Prostaglandins in Superovulation Induced Bovine Follicles During the Preovulatory Period and Early Corpus Luteum

The aim of this study was to characterize the regulation pattern of prostaglandin family members namely prostaglandin F2alpha (PTGF), prostaglandin E2 (PTGE), their receptors (PTGFR, PTGER2, PTGER4), cyclooxygenase 2 (COX-2), PTGF synthase (PTGFS), and PTGE synthase (PTGES) in the bovine follicles during preovulatory period and early corpus luteum (CL). Ovaries containing preovulatory follicles or CL were collected by transvaginal ovariectomy (n = 5 cows/group), and the follicles were classified: (I) before GnRH treatment; (II) 4 h after GnRH; (III) 10 h after GnRH; (IV) 20 h after GnRH; (V) 25 h after GnRH, and (VI) 60 h after GnRH (early CL). In these samples, the concentrations of progesterone (P4), estradiol (E2), PTGF and PTGE were investigated in the follicular fluid (FF) by validated EIA. Relative mRNA abundance of genes encoding for prostaglandin receptors (PTGFR, PTGER2, PTGER4), COX-2, PTGFS and PTGES were quantified by RT-qPCR. The localization of COX-2 and PTGES were investigated by established immunohistochemistry in fixed follicular and CL tissue samples. The high E2 concentration in the FF of the follicle group before GnRH treatment (495.8 ng/ml) and during luteinizing hormone (LH) surge (4 h after GnRH, 574.36 ng/ml), is followed by a significant (P<0.05) downregulation afterwards with the lowest level during ovulation (25 h after GnRH, 53.11 ng/ml). In contrast the concentration of P4 was very low before LH surge (50.64 mg/ml) followed by a significant upregulation (P < 0.05) during ovulation (537.18 ng/ml). The mRNA expression of COX-2 increased significantely (P < 0.05) 4 h after GnRH and again 20 h after GnRH, followed by a significant decrease (P < 0.05) after ovulation (early CL). The mRNA of PTGFS in follicles before GnRH was high followed by a continuous and significant downregulation (P < 0.05) afterwards. In contrast, PTGES mRNA abundance increased significantely (P < 0.05) in follicles 20 h after GnRH treatment and remained high afterwards. The mRNA abundance of PTGFR, PTGER2, and PTGER4 in follicles before GnRH was high, followed by a continuous and significant down regulation afterwards and significant increase (P < 0.05) only after ovulation (early CL). The low concentration of PTGF (0.04 ng/ml) and PTGE (0.15 ng/ml) in FF before GnRH, increased continuously in follicle groups before ovulation and displayed a further significant and dramatic increase (P < 0.05) around ovulation (101.01 ng/ml, respectively, 484.21 ng/ml). Immunohistochemically, the granulosa cells showed an intensive signal for COX-2 and PTGES in follicles during preovulation and in granulosa-luteal cells of the early CL. In conclusion, our results indicate that the examined bovine prostaglandin family members are involved in the local mechanisms regulating final follicle maturation and ovulation during the folliculo-luteal transition and CL formation

Pojavnost aflatoksina M1 u uzorcima jogurta pronađenima na tržištu u Kosovu tijekom proljeća 2023.

