Martin Nikolovski, Monika Dovenska, Ksenija Ilievska, Nikola Adamov, Branko Atanasov, Miroslav Radeski, Daniela Kirovski, Vladimir Petkov and Toni Dovenski
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.
Dmytro O. Minchenko, Daria O. Tsymbal, Olena O. Riabovol, Yuliia M. Viletska, Yuliia O. Lahanovska, Myroslava Y. Sliusar, Borys H. Bezrodnyi and Oleksandr H. Minchenko
Objective. The aim of the present investigation was to study the effect of hypoxia on the expression of genes encoding endothelin-1 (EDN1) and its cognate receptors (EDNRA and EDNRB) as well as endothelin converting enzyme 1 (ECE1) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of glioma growth through ERN1 and hypoxia.
Methods. The expression level of EDN1, EDNRA, EDNRB, and ECE1 genes as well as micro-RNA miR-19, miR-96, and miR-206 was studied in control and ERN1 knockdown U87 glioma cells under hypoxia by quantitative polymerase chain reaction.
Results. It was shown that the expression level of EDN1, EDNRA, EDNRB, and ECE1 genes was up-regulated in ERN1 knockdown glioma cells in comparison with the control glioma cells, being more significant for endothelin-1. We also observed down-regulation of microRNA miR-206, miR-96, and miR-19a, which have specific binding sites in mRNA EDN1, EDNRA, and EDNRB, correspondingly, and can participate in posttranscriptional regulation of these mRNA expressions. Furthermore, inhibition of ERN1 endoribonuclease lead to up-regulation of EDNRA and ECE1 gene expressions and down-regulation of the expression level of EDN1 and EDNRB genes in glioma cells. Thus, the expression of EDNRA and ECE1 genes is regulated by ERN1 endoribonuclease, but EDN1 and EDNRB genes preferentially by ERN1 protein kinase. We have also shown that hypoxia enhanced the expression of EDN1, EDNRA, and ECE1 genes and that knockdown of ERN1 signaling enzyme function significantly modified the response of all studied gene expressions to hypoxia. Thus, effect of hypoxia on the expression level of EDN1 and ECE1 genes was significantly or completely reduced in ERN1 knockdown glioma cells since the expression of EDNRA gene was down-regulated under hypoxia. Moreover, hypoxia is induced the expression of EDNRB gene in ERN1 knockdown glioma cells.
Conclusions. Results of this investigation demonstrate that ERN1 knockdown significantly increased the expression of endothelin-1 and its receptors as well as ECE1 genes by different mechanisms and that all studied gene expressions were sensitive to hypoxia. It is possible that hypoxic regulation of the expression of these genes is a result of complex interaction of variable ERN1 related transcription and regulatory factors with HIF1A and possibly contributed to the control of glioma growth.
Furkan Alaraji, Hussam Muhsen, Abdullah O. Alhatami and Yahia Ismail Khudhair
For the first time in Iraq, we identified in March, 2018 the presence of a highly virulent avian influenza virus (AIV), H5N1 (Clade 18.104.22.168c), causing highly pathogenic avian influenza (HPAI) in poultry farms, Iraq,. The identification of the virus was done using a rapid serological test, a real time-qPCR, and glycoprotein gene sequencing. Using sequencing and phylogenetic analyses, the clade 22.214.171.124c virus was recorded to be clustered, with high similarity to Asian and West African AIV, HPAI H5N1 from Ivory Coast identified in 2015. According to our knowledge, there was no previous detection of the clade 126.96.36.199c made in Iraq. Our results provide evidence that high risk of HPAI H5 outbreaks might be present in Iraq, and this needs to lead to high quality surveillance targeting of wild and domestic birds for early diagnosis of HPAI. The current work provides feasible and accurate approaches for understanding the evolution of HPAI H5 virus in different countries around the world.
A 15 year old female African lion (Panthera leo) was necropsied after its sudden death. The necropsy showed a mammary gland lesion measuring 10 cm in diameter and numerous white nodules with variable size in the liver, spleen, uterus, lungs and the heart. The histopathological examination showed that the neoplastic formation in the mammary region was a simple tubular carcinoma with metastases on the other organs. Upon immunohistochemical examination, the neoplastic cells expressed cytokeratins while the stroma of the tumour expressed vimentin. The proliferation index Ki-67 was moderate. Based on the macroscopic, histopathological and immunohistochemical findings, the neoplasia was diagnosed as a simple tubular mammary carcinoma.
