Effect of TGF‐β1 Superfamily Members on Survival of Buffalo (Bubalus bubalis) Embryonic Stem‐like Cells

This study examined the effects of supplementation of ES‐like cell culture medium with bone morphogenetic protein (BMP)‐4 (0, 10, 20 or 100 ng/ml) or Noggin (250, 500 or 750 ng/ml) or TGF‐β1 (0, 0.1, 1 or 10 ng/ml) or SB431542 (0, 10, 25 or 50 μm ), an inhibitor of TGF‐β1 signalling, on survival, colony area and expression level of pluripotency genes in buffalo ES‐like cells at passage 40–80, under different culture conditions. BMP‐4 supplementation significantly reduced (p < 0.05) colony survival rate, percentage increase in colony area and relative mRNA abundance of OCT4, whereas that of NANOG and SOX‐2 was increased significantly (p < 0.05). Noggin supplementation did not affect the colony survival rate and percentage increase in colony area in the presence of FGF‐2 and LIF. In the presence of FGF‐2 alone, it significantly reduced (p < 0.05) the relative mRNA abundance of OCT4 and SOX‐2 and increased (p < 0.05) that of NANOG. Supplementation with TGF‐β1 at 1.0 ng/ml but not at other concentrations increased colony survival rate but had no effect on percentage increase in colony area at any concentration. Supplementation with SB‐431542 decreased (p < 0.05) colony survival rate at 50 μm but not at other concentrations. The percentage increase in colony area was lower (p < 0.05) with 10 μm SB‐431542 than that in the controls, whereas at higher concentrations of 25 or 50 μm , SB‐431542 decreased (p < 0.05) the colony size instead of increasing it. In conclusion, these results suggest that BMP‐4 induces differentiation in buffalo ES‐like cells, whereas TGF‐β/activin/nodal pathway may not be playing a crucial role in maintaining pluripotency in these cells.     

Transient hyperglycaemia in a prediabetic dog treated with prednisone and cyclosporin A

A dog with immune-mediated haemolytic anaemia developed transient hyperglycaemia and glucosuria requiring insulin therapy in association with prednisone and cyclosporin A therapy. Following short-term therapy with insulin and cyclosporin A, the dog remained on prednisone therapy but required no further insulin therapy for 12 weeks, at which time the dog became permanently diabetic. We hypothesise that prednisone and cyclosporin A contributed to insulin resistance in a prediabetic dog with suboptimal endogenous insulin concentration and that the degree of insulin resistance decreased when cyclosporin A therapy was discontinued.     

Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation

Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer‐assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.     

Use of protocatechuic acid as the sole antioxidant in the base medium for in vitro culture of ovine isolated secondary follicles

This study evaluated the effect of the protocatechuic acid (PCA) as the sole antioxidant in the base medium for in vitro culture of ovine secondary follicles. Secondary follicles (200‐230 μm) were isolated and cultured in α‐minimal essential medium supplemented with BSA, insulin, glutamine and hypoxanthine (α‐MEM: antioxidant‐free medium) or α‐MEM also added by transferrin, selenium and ascorbic acid (α‐MEM+: with antioxidant) or α‐MEM added by PCA (56.25; 112.5; 225; 450; or 900 μg/ml). Moreover, after culture, oocytes were matured and the chromatin configuration and DNA fragmentation were evaluated. After 12 days, the treatment containing 56.25 μg/ml PCA showed higher percentage of normal follicles than control medium or the other treatments (p < .05), except for 900 μg/ml PCA (p > .05). The antrum formation was significantly higher in treatments containing 56.25, 112.5 or 900 μg/ml PCA, compared to the α‐MEM and similar (p > .05) to the other treatments. The rates of fully grown oocytes (≥110 μm) were similar (p > .05) among all treatments containing PCA and α‐MEM+, and those were superior (p < .05) than α‐MEM, except for 450 μg/ml PCA (p > .05). GSH levels and mitochondrial activity were higher (p < .05) in α‐MEM+ than in α‐MEM and similar (p > .05) to all PCA treatments. The rates of meiotic resumption and DNA fragmentation were similar (p > .05) among α‐MEM+ and 56.25 μg/ml PCA. In conclusion, PCA at 56.25 μg/ml as the sole antioxidant added to the medium for ovine isolated secondary follicle culture maintains follicular survival, GSH and active mitochondria levels, meiotic developmental competence and DNA integrity of cultured oocytes.     

Prevalence of proliferative enteritis on pig farms in Australia

SUMMARY Three surveys, undertaken to assess the prevalence of proliferative enteritis (PE) on pig farms in Australia and to investigate risk factors associated with clinical disease, indicated that PE was a common disease in pig farms. Forty of the 71 (56%) randomly-selected producers had either observed PE or had a veterinarian diagnose the disease in their herd during 1988 to 1990. A relatively low prevalence of the disease was recorded at veterinary diagnostic laboratories, and this suggested that diagnoses of PE were often not confirmed by histopathological examination of the intestines of affected pigs. Non-haemorrhagic PE occurred most often in six- to 24-week-old pigs, but was also reported in 52-week-old pigs. Proliferative haemorrhagic enteropathy usually affected pigs over 16 weeks of age, but was also reported in pigs as young as six weeks and as old as four years of age. A survey of pig-specialist veterinarians indicated that most veterinarians diagnosed PE based on clinical and gross pathological examination of affected pigs, without laboratory confirmation. There were difficulties associated with measuring the prevalence of PE among herds, including the effectiveness of antibacterials for its prevention and control, its subclinical nature and probable mis-diagnoses. This study highlighted the need for an ante-mortem diagnostic test to measure the prevalence of PE more accurately.     

m‐carboxycinnamic acid bishydroxamide improves developmental competence,reduces apoptosis and alters epigenetic status and gene expression pattern in cloned buffalo (Bubalus bubalis) embryos

Incomplete or aberrant reprogramming of nuclear genome is one of the major problems in somatic cell nuclear transfer. In this study, we studied the effect of histone deacetylase inhibitor m‐carboxycinnamic acid bishydroxamide (CBHA) on in vitro development of buffalo embryos produced by Hand‐made cloning. Cloned embryos were treated with CBHA (0, 5, 10, 20 or 50 μM) for 10 hr from the start of reconstruction till activation. At 10 μM, but not at other concentrations examined, CBHA increased (p < .05) the blastocyst rate (63.77 ± 3.97% vs 48.63 ± 3.55%) and reduced (p < .05) the apoptotic index of the cloned blastocysts (8.91 ± 1.94 vs 4.36 ± 1.08) compared to untreated controls, to levels similar to those in IVF blastocysts (4.78 ± 0.74). CBHA treatment, at all the concentrations examined, increased (p < .05) the global level of H3K9ac in cloned blastocysts than in untreated controls to that observed in IVF blastocysts. Treatment with CBHA (10 μM) decreased (p < .05) the global level of H3K27me3 in cloned blastocysts than in untreated controls but it was still higher (p < .05) than in IVF blastocysts. CBHA (10 μM) treatment increased (p < .05) the relative expression level of pluripotency‐related genes OCT‐4 and NANOG, and anti‐apoptotic gene BCL‐XL, and decreased (p < .05) that of pro‐apoptotic gene BAX than in untreated controls but did not affect the relative expression level of apoptosis‐related genes p53 and CASPASE3 and epigenetics‐related genes DNMT1, DNMT3a and HDAC1. These results suggest that treatment of cloned embryos with 10 μM CBHA improves the blastocyst rate, reduces the level of apoptosis and alters the epigenetic status and gene expression pattern.