Tayler Catherine Kent

This study aimed to determine the prevalence and associated plasmid resistant genome of extended spectrum β-lactamase (ESBL) and Ambler class C cephalosporinase (AmpC) producing commensal Escherichia coli, using a targeted pDNA approach, from lactating cows and pre-weaned calves on dairy farms in Western Australian (WA). Following culture and modified double disc diffusion, ESBL and/or AmpC producing commensal E. coli were isolated from faecal samples and phenotypic antimicrobial resistance (AMR) determined. Plasmid DNA (pDNA) was extracted, amplified, and sequenced to identify the AMR and replicon composition. Phenotypic analysis of 1,117 faecal samples from 26 Western Australian dairy farms revealed a low overall prevalence (7.3%) of ESBL and/or AmpC-producing commensal E. coli, with 3.2% in cows (n = 633) and 12.8% in calves (n = 484). All isolates were classified ‘non-wildtype’ for ampicillin and ceftiofur, with 95.1% classified as ‘non-wildtype’ multidrug resistant (MDR). While blaTEM was common in the pDNA (total: 60.6%; cow: 76.9%; calf: 57.7%), a total of 13 AMR genes were identified across 126 plasmids (cow: n = 30; calf: n = 96) isolated. Multiple correspondence analysis (with chi-square–based 95% confidence ellipses) showed variation in pDNA AMR genes and replicons by farm, with generally distinct plasmid profiles, though some clustering suggests possible farm-to-farm plasmid transmission. In conclusion, though low in prevalence, ESBL and/or AmpC producing commensal E. coli having high phenotypic AMR, MDR, and pDNA diversity were isolated from dairy cattle on 65.4% of screened farms.
•Low prevalence of commensal E. coli producing ESBL and/or AmpC enzymes.•ESBL and/or AmpC producing commensal E. coli were isolated from dairy cattle on 65.4% of the 26 farms screened.•Isolates had high phenotypic resistance, multiple drug resistance, and plasmid diversity.•Targeted pDNA approach appreciates AMR diversity with lower cost, time, and data complexity.

Herbal medicines and dietary supplements are used by around 63% of Australians, despite known risks to consumers. In Australia, these products are deemed ‘low risk’, and as such their regulation by the Therapeutic Goods Administration (TGA) is largely hands-off compared to registered medicines, resulting in widespread use despite a lack of safety and efficacy data. Particularly common are ‘fertility-boosting’ supplements, which are heavily marketed towards and have a vulnerable audience in couples trying to conceive. In addition, the use of these supplements has more recently expanded into veterinary assisted reproduction, especially as an additive to frozen spermatozoa, for their purported antioxidant effects.

Using ram spermatozoa as a model for both human and veterinary applications, this study characterised the biochemical changes in spermatozoa after exposure to three common herbal supplement ingredients: Panax ginseng (ginseng), Lepidium meyenii (maca) root and Camellia sinensis (green tea) extract (GTE). A suite of techniques including flow cytometry, gas chromatography-mass spectrometry, computer-assisted sperm analysis (CASA) and enzyme-linked immunosorbent assay (ELISA) was used to demonstrate that exposure to varying concentrations of these compounds induced significant biochemical changes in spermatozoa. These included altered energy metabolism and cell signalling pathways, as well as increased frequency of acrosome reaction, an increase in mitochondrial oxidative stress, and increased DNA fragmentation. Additionally, isolated active ginsenosides and catechins (from ginseng and GTE respectively) were found to induce changes similar to the parent compounds when spermatozoa were exposed.

The effect of herbal compounds on cultured testicular cell metabolism was also investigated. Murine Leydig and Sertoli cells were exposed to the compounds listed above, and select biochemical changes measured. Testicular cells also exhibited altered energy metabolism, cell signalling, and hormone changes in the presence of these supplements.


