Detection and localization of regucalcin in spermatozoa of water buffalo (Bubalus bubalis): A calcium‐regulating multifunctional protein

Regucalcin (RGN) is a calcium‐regulating, anti‐apoptotic, antioxidative and antiproliferative multifunctional protein predominantly seen in liver and kidney. All these functions are very crucial during spermatogenesis and sperm maturation process until fertilization of the ovum. Although many studies have reported the wide distribution of regucalcin in the male reproductive tract of the rat, human and bovine, its presence in spermatozoa is yet to be demonstrated wherein calcium has a pivotal role in the transport, capacitation, acrosomal reaction and further fusion with ova. Here, we detected the expression of regucalcin mRNA and protein in buffalo spermatozoa using real‐time PCR and Western blot, respectively. The study detected two new regucalcin isoforms of 44 kDa and 48 kDa size along with the reported 34‐kDa, 28‐kDa and 24‐kDa isoforms, wherein the 34‐kDa isoform was found to be membrane associated in spermatozoa. Further, immunocytochemistry study localized the regucalcin protein in the acrosomal region of the caudal and ejaculated buffalo spermatozoa while it was detected in both cytoplasm and acrosomal region of testicular spermatozoa. This discovery of RGN in spermatozoa and localization in the acrosomal region will help to focus researchers to see its role in calcium‐related functions like capacitation, acrosomal reaction and membrane fusion. Overall, regucalcin may be a new fertility marker in buffalo and can be utilized for infertility treatments.     

Identification and changes in the seasonal concentrations of heat shock proteins in roe deer (Capreolus capreolus) epididymides

Heat shock proteins (HSPs) act as molecular chaperones with important regulatory functions. HSPs are considered to be essential factors in animal reproduction. In view of seasonal variations in the secretory activity of the reproductive tract of mature roe deer (Capreolus capreolus), the aims of this study were to identify HSPs in the epididymides and compare the expression of the identified proteins in three periods of the reproductive season. Two‐dimensional polyacrylamide gel electrophoresis revealed the highest number of polypeptides in homogenates of epididymal tissues and in caput, corpus and cauda epididymal fluids throughout the reproductive season. Epididymal tissue homogenates and epididymal fluids were analysed by tandem mass spectrometry (MS/MS) to reveal 31 polypeptides with enzymatic activity, including polypeptides with antioxidant properties, structural and cell signalling functions. Moreover, among the identified polypeptides, five of them were similar to heat shock proteins: endoplasmin (Grp94); heat shock protein 90 kDa (HSP90); 78‐kDa glucose‐regulated protein (Grp78); chain A, the crystal structure of the human HSP70 ATPase domain and heat shock protein beta‐1 isoform X. The concentrations of the analysed polypeptides, expressed in optical density units (ODU), differed significantly (p ≤ .05) across the examined periods of the reproductive season. The highest ODU values for almost all analysed proteins were observed during the rutting period. The presence of HSPs in the epididymal tissues and fluids of roe deer in different periods of the reproductive season could indicate that those proteins play an important role in sperm maturation in the epididymis.     

Toxicity evaluation of copper oxide bulk and nanoparticles in Nile tilapia, <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>, using hematological,bioaccumulation and histological biomarkers

The increased industrial applications of nanoparticles (NPs) augment the possibility of their deposition into aquatic ecosystems and threatening the aquatic life. So, this study aimed to provide a comparable toxicological effects of nano-CuO and bulk CuO on a common freshwater fish, Oreochromis niloticus. Fish were exposed to two selected doses (1/10 and 1/20 of the LC₅₀/96 h) of both nano-/bulk CuO for 30 days. Based on the studied hematological parameters (RBCs count, hemoglobin content and hematocrit%), the two selected concentrations of CuO in their nano- and bulk sizes were found to induce significant decrease in all studied parameters. But, nano-CuO-treated fish showed the maximum decrease in all recorded parameters among the all studied groups especially at the low concentration of 1/20 LC₅₀/96 h. Hematological status was also confirmed using the calculated blood indices (MCV, MHC and MCHC). In case of bulk CuO-treated groups, the significant decrease in the studied hematological parameters was not followed by any change in MCV and MCH (normocytic anemia), while fish that exposed to NPs showed a significant increase in all calculated blood parameters reflecting erythrocytes swelling which is related to the intracellular osmotic disorders (macrocytic anemia). Regarding metal bioaccumulation factor, the results showed that CuO NPs had more efficiency to internalize fish tissues (liver, kidneys, gills, skin and muscle). The accumulation pattern of Cu metal was ensured by histopathological investigation of liver, kidneys and gills. The histopathological analysis revealed various alterations that varied between adaptation responses and permanent tissue damage.     