Hayder N. Ayyez, Yahia I. Khudhair and Qassim Haleem Kshash
Anaplasma spp. are widely spread rickettsial bacteria transmitted by ticks and placing high impacts on veterinary and public health. A limited number of studies have been carried out on Anaplasmosis in the central part of Iraq. This study was conducted to determine the presence of Anaplasma spp. in cattle in Al-Qadisiyah province, Iraq. A total of 400 blood specimens were collected from cattle suffering from heavy tick infestation. Cattle were blood-sampled from four hyper-endemic areas with ticks. Blood samples were screened using microscopic and polymerase chain reaction (PCR) methods. Diff-quick stained blood smears revealed Anaplasma-like inclusion bodies in 254 (63.5%) samples. According to the 16S rRNA-gene-based PCR analysis, Anaplasma spp. was detected in 124 of the 400 (31%) samples, divided as 96/254 (37.8%) among the microscopical positive samples and 28/146 (19.17%) among the microscopical negative samples. Phylogenetic analysis based on the partial 16S rRNA gene sequencing of ten-PCR positive samples were 99–97% identical to sequences deposited in the GenBank, revealing presence of A. phagocytophilum, A. marginale and unnamed Anaplasma spp. in 40%, 20%, and 40% samples, respectively. Relationships among Anaplasma spp. infections and cattle breed, age, and sex were analyzed. Calves less than one year old showed significantly higher rates (p<0.005) than those from other age groups, whereas sex and breed demonstrated no significant differences (p˃0.001). This study shows that a variety of Anaplasma spp., were endemic in central part of Iraq and is still a hidden problem in cattle in the hyperendemic areas of tick, which requires serious control strategies.
Alhaji Zubair Jaji, Adamu Saleh Saidu, Mohammed Bakari Mahre, Mbaya Pindar Yawulda, Ibrahim Alhaji Girgiri, Piyush Tomar and Faruk Da’u
Prenatal gross morphologic, morphometric and histologic developmental features of the dromedary spleen were studied. The dromedary gestation period (13 months) was categorized into four (1-4) phases and ten developing spleens per growth phase were sampled. Splenic topographical anatomy was noted before being eviscerated from each foetus. Morphologic and morphometric features of the eviscerated spleens were immediately documented and 2 – 4 mm thick samples were collected for histological analysis. The developing spleen was dark brown in colour, semilunar shaped and significantly increased (p<0.05) in size and weight across the four phases of prenatal development. The full-term dromedary spleen was observed to have unique histological features. Its capsule had an inner smooth muscle and an outer predominant connective tissue layer. The pumping of stored blood from the muscular capsule and trabeculae was proportionate to the body’s requirement. The splenic venous return was characterized by blood flow from the red pulp (venous sinusoids) to the peritrabecular sinuses, subcapsular sinuses and finally to the splenic veins. The dromedary has a sinusal type of spleen and has both open and closed types of circulation. The presence of closed circulation and absence of marginal sinus could be the reason for dromedary main health problems of blood parasites; Trypanosoma evansi. It was concluded that most of the salient features of the postnatal spleen were already evident in the first growth phase and became developed by the second phase. Other growth phases were mainly characterized by increase in sizes.
The vascular endothelial growth factor (VEGF) is a multifunctional cytokine stimulating the growth of vascular endothelial cells, survival and proliferation, inhibiting apoptosis. It is one of the most potent stimulants of vascular permeability. VEGF is found at high levels in inflammatory and tumour-associated pleural and abdominal effusions and is involved in their occurrence. In the present study, the blood plasma and abdominal fluid VEGF levels were assayed in thirty-one client-owned dogs with neoplastic and non-neoplastic diseases by means of enzyme-linked immunosorbent assay (ELISA). The VEGF concentration in abdominal fluid of dogs (n=6) with ascites was 190.70±34.35 pg/ml, in dogs (n=6) with peritonitis: 1449.81±365.42 pg/ml and in dogs (n=9) with tumour-associated effusion: 1993.13±202.56 pg/ml. Blood plasma VEGF of healthy dogs (control group, n=10) was 36.79±5.72 pg/ml, in dogs with ascites: 57.92±2.88 pg/ml, in dogs with peritonitis: 76.98±7.24 pg/ml and in dogs with tumour-associated effusion: 173.50±40.9 pg/ml. There were substantial differences between blood plasma and abdominal fluid VEGF levels.