Despite the reported antioxidant activity of these compounds, the increase in mitochondrial reactive oxygen species (ROS) production and DNA damage in spermatozoa indicates that, at the concentrations tested, these compounds had a pro-oxidant effect on spermatozoa, which may lead to decreased fertility. Significant DNA damage, caused by oxidative stress, can reduce the capacity of a spermatozoon to fertilise an ovum or compromise the integrity of a zygote if fertilisation does occur. Additionally, the increase in spontaneous acrosome reaction, normally the final step in fertilisation, as well as increased oestrogen concentration in testicular cells, suggest that these compounds may act as endocrine disruptors and alter normal reproductive function. These results indicate that herbal medicines and dietary supplements containing ginseng, maca or GTE should be used with caution until their effects on male fertility are further elucidated. Future research in this area should target the mechanism by which these compounds induce DNA damage and endocrine disruption.

This study aimed to determine the prevalence of antimicrobial resistance (AMR) in commensal E. coli from healthy lactating cows and calves in the Mediterranean pasture-based feeding dairy system of Western Australia (WA). Fecal samples were collected from healthy adult lactating cows and healthy calves from dairy farms in WA. Presumptive commensal E. coli was isolated from these samples and confirmed using matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Broth microdilution was used to assess the prevalence and the phenotypic AMR profiles of the E. coli isolates to 8 antimicrobial agents of dairy industry and human importance. The minimum inhibitory concentration (MIC) for each isolate was interpreted using the Epidemiologic Cutoff (ECOFF) and Clinical and Laboratory Standards Institute (CLSI) breakpoints. Genomic characterization provided multi-locus sequence types and AMR genes for a selection of isolates categorised as non-wild type (NWT) by ECOFF values for the combination of ampicillin, trimethoprim-sulfamethoxazole, and tetracycline. From a total of 1,117 fecal samples (633 adult, 484 calf) collected across 26 randomly selected farms, 891 commensal E. coli isolates were recovered (541 adult, 350 calf). Commensal E. coli classified as NWT was highest for ampicillin for both adult (68.8%; 95% CI = 64.7 – 72.7) and calf feces (67.1%; 95% CI = 62.0 – 72.0). A large proportion of tetracycline NWT and trimethoprim-sulfamethoxazole NWT organisms were also identified from calf feces, being 44.0% (95% CI = 38.7 – 49.4) and 24.6% (95% CI = 20.2 – 29.4) respectively. Clinical resistance prevalence was low, being higher for calves than for adult feces (ampicillin (adult: 7.8% (95% CI = 5.7 – 10.3); calf: 30.0% (95% CI = 25.2 – 35.1), tetracycline (adult: 6.3% (95% CI = 4.4 – 8.7); calf: 40.3% (95% CI = 35.1 – 45.6), and trimethoprim-sulfamethoxazole (adult: 2.6% (95% CI = 1.4 – 4.3); calf: 22.0% (95% CI = 17.7 – 26.7)). Commensal E. coli originating from calf feces was significantly higher in NWT prevalence compared with adult feces for ciprofloxacin (P = 0.023), gentamicin (P = 0.02), tetracycline (P < 0.001), and trimethoprim-sulfamethoxazole (P < 0.001). The overall number of antimicrobials an isolate was classified as NWT toward varied among farms and was significantly higher for isolates originating from calf than adult feces (P < 0.001). The strain type and sampling source of the commensal E. coli investigated were both associated with the commonality of the resultant resistance genome. Clinical resistance and NWT classification were highest for ampicillin, tetracycline, and trimethoprim-sulfamethoxazole, all antimicrobials commonly used in the treatment of dairy cattle in Australia. Though highly variable across farms, commensal E. coli isolated from healthy dairy calf feces had significantly higher NWT and multidrug resistance (MDR) prevalence compared with feces from healthy adult lactating dairy cows. The resistant genome identified in MDR isolates, though not always consistent with the phenotype, included QnrS1 and genes encoding AmpC β-lactamase and aminoglycoside phosphotransferase.

Introduction: Herbal medicines are popular, despite a lack of regulation and research into safety and efficacy [1]. Supplements marketed for fertility are prevalent, but most studies regarding sperm focus on motility, with little research regarding toxicity. This study aimed to characterise the biochemical changes that occur in sperm after in vitro exposure to three common herbal supplements; green tea extract (GTE), maca root and Korean ginseng.

