Faculty of Veterinary Medicine

Rhodococcus equi is an encapsulated gram-positive , pleomorphic rod which exists in the soil and frequently is isolated from the feces of normal herbivores. It is the primary cause of pneumonia in foals. This organism has been isolated from various animals, but isolation from the wound is rarely reported. In May 2013, a 7-month-old Janet (female mule) was referred with some ulcers on the face below the zygomatic process in the right side in dimensions of 2×2 cm. Swab samples were taken from the ulcer for bacteriological cultures. The isolated bacteria grew on cysteine tellurite agar. The tests of catalase and nitrate reduction were positive, but urea hydrolysis was negative. The result of the Christie-Atkins-Munch-Petersen (CAMP) test was positive and " spade showel-shaped " hemolytic pattern was seen in blood agar. The bacteria were identified as R. equi. This organism was susceptible to gentamycin, azithromycin, and erythromycin. The presence of R. equi as a pathogen without pulmonary disease is important because it can cause wound contamination and leads to persistence of the organism in the environment. An exposure to soil contaminated with livestock manure seems to be the most probable mode of infection for this case.

Successful Embryo Transfer (ET) technique is a fateful step of all efforts to achieve live births from in vitro produced embryos in assisted reproductive techniques or in knockout, transgenic or cloned animal projects. Small reproductive tract of mice and limitation of current techniques may not well satisfy the requirements for mass production of genetically modified mice. Genetic abnormalities of embryos, receptivity and uterine contractions, expulsion of embryos, blood, mucus or bacterial contamination on the transfer pipette tip, technical problems and even animal strain may affect embryo transfer outcome. In this study, two techniques of embryo transfer in mice were compared. In conventional technique the oviduct wall was punctured with a 30-gauge needle and the loaded Pasteur pipette with embryos and medium was inserted into the hole. In new technique, embryos that were loaded in modified micropipette with minimal medium were transferred directly to the oviduct by manual pisto...

Purpose The present study by using different growth factors was aimed to develop the best practical culture condition for purification of goat undifferentiated SSCs and their colonization under in vitro and in vivo conditions. Methods The enzymatically isolated SSCs obtained from one month old goat testes were cultured in DMEM plus FCS supplemented with different sets of growth factors (GDNF, LIF, bFGF, and EGF) for 2 weeks. At the end of each week, the morphological characteristics of cells and colonies alongside with purification rate of undifferentiated type A spermatogonia were evaluated by immunocytochemical staining and flow cytometry. Results The number and size of colonies in treatment groups were significantly (P<0.01) higher than corresponding values in control group. In immunocytochemical evaluation, the proportion of KIT and PGP9.5 positive cells were significantly (P < 0.001) higher in control and treatment groups, respectively. Conclusions The culture medium comprising all four growth factors, especially the one supplemented with the higher concentration of GDNF, was superior to the other groups with respect to the population of undifferentiated type A spermatogonia and its propagation in culture system. Additionally, goat SSCs could colonize within the mouse testis following xenotransplantation. Keywords PGP9.5. KIT. Goat. Spermatogonial stem cell. Growth factors. Mouse Mohammad Sadra Shirazi and Banafsheh Heidari these authors contributed equally to the work. Capsule Spermatogonial stem cells are the foundation of spermatogenesis. Addition of growth factors to the culture medium could effectively retain the undifferentiation status of goat SSCs. The culture medium containing higher concentration of GDNF was superior to the other groups comprising either the lower concentration of GDNF or other combinations of growth factors. The colonization of goat SSCs within the mouse testis following xenotransplantation, was promising in male fertility preservation and production of transgenic animals.

Media supplementation with various compounds in order to increase the oocyte developmental competence is of particular importance. This experiment was conducted to evaluate the effect(s) of media supplementation with aldosterone on embryo development and Na + /K + /ATPase expression following sheep oocyte vitrification. The abattoir-derived sheep COCs (cumulus oocyte complexes) were randomly allocated into six experimental groups: IVM of fresh and vitrified COCs in the presence of aldosterone followed by IVF/IVC (F-IVM and Vit-IVM groups, respectively); IVM/IVF of fresh and vitrified COCs followed by IVC wherein the embryos were exposed to aldosterone on Day 4 of IVC (F-D4 and Vit-D4 groups, respectively); and IVM/IVF and IVC of fresh and vitrified COCs in the absence of aldosterone (F-Cont and Vit-Cont groups, respectively). The expression of Na + /K + /ATPase α 1 and β 1 subunits in embryos were assessed at morula and blastocyst stages with related primary antibodies. The hatching rate was greater in aldosterone supplemented groups in both fresh and vitrified COCs compared to their controls. The expression of Na + /K + /ATPase β 1 subunit was significantly greater in F-D4 and Vit-D4 groups compared to other groups. The ICM / Total ratio in F-IVM was greater compared to the other groups. In conclusion, addition of aldosterone to the IVM and IVC media could improve the hatching rate of blastocysts derived from both fresh and vitrified COCs. These improvements, in D4 supplemented groups, might be related to the greater expression of Na + /K + /ATPase β 1 subunit induced by aldosterone supplementation.

