Hans Laevens

Prevalence of apoptosis and inner cell allocation in bovine embryos cultured under different oxygen tensions with or without cysteine addition

Supraphysiological oxygen tension during embryo culture can generate reactive oxygen species (ROS), which can induce apoptosis. Antioxidants such as thiol compounds (cysteine, cysteamine) can be used to prevent ROS damage to the embryo. The purpose of this study was to evaluate the prevalence of apoptosis during bovine embryo development and to evaluate the effect of the presence or absence of cysteine 0.6 mM in modified synthetic oviduct fluid (mSOF) on in vitro produced cattle embryos cultured under two different oxygen tensions (5% O2 versus 20% O2). Effects were assessed by checking embryo development at Days 7, 8 and 9 and by evaluating Day 9 hatched blastocysts for differentiation by means of differential staining and for apoptosis by means of TUNEL-assay. Apoptotic cells were present in 94% of Day 7 blastocysts and in 100% of Days 8 and 9 blastocysts. Cysteine addition affected Day 8 blastocyst rates in a negative way (P < 0.05) regardless of the oxygen tension. In fact, cysteine addition to the mSOF culture medium had a negative effect upon embryo development in terms of blastocyst rates, hatching rates and apoptotic cell ratio. Embryos cultured under 5% O2 in the presence of cysteine, however, possessed significantly higher numbers of ICM cells. This finding corroborates the theoretical assumption that antioxidants are beneficial for ICM development.

Prepartum teat apex colonization with Staphylococcus chromogenes in dairy heifers is associated with low somatic cell count in early lactation

A high number of dairy heifers freshen with udder health problems. The prevalence of teat apex colonization (TAC) with Staphylococcus chromogenes, one of the most widespread coagulase-negative staphylococci (CNS) in milk samples from freshly calved dairy heifers, was measured cross-sectionally in non-lactating heifers on eight commercial dairy farms in Belgium. The influence of age on this prevalence, and the association between teat apex colonization with S. chromogenes prepartum and quarter milk somatic cell count (SCC) in early lactation were studied. In total, 492 teat apices were sampled from 123 heifers. The age of the heifers varied from 8 to 34 months. Overall, 20% of the heifers had at least one teat apex colonized with S. chromogenes. Of all teats sampled, 10% were colonized with S. chromogenes. The chance of having at least one teat apex colonized with S. chromogenes increased with age of the heifer. The presence of prepartum teat apex colonization with S. chromogenes was not associated with intramammary infection (IMI) early postpartum with the same bacterium. On the contrary, teat apex colonization with S. chromogenes prepartum appeared to protect quarters in the first few days of lactation from having somatic cell count >or=200000cells/ml milk, commonly accepted as the threshold for intramammary infection.

Identification of aesculin-hydrolyzing streptococci, lactococci, aerococci and enterococci from subclinical intramammary infections in dairy cows

Aesculin-hydrolyzing, catalase-negative, gram-positive cocci isolated from subclinical intramammary infections in dairy cows were identified to species level using growth characteristics and biochemical activity. The results indicated that the aesculin-hydrolyzing cocci associated with this type of infection are a very heterogenic group. S. uberis strains, including inulin- or beta-glucuronidase-negative isolates, accounted for only about one-third of the collection, and Enterococcus faecalis strains for one-fifth. Other species of some importance included (in descending order of isolation frequency) Aerococcus viridans, Streptococcus pluranimalium, Lactococcus garvieae, Streptococcus bovis and Streptococcus gallolyticus.

Experimental infection of slaughter pigs with classical swine fever virus: transmission of the virus, course of the disease and antibody response.

