First Results on the Presence of Mycotoxins in the Liver of Pregnant Fallow Deer (Dama dama) Hinds and Fetuses.

Reproductive abnormalities have been observed in fallow deer populations in Hungary. We supposed mycotoxin contamination to be one of the possible causes because multi-mycotoxin contamination is known to be dangerous even at low toxin levels, especially for young animals. We investigated the spatial pattern of mycotoxin occurrences and the relationship between maternal and fetal mycotoxin levels. A total of 72 fallow deer embryos and their mothers were sampled in seven forested regions in Hungary in the 2020/2021 hunting season. We analyzed Aflatoxin (AF), Zearalenone (ZEA), Fumonizin B1 (FB1), DON, and T2-toxin concentrations in maternal and fetal livers by ELISA. AF was present in 70% and 82%, ZEA in 41% and 96%, DON in 90% and 98%, T2-toxin in 96% and 85%, and FB1 in 84% and 3% of hind and fetus livers, respectively. All mycotoxins passed into the fetus, but only Fumonizin B1 rarely passed. The individual variability of mycotoxin levels was extremely high, but the spatial differences were moderate. We could not prove a relation between the maternal and fetal mycotoxin concentrations, but we found an accumulation of ZEA and DON in the fetuses. These results reflect the possible threats of mycotoxins to the population dynamics and reproduction of wild fallow deer.

[Possible applications of poultry immunoglobulins focusing on mycotoxin environmental loads and human influence]. / Baromfi-immunglobulinok lehetséges alkalmazásai a mikotoxin-környezetterhelések és a humánérintettség fókuszában.

In addition to their role, immunoglobulins can be used in animal and human diagnostic (immunoassay-based) measurements, prophylaxis and (immuno)therapy. For these purposes, today's "alternative" that is advantageous from an animal ethical point of view is the bird immunoglobulin Y isolated from egg yolk. Its development and production are cost-effective, the complexity is low, and due to its advantageous properties, it can be used in assays or even more so in medical therapies (primarily passive immunization). It is widely used (against pathogens or their toxins) in treatments of intestinal or metabolic diseases and inflammations. Its application in human diagnostics is limited, some markers are measured using immunoglobulin Y as assay component. In this study, a possible application, which is less common today, is presented. The problem of environmental impacts is becoming significant. Due to human activities, industrialization, environmental changes increase the appearance of natural environmental pollutants, including the effects of mycotoxins produced by molds locally and/or globally, which (mainly through nutrition) affect humans. Such agents often appear together, several mycotoxins affect the individual. As a result of their persistence, mycotoxins absorbed in the intestinal tract and accumulated in organs, can already reach levels that can cause physiological and/or behavioral effects. Although the examination of sources (contaminated foods) is regulated by law, the extent of accumulation has not been or cannot be examined and is often insufficiently taken into account. Due to the nature of the technique, the anti-mycotoxin avian immunoglobulin Y could be used both for detection of (deposited) mycotoxin(s) and/or even for immunotherapy (e.g., mycotoxin neutralization). Demonstrating the endocrine-disrupting mycotoxins using the example of zearalenone (with an explanation of its reproductive and immunological effects), we present generation of zearalenone (and mycotoxin-specific) avian immunoglobulin developments, advocate its use in human detection, urging the development of measurements that are suitable for detecting (multiple) accumulation. Orv Hetil. 2023; 164(39): 1527-1536.

Elaboration of massage technique for semen collection and examination of semen characteristics in chinchilla (Chinchilla lanigera).