Somchit Eiam-Ong, Mookda Chaipipat, Krissanapong Manotham and Somchai Eiam-Ong
Objectives. Aldosterone rapidly enhances protein kinase C (PKC) alpha and beta1 proteins in the rat kidney. The G protein-coupled receptor 30 (GPR30)-mediated PKC pathway is involved in the inhibition of the potassium channel in HEK-239 cells. GPR30 mediates rapid actions of aldosterone in vitro. There are no reports available regarding the aldosterone action on other PKC isoforms and GPR30 proteins in vivo. The aim of the present study was to examine rapid actions of aldosterone on protein levels of phosphorylated PKC (p-PKC) delta, p-PKC epsilon, and GPR30 simultaneously in the rat kidney.
Methods. Male Wistar rats were intraperitoneally injected with normal saline solution or aldosterone (150 µg/kg body weight). After 30 minutes, abundance and immunoreactivity of p-PKC delta, p-PKC epsilon, and GPR30 were determined by Western blot analysis and immunohisto-chemistry, respectively.
Results. Aldosterone administration significantly increased the renal protein abundance of p-PKC delta by 80% (p<0.01) and decreased p-PKC epsilon protein by 50% (p<0.05). Aldosterone injection enhanced protein immunoreactivity of p-PKC delta but suppressed p-PKC epsilon protein intensity in both kidney cortex and medulla. Protein abundance of GPR30 was elevated by aldosterone treatment (p<0.05), whereas the immunoreactivity was obviously changed in the kidney cortex and inner medulla. Aldosterone translocated p-PKC delta and GPR30 proteins to the brush border membrane of proximal convoluted tubules.
Conclusions. This is the first in vivo study simultaneously demonstrating that aldosterone administration rapidly elevates protein abundance of p-PKC delta and GPR30, while p-PKC epsilon protein is suppressed in rat kidney. The stimulation of p-PKC delta protein levels by aldosterone may be involved in the activation of GPR30.
Objective. While dulaglutide has been approved inpatients with type 2 diabetes (T2DM) in combination with insulin, it has not been studied in insulin-deficient patients, not whether they have type 1 diabetes (T1DM) or T2DM. The aim of this study is to assess the efficacy and safety of dulaglutide 0.75 mg/once weekly (QW) in patients with absolute insulin deficiency (n=10).
Subjects and Results. Significant reductions of HbA1c (9.30±1.03% to 8.61±1.21%; p<0.02) and body mass index (BMI; 23.61±3.95 to 23.41±4.24; p<0.02) levels were observed at 3 months with the addition of dulaglutide to the existing pharmacotherapy. However, in all the patients, post-meal C-peptide levels remained undetectable. One patient had gastrointestinal adverse events and discontinue dulaglutide within the first month. One patient was a non-responder, who had little if any changes in HbA1c levels at 3 months.
Conclusions. The results indicate that dulaglutide is effective in patients with T1DM or T2DM with absolute insulin deficiency, though gastrointestinal adverse events might be of concern. The improvements in glycemic control could not be due to enhanced insulin secretion, but may be as a result of a combination of the other effects of glucagon like peptide 1 (GLP-1), such as postprandial glucagon suppression, delayed gastric emptying, and weight loss.
Tolulope Oyesola, Bolanle Iranloye and Olufeyi Adegoke
Objective. This study was designed to investigate the effect of sublethal doses (10, 60, and 120 mg/kg of pirimiphos-methyl on implantation and pregnancy in female Sprague-Dawley rats. Pirimiphos-methyl is a pesticide widely used worldwide, especially in Africa to protect food against pests and has gained widespread acceptance.
Methods. Pregnant Sprague-Dawley rats used for this study had access to food and water ad libitum and were divided into a control group and three experimental groups based on dose of chemical given. The pregnant rats were given pirimiphos-methyl orally on days 1–5, 1–7, 7–18th day of gestation and from day 1 to term. Implantation studies were carried out on days 6 and 8 of pregnancy, while the fetal parameters were ascertained on day 19 of pregnancy and at term. Serum levels of progesterone and estradiol were measured on days 6, 8 and 19 of pregnancy.
Results. Sublethal administration of pirimiphos-methyl showed decreased number of implantation sites on days 6 and 8, fetal weight, crown-to-rump length, length of umbilical cord and placenta weight (day 19), birth weight, litter size and total number (at term) in rats administered with pirimiphos-methyl when compared with control.
Conclusion. Administration of pirimiphos-methyl resulted in a reduced implantation rate due to decreased uterine receptivity caused by an imbalance in the level of estradiol and progesterone and impaired reproductive outcome during pregnancy.