Methods: 12 ejaculates from three Merino rams were collected for each supplement, and sperm incubated with 0, 0.1, 0.5, 1 or 5 mg/ml of GTE, Korean ginseng (Paanax ginseng) or maca root (Lepidium meyenii). Supplements were dissolved in DMSO and PBS with a final concentration of 1% DMSO in all samples. At 0.5, 3 and 6 h post-exposure, computer-assisted sperm analysis (CASA) and flow cytometry were used to assess motility, viability, acrosome reaction, membrane lipid disorder, mitochondrial superoxide production, intracellular reactive oxygen species (ROS) and DNA fragmentation.

Results: Treatment with each supplement induced acrosome reaction in treated cells (2.02%± 0.39 n=180 p < .001), but showed no difference in viability(73.3%±1.49 n=180 p > .05). There was increased mitochondrial superoxide production across all concentrations and time points for each sup-plement (9.72%± 0.79% n=180 p < .001), as well as membrane lipid disorder across all supplements (17.28%±0.85 n=180 p < .001). All concentrations of GTE, and 0.5, 1 and 5 mg/ml ginseng and maca increased DNA fragmentation (7.71% ± 0.39% n=180 p < .001). Intracellular ROSproduction increased with 0.1, 0.5 and 5 mg/ml GTE (7.42%±0.45% p < .001).

Conclusions: Despite the reported antioxidant activity of herbal supplements, the increase in mitochondrial superoxide production and DNA fragmentation suggests that exposure to these concentrations of ginseng, maca and GTE increased oxidative stress in sperm [2]. The higher membrane fluidity and reduced acrosome integrity suggests potential oestrogenic activity of herbal supplements, as oestrogen can induce capacitation-like changes[3]. Despite the popularity and perceived safety of herbal supplements, this research indicates they should be used with caution until their effects are further elucidated.

Compounds with oestrogen-like actions are now common in both the Western diet. The long-term impacts and underlying mechanisms by which oestrogenic compounds alter male reproduction, however, are unclear. To investigate this, we used a longitudinal sheep model examining the impact of oestrogenic pasture consumption on semen quality and production, testicular size, sexual behaviour and the seminal plasma proteome of Merino rams (n = 20), over a full spermatogenic cycle and in the subsequent breeding season. Throughout the study period, sexual behaviour, sperm production and motility were similar between the exposed and non-exposed rams (P > 0.05). However, between 5 and 8 weeks of exposure to dietary phytoestrogens, rams produced a higher percentage of spermatozoa with a specific malformation of the sperm midpiece and reduced DNA integrity, compared to non-exposed rams (P < 0.001). Investigation into the seminal plasma proteome revealed 93 differentially expressed proteins between phytoestrogen-exposed and control rams (P < 0.05). Exposure to phytoestrogens increased the expression of proteins involved in cellular structure development, actin cytoskeleton reorganisation, regulation of cell function and decreased expression in those related to catabolic processes. The greatest fold changes were in proteins involved in the assembly of the sperm flagella, removal of cytoplasm, spermatid development and maintenance of DNA integrity. After returning to non-oestrogenic pasture, no differences in any measure were observed between treatment groups during the subsequent breeding season. We conclude that dietary phytoestrogens can transiently disrupt specific stages of ram spermatogenesis, causing subtle decreases in sperm quality by affecting the expression of pathways involved in the structural integrity of the spermatozoa.

Introduction. Korean ginseng is a common component of traditional herbal medicine, and is included in popular antioxidant supplements, including those marketed as ‘fertility-boosting’. Previous studies have indicated that ginseng consumption may increase sperm motility, though there is little research elucidating its effects on other functional or metabolic parameters in sperm.

Aims. This study aimed to elucidate the biochemical changes that occur in ovine sperm after in vitro exposure to ginseng.
Methods. Sperm was exposed to 0, 0.1, 0.5, 1 or 5 mg/mL ginseng in vitro, At 0.5, 3 and 6 hours post-exposure, computer-assisted sperm analysis (CASA) and flow cytometry were used to assess motility, viability, acrosome reaction, membrane lipid disorder, mitochondrial superoxide production, intracellular reactive oxygen species (ROS) and DNA fragmentation.