The spread of classical swine fever virus was investigated in an isolation unit containing four pens, each containing six slaughter pigs. One pig in the middle pen of three adjacent pens was inoculated intramuscularly and intranasally with the virus. The fourth pen was located in a separate compartment. containing six slaughter pigs. One pig in the middle pen of three adjacent pens was inoculated intramuscularly and intranasally with the virus. The fourth pen was located in a separate compartment. The pens were visited in a strict order to study, first, the effect of indirect contact via contaminated clothing and footwear on the spread of the virus to adjacent pens and, secondly, the airborne transmission of the virus between compartments. The pigs were examined and blood samples were taken every other day for 62 days for virological and serological analyses. The virus was highly contagious for the five pigs that were in direct contad with the inoculated pig, but spread to the other pens only after all the pigs in the originally infeded pen had become viraemic. The spread of the virus was promoted by contaminated clothing and footwear, but airbome transmission contributed considerably to the spread of the virus within the pighouse. The first clinical signs observed after the virus was introduced into a pen were decreased feed intake, increased mean rectal temperature and apathy. Neither the clinical course of the infection, nor the pattem of seroconversion observed over time, was affected by the differences in the intensity of contact with the virus between the pigs in the different pens.

An E2 sub-unit marker vaccine does not prevent horizontal or vertical transmission of classical swine fever virus.

An experimental infection with classical swine fever (CSF) virus in E2 sub-unit marker vaccine vaccinated gilts was conducted in order to evaluate the effect of vaccination on virus transmission and course of the disease. Therefore, clinical signs as well as horizontal and vertical virus transmission were monitored in two inoculated, non-vaccinated and 10 vaccinated conventional gilts, housed in individual sow boxes. Within 10 days post-inoculation, all vaccinated gilts became infected. Depending on the definition of the infectious period, two different estimates of R0 were calculated (R0=14.8 and 3.3), both significantly larger than 1 (P<0.01). In three out of the eight vaccinated pregnant gilts vertical virus transmission occurred, resulting in infected offspring. Based on the results of this experiment, it can be concluded that double vaccination with an E2 sub-unit marker vaccine only protects pregnant gilts from the clinical course of the disease but does not prevent horizontal nor vertical spread of the CSF virus.

Sperm Binding to Epithelial Oviduct Explants in Bulls with Different Nonreturn Rates Investigated with a New In Vitro Model

Abstract A new in vitro method was developed for analyzing the capacity of sperm to bind to oviductal epithelium to determine whether this binding capacity could be used to predict nonreturn rates (NRR). Sperm binding was evaluated by counting 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1)-labeled spermatozoa attached to oviductal epithelium and by measuring the surface area of the oviduct explants by means of an image analysis program. Hepes + Tyrode albumin lactate pyruvate (TALP) was a more useful medium than in vitro fertilization (IVF)-TALP, TCM-199 medium + 10% fetal calf serum, and TCM-199 medium alone for the investigation of sperm binding to oviductal explants. Oviduct explants with a surface area of ;lt20 000 μm2 provided more consistent results than did explants with a surface area of >100 000 μm2. A positive association was found between the loge transformed number of spermatozoa bound to 0.1 mm2 oviductal epithelium and the NRR of the respective sires after 24 h of coincubation, provided that the membrane integrity of the sperm sample was >60%. Determination of the capacity of sperm to bind to oviductal explants could become a reliable in vitro method for predicting the NRR of a given sire.

Insulin resistance: the link between metabolic disorders and cystic ovarian disease in high yielding dairy cows?

A preliminary investigation was performed to examine whether insulin resistance is a factor in the pathogenesis of cystic ovarian disease (COD) in high-yielding dairy cows. In total 30 cows, of which 15 were diagnosed as suffering from COD based on the anamnesis and clinical examination, and the other 15 served as matched controls, were subjected to an intravenous glucose tolerance test (IVGTT). The aim of the study was to investigate whether insulin activity was altered in COD cows. Differences in glucose clearance between the COD cows and their controls were analyzed comparing the fractional turnover rate (k), the glucose half-time (T1/2), and the area under the curve (AUC) 60 and 120 min after infusion. Differences in insulin response were analyzed comparing the insulin increment, the insulin peak concentration, and the AUC 60 and 120 min after glucose infusion. Although insulin resistance, attended by a secondary hyperinsulinemia, is stated to directly contribute to the ovarian abnormalities that characterize the polycystic ovary syndrome (PCOS) in human medicine, this was not observed in COD cows. On the contrary, COD cows appeared to have a low insulin response following an intravenous glucose load as compared with their matched controls. This was illustrated by significantly lower insulin increments (P = 0.04) and lower insulin peak concentrations (P = 0.04). As COD cows had a significantly lower insulin response to a standard glucose load, it was concluded that insulin could be a factor in the pathogenesis of COD in dairy cows.