Effect of exogenous circulating anti-bPL antibodies on bovine placental lactogen measurements in foetal samples

Background

The involvement of placental lactogen (PL) in the regulation of foetal growth has been investigated in different species by in vivo immunomodulation techniques. However, when circulating antibodies are present together with the hormone, the procedure for hormonal measurement becomes considerably complex. The aim of this study was the immunoneutralization of bovine placental lactogen (bPL) concentrations in bovine foetal circulation by direct infusion of rabbit anti-bPL purified immunoglobulins (IgG) via a foetal catheter (in vivo study). The ability of a RIA based on guinea pig anti-bPL antiserum, for the measurement of bPL concentrations in samples containing exogenous rabbit anti-bPL immunoglobulins, was also analyzed in in vitro and in vivo conditions.

Methods

Six bovine foetuses were chronic cannulated on the aorta via the medial tarsal artery. Infusion of rabbit anti-bPL IgG was performed during late gestation. Pooled rabbit anti-bPL antisera had a maximal neutralization capacity of 25 μg bPL/mL of immunoglobulin. Interference of rabbit anti-bPL immunoglobulin with radioimmunoassay measurement using guinea pig anti-bPL as primary antibody was first evaluated in vitro. Polyclonal anti-bPL antibodies raised in rabbit were added in foetal sera to produce 100 samples with known antibodies titers (dilutions ranging from 1:2,500 till 1:1,280,000).

Result(s)

Assessment of the interference of rabbit anti-bPL antibody showed that bPL concentrations were significantly lower (P < 0.05) in samples added with dilutions of rabbit antiserum lower than 1:80,000 (one foetus) or 1:10,000 (four foetuses). It was also shown that the recovery of added bPL (12 ng/mL) was markedly reduced in those samples in which exogenous rabbit anti-bPL were added at dilutions lower than 1:20,000. Concentrations of foetal bPL were determined in samples from cannulated foetuses. In foetuses 1 and 6, bPL concentrations remained almost unchanged (<5 ng/mL) during the whole experimental period. In Foetus 3, bPL concentrations decreased immediately after IgG infusion and thereafter, they increased until parturition.

Conclusion(s)

The use of a bPL RIA using a guinea pig anti-bPL as primary antiserum allowed for the measurement of bPL concentrations in foetal plasma in presence of rabbit anti-bPL IgG into the foetal circulation. Long-term foetal catheterization allowed for the study of the influence of direct infusion of anti-bPL IgG on peripheral bPL concentrations in bovine foetuses.     

Bacterial septic arthritis in 19 dogs

OBJECTIVE: To provide information on the clinical features, diagnosis and treatment of bacterial septic arthritis in dogs. DESIGN: A retrospective study examining case records of all dogs diagnosed with bacterial septic arthritis at Murdoch University Veterinary Hospital between 1988 and 1997. RESULTS: Nineteen dogs were diagnosed with bacterial septic arthritis, which most commonly occurred after surgery involving the stifle joint. Haematogenous infection occurred in only five dogs. Diagnosis was based on clinical signs, joint fluid analysis, radiography, microbiology and/or response to treatment. Chronic lameness was the most common problem at presentation. Analysis of joint fluid invariably revealed large number of nucleated cells, which consisted primarily of neutrophils. In all but one case the neutrophils were nondegenerate. Culture of joint fluid was frequently successful. Staphylococcus spp were the most common bacteria isolated. Treatment involved antimicrobial drugs only in five dogs. Other dogs received antimicrobial drugs in combination with surgical procedures such as joint lavage and removal of nonabsorbable suture material (eight), arthrodesis (two) or amputation (one). Two dogs were euthanased. Most dogs responded well to treatment and were free of signs of septic arthritis at follow-up. CONCLUSION: Bacterial septic arthritis may often be mild and manifest as chronic lameness. Analysis of joint fluid will detect an inflammatory arthropathy but the presence of toxic neutrophils should not be relied on as an indicator of sepsis. Culture of infected joint fluid is likely to be successful if antimicrobials are not given prior to collection and if the sample is inoculated into enrichment broth. Treatment should involve antimicrobial drugs, open-joint lavage and removal of joint prostheses if the infection is associated with previous surgery.     

Disc extrusion in a Rottweiler dog with caudal cervical spondylomyelopathy after failure of intervertebral distraction/stabilisation

A Rottweiler dog was presented with an 8 week history of hindlimb ataxia. Neurological examination localised the lesion to the cervical spinal cord. Myelography demonstrated dynamic compressive lesions at C5-6 and C6-7 consistent with a diagnosis of caudal cervical spondylomyelopathy. Distraction/stabilisation of both discs was performed using interbody polymethyl methacrylate. Both implants subsequently failed leading to extrusion of the remaining dorsal annulus fibrosus of the C5-6 intervertebral disc and nonambulatory tetraparesis. A ventral slot combined with distraction/stabilisation using screws and polymethyl methacrylate was performed and resulted in nearly full neurological recovery.     

