Motility and Plasma Membrane Integrity of Spermatozoa in Fractionated Stallion Ejaculates after Storage

With the aim of investigating properties of stallion seminal plasma to eventually improve semen-handling techniques, sperm motility and plasma membrane integrity were analysed in different fractions of the ejaculates after storage. Semen was collected using a computer-controlled automated phantom that separates the ejaculates into five successive cups. Samples containing seminal plasma and skim milk extender were compared with samples stored in skim milk extender after the removal of seminal plasma by centrifugation. Fractionated ejaculates were stored cooled for 24 h after dilution with extender (Expt 1) or frozen in liquid nitrogen (Expt 2). In Expt 1, cup 1 was pre-sperm fluid, cups 2 and 3 sperm-rich fractions, and cup 4 sperm-poor fractions. In Expt 2, cups 1 and 2 were sperm-rich fractions, and cups 3 and 4 sperm-poor fractions. One sample (WE) represented the whole ejaculate in both experiments. Motility parameters were determined with a Hamilton-Thorn Motility Analyzer, and plasma membrane integrity was assessed using carboxyfluorescein diacetate and propidium iodide staining and fluorescence microscopy. The removal of seminal plasma lowered motility values, but not plasma membrane integrity, in both experiments. No significant differences between cups were observed after cooled storage. The cups differed significantly in most post-thaw motility parameters, and the sperm-rich fraction showed higher post-thaw motility than the whole ejaculate.     

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.     

Bovine thyroglobulin gene polymorphisms and their association with sexual precocity in Guzerat bulls

Puberty is a stage of sexual development determined by the interaction of environmental factors and genetic mechanisms. Among them, thyroid function plays a key role in sexual development and spermatogenic function and is under the control of several genes, including the well‐described thyroglobulin gene (TG). Previous reports have shown genetic association between thyroid function and selected single nucleotide polymorphisms (SNPs) in taurine cattle. Therefore, the identification of genetic mechanisms involved in the regulation of this trait can assist with the selection for early pubertal bulls, thus improving genetic progress in livestock breeding. The aim of this study was to validate the association between TG SNPs and age at puberty in zebuine bulls. Three SNPs (rs110406764, rs109662686, rs109057985) were genotyped in 159 Guzerat animals using SEQUENOM technology. Results showed a significant association (p < .05) between the studied SNPs and puberty age, in agreement with our previous reports in a taurine breed. Interestingly, allele frequencies were different from those already reported, being GAT the most favourable allele for age at puberty in Guzerat (94.4 days lower). Overall, our findings corroborate previous reports and reinforce the importance of genetic influence in the regulation of sexual development and puberty through a thyroid pathway in zebuine cattle.     

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.     

Carbon dioxide and energy flux partitioning between the understorey and the overstorey of a maritime pine forest during a year with reduced soil water availability

Carbon dioxide, water vapour and energy fluxes were measured above and within a maritime pine forest during an atypical year with long-lasting reduced soil water availability. Energy balance closure was adequately good at both levels. As compared with what is usually observed at this site the ecosystem dissipated less energy via latent heat flux and more via sensible heat flux. The understorey canopy was responsible for a variable, significant component of the whole canopy fluxes of water vapour and carbon dioxide. The annual contribution of the understorey was 38% (154 mm) of the overall evaporation (399 mm) and 32% (89 mm) of the overall sensible heat flux (274 mm). The participation of the understorey reached 45% of the overall evaporation and 30% of the daytime overall assimilation during significant soil water deficit periods in summertime. Even during winter, understorey photosynthesis was consistent as it compensated soil and understorey respiration. The ecosystem behaved as a sink of carbon, with a negative annual carbon budget (−57 g C m⁻²). However, due to high soil water deficit, the annual ecosystem GPP was 40% less than usually observed at this site. This budget resulted from a sink of −131 g C m⁻² for the overstorey and a source of +74 g C m⁻² for the understorey. Moreover, on an annual basis the overstorey layer contributed to almost two-thirds of the ecosystem respiration. Finally, the effect of long-lasting soil water deficit on the maritime pine forest was found more important than the effect of the heat wave and drought of summer 2003.     

Evaluation of zebrafish (Danio rerio) as an animal model for the viral infections of fish

Zebrafish (Danio rerio) is a laboratory model organism used in different areas of biological research including studies of immune response and host–pathogen interactions. Thanks to many biological tools available, zebrafish becomes also an important model in aquaculture research since several fish viral infection models have been developed for zebrafish. Here, we have evaluated the possible use of zebrafish to study infections with fish viruses that have not yet been tested on this model organism. In vitro studies demonstrated that chum salmon reovirus (CSV; aquareovirus A) and two alloherpesviruses cyprinid herpesvirus 1 (CyHV‐1) and cyprinid herpesvirus 3 (CyHV‐3) are able to replicate in zebrafish cell lines ZF4 and SJD.1. Moreover, CSV induced a clear cytopathic effect and up‐regulated the expression of antiviral genes vig‐1 and mxa in both cell lines. In vivo studies demonstrated that both CSV and CyHV‐3 induce up‐regulation of vig‐1 and mxa expression in kidney and spleen of adult zebrafish after infection by i.p. injection but not in larvae after infection by immersion. CyHV‐3 is eliminated quickly from fish; therefore, virus clearing process could be evaluated, and in CSV‐infected fish, a prolonged confrontation of the host with the pathogen could be studied.     

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.