Viral respiratory disease of the horse.

The diagnosis of any viral respiratory disease relies on laboratory procedures to isolate the virus and demonstrate a significant rise in serum antibody titers. To isolate viruses from the upper respiratory tract, it is imperative that nasopharyngeal swabs are obtained from animals in the early acute stage of illness, i.e., during the pyrexic phase when the virus is replicating. Nasopharyngeal swabs must be placed in a virus transport medium and forwarded immediately to the laboratory at refrigerated temperature. Equine influenza, rhinopneumonitis, and equine viral arteritis are the three viral infections causing outbreaks of respiratory disease in North America. African horse sickness, although foreign to North America, could be introduced despite stringent horse importation regulations. Specific antiviral therapy is not available to treat viral respiratory disease in the horse. A variety of inactivated and modified live vaccines, however, are available to prevent clinical disease and the spread of infection caused by the common viral respiratory pathogens. A considerable amount of research is underway to enhance the potency and duration of immunity of the present vaccines against influenza and rhinopneumonitis. This research is directed at defining and characterizing the importance of specific glycoprotein antigens on the surface of the virus, which trigger the various host immune responses, and determining whether they are stimulatory or suppressive.     

Detection of Toxoplasma gondii in the milk of experimentally infected lactating cats.

Tachyzoites of Toxoplasma gondii have been found in the milk of sheep, goats, cows and mice and infection by ingestion of raw goat milk has been documented in humans. Lactational transmission from infected cats to their kittens is suspected but the organism has not been detected in the milk. The purpose of this study was to demonstrate the presence of T. gondii in the milk of experimentally infected cats. Pregnant specific pathogen free cats were inoculated orally with T. gondii at various times prior to parturition. Feces were examined for oocyst shedding after sugar solution centrifugation. Milk was collected for polymerase chain reaction (PCR) and bioassay in mice. T. gondii was detected in the milk of five of six cats by either bioassay or PCR.     

The Influence of Oxygen Tension on Cumulus Cell Viability of Canine COCs Matured in High-Glucose Medium

High in vitro oxygen (O₂) tensions are associated with enhanced levels of reactive oxygen species and cumulus oocyte complex (COC) apoptosis. The objective of this study was to determine the influence of O₂ tension on cumulus cell (CC) viability from canine oocytes. Cumulus oocyte complexes were distributed into three groups (CG, T20 and T5) and two O₂ tension levels (20% and 5%). The control group (CG) was matured in vitro in a humidified atmosphere with 5% CO₂ in air in TCM199 with 26.19 m m sodium bicarbonate, 10% (v/v) foetal calf serum (FCS), 0.10 m m gentamicin, 0.20 m m pyruvic acid, 20 μg/ml oestradiol, 0.5 μg/ml follicle-stimulating hormone, 0.03 IU/ml human chorionic gonadotropin, and 1.0 μg/ml human somatotropin. Groups T20 and T5 were matured under 20% or 5% O₂ tensions respectively in a high-glucose medium, without FCS. T20 and T5 were as CG, and supplemented with 0.1% Polyvinyl Alcohol, and 5.5 m m glucose. After 48 h of IVM, CCs from COCs were stained with propidium iodide (1.50 m m ). The results showed that viability of CCs (cytoplasmic features and nuclear morphological integrity) was different for the three groups. Rates of apoptosis were at 57.9% (521/900) for CG, 54.4% (490/900) for T20 and 38.9% (350/900) for T5 (p < 0.001). Predominant features in apoptotic cells (n = 1361) were DNA nuclear fragments (94.0%). It was concluded that CCs of canine COCs cultured in high-glucose medium showed significantly less apoptosis than those cultured in medium with FCS. Low O₂ tension was efficient in reducing apoptosis in canine CCs.     

Meiotic Response of In vitro Matured Canine Oocytes under Different Proteins and Heterologous Hormone Supplementation

