Identification of Apoptotic Bodies in Equine Semen

Apoptosis in the testis is required to ensure an efficient spermatogenesis. However, sometimes, defective germ cells that are marked for elimination during this process escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa and a high percentage of apoptotic bodies. Apoptosis markers in the ejaculate have been associated with low fertility, either in animals or humans. Therefore, the goal of this study was to investigate whether fresh equine semen contains apoptotic bodies [initially named Merocyanine 540 (M540) bodies] and to study the relationship between the quantity of these bodies and cell concentration, the volume of ejaculate, viability and motility. Moreover, we also studied whether the presence apoptotic bodies in fresh semen was related to the resistance of the stallion spermatozoa to being incubated at 37°C or being frozen and thawed. Fresh equine semen was stained with fluorescent dyes such as M540 and Annexin‐V. Active Caspase 3 was studied in fresh semen through Western blotting and immunofluorescence with a specific antibody. Sperm kinematics was assessed in fresh, incubated and thawed samples using computer‐assisted semen analysis, and viability was evaluated with the LIVE/DEAD Sperm Viability Kit. Overall, our results demonstrate for the first time the presence of apoptotic bodies in equine semen. The quantity of apoptotic bodies was highly variable among stallions and was positively correlated with Caspase 3 activity in fresh samples and negatively correlated with the viability and motility of stallion spermatozoa after the cryopreservation process.     

Effect of Overnight Staining on the Quality of Flow Cytometric Sorted Stallion Sperm: Comparison with Tradtitional Protocols

Flow cytometry is considered the only reliable method for the separation of X and Y chromosome bearing spermatozoa in equines. The MoFlo SX DP sorter is highly efficient, allowing the production of foals of the desired sex. However, to achieve acceptable pregnancy rates the currently used protocol requires working with fresh semen obtained close to, or at, the sorting facility. An alternative protocol was tested during two consecutive breeding seasons. Fresh stallion semen was cooled for 20 h, during which staining with Hoechst 33342 took place. On the following day, this sample was flow sorted and compared with spermatozoa from the same ejaculate that had been sexed on the previous day. All sperm parameters evaluated remained unchanged when fresh sorted and refrigerated sorted semen were compared. Pre‐sorting storage at 5°C did not alter sperm velocities nor kinetics, viability or membrane permeability, production of reactive oxygen species, mitochondrial membrane potential or DNA fragmentation index of the sorted sample. The findings open for the possibility of using semen from stallions housed far from the sorting facilities. Processed and stained sperm could be shipped refrigerated on the previous day, sorted and inseminated on the next day.     

The Effect of Glycerol Concentrations on the Post‐thaw In Vitro Characteristics of Cryopreserved Sex‐sorted Boar Spermatozoa

The objective of this study was to optimize protocols for the cryopreservation of sex‐sorted boar spermatozoa. In the experiment 1, we evaluated the effects of a standard boar sperm cryopreservation procedure (3% final glycerol concentration) on the in vitro characteristics of sex‐sorted sperm frozen at low sperm concentrations (20 × 10⁶ sperm/ml; S20 group). Non‐sorted spermatozoa frozen at 1000 × 10⁶ (C1000 group) and 20 × 10⁶ (C20 group) sperm/ml were used as the freezing control groups. In experiment 2, the effects of different final glycerol concentrations (0.16%, 0.5%, 1.0%, 2.0% and 3.0%) on post‐thaw quality of the S20 and C20 groups were evaluated. In both experiments, the samples were evaluated prior to freezing (5°C) and at 30, 90 and 150 min after thawing. Experiment 1 indicated that freezing sperm at low concentrations decreased (p < 0.05) the total motility (TM) and progressive motility (PM) at 90 and 150 min after thawing regardless of whether the sperm were sorted or not. However, the sperm membrane integrity was not affected at any evaluation step. Inexperiment 2, significant effects on the TM and PM because of increased glycerol concentrations in the S20 and C20 groups were observed only at 90 and 150 min after thawing. The samples frozen in 3% glycerol showed lower (p < 0.05) TM and PM values when compared to those frozen in the presence of 0.5% and 1% glycerol. In both experiments, non‐sorted control samples displayed higher percentages of spermatozoa with damaged DNA than sorted spermatozoa. In conclusion, the optimization of cryopreservation conditions by decreasing the glycerol concentrations can improve post‐thaw motility of sex‐sorted spermatozoa frozen at low concentrations.     

