beneficial or adverse. The practical applications of these compounds in agriculture and phytomedicine will be also demonstrated, as well as the future prospects for related research.

A rapid UHPLC-MS/MS method for quantitation of phytoestrogens and the distribution of enterolactone in an Alabama estuary.

Endocrine disrupting chemicals (EDCs) are natural or synthetic compounds that can interfere with the endocrine systems of humans and wildlife. EDCs can pass through wastewater treatment systems, or run off from urban areas or agricultural operations, into natural water bodies, exposing resident and migratory organisms to complex EDC mixtures. Some phytoestrogenic polyphenolics (PEPP) are known or suspected EDCs; however, their contribution to total EDC burden in natural surface water systems is largely unknown. We describe a rapid, sensitive, and reproducible quantitative method for analysis of 15 PEPP in estuarine sediment and water, using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS/MS). The method provides excellent peak resolution, peak separation, and rapid run times (method separation/total run time: 8/12.5 min). With two exceptions, spiking experiments demonstrated that the percent recoveries for target PEPP in sediment and water samples were within acceptable analytical validation limits. LOD and LOQ values ranged from 0.004 to 0.010 ng/injection and 0.013-0.032 ng/injection, respectively. The validated method was used for PEPP analysis of sediment and water samples collected from 11 locations within the Perdido Bay estuary in coastal Alabama. No PEPP above the LOD were detected in sediment samples. The mammalian-derived lignin enterolactone was observed at low concentrations in water throughout the estuary, and significantly, at elevated concentrations at two locations associated with small-scale septic systems (3.66 ±â€¯0.27 ng L-1 and 4.01 ±â€¯0.33 ng L-1) and a large wastewater treatment system (4.56 ±â€¯0.24 ng L-1 and 5.69 ±â€¯0.43 ng L-1).

Concentrations of phytoestrogens in conventional, organic and free-range retail milk in England.

The effect of dairy management system (conventional, CNV; organic, ORG; free-range, FRG) and month on retail milk phytoestrogen composition was assessed for 12 consecutive months. ORG milk contained more secoisolariciresinol, matairesinol, lariciresinol, sum of plant lignans, daidzein, genistein, formononetin, naringenin, equol, sum of isoflavones and coumestrol, than CNV and FRG milk. This may be explained by the higher supply of pasture, and grazed or ensiled clover, in ORG dairy diets. Seasonal variation in milk phytoestrogen concentrations was higher for ORG than CNV and FRG systems. Phytoestrogen composition did not vary between FRG and CNV milk. Consuming organic milk can increase intake of potentially beneficial lignans and isoflavonoids, and in particular equol; but, any effects on human health from such milk compositional differences cannot be implied.

Gut Microbiota and Phytoestrogen-Associated Infertility in Southern White Rhinoceros.

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.

Solid-phase microextraction and on-fiber derivatization for assessment of mammalian and vegetable milks with emphasis on the content of major phytoestrogens.

A new solvent-free method for the simultaneous determination of some major phytoestrogens (equol, enterodiol, daidzein, genistein, glycitein) in different commercial milks (cow, goat and soy-rice) was developed. After solid phase microextraction, performed by direct immersion of a 65 µm-polydimethylsiloxane-divinylbenzene fiber in diluted (1:100 with 0.2% formic acid - 30% sodium chloride) milk samples (18 °C for 20 min under stirring), a direct on-fiber silylation with N,O-bis (trimethylsilyl)trifluoroacetamide) containing 1% trimethylchlorosilane (70 °C for 20 min) was performed prior to gas chromatography-mass spectrometry analysis. Since the target compounds were determined as aglycones, the hydrolytic removal of the aglycone from the glycosides was performed. The method permitted the determination of the target analytes in all the considered milk samples as well as the detection of some major amphipathic fats indicating that the approach could potentially be applied in the future for further applications, such as milk profiling.