Demi-embryos were successfully produced by bisection of ICR mouse embryos at preimplantation stages. They were microsurgically bisected using a microsurgical blade attached to a micromanipulator after pretreatment with 0.5 % pronase in PBS for two minutes or not. Embryos with softened zona pellucida were more easily bisected and less damaged than intact embryos. The highest success rate in bisection has been achieved by selecting blastocysts (94.1 % in success rate with intact blastocysts and 100 % in success rate with zona softened blastocysts). Demi-embryos without zona pellucida were cultured in D-PBS or M-16 medium at 37deg C, 5 % CO2 in air for 72 hours for 2-cell stage embryos, 48 hours for 4-to 8-cell stage embryos, 24 hours for morula stage embryos and 6-12 hours for blastocyst stage embryos. For the in vitro culture of 2-cell stage embryos, 100 micro M 2Na-EDTA was added to the media. M-16 medium was better for the in vitro development of mouse embryos than PBS, and PBS is not considered to be suitable for long-term culture of embryos, especially at early stage of cleavage. In M-16 medium, developing rate of demi-embryos of which pair underwent development to form eublastocysts was 15.8 % at 2-cell stage, 16.8 % at 4-cell stage, 38 % at 8-cell stage, 89.6 % at morula stage and 94.4 % at blastocyst stage, respectively. The more rapid and efficient production of demi-embryos and higher viability after bisection can be expected by softening zona pellucida with pronase and by selecting morulae or blastocysts rather than embryos at early stage of cleavage.

Introduction: The objective of the study was immunophenotypic and cytogenetic analysis of mesenchymal stem cells from equine bone marrow and foal umbilical cords during in vitro culture.Material and Methods: The mesenchymal stem cells were obtained from equine bone marrow of three horses and from foal umbilical cords of six foals. The cells were cultured in CO₂ incubators by standard procedure. Quantitative abnormalities of chromosomes, i.e. aneuploidy and polyploidy, and structural aberrations, i.e. breaks in chromosomes and chromatid, were taken into account during the study.Results: The results of cytogenetic analysis of equine bone marrow mesenchymal stem cells at the third and fourth passages indicated that the level of karyotype variability of these cells corresponded to the spontaneous level of karyotype variability typical of the peripheral blood lymphocytes of this species. Equine bone marrow contained several clones of stem cells that differed in the expression of specific nuclear markers characteristic of proliferating cells.Conclusion: Mesenchymal stem cells from foal umbilical cords during in vitro cultivation are characterised by quantitative abnormalities of the chromosomal apparatus.

Adipose-derived stem cells (ADSCs) are multipotent, and can be differentiated into many cell types in vitro. In this study, tissues from pigs were chosen to identify and characterize ADSCs. Primary ADSCs were sub-cultured to passage 28. The surface markers of ADSCs: CD29, CD71, CD73, CD90, and CD166 were detected by reverse-transcription polymerase chain reaction assays and the markers CD29, CD44, CD105, and vimentin were detected by immunofluorescence. Growth curves and the capacity of clone-forming were performed to test the proliferation of ADSCs. Karyotype analysis showed that ADSCs cultured in vitro were genetically stable. To assess the differentiation capacity of the ADSCs, cells were induced to differentiate into osteoblasts, adipocytes, epithelial cells, neural cells, and hepatocyte-like cells. The results suggest that ADSCs from pigs showed similar biological characteristics with those separated from other species, and their multi-lineage differentiation shows potential as an application for cellular therapy in an animal model.

