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There is little data on the use of the immunohistochemical method in the observation of the T lymphocytes and macrophages distribution within the mesenteric lymph nodes in canine parvovirus (CPV) enteritis. The virus initially replicates in the systemic lymphoid tissue and causes highest changes in the small intestine. This current study aimed to demonstrate the CD3 and MAC387 antibodies detection and distribution in mesenteric lymph nodes and small intestines in dogs which had positive clinical, pathological, and histological findings of CPV. Twenty dogs that were clinically confirmed on CPV, had been autopsied following onset of death. Tissue samples (lymph nodes and small intestines) from each dog was processed for histology and immunohistochemistry utilizing hematoxylin-eosin and peroxidase/DAB (3,3′-diaminobenzidine) staining methods, respectively. The virus distribution was greatest in the tissue areas of the small intestine and the lymph nodes where the virus had made the most severe tissue damage. The distribution of macrophage was highest in the necrotic areas of the small intestine and the mesenteric lymph nodes. The amount of CD3 positive lymphocytes was greatly reduced in the mesenteric lymph nodes and the lymphoid tissue of the small intestine.

Brilliant cresyl blue (BCB) staining is a method used for selection of developmentally competent porcine oocytes, in addition to the standard morphological classification. The aim of the current study was to assess the relationship between BCB staining in porcine oocytes with high and low morphological characteristics and its maturation rate. In the first part of the study, cumulus-oocytes complexes (COCs) (n=434) were aspirated from 60 ovaries. According to the morphological classification and BCB staining, they were divided in 4 groups: COC+/BCB+, COC-/BCB+, COC+/BCB-, and COC-/BCB-. In the second experiment, COCs (n=445) were categorized into 3 groups: control, BCB+, and BCB-. Significant differences in the maturation rate were observed between COC+/BCB+ and COC-/BCB- (66% vs. 23%), and between COC+/BCB+ and COC-/BCB+ (66% vs. 38%) (p

The aim of the present study was to identify the adaptability of Normandy cows raised in a loose system to the climatic and technological conditions and to compare their adaptability with already acclimatized Simmental cows in one farm in Southwestern Bulgaria. Total of 20 cows at same age (II – IV lactation) and same body weight (630-660 kg) from both breeds were classified into 2 groups (each having 10 cows) and subjected to skin thickness measurement, fibers total weight determination and fiber categorization during the winter and summer season. The results have shown that during the winter season, the Normandy breed had significantly higher skin thickness at the elbow compared to the Simmental breed, whereas no differences were observed in the skin thickness between both breeds either at the neck or at the middle of the last rib. During the summer season, the differences on the skin thickness compared to the Simmental breed were observed at the neck (p0.05). During the winter seasons, the coat of Normandy cows contained more soft fibers in comparison to the Simmentals cows. Furthermore, the changes in the observed parameters, influenced by climatic conditions showed similar pattern in both breeds. It can be concluded that the two breeds have emphasized their genetic potential and have a good adaptability to the temperate continental climate of the region.

The aim of this study was to construct two plasmid-specific shRNA transcripts of the bcl-2 gene in order to prepare for reverse of cell apoptosis. The plasmid was designed according to a previously published sequence of interfering RNA following an appropriate reference, using appropriate software. By annulling complementary oligonucleotides, doublestranded inserts were formed. Recombinant shRNA-encoding plasmids were constructed by digestion of psiRNAx7SKGFPzeo plasmid (psiRNA-x7SKGFPzeo, with restrictive endonuclease BbsI electrophoresis in ultra-pure agarose with low melting point (LMP-Agarose). For each of the constructs, a suitable double-stranded insert downstream of x7SK (strong RNA III promoter) with T4 DNA ligase was cloned. The control plasmid psiRNAScr was used directly for transformation. The PC-3 cell lines were transfected with 2 plasmids, psiRNA-Bcl-2 and psiRNAScr to suppress the bcl-2 gene construct. The results have shown that the lowest level of bcl-2 genes was 48 h, and even lower 72 h after the transfer, and the mRNA levels returned to normal in 120 h. An increase in the percentage of cells with spontaneous apoptosis has been observed with successful inhibition of the bcl-2 gene. The induction of apoptosis in transfected cells increased the percentage of necrotic cells proportionally. The percentage of apoptotic cells transfected with psiRNA-bcl-2 plasmid increased proportionally to the increase of hydrogen peroxide concentration. The transfection of the PC-3 cell line from prostate cancer with constructed shRNA plasmid has induced suppression of bcl-2 gene expression versus control Scr plasmid. Suppression of bcl-2 gene expression significantly increased cell sensitivity to apoptosis induction.