The practice of artificial insemination for the long-tailed chinchilla has not been fully elaborated to date, and existing data available regarding their reproduction properties is contradictory. Until now, the collection of semen for chinchillas has been most-commonly obtained using electro-ejaculation methods exclusively. The primary objective of this study was the development of a manual technique for semen collection which meets all animal welfare requirements. An additional aim was to determine the basic spermatological parameters, such as motility, concentration, type and ratio of morphological abnormalities and live/dead cell ratio, under typical northern-hemisphere conditions, in Hungary. Over a 3 month period, a special massage technique was developed for the study, and using this method, the sperm parameters of 46 males were subsequently analyzed weekly for a period of one year. Approximately 66% of chinchillas responded positively to this technique, with the success rate of semen-collection attempts showing no variation between seasons. Average sperm concentration for the whole year was 935.17 million/ml using this method. Total cell motility was the highest in winter (90.3%), and the lowest in spring (84.3%). The proportion of live, intact cells were above 80% on average for the year, while the ratios of live, morphologically abnormal and dead cells were 6% and 14%, respectively. We found that midpiece abnormalities occurred in the highest proportion (0.95%-3.38%), while the head abnormalities showed the lowest ratio (0.01%-0.15%). Standard deviation among the parameters was relatively high, with the spring season proving to be the weakest in terms of sperm quality. This study has demonstrated that, semen can be successfully collected without the use of electro-ejaculation or anesthesia. Furthermore, although spermatological parameters do exhibit some fluctuation for the different times of the year, semen collected is nonetheless suitable for the purpose of artificial insemination of chinchillas at any time.

Analysis and Comparison of Rapid Methods for the Determination of Ochratoxin a Levels in Organs and Body Fluids Obtained from Exposed Mice.

Mycotoxins are bioaccumulative contaminants impacting animals and humans. The simultaneous detection of frequent active exposures and accumulated mycotoxin level (s) in exposed organisms would be the most ideal to enable appropriate actions. However, few methods are available for the purpose, and there is a demand for dedicated, sensitive, reliable, and practical assays. To demonstrate the issue, mice were exposed to a relevant agent Ochratoxin A (OTA), and accumulated OTA was measured by fine-tuned commercial assays. Quantitative high-performance liquid chromatography with fluorescence detection, enzyme-linked immunosorbent assay, and flow cytometry assays have been developed/modified using reagents available as commercial products when appropriate. Assays were performed on excised samples, and results were compared. Accumulated OTA could be detected and quantified; positive correlations (between applied doses of exposure and accumulated OTA levels and the results from assays) were found. Dedicated assays could be developed, which provided comparable results. The presence and accumulation of OTA following even a short exposure could be quantitatively detected. The assays performed similarly, but HPLC had the greatest sensitivity. Blood contained higher levels of OTA than liver and kidney. We demonstrate that specific but flexible and practical assays should be used for specific/local purposes, to measure the exposure itself and accumulation in blood or organs.

Transcriptome profiling of kisspeptin neurons from the mouse arcuate nucleus reveals new mechanisms in estrogenic control of fertility.

Kisspeptin neurons in the mediobasal hypothalamus (MBH) are critical targets of ovarian estrogen feedback regulating mammalian fertility. To reveal molecular mechanisms underlying this signaling, we thoroughly characterized the estrogen-regulated transcriptome of kisspeptin cells from ovariectomized transgenic mice substituted with 17ß-estradiol or vehicle. MBH kisspeptin neurons were harvested using laser-capture microdissection, pooled, and subjected to RNA sequencing. Estrogen treatment significantly (p.adj. < 0.05) up-regulated 1,190 and down-regulated 1,139 transcripts, including transcription factors, neuropeptides, ribosomal and mitochondrial proteins, ion channels, transporters, receptors, and regulatory RNAs. Reduced expression of the excitatory serotonin receptor-4 transcript (Htr4) diminished kisspeptin neuron responsiveness to serotonergic stimulation. Many estrogen-regulated transcripts have been implicated in puberty/fertility disorders. Patients (n = 337) with congenital hypogonadotropic hypogonadism (CHH) showed enrichment of rare variants in putative CHH-candidate genes (e.g., LRP1B, CACNA1G, FNDC3A). Comprehensive characterization of the estrogen-dependent kisspeptin neuron transcriptome sheds light on the molecular mechanisms of ovary-brain communication and informs genetic research on human fertility disorders.

Effects of Single and Repeated Oral Doses of Ochratoxin A on the Lipid Peroxidation and Antioxidant Defense Systems in Mouse Kidneys.