The effect of a dietary supplement, choreito, on in vitro struvite crystal growth in feline urine was evaluated. Adult specific-pathogen-free cats (4 females, 4 males) considered to be clinically normal on the basis of physical examination findings and normal results of CBC, serum biochemical analyses, and urinalyses obtained before the beginning of the study were used. Before 24-hour urine sample collections were made, cats were fed a commercial canned diet with 0 or 500 mg of choreito supplement/kg of body weight for at least 2 weeks in a cross-over design with 4 cats/treatment. Filtered urine samples were analyzed for urine pH, specific gravity, osmolality, and urine electrolytes. The struvite activity product was calculated, using a statistical software program that calculates urine saturation. Urine samples were placed in wells of cell culture plates, increasing concentrations of ammonium hydroxide were added to adjacent wells to stimulate struvite crystal growth, and the plates were incubated at 37 C. Crystal growth was assessed by determination of number of crystals and supersaturation index by direct visualization, using an inverted microscope. Supplementation of the diet with choreito (at this concentration) did not change urine pH, specific gravity, osmolality, urine electrolyte composition, or calculated struvite activity product. However, supplementation significantly (P < 0.05) reduced crystal number and supersaturation index. These results indicate that direct observation of struvite crystal formation in whole urine may more accurately predict the effects of treatments to prevent or treat struvite urolithiasis than do calculations based on electrolyte concentration that do not account for the effect of urine macromolecules. It also may mean that choreito consumption affects the concentration of inhibitors or promoters in urine. It was concluded that choreito significantly (P < 0.05) reduced growth of struvite crystals in feline urine, and thus may have a role in prevention of feline struvite urolithiasis. In vivo studies will be necessary to test this hypothesis.

Cultured rat pituitary cells were studied to: determine the effects of ergovaline and loline on in vitro prolactin release; delineate the agonistic activity of these alkaloids at the D2 dopamine receptor, using 2 selective D2 dopamine receptor antagonists; and compare the efficacy of 2 dopamine receptor antagonists in reversing effects of the treatments on in vitro prolactin secretion. Ergovaline reduced in vitro prolactin release by at least 40% (P < 0.05) at concentrations of 10(-4),10(-6), and 10(-8) M. However, loline reduced (P < 0.05) prolactin release only at the highest concentration, 10(-4) M. Two standard dopamine agonists, dopamine and alpha-ergocryptine, were used to verify that the inhibitory control mechanisms of in vitro prolactin release were intact. Both compounds reduced prolactin release by at least 40% for concentrations of 10(-4), 10(-6), or 10(-8) M. Selective D2 dopamine receptor antagonists 10(-6) M, domperidone and sulpiride, reversed (P < 0.05) the effect of loline on in vitro prolactin release. However, only domperidone (10(-6) M) was able to reverse (P < 0.05) the effect of ergovaline and only at the lowest ergovaline concentration (10(-8) M). Domperidone was more effective (P < 0.05) in reversing the prolactin-suppressing effect of alpha-ergocryptine than was sulpiride. The dose-response curve for domperidone (cubic fit, P < 0.0001) indicated a threshold concentration (10(-7)M) for reversal of alpha-ergocryptine's (10(-8)M) effect on prolactin release. However, at similar concentration of sulpiride (quadratic fit, P < 0.007), a threshold level was not obtained. These data indicate that ergovaline and loline mayact as D2 dopamine receptor agonists. Additionally, domperidone seems to be a more potent drug for reversal of the alkaloids hypoprolactinenic effect in vitro than does sulpiride.

Reliable capacitation of equine spermatozoa has been a major obstacle in the development of equine in vitro fertilization. Experiments were done to compare in vitro capacitation of equine spermatozoa by use of heparin/caffeine, calcium ionophore, uterine tube epithelial cell (UTEC)-conditioned medium, and direct culturing of spermatozoa with UTEC (coculturing). Capacitation-like changes, as determined by chlortetracycline membrane staining patterns, developed with UTEC-conditioned medium and coculturing, equivalent to that with calcium ionophore. Both of these treatments induced more (P < 0.05) capacitation-like changes than did the control, a modified Tyrode's medium. More (P < 0.05) spermatozoa were viable after 24 hours of UTEC coculturing than in the control incubation.