Growing usage of animals in the research projects has drawn more attention to their welfare and ethics surrounding this practice. Dissemination of information about the existing ethical consideration and alternatives in animal experiments has two important functions; first, it increases the researcher&#39;s awareness of the possible methods of using animals in the experiment, and second, to ensure that potential users are aware of the established alternatives. For example, legislations enacted in many countries during the 1980s state that laboratory animal applications should be reduced, refined and replaced wherever possible according to principles of the 3Rs. Thus, scientists around the world tried to apply the 3Rs in their biomedical researches regarding welfare of the laboratory animals. However, the Qur&#39;an, the holy book of Muslims, and also Hadiths contain the obligatory ways to keep and treat animals since their revelations. According to Islamic viewpoint, animals represe...

The basal medium that supports Isolated Mouse Oviduct (IMO) is important for supporting embryo development and quality. The culture of ovine IVM/IVF zygotes was done in IMO using SOFaaciBSA and SOFaaBSA as basal medium of IMO and in SOFaaBSA alone as control. For preparation of IMO mature inbred strain C57BL/6 female mice were synchronized and mated with vasectomized males. The females with vaginal plug were sacrificed and the zygotes were transferred in to the isolated oviduct at 20 hpi. The oviducts were cultured with SOFaaciBSA and SOFaaBSA for 6 days. Another group of zygotes were cultured in SOFaaBSA alone as control. Culture of zygotes in the IMO with SOFaaciBSA and SOFaaBSA, did not significantly affect the development and quality of embryos (p &gt; 0.05). The hatching rate, total and trophectoderm cells number in IMO groups&#39; blastocysts were significantly higher than SOFaaBSA alone. The morphological appearance of IMO blastocysts was superior to SOFaaBSA alone. When the ...

The well documented source for adult multipotent stem cells is Spermatogonial Stem Cells (SSCs). They are the foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. The aim of this study was to assess the effect of percoll density gradient and differential plating on enrichment of undifferentiated type A spermatogonia in dissociated cellular suspension of goat testes. Additionally, we evaluated the separated fractions of the gradients in percoll and samples in differential plating at different times for cell number, viability and purification rate of goat SSCs in culture. Testicular cells were successfully isolated from one month old goat testis using two-step enzymatic digestion and followed by two purification protocols, differential plating with different times of culture (3, 4, 5, and 6 hr) and discontinuous percoll density with different gradients (20, 28, 30, and 32%). The difference of percentage of undifferentiated SS...

Assisted reproductive techniques involving isolation, culture, and transplantation of spermatogonial stem cells offer unique approach to manipulate the male germline. The application of these techniques in farm animals has been the subject of an increasing number of studies, mostly because of its potential as an alternative strategy in producing transgenic livestock with higher efficiency and less time and capital requirement than the current methods. The aim of this study was to assess the colonization and differentiation potentials of enriched goat spermatogonia into the mouse testes. Moreover, because stem cells may need to be preserved for several years before re-introduction to the recipient testes, we developed the efficient cryopreservation technique for type A spermatogonoa. The enzymatically isolated SSCs obtained from one month old goats' testes were enriched by using discontinuous percoll density, and followed by cryopreservation protocol. After xenotransplantation of prepared goat testicular cells into the mouse rete testis, the proliferative activity and stemness potential of SSCs were evaluated and compared with in vitro culture condition. We demonstrated that the viability of testicular cells after cryopreservation was significantly lower than fresh cells, although these cells had normal structural and functional characteristics (P <0.001). Donor goat spermatogonia were able to survive and colonize in depleted recipient's testis at 80 days after transplantation, but later stages of donor-derived spermatogenesis were not observed at this time. Although cross-species spermatogonial transplantation did not have the envisioned immediate practical application, it nonetheless provides a bioassay for stem cell potential of germ cells isolated from other species.

