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.

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.

Food adulteration and fraudulent practices are widely observed in the food industry worldwide and are of great concern for Balkan countries. This study aims at investigating the level of undeclared pork meat in commercial beef and chicken meat products sold in Kosovo by implying one commercial enzyme-linked immunosorbent assay (ELISA) and two confirmatory real-time polymerase chain reaction (PCR) approaches [ready-to-use real-time PCR and real-time PCR with primers specific for pork mitochondrial deoxyribonucleic acid (DNA)]. In supermarkets in the capital city, Prishtina, 62 meat products were randomly sampled, and the three methods were applied. Additionally, these three approaches were evaluated for their practicability, reproducibility, and cost. The results showed that pork was present in 32% of beef- and 8% chicken-based products. ELISA and real-time PCR with pork specific primers showed 100% of reproducibility for beef- and chicken-based products. In contrast, the ready-to-use real-time PCR kit showed 100% reproducibility in chicken-, but only 75% in beef-based samples. ELISA was more rapid than both real-time PCR approaches, but it was more challenging when large numbers of samples were processed. The real-time PCR approach with pork specific primers was the cheapest, while the ready-to-use real-time PCR was the most practical method. Commercial ELISA, in combination with real-time PCR with pork specific primers, provides a reliable and affordable testing methodology that can be implemented for rapid detection and monitoring of pork adulteration in diverse commercial foods.

This study was aimed for the evaluation of somatic cell count (SCC), physicochemical, and microbiological parameters during the end of lactation in the raw milk of Alpine and native Red goat breed. In the experiment, 102 milk samples from Alpine and native Red goats were included. Two different groups within the same breed were analyzed: a group consisting of animals in their first lactation and the second group consisting of animals from the fifth lactation. The milk samples were individually and daily collected during late lactation for three consecutive weeks, and milk fat, protein, lactose, SCC, and total bacteria with enterobacteria were assessed. Fresh milk of goats from late lactation period had a number of somatic cells (SC) within the expected value with log10 of 5.8–6.18 cells/ml for the compared groups. In both breeds, the total mesophilic bacteria were fewer in numbers, however, in the native Red goat, a larger population of such bacteria was enumerated. The number of coliforms and enterobacteria was below 100 cfu/ml. In the current study, we were able to show a significant difference among the studied breeds depending on lactation and season for fat (p = 0.002), but not for lactose and protein content. A positive correlation for total protein (TP), lactose, and fat as well as for lactose and SCC was found in the native Red goat breed. In the Alpine goat breed, a strong positive correlation (0.821**) was found for lactose and enterobacteria count (EC). In conclusion, these findings evaluate different goat milk parameters during late lactation period and provide an indirect measure to monitor goat mammary gland health for both breeds.

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. Keywords: in vivo gene knockdown, conditional gene knockdown, shRNA, Cre-lox genetics, lentivirus-mediated transgenesis INTRODUCTIONGenetic models in the mouse have significantly advanced the understanding of gene functions, both in physiological and pathological conditions. Most of the existing models have been generated by genetic manipulations that lead to a gain-or loss-offunction of the candidate gene (Aronoff and Petersen, 2006). This is usually achieved with vector-based overexpression systems, or by gene targeting like gene knockout or knockin (Capecchi, 2005;Glaser et al., 2005). Recently, RNA interference (RNAi) has emerged as an alternative approach with the advantage of decreasing rather than fully eliminating the expression of target gene(s) Paddison and Hannon, 2002). RNAi-based gene knockdown relies on short doublestranded RNAs (19-21 nucleotides) that target complementary mRNA sequences, and promote their degradation or inhibit their translation Dykxhoorn et al., 2003). It can be used in rodents by local or systemic delivery of shortinterfering RNAs (siRNAs) (Thakker et al., 2004), or through virus-based shRNA expression vectors (Ralph et al., 2005;Singer et al., 2005;Sapru et al., 2006). Although this approach can be extremely useful, its efficiency is subjected to variability and depends on several factors, in particular the degree of sequence matching with the target DNA, and the level of expression of the shRNA and the targeted gene. Further, even if it has been used successfully in cell culture (Elbashir et al., 2001;Brummelkamp et al., 2002;Lee et al., 2002), it remains challenging in vivo. Thus, overall, genetic systems for gene silencing have been reported in the literature (Rubinson et al., 2003;Tiscornia et al., 2003;Coumoul et al., 2005;Szulc et al., 2006;Dickins et al., 2007;Delic et al., 2008;Seidler et al., 2008) but are often difficult to use due to their limitations s...