Salmonella Enteritidis: the egg and I

SUMMARY: The world-wide clinical incidence of Salmonella Enteritidis has increased markedly. The increase is associated with the enhanced ability of the bacterium to systemically colonise layer chickens. Subsequent contamination and consumption of intact shell eggs from colonised layer hens, either directly or in foods containing raw or lightly cooked eggs, causes human disease. Despite investigation, no change in the biology of the bacterium has been correlated with increased colonisation in chickens. To date, no method of control at the production level has proven effective; consumer education is the best means of minimising the public health risk.     

Hormonal response to dobutamine cardiac stress testing in a conscious canine model of early left ventricular dysfunction induced by chronic rate overload

The aims of this study were to investigate (1) hormonal activation before and during dobutamine cardiac stress testing (DST) in a canine model of early left ventricular dysfunction (ELVD) induced by rapid right ventricular pacing (RRVP) and (2) the relationship between this hormonal profile and carnitine concentrations.Before the pacing period, the 6 dogs were assigned to 2 groups according their baseline total plasma carnitine concentration. A DST was performed on each dog before activation of the pacemaker and every 3 to 4 days during development of 3 progressive stages of ELVD (stages 1, 2 and 3). Plasma atrial natriuretic factor (ANF), angiotensin II (ANG II) and endothelin-1 (ET-1) levels were measured at the start and at the end of each DST. Effects of ELVD, DST and plasma carnitine concentration on these measurements were tested.The RRVP induced a significant increase of ANF and ANG II and a non significant trend toward increase of ET-1 in all dogs.Before the pacing period, ANF remained constant during the DST in dogs with normal total plasma carnitine concentration, while it significantly decreased in dogs with low total plasma carnitine concentration. Dobutamine stress testing induced a significant decrease in ANF in all dogs in ELVD. Dobutamine infusion induced a significant increase in ANG II in all dogs before as well as during the pacing period while ET-1 was unchanged.These results suggest that investigation of the hormonal profile before and after a dobutamine challenge might provide important diagnostic information in dogs with asymptomatic or mildly symptomatic cardiac dysfunction of different origins.     

Effect of Semen Extender and Density Gradient Centrifugation on the Motility and Fertility of Turkey Spermatozoa

In the absence of commercially viable methods for cryopreserving turkey spermatozoa, new processing methods are required to extend the functional life of stored turkey spermatozoa for artificial insemination. The present study evaluates the efficacy of a new extender (Turkey Semen Extend) and investigates the use of density gradient centrifugation in processing turkey spermatozoa for artificial insemination. The new extender is compared with two commercially available turkey semen extenders, Beltsville Poultry Semen Extender and Ovodyl. Turkey spermatozoa in Turkey Semen Extend were still motile 20 h after collection, representing a considerable improvement over the other semen extenders (40%, 0% and 8% for Turkey Semen Extend, Beltsville Poultry Semen Extender and Ovodyl, respectively). A field trial on a commercial turkey farm showed improved fertilization rates following insemination of turkey hens with semen extended in Turkey Semen Extend (89.7%) compared with Beltsville Poultry Semen Extender (86.9%). This difference is statistically significant (p < 0.05). Processing on a density gradient, optimized for turkey spermatozoa, also increased sperm survival (50% gradient-prepared spermatozoa still motile after 18 h compared with <10% non-processed spermatozoa). Preliminary studies indicate that gradient preparation of spermatozoa may aid survival during cryopreservation.     

