Determination of Veterinary Drug Residues, Mycotoxins, and Pesticide Residues in Bovine Milk by Liquid Chromatography Electrospray Ionisation -tandem Mass Spectrometry.

Introduction: Multi-class and multi-residue analyses are very complex procedures because of the physico-chemical properties of veterinary drug residues and other contaminants. The purpose of the study was to develop an analytical method for the sensitive determination of 69 analytes in bovine milk by liquid chromatography electrospray ionisation-tandem mass spectrometry. Material and Methods: Antimicrobial, anabolic hormone, lactone, ß-agonist, mycotoxin and pesticide residues were analysed in 120 raw milk samples from different dairy farms in North Macedonia. Stable isotopically labelled internal standards were used to facilitate effective quantification of the analytes. Results: The linear regression coefficients were higher than 0.99, the limits of detection ranged from 0.0036 to 47.94 µg/L, and the limits of quantification ranged from 0.053 to 59.43 µg/L. The decision limit values ranged from 0.062 to 211.32 µg/L and the detection capability from 0.080 to 233.71 µg/L. Average recoveries of the analytes spiked in raw milk were in the range of 70.83% to 109%, intra-day coefficient of variation (CV) values from 2.41% to 22.29%, and inter-day CV values from 3.48% to 23.91%. The method was successfully applied in the testing of bovine milk samples. In five samples residues were detected. They were sulfadimethoxine (in two samples), enrofloxacin, tetracycline and oxytetracycline and were at concentrations below the EU maximum residue limit. Conclusion: The method is useful for routine testing for this group of chemical hazards in bovine milk.

A new LC-MS/MS method for multiple residues/contaminants in bovine meat.

A multi-class and multi-residue/contaminant method for the determination of veterinary drug and pesticide residues and mycotoxins in bovine meat has been developed and validated. The veterinary drug residues/contaminants included antimicrobials, anabolic hormones, lactones, ß-agonists, mycotoxins, and pesticides. Isotopic labeled internal standards were included to compensate residual matrix effects. The calibrators used in the method demonstrated linearity with the R2 > 0.98. The decision limit (CCα) values were in the range from 0.067 to 2103.84 µg/kg, while the range for detection capability (CCß) was from 0.083 to 2482.13 µg/kg. The limit of detection (LOD) and limit of quantification (LOQ) were in the range from 0.059 to 291.36 µg/kg, and 0.081 to 328.13 µg/kg, respectively. The recovery of analytes ranged from 61.28% to 116.20%. The intra-day coefficient of variation (CV) was from 0.97 to 25.93% and the inter-day CV was 2.30-34.04%. The method has been used for the determination of 49 residues/contaminants in bovine meat. Application of the method in routine analysis in bovine samples, revealed in limited samples the presences of enrofloxacin, oxytetracycline and sulfadiazine at the concentration of 35.22 µg/kg, 27.35 µg/kg, and 36.20 µg/kg, respectively.

Prenatal dexamethasone exposure and developmental programming of the ovary of the offspring: a structural study in the rat.

Overexposure to glucocorticoids during fetal development alters fetal organ growth and maturation patterns, which can result in adverse programming outcomes in adulthood. The aim of this study was to determine whether exposure to dexamethasone (Dx) during the fetal period programmed ovary development and function in infant (16-day-old) and peripubertal (38-day-old) female offspring. Pregnant Wistar rats were separated into control and Dx-treated (0.5mg kg-1) groups and were injected with Dx or an equivalent volume of vehicle on Days 16, 17 and 18 of gestation. Ovaries from 16- and 38-day-old female offspring were prepared for histological and stereological examination. The volume of the ovary and the number of primordial and primary follicles were significantly reduced in prenatally Dx-exposed infant and peripubertal female offspring compared with control offspring. The number of multilaminar follicles was decreased in infant female offspring. In peripubertal females, prenatal exposure to Dx increased the number of multilaminar and large follicles of all classes. Because vaginal opening did not occur up to Day 38 postpartum in the Dx-exposed offspring, the absence of ovulation and corpora lutea is confirmation that the onset of puberty had been delayed. We can conclude that overexposure to glucocorticoids early in life programs ovary development, which may affect fertility in adulthood.

