Praktična primjena brzog progesteronskog P4 testa za rano otkrivanje gravidnosti iz kravljeg mlijeka

Pregnancy diagnosis is an essential part of fertility management in cattle. Early detection of pregnancy 21 days after artificial insemination (AI) allows for early recognition and rebreeding of non-pregnant animals with a minimum delay, which benefits the economic interest for farmers. Rapid progesterone P4 tests are widely used in fertility management strategies, yet there is limited data about the efficiency of tests for P4 measurements in cow’s milk. By measuring P4 concentrations in the milk of different breeds: Red Holstein, Black-Holstein, Simmental, and local crossbreeds, this study aimed to assess the practical application and efficiency of an early and rapid pregnancy detection test from cow’s milk. The study was conducted in 2017–2018 and milk samples were taken from animals at day 21 after artificial insemination (AI) to diagnose pregnancy. The test indicated that of the total cows (n=400) included in this study, 69% were pregnant and 31% were not pregnant, as interpreted based on the P4 Rapid (Ridgeway Science UK) test results. Accuracy of the early diagnosis was verified 60 days after AI (without further AI of cows) by rectal palpation. Based on this examination, 263 (66%) cows were diagnosed as pregnant, while 14 (34%) were diagnosed as non-pregnant. The highest accuracy in pregnancy detection was found in the local crossbreeds and the Simmental breed, with 97% correct diagnosis of cows, followed by Red Holstein with 94% and Black Holstein with an accuracy of 90%. This study showed that the use of progesterone P4 rapid tests in milk samples is an effective, fast and accurate method for the early detection of pregnancy in cows, with an accuracy rate of 90 to 97% based on cattle breed.Dijagnoza gravidnosti bitan je dio menadžmenta plodnosti u goveda. Rana dijagnostika gravidnosti 21 dana nakon osjemenjavanja omogućava rano prepoznavanje i ponovno umjetno osjemenjivanje (UO) negravidnih krava s minimalnim produžetkom servisnog razdoblja te je od ekonomskog interesa za poljoprivrednike. Brzi progesteronski P4 testovi puno se koriste u strategijama upravljanja plodnosti, ali postoji ograničen broj podataka o učinkovitosti tih testova za mjerenje P4 iz kravljeg mlijeka. Mjereći koncentraciju progesterona P4 iz mlijeka različitih pasmina krava kao što su: crveni holstein, crni holstein, simentalska pasmina i lokalne križane pasmine, ova studija ima za cilj procjenu praktične primjene i učinkovitosti ranog i brzog testa dijagnostike gravidnosti iz kravljeg mlijeka. Studija je provedena u razdoblju od 2017. i 2018., a uzorci mlijeka uzorkovani su životinjama 21. dana nakon UO u svrhu dijagnostike gravidnosti. Od 400 krava uključenih u ovo istraživanje, interpretacijom rezultata na temelju uporabe progesteronskog P4 brzog testa (Ridgeway Science, VB) 69 % testiranih krava je bilo gravidno, dok 31 % krava nisu bile gravidne. Točnost rane dijagnoze potvrđena je 60 dana nakon UO krava (bez daljnjih UO krava) rektalnom palpacijom. Na temelju ovog ispitivanja 263 krave, odnosno njih 66 % je dijagnosticirano gravidnima, dok 14 krava ili 34 % nisu bile gravidne. Najveća preciznost u otkrivanju gravidnosti ovom metodom ustvrđena je u lokalnih križanih pasmina i krava simentalske pasmine s 97 % točno dijagnosticiranih krava, a slijede ih pasmina crveni holstein s 94 % i crni holstein s 90 % točnosti. Naše istraživanje pokazalo je da se s preciznošću od 90 % do 97 % za različite testirane pasmine krava uporaba brzih progesteronskih P4 testova u uzorcima mlijeka pokazala učinkovitom, brzom i točnom metodom za rano otkrivanje gravidnosti krava

Bacteriocin production by lactic acid bacteria (LAB) isolated from traditional cheese