Aflatoxin M1 (AFM1), a toxic byproduct of aflatoxin B1 (AFB1) produced by Aspergillus fungi, is a carcinogenic mycotoxin that can contaminate various agricultural commodities. It can be transferred from AFB1-contaminated feed to milk and dairy products, including yogurt, posing a potential health risk to consumers. In spring 2023, a total of 74 yogurt samples were collected from the largest food suppliers in Kosovo for analysis, including samples produced in Kosovo and seven other countries: Albania, North Macedonia, Bosnia and Herzegovina, Slovenia, Greece, Italy, and Germany. A rapid and sensitive analytical method, Enzyme-linked immunosorbent assay (ELISA), was used for the analysis. The results of the study highlight discernible differences in the maximum tolerable levels of AFM1 between the countries. More specifically, yogurt samples from Slovenia and Germany had lower levels than those from other countries. Additionally, the median levels of AFM1 in samples from Slovenia and Germany were significantly lower. The mean concentrations of AFM1 in yogurt samples from Kosovo and other countries were 0.071 μg/kg and 0.080 μg/kg, respectively. Out of all samples, 66 (89%) exceeded the maximum tolerable limit of 0.05 μg/kg. Among the exporting countries, Albania had the highest median AFM1 level of 0.085 μg/kg and the highest maximum level of 0.195 μg/kg. Slovenia had the lowest median AFM1 level, while Germany had the lowest maximum AFM1 level. All samples from Albania, Greece, and Bosnia and Herzegovina exceeded the maximum tolerable limit. High prevalence was also observed in samples from Kosovo, North Macedonia, and Slovenia. Considering the average daily consumption of about 250 grams of yogurt, and the total median value of Aflatoxin M1 concentration (0.071 μg/kg), the estimated daily intake was calculated to be 0.017 μg. These findings highlight the importance of monitoring and enforcing regulatory limits to ensure yogurt safety and to protect public health. Efforts should be focused on mitigating AFM1 contamination and implementing measures to minimise its presence in dairy products, especially in regions where levels exceed the established limits.Aflatoksin M1 (AFM1), toksični nusproizvod aflatoksina B1 (AFB1) kojeg proizvode gljivice Aspergillus kancerogeni je mikotoksin koji može kontaminirati različite poljoprivredne proizvode. Može se prenijeti iz hrane za životinje kontaminirane s AFB1 na mlijeko i mliječne proizvode, uključujući jogurt, predstavljajući potencijalni rizik po zdravlje potrošača. U proljeće 2023. godine prikupljeno je ukupno 74 uzoraka jogurta od najvećih dobavljača hrane na Kosovu za analizu, uključujući uzorke proizvedene na Kosovu i u sedam drugih zemalja: Albaniji, Sjevernoj Makedoniji, Bosni i Hercegovini, Sloveniji, Grčkoj, Italiji i Njemačkoj. Za analizu je rabljena brza i osjetljiva analitička metoda, Enzimski povezani imunosorbentni test (ELISA). Rezultati studije ukazuju da nalazi ove studije naglašavaju zamjetne razlike u maksimalno dopuštenim razinama AFM1 između različitih zemalja. Točnije, u uzorcima jogurta iz Slovenije i Njemačke zamijećene su niže razine od onih iz drugih zemalja. Uz to, srednje razine AFM1 u uzorcima iz Slovenije i Njemačke bile su značajno niže. Srednje koncentracije AFM1 u uzorcima jogurta s Kosova i iz drugih zemalja bile su 0,071 μg/kg, odnosno 0,080 μg/kg. Od svih uzoraka, 66 (89 %) prekoračilo je maksimalno dopušteno ograničenje od 0,05 μg/kg. Od zemalja izvoznica, Albanija je imala najveću srednju razinu AFM1 od 0,085 μg/kg i najveću maksimalnu razinu od 0,195 μg/kg. Slovenija je imala najnižu srednju razinu AFM1, a Njemačka najnižu maksimalnu razinu AFM1. Svi uzorci iz Albanije, Grčke i Bosne i Hercegovine prekoračili su maksimalnu dopuštenu razinu. Visoka prevalencija zamijećena je i u uzorcima s Kosova, iz Sjeverne Makedonije i Slovenije. Razmatrajući prosječnu dnevnu konzumaciju od jedne čašice od oko 250 g jogurta i ukupnu srednju vrijednost koncentracije aflatoksina M1 (0,071 μg/kg), izračunat je procijenjeni dnevni unos od 17,75 μg. Ovi nalazi naglašavaju važnost nadziranja i provođenja regulatornih ograničenja kako bi se osigurala sigurnost jogurta i zaštitilo javno zdravlje. Potrebno je uložiti napore za smanjenje kontaminacije AFM1 i provedbu mjera za smanjenje njegove prisutnosti u mliječnim proizvodima, posebice u regijama gdje razine prekoračuju utvrđena ograničenja

Pojavnost citrinina u zrnju pšenice uzgojenoj na Kosovu i u Albaniji tijekom 2021. godine