A Moderate Increase in Ambient Temperature Influences The Structure and Hormonal Secretion of Adrenal Glands in Rats.

OBJECTIVE: As a consequence of global warming, the increase in the average annual temperature is observed, while the living organisms actively adapt to these changes. High environmental temperature initiates numerous physiological, autonomic, and behavioral responses, and activates the stress response. Thus, the aim of the study was to investigate effect of a moderate increase in ambient temperature on the activity of the hypothalamic-pituitary-adrenocortical (HPA) axis by determining histological changes in adrenal glands and hormonal levels in adult male rats. MATERIALS AND METHODS: In this experimental study, the morpho-functional state of adrenal glands was estimated by stereological evaluation of parameters, including the adrenal volume, adrenocortical cell/nuclear size and number, and the volume density of vascular tissues after four days of exposure to a moderate increase in ambient temperature of 35 ± 1˚C. Novelli histochemical and vascular endothelial growth factor (VEGF) immunohistochemical staining provided insight into the adrenal gland vascular network. Additionally, the adrenal levels of aldosterone, corticosterone, and pituitary adrenocorticotropic hormone (ACTH) were determined as crucial indicators of the hypothalamic-pituitaryadrenocortical (HPA) axis activity. RESULTS: Prolonged exposure to a moderate increase in ambient temperature for four days resulted in a significant increase in ACTH level up to 24%, which altered adrenal glands both structurally and functionally. The adrenocortical volume and number of cells in all cortical zones were markedly increased (P<0.05). A statistically significant increase was shown in the level of aldosterone (16%) and corticosterone (25%) in serum levels of individuals. CONCLUSION: Increased activity of the HPA axis reflects the response to a moderate increase in ambient temperature during four days, showing the capacity of the HPA axis to adapt the organism to daily temperature changes.

Global transcriptome analysis of porcine oocytes in correlation with follicle size.

Although our knowledge regarding oocyte quality and development has improved significantly, the molecular mechanisms that regulate and determine oocyte developmental competence are still unclear. Therefore, the objective of this study was to identify and analyze the transcriptome profiles of porcine oocytes derived from large or small follicles using RNA high-throughput sequencing technology. RNA libraries were constructed from oocytes of large (LO; 3-6 mm) or small (SO; 1.5-1.9 mm) ovarian follicles and then sequenced in an Illumina HiSeq4000. Transcriptome analysis showed a total of 14,557 genes were commonly detected in both oocyte groups. Genes related to the cell cycle, oocyte meiosis, and quality were among the top highly expressed genes in both groups. Differential expression analysis revealed 60 up- and 262 downregulated genes in the LO compared with the SO group. BRCA2, GPLD1, ZP3, ND3, and ND4L were among the highly abundant and highly significant differentially expressed genes (DEGs). The ontological classification of DEGs indicated that protein processing in endoplasmic reticulum was the top enriched pathway. In addition, biological processes related to cell growth and signaling, gene expression regulations, cytoskeleton, and extracellular matrix organization were among the highly enriched processes. In conclusion, this study provides new insights into the global transcriptome changes and the abundance of specific transcripts in porcine oocytes in correlation with follicle size.

Intranuclear characteristics of pig oocytes stained with brilliant cresyl blue and nucleologenesis of resulting embryos.