Ochratoxin-A (OTA) is a carcinogenic and nephrotoxic mycotoxin, which may cause health problems in humans and animals, and it is a contaminant in foods and feeds. The purpose of the present study is to evaluate the effect of oral OTA exposure on the antioxidant defense and lipid peroxidation in the kidney. In vivo administration of OTA in CD1, male mice (1 or 10 mg/kg body weight in a single oral dose for 24 h and repeated daily oral dose for 72 h or repeated daily oral dose of 0.5 mg/kg bodyweight for 21 days) resulted in a significant elevation of OTA levels in blood plasma. Some histopathological alterations, transcriptional changes in the glutathione system, and oxidative stress response-related genes were also found. In the renal cortex, the activity of the glutathione-system-related enzymes and certain metabolites of the lipid peroxidation (conjugated dienes, trienes, and thiobarbituric reactive substances) also changed.

Flow cytometry based rapid duplexed immunoassay for fusarium mycotoxins.

At small food processing facilities, the most frequently used test to determine if grain-derived mycotoxin concentrations are compliant with legal limits is the enzyme-linked immunosorbent assay (ELISA). Each kit is designed to detect one of the six dangerous mycotoxins. With the increasing occurrence of coinfection of grain with multiple-mycotoxins in the field and/or during storage, ELISA is no longer a cost effective best assay option. With ELISA, each species of mycotoxin requires different sample preparation/extraction and a 45 min incubation. The alternative multiplexed assay presented here, the competitive fluorescent microsphere immunoassay (CFIA), follows current food safety standards. It handles several toxins simultaneously with a single universal extraction protocol. The authors' objective was to modify an existing commercial CFIA kit developed for bench top flow cytometry and extend its utility for point-of-need (PON) applications. The accelerated protocol offers over 60% reduction in total processing time and it detects dual mycotoxin contamination simultaneously. The observed enhanced binding kinetics equations reported here utilizing suspended solid phase particles in liquid phase, are also supported by published theoretical calculations. In the near future portable cytometry may bring rapid multiplexed PON testing to assure the safety of small food processing installations. © 2016 International Society for Advancement of Cytometry.

Xenoestrogens Ethinyl Estradiol and Zearalenone Cause Precocious Puberty in Female Rats via Central Kisspeptin Signaling.

Xenoestrogens from synthetic or natural origin represent an increasing risk of disrupted endocrine functions including the physiological activity of the hypothalamo-pituitary-gonad axis. Ethinyl estradiol (EE2) is a synthetic estrogen used in contraceptive pills, whereas zearalenone (ZEA) is a natural mycoestrogen found with increasing prevalence in various cereal crops. Both EE2 and ZEA are agonists of estrogen receptor-α and accelerate puberty. However, the neuroendocrine mechanisms that are responsible for this effect remain unknown. Immature female Wistar rats were treated with EE2 (10 µg/kg), ZEA (10 mg/kg), or vehicle for 10 days starting from postnatal day 18. As a marker of puberty, the vaginal opening was recorded and neuropeptide and related transcription factor mRNA levels were measured by quantitative real time PCR and in situ hybridization histochemistry. Both ZEA and EE2 accelerated the vaginal opening, increased the uterine weight and the number of antral follicles in the ovary, and resulted in the increased central expression of gnrh. These changes occurred in parallel with an earlier increase of kiss1 mRNA in the anteroventral and rostral periventricular hypothalamus and an increased kisspeptin (KP) fiber density and KP-GnRH appositions in the preoptic area. These changes are compatible with a mechanism in which xenoestrogens overstimulate the developmentally unprepared reproductive system, which results in an advanced vaginal opening and an enlargement of the uterus at the periphery. Within the hypothalamus, ZEA and EE2 directly activate anteroventral and periventricular KP neurons to stimulate GnRH mRNA. However, GnRH and gonadotropin release and ovulation are disrupted due to xenoestrogen-mediated inhibitory KP signaling in the arcuate nucleus.