We hypothesized that a single dose of PGF2α belatedly injected on day 8 after GnRH-1 in cows receiving a 7-day Ovsynch-56 protocol (GnRH – 7 days – PGF2α – 56h – GnRH – 16h – timed AI) will increase the proportion of cows with complete luteolysis. At day 35±3 postpartum, 70 lactating Holstein cows from one herd were scored for body condition and pre-synchronized with PGF2α and GnRH (3 days apart) and 7 days later submitted to an Ovsynch-56 protocol for first AI after random assignment to two treatments: (1) OV-7 (n=35) with an injection of PGF2α either on day 7; or (2) OV-8 (n=35) on day 8 after G1, respectively. Blood was collected before the first PGF2α, at day 7 and day 8 in OV-7 and OV-8, respectively, at AI and at 7 days after AI to assess progesterone concentration. Ten cows were classified as acyclic and were excluded from the analysis resulting in 60 cows (OV-8, n=27; OV-7, n=33). In total, more (P=0.01) OV-8 cows and more (P=0.04) primiparous OV-8 cows had complete luteolysis compared with their OV-7 herd mates. In addition, more (P=0.008) OV-8 cows with BCS

The present study aimed to evaluate whether the induction and the formation of an accessory corpus luteum (CL) after AI might increase the pregnancy per AI (P/AI) in heat stressed dairy cows. Starting at d 50±3 post-partum, 113 lactating Holstein cows from one commercial herd during summer were scored for body condition, blood sampled and examined by ultrasound. Those bearing a CL>25mm and progesterone (P4) level>2ng/mL were synchronized using a double PGF2α injection given 12 h apart and AI-ed at detected estrus. In total 18 cows, there were not any signs of estrus (n=10) nor a P4 level 0.05). In conclusion, the induction of an accessory CL at d 5 after AI might increase P/AI in heat stressed dairy cows.

The bovine kappa-casein (κ-CN) is a phospho-protein with 169 amino acids encoded by the CSN3 gene. The two most common gene variants in the HF breed are CSN3*A and CSN3*B while CSN3*E has been found with lower frequency. The aim of this study was to optimize a laboratory method for genotyping of these three alleles as well as to determine their genotype and allele frequencies in the HF cattle population in the Republic of North Macedonia. Genomic DNA was extracted from full blood from 250 cows. The target DNA sequence was amplified with newly designed pair of primers and the products were subjected to enzymatic restriction with HindIII and HaeIII endonucleases. Genotype determination was achieved in all animals. The primers successfully amplified a fragment of 458 bp and the digestion of this fragment with both endonucleases enabled differentiation of five different genotypes with the following observed frequencies: AA (0.39), AB (0.29), BB (0.16), AE (0.10), and BE (0.06). The estimated allele frequencies were: CSN3*A (0.584), CSN3*B (0.336) and CSN3*E (0.08). The observed genotype frequencies differed significantly (P

The objective of the present study was to determine the prevalence of postpartum dysgalactia syndrome (PDS) and associated clinical signs in farmed sows in the Republic of Macedonia (RM) in the first 12-24 h postpartum. A total of 202 sows of different parity and different genetic lines from 5 pig farms in RM were included in the study. The sows and their litters were clinically examined 12-24 hours after farrowing. Postpartum dysgalactia syndrome was detected in 23.3% of all clinically examined sows, while prevalence between farms ranged from 14.8% to 38.1%. Altered piglet’s behavior was the most frequent clinical pattern observed in 68.1% of the PDS–affected (PDSA) sows. Regarding the clinical signs in PDSA sows detected among farms, significant differences were observed in the altered piglet’s behavior (p