Brilliant cresyl blue (BCB) vital labelling is a powerful method for analyzing the quality of porcine cumulus-oocyte complexes. Our aim was to investigate the correlation between the selection of porcine oocytes using BCB labelling and selected intranuclear characteristics of porcine oocytes and parthenotes. Moreover, BCB labelling was correlated with the diameter of the oocyte and the developmental potential of the parthenotes. The following methods were used: BCB labelling, measurement of the diameter of the oocyte, parthenogenetic activation, immunocytochemistry, transmission electron microscopy, enucleation and relative protein concentration (RPC) analysis. We determined that the diameter of the oocytes in the BCB-positive (BCB+) group was significantly larger than in the BCB-negative (BCB-) group. Immediately after oocyte selection according to BCB labelling, we found significant difference in chromatin configuration between the analyzed groups. BCB+ oocytes were significantly better at maturation than BCB- oocytes. BCB+ embryos were significantly more competent at cleaving and in their ability to reach the blastocyst stage than BCB- embryos. Ultrastructural analyses showed that the formation of active nucleoli in the BCB+ group started at the 8-cell stage. Conversely, most BCB- embryos at the 8-cell and 16-cell stages were fragmented. No statistically significant difference in RPC in nucleolus precursor bodies (NPBs) between BCB+ and BCB- oocytes was found. We can conclude that BCB labelling could be suitable for assessing the quality of porcine oocytes. Moreover, the evaluation of RPC indicates that the quantitative content of proteins in NPB is already established in growing oocytes.

microRNA expression profile in porcine oocytes with different developmental competence derived from large or small follicles.

Oocyte developmental competence is acquired during folliculogenesis and regulated by complex molecular mechanisms. Several molecules are involved in these mechanisms, including microRNAs (miRNAs) that are essential for oocyte-specific processes throughout the development. The objective of this study was to identify the expression profile of miRNAs in porcine oocytes derived from follicles of different sizes using RNA deep sequencing. Oocytes were aspirated from large (LO; 3-6 mm) or small (SO; 1.5-1.9 mm) follicles and tested for developmental competence and chromatin configurations. Small RNA libraries were constructed from both groups and then sequenced in an Illumina NextSeq. 500. Oocytes from the LO group exhibited higher developmental competence and different chromatin configuration compared with oocytes from the SO group. In total, 167 and 162 known miRNAs were detected in the LO and SO groups, respectively. MiR-205, miR-16, miR-148a-3p, and miR-125b were among the top 10 highly expressed miRNAs in both groups. Eight miRNAs were differentially expressed (DE) between both groups. Target gene prediction and pathway analysis revealed 46 pathways that were enriched with miRNA-target genes. The oocyte meiosis pathway and signaling pathways including FoxO, PI3K-Akt, and cAMP were predictably targeted by DE miRNAs. These results give more insights into the potential role of miRNAs in regulating the oocyte development.

Adverse effect of dexamethasone on development of the fetal rat ovary.

Dexamethasone (Dx) is often used in obstetric practice to promote fetal lung maturation and to prevent respiratory distress syndrome when the risk of preterm delivery persists. This therapy enables survival of the newborn, but also is associated with deleterious effects on the offspring, such as reproductive disorders. The aim of this study was to determine specifically whether prenatal exposure to Dx disturbs the physiological balance between proliferation and apoptosis of germinative cells (GC) in the ovary of 19- and 21-day-old fetuses and thus induces developmental programming of the female reproductive system. Pregnant Wistar rats (n = 10/group), separated into control (vehicle) and Dx-treated (0.5 mg/kg body mass) groups, received injections on gestational days 16, 17, and 18. Exposure to Dx lowered the volume of the fetal ovary by 30% (P < 0.05) in 21-day-old fetuses, as well as the total number of GC in the ovary by 21% (P < 0.05). When compared to the controls, in Dx-exposed fetuses, the total number of PCNA-positive GC was 27% lower at 19 days and 71% lower at 21 days old (P < 0.05), while total numbers of caspase-3-positive GC were 2.3-fold and 34% higher, respectively (P < 0.05). Our results demonstrate that prenatal exposure to Dx diminished proliferation but increased the rate of germinative cell apoptosis, with consequently reduced total germinative cell number and ovary volume. Impairment of fetal oogenesis and fewer GC in the fetal ovary compromise the oogonial stock and thus may constitute a risk of female fertility.

Maternally inherited rRNA triggers de novo nucleolus formation in porcine embryos.