A new ochratoxin A biodegradation strategy using Cupriavidus basilensis Or16 strain.

Ochratoxin-A (OTA) is a mycotoxin with possibly carcinogenic and nephrotoxic effects in humans and animals. OTA is often found as a contaminant in agricultural commodities. The aim of the present work was to evaluate OTA-degrading and detoxifying potential of Cupriavidus basilensis OR16 strain. In vivo administration of OTA in CD1 male mice (1 or 10 mg/kg body weight for 72 hours or 0.5 mg/kg body weight for 21 days) resulted in significant elevation of OTA levels in the blood, histopathological alterations- and transcriptional changes in OTA-dependent genes (annexinA2, clusterin, sulphotransferase and gadd45 and gadd153) in the renal cortex. These OTA-induced changes were not seen in animals that have been treated with culture supernatants in which OTA was incubated with Cupriavidus basilensis OR16 strain for 5 days. HPLC and ELISA methods identified ochratoxin α as the major metabolite of OTA in Cupriavidus basilensis OR16 cultures, which is not toxic in vivo. This study has demonstrated that Cupriavidus basilensis OR16 efficiently degrade OTA without producing toxic adventitious metabolites.

Comparison and evaluation of seven different bench-top flow cytometers with a modified six-plexed mycotoxin kit.

Many bench-top flow cytometers (b-FCs) are compatible with microsphere-based multiplexed assays. Disciplines implementing b-FCs-based assays are expanding; they include monitoring and validating food quality. A multiplexed platform protocol was evaluated for poly-mycotoxin assays, which is compatible with a variety of b-FC models. The seven instruments included: BD FACSCalibur(™) , BD FACSArray(™) Bioanalyzer, Accuri C6, Partec CyFlow(®) Space, Beckman Coulter FC 500, Guava EasyCyte Mini, and Luminex 100 (™) . Current reports related to the food industry describe fungal co-infections leading to poly-mycotoxin contamination in grain (Sulyok M, Berthiller F, Krska R, Schuhmacher R, Rapid Commun Mass Spectrom 2006;20:2649-2659). It is imperative to determine whether b-FC-based assays can replace traditional single-mycotoxin enzyme-linked immunosorbent assay (ELISA). A six-plexed poly-mycotoxin kit was tested on seven different b-FCs. The modified kit was initially developed for the BD FACSArray(™) Bioanalyzer (BD Biosciences) (Czeh A, Mandy F, Feher-Toth S, Torok L, Mike Z, Koszegi B, Lustyik G, J Immunol Methods 2012;384:71-80). With the multiplexed platform, it is possible to identify up to six mycotoxin contaminants simultaneously at regional grain collection/transfer/inspection facilities. In the future, elimination of contaminated food threat may be better achieved with the inclusion of b-FCs in the food protection arsenal. A universal protocol, matched with postacquisition software, offers an effective alternative platform compared to using a series of ELISA kits. To support side-by-side evaluation of seven flow cytometers, an instrument-independent fluorescence emission calibration was added to the protocol. All instrument performances were evaluated for strength of agreement based on paired sets of evaluation to predicate method. The results suggest that all b-FCs were acceptable of performing with the multiplexed kit for five of six mycotoxins. For OTA, the detection sensitivity was consistent only for five of the seven instruments.

Effect of sex ratios, spiking and extra artificial insemination on the breeding efficiency of broiler breeders.

Since early fertility decline is a permanent problem of broiler breeders, the aim of this study was to test the effects of various sex ratios, spiking strategies and additional artificial inseminations (AI) on their breeding efficiency. Six breeder flocks were analysed during the whole reproduction cycle. In Flock A the sex ratio was maintained at 10% during the whole cycle (control), while in Flock B the number of males was increased to a final ratio of 16%. In Flocks C (technological control), D, E and F the ratio of males was gradually decreased from 10% to 6.5% until the end of the cycle. Moreover, at the age of 44 weeks in Flocks D and E 50 and 100% of cockerels were replaced by young ones, respectively, while in Flock F additional artificial inseminations were applied in the second half of the reproduction cycle. The increase of sperm transport was successful only in Groups B (increase in male numbers) and D (50% replacement of old cockerels with young ones); however, it was not sufficient for increasing the fertility rates in either group. Nor did additional artificial inseminations (Flock F) have an effect on fertility. As a conclusion, it can be established that increasing the sperm count in the hens' oviducts in any way could not improve fertility in the last third of the production cycle. The results also suggest that the expensive and labour-intensive spiking technique used in broiler breeder management is useless. The prime factor responsible for the shortened persistence of fertility may be the reduced ability of the female oviduct to accept and store sperm.

