The effect of mesenchymal stem cells as co-culture in in vitro nuclear maturation of ovine oocytes

This study compared the effects of ovine mesenchymal stem cells (MSCs) and ovine oviductal epithelial cells (OECs) as feeder cells in cell free culture systems (HEPES-modified tissue culture medium, TCM199) supplemented with polyvinyl alcohol (PVA) or fetal calf serum (FCS) on in vitro oocyte maturation and subsequent embryo development (IVM/IVC). Cumulus-oocyte complexes (COCs) were harvested from ovine ovaries and subjected to IVM in the above-mentioned culture media. After culture for 24 h, nuclear maturation of the oocytes was evaluated by 4, 6-diamino- 2-phenylindole (DAPI) staining. After fertilization the presumptive zygotes were cultured under identical culture conditions and embryo development was evaluated. The percentage of oocytes at nuclear maturation (metaphase II) cultured in the MSC group was higher than for the IVM medium + PVA group (P<0.05), while between MSCs, OECs and IVM medium + FCS it was non-significant. The rates (%) of cleavage and the percentage of total blastocysts in MSCs and the IVM medium + FCS group were higher than for OECs and the IVM medium + PVA group (P<0.05). These rates were non-significant between MSCs and the IVM medium + FCS group or between OECs and the IVM medium + PVA group. The percentage of hatched blastocysts (%) was significantly increased in MSCs and the IVM medium + FCS group when compared to OECs and the IVM medium + PVA group (P<0.05). In conclusion, the effects of mesenchymal stem cells as co-culture on oocyte maturation and the successive embryo development in vitro are similar to those in the medium supplemented with FCS. This study suggests that co-culturing with mesenchymal stem cells may be a promising alternative to FCS-medium

A Comparison of Bacteriological Culture, Serology, and Quantitative PCR for Detecting Brucellosis in Ewes with a History of Abortion

The zoonotic disease brucellosis is a serious public health and livestock industry concern. In the present study, we used bacteriological culture, RBT, and qPCR to determine the prevalence of brucellosis in the serum and milk samples of sheep with a history of abortion. Serum and milk samples were obtained from 100 sheep aged 3-5 years. In order to determine the prevalence of brucellosis, a modified RBT was performed on serum samples, Brucella was isolated from milk by bacteriological culture, and qPCR was applied to detect bacterial DNA in milk. The prevalence of brucellosis using modified RBT, bacteriological culture, and qPCR was 32%, 42%, and 44%, respectively. By considering qPCR as the standard, modified RBT showed a sensitivity of 95%, a specificity of 100%, an accuracy of 98%, a PV+ of 100%, and a PV- of 97%. The sensitivity, specificity, accuracy, PV+, and PV- for bacteriological culture were 77%, 100%, 90%, 100%, and 85%, respectively. The agreement between qPCR and modified RBT was 0.959 (95% CI: 0.896-1), between qPCR and bacteriological culture was 0.792 (95% CI: 0.667-0.897), and between modified RBT and bacteriological culture was 0.831 (95% CI: 0.709-0.38). Based on the results, bacterial isolation from sheep milk is not recommended except in specific cases due to its low sensitivity, as well as its time-consuming and hazardous nature. However, the modified RBT can be used as a routine method because of its cost-effectiveness, higher sensitivity, and higher accuracy compared to bacterial isolation. Moreover, qPCR is recommended as the gold standard test for detecting brucellosis in sheep milk, especially in those with a history of abortion

Effect of different oocyte retrieval and culture methods on in vitro maturation of bovine oocytes derived from vitrified ovarian tissue slices