Diagnosing feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) infection: an update for clinicians

With the commercial release in Australia in 2004 of a vaccine against feline immunodeficiency virus (FIV; Fel‐O‐Vax FIV®), the landscape for FIV diagnostics shifted substantially. Point‐of‐care (PoC) antibody detection kits, which had been the mainstay for diagnosing FIV infection since the early 1990s, were no longer considered accurate to use in FIV‐vaccinated cats, because of the production of vaccine‐induced antibodies that were considered indistinguishable from those produced in natural FIV infections. Consequently, attention shifted to alternative diagnostic methods such as nucleic acid detection. However, over the past 5 years we have published a series of studies emphasising that FIV PoC test kits vary in their methodology, resulting in differing accuracy in FIV‐vaccinated cats. Importantly, we demonstrated that two commercially available FIV antibody test kits (Witness? and Anigen Rapid?) were able to accurately distinguish between FIV‐vaccinated and FIV‐infected cats, concluding that testing with either kit offers an alternative to PCR testing. This review summarises pertinent findings from our work published in a variety of peer‐reviewed research journals to inform veterinarians (particularly veterinarians in Australia, New Zealand and Japan, where the FIV vaccine is currently commercially available) about how the approach to the diagnosis of FIV infection has shifted. Included in this review is our work investigating the performance of three commercially available FIV PoC test kits in FIV‐vaccinated cats and our recommendations for the diagnosis of FIV infection; the effect of primary FIV vaccination (three FIV vaccines, 4 weeks apart) on PoC test kit performance; our recommendations regarding annual testing of FIV‐vaccinated cats to detect ‘vaccine breakthroughs’; and the potential off‐label use of saliva for the diagnosis of FIV infection using some FIV PoC test kits. We also investigated the accuracy of the same three brands of test kits for feline leukaemia virus (FeLV) diagnosis, using both blood and saliva as diagnostic specimens. Based on these results, we discuss our recommendations for confirmatory testing when veterinarians are presented with a positive FeLV PoC test kit result. Finally, we conclude with our results from the largest and most recent FIV and FeLV seroprevalence study conducted in Australia to date.     

Modulation of diversity by grazing and mowing in native tallgrass prairie

Species diversity has declined in ecosystems worldwide as a result of habitat fragmentation, eutrophication, and land-use change. If such decline is to be halted ecological mechanisms that restore or maintain biodiversity are needed. Two long-term field experiments were performed in native grassland to assess the effects of fire, nitrogen addition, and grazing or mowing on plant species diversity. In one experiment, richness declined on burned and fertilized treatments, whereas mowing maintained diversity under these conditions. In the second experiment, loss of species diversity due to frequent burning was reversed by bison, a keystone herbivore in North American grasslands. Thus, mowing or the reestablishment of grazing in anthropogenically stressed grasslands enhanced biodiversity.     

Testosterone serum profile,semen characteristics and testicular biometry of Mangalarga Marchador stallions in a tropical environment

This study was conducted to characterize the daily profile of testosterone secretion and its mean concentrations in the four seasons as well as to evaluate the semen characteristics and testicular biometry of Mangalarga Marchador stallions throughout the year in a tropical region. Three stallions were submitted to semen collections and evaluation of testicular biometry every 14 days along a year. Blood samples were collected once at the middle of each season, in a 20‐min interval during 24 hr in order to evaluate the testosterone secretion profiles among seasons. Testosterone concentrations along the day were higher at the beginning of the afternoon (from 12:00 to 15:00 hr), but a circadian secretion was not clearly observed. Mean testosterone concentrations did not differ among seasons (p > .05), but a pattern of secretion along the day showed variations with higher concentrations in the afternoon during the winter. Ejaculate volume was higher during summer; however, sperm motility decreased in summer and spring. Total sperm in ejaculate, sperm morphology and testicular biometry kept constant along the year showing no differences among the seasons. The results demonstrated that in a tropical region, reproductive aspects of stallions did not show a clearly defined seasonal variation, and months of autumn and winter were not unsuitable for reproduction of the males.