In vitro follicle growth is a promising strategy for female fertility preservation. This study was conducted to compare the development of ovine follicles either isolated or in the context of ovarian cortical pieces after short term (8 days) three-dimensional culture in fresh and vitrified samples. Four different experiments were conducted; I) culture of ovarian cortical pieces encapsulated in 0.5% and 1% alginate and without alginate encapsulation (CP-0.5%, CP-1% and CP, respectively), II) culture of isolated primordial and primary follicles encapsulated in 1% and 2% alginate (IF-1% and IF-2%, respectively), III) culture of fresh and vitrified-warmed cortical pieces (F-CP and Vit-CP, respectively), and IV) culture of fresh and vitrified-warmed encapsulated isolated follicles (F-IF and Vit-IF, respectively). The number of secondary follicles after culture was negatively influenced by encapsulation of ovarian cortical pieces (6.3 ± 3.3 and 10.6 ± 0.9 vs 21.5 ± 2.3 in CP-0.5% and CP-1...

The presence of rennin-angiotensin components in mammalian ovaries and their involvement in ovarian physiology have been established. In the present study, effects of angiotensin II (Ang II) on sodium-potassium adenosine triphosphatase (Na(+)/K(+)/ATPase) expression and development of sheep embryos was evaluated. The abattoir-derived Cumulus Oocyte Complexes (COC) were randomly allocated into three experimental groups; group I) in vitro Maturation (IVM) of oocytes in the presence of Ang II followed by in vitro fertilization (IVF)/in vitro Culture (IVC) (IVM group), group II) IVM/IVF of oocytes followed by IVC wherein the embryos were exposed to Ang II on day 4 of IVC (D4 group), and group III) IVM/IVF and IVC of oocytes without any angiotensin (Control). The blastocyst and hatching rates were recorded on days 6 to 8. Day 8 embryos were immunostained with primary and secondary antibodies against Na(+)/K(+)/ATPase α1 and β1 subunits. Addition of Ang II during IVM and IVC significantly...

This study was aimed to assess the effects of angiotensin II (Ang II) supplementation to the In Vitro Maturation (IVM) and In Vitro Culture (IVC) media of vitrified-warmed ovine oocytes on their developmental competence and expression of Na(+)/K(+)/ATPase in resulting embryos. The slaughterhouse-derived immature oocytes (n=1069) were randomly distributed into four experimental groups: groups I and II) IVM/IVF and IVC of fresh and vitrified oocytes without angiotensin supplementation (Control-Fresh and Control-Vit groups, respectively); group III) IVM of vitrified oocytes in the presence of Ang II followed by IVF/IVC (Vit-IVM group); and group IV) IVM/IVF of vitrified oocytes followed by IVC wherein the embryos were exposed to Ang II on day 4 of IVC (Vit-D4 group). The embryos were immunostained with primary antibodies against Na(+)/K(+)/ATPase α1 and β1 subunits. In Vit-IVM and Vit-D4 groups, the rates of expanded and total blastocysts on day 7 as well as the proportion of blastocys...

This study aimed to assess the effects of platelet-rich plasma (PRP) on growth and survival of isolated early human follicles in a three-dimensional culture system. After fresh and vitrified-warmed ovarian tissue was digested, isolated early preantral follicles and ovarian cells were separately encapsulated in 1% alginate (w/v). The encapsulated follicles and ovarian cells were cultured together in a medium supplemented with foetal bovine serum (FBS), PRP, PRP + FBS, or human serum albumin (HSA) for 10 days. Growth and survival of the follicles were assessed by measurement of diameter and staining with trypan blue. Follicular integrity was assessed by histological analysis. After culturing, all follicles increased in size, but growth rate was greater in follicles isolated from fresh samples than those from vitrified-warmed ones (P &lt; 0.001). Similarly, follicular viability of fresh samples after culturing was higher than that of vitrified-warmed ones. The growth and survival rates...