An experimental infection with a classical swine fever virus in weaner pigs

The spread of Classical Swine Fever (CSF) virus (strain Lorraine), originally isolated in the first CSF infected herd of the 1993-1994 Belgian epizootic, was examined in an isolation unit with three adjacent pens and 15 weaner pigs per pen. Virus was introduced through experimental inoculation of one weaner pig in the middle pen (pen 2). The experimentally inoculated pig became viraemic 4 days post-inoculation (dpi) and the pen mates at 12 (n = 9) and 14 dpi (n = 5). The first viraemia in pens 1 and 3 was observed 18 dpi. Pigs were found to be seropositive in pens 1, 2, and 3 from 24, 20, and 22 dpi onwards, respectively. The reproduction ratio (R0) for the pigs in pen 2, estimated according to the martingale method, was 81.3 (s.e. = 109.54). The rate ratio (Cox proportional hazard) of the first pigs to become viraemic in pen 3 (airborne contact plus contact via contaminated clothing and footwear with pen 2) versus pen 1 (airborne contact with pen 2 only) was 1.60 (P = 0.3342). Thus, the additional contact of contaminated clothing did not affect transmission of the CSF virus. The survivor function (Kaplan-Meier survival analysis) did not significantly differ per pen. The time from first detection of virus in plasma to death was not significantly different between pens. The mean rectal temperature of pigs in a pen increased 3 to 4 days prior to detection of virus. The proportion of seropositive pigs per pen (p) from the day the first weaner pig in a pen became viraemic (dpf) was examined as a function of time with a logistic regression model. The model parameter estimates did not differ between pens. Hence, the data from the three pens were pooled. The regression equation of the seroprevalence over time for the pooled data was p = 1/[1+e(4.65-0.39 *dpf)].In a companion paper a logistic regression model of seroprevalence over time was developed on the basis of data obtained during an experimental infection of weaner pigs with classical swine fever (CSF) virus. The model was applied to seroprevalence data from three outbreaks of the 1993-1994 epizootic to test whether the model could predict correctly the day of virus introduction into the herd. It was concluded that the logistic regression model has potential as a tool to estimate in retrospect the day CSF virus was introduced into a pig herd, which in turn may assist in identification of risk factors implicated in the further spread of the disease.

Airborne transmission of classical swine fever virus under experimental conditions

Sixty-one pigs were housed in an isolation unit with three compartments and five pens. Each compartment had its own ventilation system resulting in air currents flowing from compartment A (pens 1 to 3) towards compartment B (pen 4), but not towards compartment C (pen 5). Classical swine fever virus was introduced by the experimental inoculation of one pig in the middle pen (pen 2) of compartment A. The virus infected the pigs in pen 4, following the prevalent air currents, and the compartmentalisation had only a retarding effect on the transmission of the virus. The absence of infection in the pigs in pen 5, which was not different from pen 4 except for the ventilation system, indicates that the spread of virus was affected by the air currents.

Transmission of classical swine fever. A review

Summary Classical swine fever (CSF) is one of the most important viral pig diseases. Basic measures to control epidemics of CSF comprise eradication of infected herds and preventive emptying of herds at risk. Identification of these herds at risk is based on knowledge of the different transmission routes of the virus. Direct transmission of CSF is undoubtedly the most efficient way of CSF virus transmission. Data on indirect transmission are variable and often equivocal. Various indirect transmission routes like swill feeding, wild boar and artificial insemination are well described, and the importance is beyond discussion. Mechanical transmission via vehicles and persons are categorized as very important based upon epidemiological research, whereas in experiments they can only be reproduced under worst case conditions. The role of arthropods, birds, rodents, and other animals in the spread of CSF virus remains doubtful. Active transmission by these has never been demonstrated and also very sparse indications for mechanical transmission are available. Also the role of airborne transmission remains debated. However epidemiological as well as experimental data indicate that airborne spread over short distances is probable.