Results. Despite no change to sperm viability, exposure to all ginseng concentrations significantly increased the production of mitochondrial superoxide across all time points (9.72%± 0.79% n=180 p < 0.001), although general intracellular reactive oxygen species (ROS) production was not altered. DNA damage was significantly increased across all time points in cells exposed to 0.5, 1 and 5 mg/mL (7.71% ± 0.39% n= 180 p < 0.001). All ginseng concentrations at all time points significantly increased membrane fluidity (19.3% ± 1.51% n = 180 p < 0.001).

Discussion. Despite the reported antioxidant activity of ginseng, the increase in mitochondrial superoxide production, and the increase in DNA damage suggests that exposure to these concentrations of ginseng increase oxidative stress in sperm. The higher membrane fluidity of treated sperm suggests potential oestrogenic activity of ginseng, as oestrogen can induce capacitation-like changes. Despite the popularity and perceived safety of herbal fertility supplements, this research indicates they should be used with caution until their effects are further elucidated.

Introduction: Complementary and alternative medicines are used frequently, despite a lack of regulation and research into their safety and efficacy. Supplements marketed towards ‘boosting fertility’ are popular, but there is little research into their effects on spermatozoa. Previous studies looking at supplementation of sperm focus on motility rather than biochemical changes, and there is little research regarding potential toxicity in sperm.

Aim: The aim of this study was to determine whether in vitro incubation with maca root induced biochemical changes in ram spermatozoa.
Method: The function and metabolism of ram spermatozoa, incubated in media containing Lepidium meyenii (maca root) were investigated. At 0.5, 3 and 6 hours post-exposure, computer-assisted sperm analysis (CASA) and flow cytometry were used to assess motility, viability, acrosome reaction, membrane lipid disorder, mitochondrial superoxide production, intracellular reactive oxygen species (ROS) and DNA fragmentation.

Results: Treatment with maca induced acrosome reaction in treated cells (2.02% 0.39 n=3 p <0.001), but there was no difference in viability (73.3%1.49 n=180 p >0.05). There was an increase in mitochondrial superoxide production across all treatments and time points, and maca promoted membrane lipid disorder across all treatments (17.28%0.85 n= 54 p <0.001).

Discussion: The promotion of premature acrosome reaction in absence of an ovum may impact fertility of the male. It is posited that this may be driven by the oestrogenic activity of maca. Oestrogenic activity may also contribute to the higher membrane fluidity of treated sperm, as oestrogen can induce capacitation-like changes. Despite reported antioxidant activity of maca, the increase in mitochondrial superoxide production suggests increased oxidative stress in the presence of maca. Contrary to popular belief that these products enhance fertility, this research indicates that they should be used with caution due to possible negative effects on sperm, and lack of stringent safety data.

Oestrogenic pastures are known to cause infertility in the ewe, due primarily to the oestrogen-like actions of the metabolite equol. Despite strong evidence that phytoestrogens and their metabolites compromise male reproductive function in many other species, there is little information concerning the effect of oestrogenic pastures on ram sperm quality and function. To investigate this, ram spermatozoa were exposed in vitro to physiologically relevant concentrations of either 0, 0.001, 0.01, 0.1 and 1 μM equol and incubated over 6 h. Sperm motility, viability, DNA integrity, membrane lipid disorder, mitochondrial superoxide production, lipid peroxidation and intracellular reactive oxygen species were assessed via computer assisted sperm analysis and flow cytometry at 0.5, 3 and 6 h post-equol exposure. Whilst sperm viability was decreased only at 1 μM equol at 0.5 h post-exposure, exposure to equol at concentrations of 0.1 and 1 μM reduced sperm total and progressive motility (P < 0.001), increased sperm membrane fluidity (P < 0.001), increased mitochondrial superoxide production (P < 0.001) and promoted lipid peroxidation (P < 0.001) across all timepoints. At 6 h post-exposure to 0.1 and 1 μM equol, DNA fragmentation was greater compared that of non-exposed spermatozoa (P = 0.045). Intracellular reactive oxygen species did not change between treatment groups throughout the study (P > 0.05). It is concluded that even low concentrations of equol negatively impact the functionality of ram spermatozoa, these effects likely driven through increased mitochondrial superoxide production. This work indicates that equol may exert oestrogen-like actions upon ram spermatozoa, bringing into question as to whether oestrogenic pastures could influence ram fertility.