Citrinin (CIT) is a mycotoxin responsible for the contamination of many agricultural products, like wheat, barley, corn, rice and their products, as also other foodstuffs and feedstuffs used in human and animal nutrition. It is essentially produced by Penicillium citrinum, although it can also be biosynthesised from Penicillium expansum and Penicillium verrucosum and some species of Aspergillus and Monascus. However, several studies have shown that CIT is known for its genotoxic, hepatotoxic, fetotoxic and teratogenic properties. The aim of this study is to investigate the occurrence of CIT in wheat grain cultivated in Kosovo and Albania. Given the fact that wheat flour is the most consumed product in Kosovo and Albania, it is necessary to analyse the CIT in wheat in these two countries. In total, 60 wheat samples were tested from Fusha e Kosovës (Kosovo), Myzeqeja (Albania) and Fusha e Maliqit (Albania), as places with the highest wheat production. The enzyme- linked immunosorbent assay (ELISA) method was used to determine CIT concentrations. To identify moulds representing potential producers of CIT, traditional macroscopic and microscopic methods and the molecular PCR method of identification were implemented. CIT was detected in 96.6% and 86.6% of wheat grain samples collected in Kosovo and Albania, respectively. The maximum amount of CIT detected in wheat grain was 53.12 μg/ kg in Kosovo, and 45.74 μg/kg in Albania. The amount of CIT found in wheat grain is not comparable with the maximal limits (MLs), as the European legislation does not provide limits for this mycotoxin. However, since there is generally a lack of data about CIT in cereals in Kosovo and Albania, the results can serve as an indicator of wheat grain contamination in this part of the Balkan Peninsula.itrinin (CIT) predstavlja mikotoksin za koji je utvrđeno da je odgovoran za kontaminaciju mnogih poljoprivrednih proizvoda poput: pšenice, ječma, kukuruza, riže i njihovih proizvoda, kao i drugih namirnica i hrane za životinje, osim onih na bazi žitarica, koje se koriste za prehranu ljudi i hranidbu životinja. Najviše ga proizvodi Penicillium citrinum, iako se može biosintetizirati i iz Penicillium expansum i Penicillium verrucosum te nekih vrsta Aspergillus i Monascus. Međutim, istraživanja pokazuju da su za CIT utvrđena genotoksična, hepatotoksična, fetotoksična i teratogena svojstva. Cilj ovoga istraživanja je bio utvrditi pojavnost CIT u zrnju pšenice koje se uzgaja na Kosovu i u Albaniji. S obzirom na činjenicu da je pšenično brašno najviše konzumirani proizvod na Kosovu i u Albaniji, analize CIT u zrnju pšenice u ove dvije zemlje od velikog su značenja. Ukupno je uzorkovano 60 uzoraka zrna pšenice na području poznatom kao Fusha e Kosovës (Kosovo), Myzeqeja (Albanija) i Fusha e Maliqit (Albanija), koja predstavljaju lokalitete na kojima se proizvodi najveća količina pšenice. Za određivanje koncentracije CIT korištena je imunoenzimna metoda ELISA. Za identifikaciju plijesni koje predstavljaju potencijalne producente CIT primijenjena je tradicionalna makroskopska i mikroskopska metoda te molekularna PCR metoda identifikacije. CIT je određen u 96,6 % i 86,6 % uzoraka zrnja pšenice prikupljenih na Kosovu i u Albaniji. Najveća količina CIT-a u zrnju pšenice proizvedenom na Kosovu bila je 53,12 μg/kg, a u Albaniji 45,74 μg/kg. Količina CIT utvrđena u pšenici ne može se usporediti s najvećom dopuštenom količinom (NDK), jer njegova razina u europskom zakonodavstvu nije definirana. No budući da podatci o količinama CIT u žitaricama uzgojenim na Kosovu i u Albaniji nisu dostupni, dobiveni rezultati mogu poslužiti kao pokazatelj kontaminacije zrnja pšenice na ovom dijelu Balkanskog poluotoka

The Dynamics of microRNA Transcriptome in Bovine Corpus Luteum during Its Formation, Function, and Regression

The formation, function, and subsequent regression of the ovarian corpus luteum (CL) are dynamic processes that enable ovary cyclical activity. Studies in whole ovary tissue have found microRNAs (miRNAs) to by critical for ovary function. However, relatively little is known about the role of miRNAs in the bovine CL. Utilizing small RNA next-generation sequencing we profiled miRNA transcriptome in bovine CL during the entire physiological estrous cycle, by sampling the CL on days: d 1-2, d 3-4, and d 5-7 (early CL, eCL), d 8-12 (mid CL, mCL), d 13-16 (late CL, lCL), and d > 18 (regressed CL, rCL). We characterized patterns of miRNAs abundance and identified 42 miRNAs that were consistent significantly different expressed (DE) in the eCL relative to their expression at each of the analyzed stages (mCL, lCL, and rCL). Out of these, bta-miR-210-3p, -2898, -96, -7-5p, -183-5p, -182, and -202 showed drastic up-regulation with a fold-change of >= 2.0 and adjusted P < 0.01 in the eCL, while bta-miR-146a was downregulated at lCL and rCL vs. the eCL. Another 24, 11, and 21 miRNAs were significantly DE only between individual comparisons, eCL vs. the mCL, lCL, and rCL, respectively. Irrespective of cycle stage two miRNAs, bta-miR-21-5p and bta-miR-143 were identified as the most abundant miRNAs species and show opposing expression abundance. Whilst bta-miR-21-5p peaked in number of reads in the eCL and was significantly downregulated in the mCL and lCL, bta-miR-143 reached its peak in the rCL and is significantly downregulated in the eCL. MiRNAs with significant DE in at least one cycle stage (CL class) were further grouped into eight distinct clusters by the self-organizing tree algorithm (SOTA). Half of the clusters contain miRNAs with low-expression, whilst the other half contain miRNAs with high-expression levels during eCL. Prediction analysis for significantly DE miRNAs resulted in target genes involved with CL formation, functionalization and CL regression. This study is the most comprehensive profiling of miRNA transcriptome in bovine CL covering the entire estrous cycle and provides a compact database for further functional validation and biomarker identification relevant for CL viability and fertility