In a successful method of ovarian tissue cryopreservation, there are always a number of antral follicles remain intact, as a result of which the methods of retrieving and culturing oocytes from them becomes very important. Therefore, this study aimed to obtain an efficient method for oocyte retrieving and maturation in vitro from vitrified/warmed ovarian tissue slices. For this purpose, slaughterhouse-derived bovine ovaries were sliced and prepared in six replicates for each segment of each experiment and vitrified at 4°C and room temperature (RT) by 1.8 ml cryovial. After warming, the oocyte-cumulus complexes (COCs) were retrieved by two methods (aspiration and slicing), and matured in vitro in two conditions (in the presence or absence of oviductal epithelial cells (OECs) as feeder cells) for 24-48h, and finally, the nuclear maturation of oocytes was evaluated as the statistical analysis showed the higher degeneration rates of oocytes derived from vitrified ovarian tissue in aspiration groups (P<0.05) and the more M-II stage oocytes were obtained in the slicing groups (P<0.05), although the aspiration method was better to approach for oocyte retrieval from fresh ovarian tissue than slicing method (P<0.05). Also, the exposure temperature in the vitrification procedure of ovarian slices did not affect the percentage of M-II oocytes in the evaluated groups. It can be concluded that the slicing method is a more proper approach for oocyte retrieval from vitrified ovarian tissue than an aspiration, and the use of co-culturing of these immature oocytes with OECs during IVM is not required

Characteristics of Staphylococci isolated from mastitic goat milk in Iranian dairy herds

One hundred and fifteen cases of sub clinical mastitis were detected in a study on 510 goats from 5 herds in west central, Iran. From positive milk samples, 23 Staphylococcus spp. strains were isolated. Fourteen isolates were determined as Staphylococcus aureus (12.17%), and the other 9 (7.82%) as Staphylococcus epidermidis. Eleven and six isolates of S. aureus and S. epidermidis produced combined form of hemolysins a/&amp;#223;/d, while &amp;#223;/d hemolysins produced by 2 and 3 isolates respectively . Only one isolate of S. aureus produced single type d hemolysin. The sensitivity of all strains to 10 chemotherapeutics was tested through the disk diffusion method, 6 strains (26.08%) were determined as methicillin-resistant: out of them 4 isolates were S. aureus and 2 S.epidermidis. S. aureus and S. epidermidis isolates were 100% resistant to Cloxaciline and Kanamycin while the resistance to Penicillin was 100% in S. aureus and 33.33% in S. epidermidis isolates. [Vet. World 2010; 3(5.000): 205-208

How to maintain and transport equine adipose tissue for isolating mesenchymal stem cells?

Abstract Background Adipose tissue (AT) is one of the most important mesenchymal stem cell (MSC) sources because of its high quantities, availability and ease of collection. After being collected samples, they should be transported to a laboratory for stem cell (SC) isolation, culture and expansion for future clinical application. Usually, laboratories are distant from animal husbandry centers; therefore, it is necessary to provide suitable conditions for adipose tissue transportation, such that adipose-derived MSCs are minimally affected. In the current study, the impact of tissue maintenance under different conditions on MSCs derived from these tissues was evaluated. We aimed at finding suitable and practical transportation methods in which ASCs go through the slightest changes. Results In the current study, after being collected, equine AT was randomized into eight groups: four samples were maintained in stem cell culture media at 25 οC and 4 οC for 6 and 12 hrs. as transportation via SC media groups. Three samples were frozen at three different temperatures (− 20, − 75 and − 196 οC) as cryopreserved groups; these samples were defrosted 1 week after cryopreservation. Fresh and unfrozen AT was evaluated as a control group. The tissue samples were then initiated into enzymatic digestion, isolation and the culturing of SCs. Cells at passage three were used to evaluate the ability to form colonies, proliferation rate, plotting of the cell growth curve, and viability rate. All experiments were performed in triplicate. Stem cell isolation was successful in all groups, although purification of SCs from the first series of cryopreservation at − 196 οC and two series of − 20 οC was unsuccessful. There was no significant difference between the surface area of colonies in all groups except for − 20 οC. The growth rate of transportation via stem cell media at 25 οC for 6 hrs. was similar to that of the control group. MTT analysis revealed a significant difference between 25 οC 12 hrs. Group and other experimental groups except for control, 4 οC 12 hrs. and − 196 οC group. Conclusion Data have shown freezing at − 75 οC, transportation via stem cell media at 4 οC for 12 hrs. and 25 οC for 6 hrs. are acceptable tissue preservation and transportation methods due to minor effects on MSCs features