A new zearalenone biodegradation strategy using non-pathogenic Rhodococcus pyridinivorans K408 strain.

Zearalenone (hereafter referred to as ZEA) is a nonsteroidal estrogenic mycotoxin produced by several Fusarium spp. on cereal grains. ZEA is one of the most hazardous natural endocrine disrupting chemicals (EDC) which induces hyper estrogenic responses in mammals. This can result in reproductive disorders in farm animals as well as in humans. Consequently, detoxification strategies for contaminated crops are crucial for food safety. In this study we have developed a bacterial based detoxification system using a non-pathogen Rhodococcus pyridinivorans K408 strain. Following 5 days treatment of ZEA with R. pyridinivorans K408 strain HPLC analyses showed an 87.21% ZEA-degradation efficiency of the bacterial enzyme systems. In another approach, the strain biotransformation ability has also been confirmed by a bioluminescent version of the yeast estrogen screening system (BLYES), which detected an 81.75% of biodegradability of ZEA, in a good agreement with the chemical analyses. Furthermore, the capacity of R. pyridinivorans to eliminate the estrogenic effects of ZEA was tested by using an immature uterotrophic assay. Prepubertal female rats were treated with vehicle (olive oil), 17ß-estradiol, ZEA (0.1-1-5-10 mg/kg body weight) and LB broth containing 500 mg/l ZEA that has already been incubated with or without Rhodococcus pyridinivorans K408 strain. Uterine weights were measured and the mRNA level changes relating to apelin, aquaporin 5, complement component 2, and calbindin-3 genes were measured by qRT-PCR. These genes represent the major pathways that are affected by estromimetic compounds. Zearalenone feeding significantly increased the uterus weight in a dose dependent manner and at the same time upregulated complement component 2 and calbindin-3 expression as well as decreased apelin and aquaporin 5 mRNA levels comparable to that seen in 17ß-estradiol exposed rats. In contrast, LB broth in which ZEA was incubated with Rhodococcus pyridinivorans K408 prior to the feeding did not display any estrogenic effect neither on uterine weight nor on the expression of estrogen-regulated genes. Consequently, the identification of Rhodococcus pyridinivorans K408 strain in ZEA biodegradation proved to be a very efficient biological tool that is able to eliminate the complete estrogenic effects of ZEA. It is also remarkable that this biotransformation pathway of ZEA did not result in any residual estrogenic effects.

Differential regulation of hypothalamic neuropeptide Y hnRNA and mRNA during psychological stress and insulin-induced hypoglycemia.

Many signals reflecting energy balance and stress are integrated at the hypothalamic orexigenic NPY neurons. To determine transcriptional changes of the NPY gene in response to stress, we followed the time course and compared the expression of heteronuclear (hn)- and messenger (m)RNA levels by in situ hybridization histochemistry and by real time PCR in mice following insulin-induced hypoglycemia and restraint. Hypoglycemia in fasted mice resulted in a rapid increase of NPY hnRNA that peaked at 1h, declined thereafter by 2-4h after insulin injection and run parallel to that of NPY mRNA. Throughout the time course examined, NPY expressing cells in the medial basal hypothalamus remained overwhelmingly localized to the arcuate nucleus. Following restraint NPY mRNA slightly increased, however hnRNA levels decreased up to 2h, suggesting increased stability of mature NPY mRNA. These results highlight rapid changes and differential regulation of NPY expression in response to metabolic and stress challenges.