Expression and localization of members of the thrombospondin family during final follicle maturation and corpus luteum formation and function in the bovine ovary

The aim of this study was to characterize the expression patterns and localization of the thrombospondin family members (THBS1, THBS2) and their receptors (CD36 and CD47) in bovine ovaries. First, the antral follicles were classified into 5 groups based on the follicle size and estradiol-17beta (E2) concentration in the follicular fluid (180 E2 ng/ml). Second, the corpus luteum (CL) was assigned to the following stages: days 1–2, 3–4, 5–7, 8–12, 13–16 and >18 of the estrous cycle and of pregnancy (month 1–2, 3–4, 6–7 and > 8). Third, the corpora lutea were collected by transvaginal ovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after inducing luteolysis by injecting a prostaglandin F2alpha analog. The mRNA expression of examined factors was measured by RT-qPCR, steroid hormone concentration by EIA, and localization by immunohistochemistry. The mRNA expression of THBS1, THBS2, CD36, and CD47 in the granulosa cells and theca interna was high in the small follicles and reduced in the preovulatory follicles. The mRNA expression of THBS1, THBS2, and CD47 in the CL during the estrous cycle was high, but decreased significantly during pregnancy. After induced luteolysis, thrombospondins increased significantly to reach the maximum level at 12 h for THBS1, 24 h for THBS2, and 48 h for CD36. The temporal expression and localization pattern of the thrombospondins and their specific receptors in the antral follicles and corpora lutea during the different physiological phases of the estrous cycle and induced luteolysis appear to be compatible with their inhibitory role in the control of ovarian angiogenesis

THE EXPRESSION OF APELIN AND ITS RECEPTOR APJ DURING DIFFERENT PHYSIOLOGICAL STAGES IN THE BOVINE OVARY

<p>Recent studies implicate that apelin and its receptor APJ may have important role for the modulation of angiogenesis. The aim of this study was to further characterise the regulation of apelin/APJ system in bovine ovary. Experiment 1: corpora lutea (CL) were assigned to the following stages: days 1-2, 3-4, 5-7, 8-12, 13-16, &#62;18 (after regression) of oestrous cycle and of gravidity (month &#60;3, 3-5, 6-7 and &#62;8). Experiment 2: Follicles during maturation were divided into granulosa cells (GC) and theca interna (TI) and were examined separately. Classification of follicles occurred by follicle size and oestradiol-17&#946; (E2) concentration in the follicular fluid (FF) (&#60;0.5 ng/ml, 0.5-5 ng/ml; 5-40 ng/ml; 40-180 ng/ml; &#62;180 ng/ml). Real-time RT-PCR (qPCR) was applied to investigate mRNA expression of examined factors. In general, the expression level of apelin during the oestrous cycle was significantly higher compared to the one during pregnancy. Apelin mRNA levels were always high during the cycle with a tendency of decrease after CL regression. The APJ mRNA in the CL was significantly up regulated on days 5-7 and 8-12 followed by a decrease on days 13-16, and further on days &#62;18. The expression of APJ does not show any significant regulation in the CL throughout pregnancy. The expression of apelin and APJ was not statistically regulated in GC, but was significantly up regulated in follicles with an E2 concentration of more than 5 ng/ml and showed an increase according to growth and maturation of follicles. In conclusion, our data suggest that apelin/APJ system is involved in the mechanism regulating angiogenesis during follicle maturation as well as during CL formation and function in the bovine ovary.</p

Application of ELISA and RT-PCR for the detection of pork adulterated in beef meat products marked in Kosovo

The problem of adulterated ingredients in processed food products is widely observed in the food industry and remains a continuous concern for consumers. This problem may interfere not only with consumers’ religious ethics, but also with their health and diet. Therefore commercial foods should be monitored for the accuracy of the declared ingredients. This study aims at identification of pork matter adulterated in processed beef meat products marked in Kosovo. Commercial beef food samples were routinely collected at different markets around Kosovo. The analyzed samples consisted of sausages, salami, pate and ragu sauce and were declared as 100% beef. All samples were initially prepared into a test portion and further processed for serum albumin or genomic DNA extraction (CTAB, ISO-21571:2005). After the initial processing, samples were first tested for the presence of pork matter by the ELISA method and all positive samples were tested for confirmation by RT-PCR. From a total of 25 analyzed food products, ELISA was able to detect pork in 32% of them with an accuracy of 100% among duplicate samples. All ELISA positive samples were further confirmed by RT-PCR, either by a commercial kit or designed primers specific for pork mitochondrial DNA. The specificity of the amplified PCR products was confirmed at the end on the micro-fluidal electrophoresis. These data show that the adulteration of beef meat products is frequent in Kosovo marked and that the combination of ELISA and RT-PCR provides a very effective and reliable option that can be applied for routine monitoring of food adulteration in commercial products of animal origin in the Kosovo