The ionophore A23187 was used to facilitate release and continued development of Anaplasma marginale in short-term erythrocyte cultures. Addition of 10 micromolar A23187 to the cultures resulted in significant decrease in percentage of parasitized erythrocytes (PPE) by 24 hours after treatment; further development and increase in PPE was not observed. In contrast, the PPE of untreated cultures, those treated with dimethyl sulfoxide (DMSO) only and with 1 micromolar A23187 increased slightly during that time. Total erythrocyte count decreased in treated cultures in excess of that expected after samples of the medium were taken for analysis. The greatest cell loss and increased hemoglobin concentration in culture medium was observed in cultures treated with 10 micromolar A23187 and with an equivalent volume of DMSO. The DMSO appeared to cause hemolysis of some erythrocytes, but not of infected cells selectively. Release of A. marginale inclusion bodies was seen by electron microscopy in samples from the 10 micromolar A23187-exposed cultures. At 30 minutes after treatment, free initial bodies were frequently seen. Inclusion body membranes and individual A. marginale were associated with membranes of adjacent erythrocytes. Individual rickettsiae were seen in cell depressions and appeared to be entering erythrocytes. However, neither further invasion nor development of the parasite in erythrocytes was observed. Ionophore A23187 appeared to promote release of A. marginale from erythrocytes, but did not enhance infection of erythrocytes or development of organisms in vitro.

A total of 302 faecal specimens from animals of various species including poultry, ruminants, mammals, swine, primates, companionanimals, wild animals, and laboratory animals were examined for the presence of Blastocystis sp. These anaerobic parasites which are environmentally resistant were found in 104 specimens (34.44%), that is, from ostriches, pigs, ruminants and nonhuman primates whereas samples from other animals were completely free of the organism. There is a need to assess the impact of these infections on theproductivity of animals and its importance in human infections.

Although tumor necrosis factor (TNF) alpha is an important key factor in degeneration of equine superficial digital flexor tendon (SDFT), the dynamism of TNF receptors and associated factors on tendinocytes has not been elucidated. To reveal signaling events mediated by TNF-receptors (TNF-Rs) in tendinocytes, we focused on four signaling factors, TNF-R1, TNF-R2, TNF-Rassociated factor 2 (TRAF2) and nuclear factor-kappa B (NE-KappaB) , and investigated the distribution and production of these factors. Cultured tendinocytes were obtained from SDFTS of thoroughbred horses. The tendinocytes were treated with 10ng/ ml equine TNFAlpha medium for 6 hours and then the four factors on tendinocytes were visualized by using an immunohistochemical method, and the amounts of the four factors were determined by Western blot analysis. Although TNF-R1 and TNF-R2 co-localized on the same tendinocyte, in untreated control cells (normal condition), immunoreactivity against TNF-R1 was very weak but TNF-R2 showed a strong reaction. However, TNF-R1 showed the same high level of reaction as TNF-R2 in TNFAlpha-treated cells (inflamed condition). Intense TRAF2 and NF-KappaB were detected at inflamed condition, however both factors were also detected at normal condition. The distinct distributions of the four factors under different conditions (normal and inflamed condition) in uitro not only reflect the dynamism of the cytokines but may also provide important clues for a means to prevent from occurrence of tendonitis and progress of tendon degeneration.

Blastocystis is considered to be a zoonoses and it is believed that animals such as chicken constitute large reservoirs for human infection via the faecal-oral route. Therefore, Blastocystis infection was surveyed in free-range chicken and cage reared chicken comprising broiler birds for consumption as well as jungle fowls andsilkie chicken kept for recreation. Fresh faecal samples collected were examined by wet smear preparation and were cultured in Jones medium supplemented with 10% horse serum. Out of 107 chickens, it was found that most of the free-range chicken was positive for Blastocystis sp. with a high prevalence rate of 80%–100% in village chicken, jungle fowl and white silkie chicken. However, the cage-reared chicken, consisting of broiler chicken had no infection. The vacuolar form was the most common Blastocystis cell form found in cultures, similar to B. hominis.These cells were usually spherical and vary greatly in size, ranging from 10 μm to 30 μm in diameter. Owing to the free ranging and scavenging habits, the likelihood of acquiring the infection fromthe environment contaminated with the faecal material of animals with Blastocystis is high in free-range chicken as compared to caged chicken.