The impact of TCM‐199 supplemented with different proteins and heterologous hormones on the in vitro maturation (IVM) rate of bitch oocytes was evaluated by nuclear staining under fluorescence microscopy. Oocytes were recovered by slicing of ovaries from bitches presented at various stages of oestrous cycle to ovariohysterectomy. The basic culture medium was TCM‐199 supplemented with 25 mM Hepes/l, with 10% heat‐inactivated oestrous cow serum (ECS), 50 μg/ml gentamicin, 2.2 mg/ml sodium bicarbonate and 22‐μg/ml pyruvic acid, 1.0‐μg/ml oestradiol (E 8875; Sigma), 0.5‐μg/ml follicle‐stimulating hormone (FSH) (Folltropin‐V; Vetrepharm Inc., Ontario, Canada) and 0.03 IU/ml human gonadotropin (hCG) (Profasi HP; Serono, Aubonne, Switzerland). Oocytes were distributed randomly between basic culture medium (control) and the corresponding experimental treatment. Hormone treatments were: oocytes cultured in; (1) medium without FSH, (2) control medium supplemented with 20 μg/ml oestradiol, or (3) medium supplemented with 1 μg/ml human somatotropin (hST; Humatrope, Lilly, Saint Cloud, France). The second experiment consisted of oocytes cultured in medium supplemented with 0.4% (w/v) bovine serum albumin (BSA, fraction V; Gibco Grand Island, NY, USA) instead of ECS, or oocytes cultured in medium with 10% inactivated oestrous bitch serum (EBS) instead of ECS. Oocytes were cultured in 100 μl droplets (up to 25 oocytes per drop) under mineral oil at 37°C in a 100% humidified atmosphere containing 5% CO₂ in air. After 72 h of IVM, the highest rates (p < 0.05) of meiotic resumption were achieved with the 0.4% BSA supplementation. A positive influence on the metaphase II (MII) acquisition rate was observed with hST supplement. Oocytes cultured with 10% EBS supplementation did not develop to the MII stage. The results in this study show that the protein and hormone supplements to TCM‐199 culture medium tested did not promote the final steps of IVM of bitch oocytes.     

An Association Analysis Between a Silent C558T Polymorphism at the Pig Vascular Cell Adhesion Molecule 1 Locus and Sow Reproduction and Piglet Survivability Traits

The vascular cell adhesion molecule 1 (VCAM1) has a strong influence on embryonic development and on the formation of the umbilical cord and placenta. These developmental processes are crucial to ensure the success of pregnancy. In this work, we have identified two T306A and C558T single nucleotide polymorphisms (SNP) at exons 2 and 3 of the pig VCAM1 locus, respectively. The T306A substitution involves a non conservative Asn to Lys replacement at amino acid position 102, whereas the C558T polymorphism is synonymous. An in silico prediction of the consequences of the Asn₁₀₂→Lys₁₀₂ mutation with the PolyPhen software revealed that it is not deleterious. The T306A SNP segregated in the Iberian, Piétrain, Duroc, Large White and Landrace breeds as well as in European wild boars. The C558T SNP also segregated and most of commercial standard breeds. The genotyping of the C558T SNP in an Iberian × Meishan intercross allowed to find a suggestive association (Bonferroni threshold, p < 0.004) between C558T genotype and time the newborn piglet needs to reach the udder (p = 0.013) as well as a significant one with time to make the first ingestion of colostrum (p = 0.003). The biological basis of these associations remains unclear and they should be interpreted with caution.     

Response of Thawed Epidi dymal Red Deer Spermatozoa to Increasing Concentrations of Hydrogen Peroxide,and Importance of Individual Male Variability

Oxidative stress represents a challenge during sperm manipulation. We have tested the effect of increasing hydrogen peroxide (H₂O₂) levels on red deer spermatozoa after cryopreservation, and the role of male‐to‐male variation in that response. In a first experiment, eight thawed samples were submitted to 0, 25, 50, 100 and 200 μm H₂O₂ for 2 h at 37°C. Intracellular reactive oxygen species (H₂DCFDA‐CM) increased with H₂O₂ concentration, but we only detected a decrease in sperm function (motility by CASA and chromatin damage by sperm chromatin structure assay) with 200 μm . Lipoperoxidation assessed by the thiobarbituric acid reactive substance (TBARS) method increased slightly with 50 μm H₂O₂ and above. In a second experiment, samples from seven males were submitted to 0 and 200 μm H₂O₂ for 2 h, triplicating the experiment within each male. Males differed at thawing and regarding their response to incubation and H₂O₂ presence. We found that the kinematic parameters reflected male‐to‐male variability, whereas the response of the different males was similar for lipid peroxidation and viability. A multiparametric analysis showed that males grouped differently if samples were assessed after thawing, after incubation without H₂O₂ or after incubation with H₂O₂. Red deer spermatozoa are relatively resilient to H₂O₂ after thawing, but it seems to be a great male‐to‐male variability regarding the response to oxidative stress. The acknowledgement of this individual variability might improve the development of optimized sperm work protocols.