Reduced glutathione (GSH) and homologous ram seminal plasma (HSP), used as additives in cryopreserving (CP) media prior to freezing, showed conflicting results in retaining structural integrity and progressive motility in post-thawed ram spermatozoa. The aims of this research were: (1) to assess the effect of GSH and/or HSP supplementation via soybean-lecithin CP extender on cryopreserved ram spermatozoa viability, morphology and motility pattern; and (2) to assess the effect of incubation in the context of the previous aim. Quantitatively and qualitatively, homogenized and pooled ram ejaculates (N=10) were extended with one of the following extenders: control (C) – tris-based, GSH and HSP-free, experimental-1 (E1) – C + GSH 5 mM, experimental-2 (E2) – C + HSP 20 % and experimental-3 (E3) - GSH 5 mM + HSP 20 %. Following thawing, samples were taken at 0- and 3-hours from each group (n=10) and were assessed for spermatozoa viability, morphology, and motility pattern. C-0h samples yielded a spermatozoa population with low viability, altered head morphology and highly deviated motility pattern. E3-3h samples yielded spermatozoa with unaffected viability, head morphology and high progressive motility. In conclusion, E3 extender added to cryopreserved-thawed ram spermatozoa is most efficient in obtaining high viability, unaltered head morphology, and progressive motility.

The objectives of the present study were to examine the fatty acid (FA) profiles in serum and in the follicular fluid (FF) and the association between polyunsaturated fatty acid level (PUFA) and follicular growth dynamics following induced luteolysis in dairy cows. A total of 29 dairy cows (CL>25mm, follicle≈15mm) at d0 (start of the experiment) were submitted to ultrasound guided transvaginal follicular aspiration for FF collection from the largest follicle and were injected with 500 μg of cloprostenol. The cows were subdivided into Group A1 (n=11) and Group A2 (n=8) resuming follicular growth either from a secondary follicle less than or larger than 8.5mm, respectively, present at the moment of aspiration and Group A0 (n=10) not resuming follicular growth. Follicular development was monitored daily by ultrasonography until the next dominant follicle reached ≈15mm and was subsequently punctured in Group A1 and A2 (d1). Serum and FF samples for FA determination were taken at d0 from all cows and at d1 in Group A1 and A2. No differences were observed between the FA profile in serum nor in FF between sampling days. Regarding the PUFA levels, the serum linoleic acid (C18:2n6) levels at d0 and d1 were significantly higher than in FF, while alpha linolenic acid (C18:3n3) was lower in the serum than in FF, both at d0 and d1. At d0, a tendency for negative correlation between serum and the FF C18:2n6 with subsequent daily follicular growth rate was observed, while, at d1 there was a strong negative correlation between the serum C18:2n6 and daily growth rate (r=-0.71; p=0.0006). The present study revealed similarities of the FA profiles in the serum and in the FF and association between serum and FF PUFA content with the follicular dynamics after induced luteolysis.

BLAD and CVM are Holstein-specific recessive mutations which have had significant economic impact on dairy cattle breeding worldwide. The aim of this study was to optimize a diagnostic test for detection of these two disorders and to obtain a preliminary picture about theire existence in Macedonian HF population. Blood samples were obtained from 84 HF cows from several different farms in Macedonia. Genomic DNA was extracted using DNeasy blood and tissue kit (Qiagen) which was also used for DNA extraction from deeply frozen sperm doses from 6 different HF bulls. Genotyping was performed by PCR-RFLP analysis with TaqI and HaeIII restriction endonucleases for BLAD and with PstI for CVM. The correctness of the genotyping was evaluated by direct sequencing of the amplified products. Among 90 HF cattle tested two cows were identified as carriers of the BLAD allele and one was carrier of the CVM allele. No bulls were found to be carriers of either of the two disorders.This study demonstrates that carriers of BLAD and CVM are present in Macedonia, although at low frequency. PCR followed by RFLP is efficient and inexpensive method for detection of the carriers and should be used as laboratory test in eradication programs. In order to establish a more accurate picture of the prevalence of these disorders, it is necessary to screen a larger number of animals. It is indispensable to test each bull before entering the A.I. program to prevent accidental transmission to a large number of offspring.