SummaryThe present study examines the role of RNA polymerase I (RPI)-mediated transcription, maternally inherited rRNA and nucleolar proteins in the resumption of fibrillogranular nucleoli during embryonic genome activation (EGA) in porcine embryos. Late 4-cell embryos were incubated in the absence (control) or presence of actinomycin D (AD) (0.2 µg/ml for inhibition of RPI; 2.0 µg/ml for inhibition of total transcription) and late 2-cell embryos were cultured to the late 4-cell stage with 0.2 µg/ml AD to block EGA. Embryos were then processed for reverse-transcriptase polymerase chain reaction (RT-PCR), and for autoradiography (ARG), transmission electron microscopy (TEM), fluorescence in situ hybridization (FISH), silver staining and immunofluorescence (for RPI). Embryos in the control group displayed extranucleolar and intranucleolar ARG labelling, and exhibited de novo synthesis of rRNA and reticulated functional nucleoli. Nucleolar proteins were located in large foci. After RPI inhibition, nucleolar precursors transformed into segregated fibrillogranular structures, however no fibrillar centres were observed. The localization of rDNA and clusters of rRNA were detected in 57.1% immunoprecipitated (IP) analyzed nucleoli and dispersed RPI; 30.5% of nuclei showed large deposits of nucleolar proteins. Embryos from the AD-2.0 group did not display any transcriptional activity. Nucleolar formation was completely blocked, however 39.4% of nuclei showed rRNA clusters; 85.7% of nuclei were co-localized with nucleolar proteins. Long-term transcriptional inhibition resulted in the lack of ARG and RPI labelling; 40% of analyzed nuclei displayed the accumulation of rRNA molecules into large foci. In conclusion, maternally inherited rRNA co-localized with rDNA and nucleolar proteins can initiate a partial nucleolar assembly, resulting in the formation of fibrilogranular structures independently on activation of RPI-mediated transcription.

Soy isoflavone effects on the adrenal glands of orchidectomized adult male rats: a comprehensive histological and hormonal study.

Genistein (G) and related soy phytoestrogens have been studied for potential usefulness in different chronic diseases, and may ameliorate signs of aging. They have a profound influence on the hypothalamo-pituitary-adrenal (HPA) axis. The present study utilized the rat model of mild andropause to thoroughly evaluate the effects of G and soy extract on the adrenal gland and related blood hormones. Adult male rats were orchidectomized (Orx) or sham operated (SO). Orx rats received daily subcutaneous injections for 3 weeks of solvent, or G (Orx+G, 30 mg/kg), or commercial soy extract (Orx+Soy, 30 mg/kg). Adrenal glands and blood were harvested at the end of the treatment for hormone analyses, histology and design-based stereology. Compared to SO rats Orx evoked significant (P<0.05) changes including: the replicating cell number in the 3 adrenocortical zones; vascularity and cortical volume and blood levels of adrenocorticotropic hormone (ACTH), aldosterone and dehydroepiandrosterone (DHEA). When comparing Orx vs. Orx+G groups the following significant (P<0.05) changes were observed: a further increase in number of replicating cells in zonas glomerulosa and reticularis, vasculature network presence, cortical and zona reticularis volumes, ACTH and corticosterone concentrations, and lower DHEA levels. Comparing Orx vs. Orx+Soy resulted in elevated (P<0.05) ACTH and corticosterone levels. Structural integrity of the adrenal gland was unchanged vs. SO rats. Overall, G and soy extract treatments resulted in proliferative activity and/or vasculature support in the adrenal cortex. The data and current literature support the impression of a beneficial effect of soy components on the homeostatic response to stress.

Histological and morphofunctional parameters of the hypothalamic-pituitary-adrenal system are sensitive to daidzein treatment in the adult rat.