Considering the advent of mesenchymal stem cells (MSCs) as a new source of somatic cells in embryo co-culture system, the current study was aimed to compare in vitro embryo development using embryonic MSCs monolayer with embryonic fibroblast cells (EFCs), oviductal epithelial cells (OECs), and cell-free culture system. The IVM/IVF presumptive sheep zygotes were randomly cultured in different culture conditions as follows: (1) SOFaaBSA medium for the whole culture period (SOF, n = 371), (2) SOFaaBSA medium for the first 3 days followed by co-culturing with MSCs for the next 5 days (SOF-MSCs, n = 120), (3) co-culturing with MSCs for the first 3 days followed by culture in SOFaaBSA medium for the next 5 days (MSCs-SOF, n = 133), (4) co-culturing with MSCs for the whole culture period (MSCs, n = 212), (5) SOFaaBSA medium for the first 3 days followed by co-culturing with EFCs for the next 5 days (SOF-EFCs, n = 132), (6) co-culturing with EFCs for the first 3 days followed by culture in ...

Several studies indicate the absence of oviduct or its assistance may compromise the developmental competence and quality of the embryos produced in vitro. This study was aimed to evaluate the quality of embryos produced in vitro by culturing zygotes in isolated mouse oviduct and oviduct epithelial cell monolayer. The in vitro produced ovine zygotes were cultured for 7 days in the following culture systems i) ampullae of isolated C57BL/ 6 mouse oviduct (IMO), ii) ovine oviduct epithelial cell monolayer (OCM), and iii) chemically semi-defined medium (CSM) as control. In CSM, embryos were cultured in synthetic oviductal fluid medium supplemented with essential and non-essential amino acids and bovine serum albumin (SOFaaBSA). The cryotolerance of resultant blastocysts and their apoptotic cells (TUNEL assay) were then evaluated. The rate of blastocysts in OCM (37.9 ± 16.7) was lower than in IMO (50.6 ± 25) and CSM (49.7 ± 21.9) groups (P < 0.05). In vitrified/ warmed blastocysts ICM:TE ratio in IMO (0.33 ± 0.14) was higher than other groups (0.23 ± 0.16 and 0.22 ± 0.10 in CSM and OCM, respectively). Similarly, cryosurvival of vitrified blastocysts and hatching rates tended to be higher in IMO compared to other groups. In vitrified/warmed blastocysts the proportion of tunnel positive cells of ICM in OCM (31.7 ± 11.5) was higher than CSM and IMO groups (23.7 ± 17.9 and 23.0 ± 11.4). In conclusion, culture of ovine zygote in isolated mouse oviduct could increase the quality of resulting blastocysts.

Considering the effects of epigenetic changes on the embryonic development through altering the gene expression of pluripotency, trophectoderm, and imprinting genes, we determined the pattern of epigenetic alterations in transgenic embryos produced by injecting transgenic mESCs into the morula. To aim this, RAG1 knock-out mESCs were produced using CRISPR-Cas9 editing system and then microinjected into morulas to develop chimeric embryos. Afterward, the expression of pluripotency, trophectoderm genes, and imprinting genes in these embryos were evaluated using real-time PCR. Immunohistochemical analysis was carried out to determine the methylation rates of H3K9me3 and H3K4me3. In the following, since epigenetic alterations can be at least one of the possible reasons of male infertility and the loss of germ-line transmission capacity, the germ-line transmission ability in chimeric mice was also evaluated by breeding them and subsequent backcrosses to wild type strains. Our findings sho...

BACKGROUND: Newcastle disease virus (NDV) majorly infects the poultry, and despite high rates of vaccination, it is still circulating in different geographical regions. Due to the high mortality rate, the economic loss of Newcastle disease (ND) is enormous. OBJECTIVES: The molecular characterization of NDV isolates from chicken farms in Northern Iran, during 2017-2018, was the main goal of this study. METHODS: We isolated and characterized five NDVs from commercial broiler and backyard chicken farms during severe disease outbreak. The partial coding sequence of fusion (F) genes of isolates was determined and compared with those of other published NDVs. RESULTS: Phylogenetic analysis revealed that all of the isolates were grouped into sub-genotype VII.1.1 (formerly known as VIIl). All isolates carried multi-basic amino acid residues at the cleavage site of fusion protein, typical of virulent strains. Studied NDV isolates had high homology with the prevalent genotype NDV strains that currently circulate in China and Republic of Korea (96.94%). CONCLUSIONS: Our results suggested that NDV sub-genotype VII.1.1 (VIIl), circulating among chicken farms, may be a dominant sub-genotype. Considering the genetic variation between the used vaccine strains (B1, LaSota, and Clone 30, all belonging to genotype II) and circulating NDVs, it is recommended that a contemporary homologous virus should be developed as the vaccine strain to avert the outbreaks of genotype VII viruses.