A laser ablation method for the synthesis of crystalline semiconductor nanowires

A method combining laser ablation cluster formation and vapor-liquid-solid (VLS) growth was developed for the synthesis of semiconductor nanowires. In this process, laser ablation was used to prepare nanometer-diameter catalyst clusters that define the size of wires produced by VLS growth. This approach was used to prepare bulk quantities of uniform single-crystal silicon and germanium nanowires with diameters of 6 to 20 and 3 to 9 nanometers, respectively, and lengths ranging from 1 to 30 micrometers. Studies carried out with different conditions and catalyst materials confirmed the central details of the growth mechanism and suggest that well-established phase diagrams can be used to predict rationally catalyst materials and growth conditions for the preparation of nanowires.     

The lichens of the Scottish native pinewoods

During the last 30 years, 434 epiphytic lichen taxa have beenrecorded from the native pinewoods of Scotland. Of these lichens,about 219 have been recorded from the bark, lignum or stumpsof pine (Pinus sylvestris) itself. Eighteen species are, inthe British Isles, confined to these native pinewoods, whichare themselves confined to the Scottish Highlands. A resumeis presented of the habitats and niches available to lichensin the native pinewoods, using examples of the most characteristicspecies for each. The ‘pinewood habitat’ for epiphyticlichens is by no means restricted to pine, but also includesassociated trees, shrubs and subshrubs, especially birch, rowan,holly, aspen, juniper and heather. The strong east–westvariation in the composition of the lichen flora is outlined.By comparison of species lists from pinewood stands of knownancientness with those from mature plantations distant fromthe ‘core’ pinewoods, an additional c. 60 speciescan be considered as indicative of an ecological continuityof the pinewood habitat. From this pool of c. 77 species, aNative Pinewood Index of Ecological Continuity is presentedas a useful tool for biodiversity assessments of pinewoods.     

Managing apple scab (Venturia inaequalis) and powdery mildew (Podosphaera leucotricha) using Adem™

Current control of scab and mildew of apple in the UK requires the routine application of fungicides at 7 - 14-day intervals to achieve the blemish-free fruit required by the market. Such practices are generally effective, but with increased public concern about pesticides and rising costs to the grower, they are now less acceptable. The use of disease-warning systems offers scope for optimizing fungicide use by better timing of sprays. Adem^? is a PC-based system that warns of the risk of scab, mildew, Nectria fruit rot and canker and fireblight. In a 'key stage' strategy, warnings by Adem^? for scab and mildew integrated with practical pest and disease control resulted in similar or better control than a routine programme, with the additional benefit of reduced fungicide inputs and costs even in seasons exceptionally favourable for these diseases. Maximum savings in fungicide use were made by applying sprays curatively in response to scab warnings. However, this approach resulted in increased disease incidence to the crop and scab infection of fruit even in seasons unfavourable for scab attack.     

Comparison of bone healing,as assessed by computed tomography,following tibial tuberosity advancement in dogs with and without autogenous cancellous bone grafts

Abstract

AIMS: To objectively compare measures of bone healing, using computed tomography (CT) in dogs following bilateral tibial tuberosity advancement (TTA), between tibiae treated with and without autogenous cancellous bone grafts.

METHODS: Ten dogs with bilateral cranial cruciate ligament disease requiring surgical stabilisation were prospectively recruited to undergo single-session bilateral TTA, with only one, randomly assigned, tibia receiving bone graft in the osteotomy deficit. Bone healing at the osteotomy site was assessed using CT performed 38–70 days post-operatively. CT images were evaluated using both objective measurements of osseous bridging and subjective evaluation by six radiologists. Repeated measures ANOVA was used to compare the objective outcomes between the grafted and non-grafted tibiae.

RESULTS: The mean percentage of the osteotomy deficit bridged at the lateral cortex was greater in grafted (77.6, SD 35.2%) compared to non-grafted (63.0, SD 36.5%) tibiae (p=0.001), but did not differ at the medial cortex (p=0.1). The mean minimum callus width was greater in grafted (7.2, SD 3.3 mm) compared to non-grafted (3.6, SD 2.9 mm) tibiae (p<0.001). There was no difference in mean attenuation (measured in Hounsfield units) of the callus between grafted and non-grafted tibiae (p=0.5). The grafted tibia was deemed to have superior bone healing in 50/60 subjective assessments made by radiologists.

CONCLUSIONS: Superior osseous bridging was detected by CT analysis following TTA using autogenous cancellous bone grafts compared with no graft. This was shown by greater bridging percentage at the lateral cortex and formation of a broader callus. Qualitative assessments made by six radiologists also supported the conclusion that bone healing was improved by use of autogenous cancellous bone graft. CT was a useful method for assessing evidence of bone healing following TTA.

CLINICAL RELEVANCE: These findings justify the application of autogenous cancellous bone graft to augment healing following TTA in dogs.