The isoflavone, daidzein is a biologically active, plant-derived compound that interacts with estrogen receptors. Data from previous studies have suggested that daidzein exerts beneficial effects in many diseases; however, as an endocrine disrupter, it may also alter the functioning of the endocrine system. Data regarding the effect of daidzein on the morphofunctional and histological parameters of the hypothalamic-pituitary-adrenal (HPA) system is still lacking. Therefore, using the newCAST stereological software, we investigated the effects of chronic (21 days) daidzein treatment on corticotropin-releasing hormone (CRH) neurons within the hypothalamus and corticotropes (ACTH cells) in the pituitary, while image analysis was employed to-examine the intensity of fluorescence of CRH in the median eminence (ME) and adrenocorticotropin hormone in the pituitary in adult orchidectomized (Ovx) rats. Circulating ACTH and corticosterone levels were also analyzed. This study showed that daidzein treatment decreased the volume density of CRH neurons within the paraventricular nucleus as well as CRH immunofluorescence in the ME. The total number of ACTH cells was decreased, while ACTH cell volume and the intensity of ACTH fluorescence were increased following daidzein treatment. Both ACTH and corticosterone blood levels were increased after daidzein administration. The results of performed experiments clearly demonstrate that volume density of CRH neurons; total number and volume of ACTH cells, as well as stress hormones levels are vulnerable to the effects of daidzein.

Mouse oocytes nucleoli rescue embryonic development of porcine enucleolated oocytes.

It is well known that nucleoli of fully grown mammalian oocytes are indispensable for embryonic development. Therefore, the embryos originated from previously enucleolated (ENL) oocytes undergo only one or two cleavages and then their development ceases. In our study the interspecies (mouse/pig) nucleolus transferred embryos (NuTE) were produced and their embryonic development was analyzed by autoradiography, transmission electron microscopy (TEM) and immunofluorescence (C23 and upstream binding factor (UBF)). Our results show that the re-injection of isolated oocyte nucleoli, either from the pig (P + P) or mouse (P + M), into previously enucleolated and subsequently matured porcine oocytes rescues their development after parthenogenetic activation and some of these develop up to the blastocyst stage (P + P, 11.8%; P + M, 13.5%). In nucleolus re-injected 8-cell and blastocyst stage embryos the number of nucleoli labeled with C23 in P + P and P + M groups was lower than in control (non-manipulated) group. UBF was localized in small foci within the nucleoli of blastocysts in control and P + P embryos, however, in P + M embryos the labeling was evenly distributed in the nucleoplasm. The TEM and autoradiographic evaluations showed the formation of functional nucleoli and de novo rRNA synthesis at the 8-cell stage in both, control and P + P group. In the P + M group the formation of comparable nucleoli was delayed. In conclusion, our results indicate that the mouse nucleolus can rescue embryonic development of enucleolated porcine oocytes, but the localization of selected nucleolar proteins, the timing of transcription activation and the formation of the functional nucleoli in NuTE compared with control group show evident aberrations.

Effects of Prenatal Dexamethasone on the Rat Pituitary Gland and Gonadotropic Cells in Female Offspring.

Glucocorticoids have a strong influence on growth and maturation of fetal organ systems, but overexposure to exogenous glucocorticoids may retard fetal growth and alter developmental processes in sensitive tissues. The aim of this study was to specifically determine whether prenatal exposure to dexamethasone (Dx) altered normal development and function of pituitary gonadotropic cells in neonatal, infant and peripubertal female offspring. On day 16 of pregnancy, rat dams received 1.0 mg Dx/kg body weight (BW) s.c., followed by 0.5 mg Dx/kg BW on days 17 and 18 of gestation. Control gravid females received the same volume of saline. Female offspring were sacrificed on days 5, 16 and 38 after delivery. The volume of the pituitary gland estimated using Cavalieri's principle was significantly reduced (p < 0.05). Using a fractionator-physical disector method, we found reduced total numbers of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells (p < 0.05), accompanied by a decrease (p < 0.05) in serum concentrations of FSH and LH, while the relative intensity of FSH and LH immunofluorescence remained unchanged in neonatal, infant and peripubertal female offspring prenatally exposed to Dx. The data document that overexposure to Dx during fetal development evokes developmental programming of the female reproductive system at the pituitary cellular level, which may be associated with impaired reproductive function.