BACKGROUND: Fracture healing is one of the important issues in medicine and veterinary. Therefore, finding new techniques with fewer side effects and faster healing is taken into consideration. OBJECTIVES: This study was conducted to evaluate the effect of the composites of micro ostrich eggshell (µ-OES) and hydroxyapatite (HA) composite on the healing of bone defect in rat calvarium. METHODS: Defects of 7 mm were made by a trephine in the calvaria of 45 male Wistar rats. The animals were divided into three groups and the defects in each group were filled with micro-composites that contained ostrich eggshell or HA or were left empty. The animals were euthanized at three different time points of 14, 28, and 42 days post-operation. Histological and serological assessments, such as measuring alkaline phosphatase were carried out at the same time points. RESULTS: Significant differences were observed in the granulation tissue formation of the treatment and control groups 14 days post-operation (P≤0.05). The difference between µ-OES and HA treatment groups was not statistically significant (P>0.05). On days 28 and 42, there were no significant differences between the groups. However, in the center of the defect, the mean of healing in the µ-OES group was higher than the two other groups. CONCLUSIONS: In conclusion, the results of this study indicated the potential efficacy of µ-OES as a bone substitute in a rat calvarial defect model.

BACKGROUND: Avian Metapneumovirus (AMPV) causes mild to acute contagious infection of the upper respiratory tract in turkey and chicken with different mortality rate. OBJECTIVES: This study was aimed to molecular detection and subtyping of AMPV infection in broiler flocks using RT-PCR method in Semnan province on samples from 2016 to2020. METHODS: Sampling was carried out from the upper part of the trachea, choana, and sinuses of broiler chickens from the 85 broiler flocks. All flocks were more than 3 weeks of age. In total 10 swabs were taken from each flock, while each 5 were pooled as one sample (total two samples per flock). The samples were transferred to the laboratory for RNA extraction and RT-PCR amplification. RESULTS: Out of 85 tested broiler flocks, 30 (35.3%) were positive for AMPV using the Nd/Nx primer set. In addition, 28 positive samples were found to be of subtype B using the Ga/G12 primer set and 2 remaining positive samples were non-subtype B, probably A, C or D subtypes. CONCLUSIONS: Since AMPV vaccination was not performed in Semnan province, it can be concluded that some cases were infected with the natural viruses. Therefore, vaccination could be effective in controlling AMPV-induced respiratory distress.

BACKGROUND: A large part of our country's waste is organic matter, so researchers are studying fertilizer production from organic waste in various ways, including compost and vermicompost. On the other hand, glycolipoprotein extract of Eisenia fetida (G-90) has been defined to show numerous biological activities, e.g., anticoagulation, fibrinolysis, and anti-oxidative, etc. OBJECTIVES: The purpose of the present study was to determine phylogenetic relationship of the Iranian isolate of Eisenia fetida (E. fetida) with the other available taxa and to determine the G-90 protein complex for evaluation of its biological activities. METHODS: A piece of 1×1cm clitellum was separated and used for DNA extraction after homogenate preparation (by two different methods). The second internal transcribed spacer of the nuclear ribosomal DNA (rDNA-ITS2) and cytochrome C oxidase subunit 1 of the mitochondrial DNA (mtDNA-COX1) from adult E. fetida were amplified using polymerase chain reaction (PCR). Subsequently, PCR products were sequenced and their phylogenetic relationships were determined. Furthermore, The G-90 protein complex was extracted from adult worms and electrophoretic pattern of proteins was obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The electrophoretic pattern of glycolipoprotein G-90, a protein complex, showed 10 bands with molecular weights of 14-130 kDa. ITS2 and COX1 sequences were 516 bp and 277 bp long, respectively. The amplified DNA sequences from both ribosomal and mitochondrial sequences had 88%-99% and 99% similarity to relevant sequences in GenBank, respectively. CONCLUSIONS: mtDNA-COX1 showed no considerable sequence variations as compared to other isolates in GenBank, while rDNA-ITS2 exhibited more variations, in comparison with other isolates, indicating more variations among some of these isolates. Our results revealed that rDNA-ITS2 of our E. fetida was in a separate subclade, showing the greatest similarity with EF534709.1 isolate (99%) provided in GenBank, followed by JX531618.1 (94%), and KU708469.1 (88%). Our COX1 sequence demonstrated the high similarity of 99% when compared with five isolates from GenBank (MH475674.1, MH475673.1, MH475672.1, MH475670.1, and MH475666.1). The G-90 glycolipoprotein showed different proteins that can be assessed for their potential biological activities in medicinal properties.