Lactic Acid Bacteria (LAB) are a group of bacteria that are found as natural microbiota in various ecosystems. They are used ato producea huge variety of fermented foods, they occur in pharmaceutical formulations and as probiotics in functional foods. They can produce a number of antimicrobial metabolites, including organic acids and other organic components, hydrogen peroxide and bacteriocins. The aim of this study was the evaluation of antibacterial activity of LAB isolated during production and maturation of traditional Rugova cheese. Samples for analysis were collected from different points of Rugova region and were transported to the laboratory under constant cooling conditions. The bacterial isolation was performed using standard methods and the isolates of LAB were identified down to the species level using a Biotyper Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). Out of 140 tested isolates 105 had the ability to produce bacteriocins. The large number of bacteriocin producers demonstrates the great assertiveness of the natural LAB microbiota over potentially existing pathogens. Thus, the ability of bacteriocin production by LAB isolated from Rugova cheese can be taken as a measure of quality and safety of this traditional product

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

Microbiological and physicochemical analysis of traditional Rugova cheese

The aim of this study was the evaluation of microbial and physicochemial parameters during production and maturation of traditional Rugova cheese. Samples were collected from different points of Rugova region and transported to the laboratory under constant cooling conditions. The average number of total mesophilic bacteria was 5.1 x 10 8 cfu/ml in milk and 2.4 x 10 8 cfu/gr in maturated cheese. The number of lactic acid bacteria in milk and cheese were 3.5 x 10 6 cfu/ml, respectively 1.7 x 10 8 cfu/gr. All physicochemical parameters in milk and cheese have been within the allowed values for milk and cheese and in accordance with the standards used by the Institute of Public Health of Kosovo

Heritable and inducible gene knockdown in astrocytes or neurons in vivo by a combined lentiviral and RNAi approach

Gene knockout by homologous recombination is a popular method to study gene functions in the mouse in vivo. However, its lack of temporal control has limited the interpretation of knockout studies because the complete elimination of a gene product often alters developmental processes, and can induce severe malformations or lethality. Conditional gene knockdown has emerged as a compelling alternative to gene knockout, an approach well-established in vitro but that remains challenging in vivo, especially in the adult brain. Here, we report a method for conditional and cell-specific gene knockdown in the mouse brain in vivo that combines Cre-mediated RNA interference (RNAi) with classical and lentivirus-mediated transgenesis. The method is based on the inducible expression of a silencing short hairpin RNA (shRNA) introduced in mice by lentivirus-mediated transgenesis, and on its activation by excision of a floxed stop EGFP reporter with an inducible Cre recombinase expressed in astrocytes or in neurons. This dual system should be of broad utility for comparative studies of gene functions in these two cell types in vivo

Crimean–Congo Hemorrhagic Fever Virus and Borrelia burgdorferi sensu lato in Ticks from Kosovo and Albania

Tick-borne diseases pose a serious threat to human health in South-Eastern Europe, including Kosovo. While Crimean–Congo hemorrhagic fever (CCHF) is a well-known emerging infection in this area, there are no accurate data on Lyme borreliosis and tick-borne encephalitis (TBE). Therefore, we sampled and tested 795 ticks. Ixodes ricinus (n = 218), Dermacentor marginatus (n = 98), and Haemaphysalis spp. (n = 24) were collected from the environment by flagging (all from Kosovo), while Hyalomma marginatum (n = 199 from Kosovo, all from Kosovo) and Rhipicephalus bursa (n = 130, 126 from Albania) could be collected only by removal from animal pasture and domestic ruminants. Ticks were collected in the years 2014/2015 and tested for viral RNA of CCHF and TBE viruses, as well as for DNA of Borrelia burgdorferi sensu lato by real-time PCR. In Kosovo, nine ticks were positive for RNA of Crimean–Congo hemorrhagic fever virus and seven for DNA of B. burgdorferi s. l. None of the ticks tested positive for TBEV. CCHF virus was detected in one H. marginatum male specimen collected while feeding on grazing cattle from the Prizren region and in eight R. bursa specimens (five females and three males collected while feeding on grazing sheep and cattle) from the Prishtina region (Kosovo). B. burgdorferi s. l. was detected in seven questing ticks (four male and one female D. marginatus, two I. ricinus one female and one male) from the Mitrovica region (Kosovo). Our study confirmed that CCHF virus is circulating in Kosovo mainly in H. marginatum and R. bursa in the central areas of the country. B. burgdorferi s. l. was found in its major European host tick, I. ricinus, but also in D. marginatus, in the north of the Kosovo. In order to prevent the spread of these diseases and better control of the tick-borne infections, an improved vector surveillance and testing of ticks for the presence of pathogens needs to be established

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 &gt; 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 &lt; 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