Bart Mateusen

Diseases in swine transmitted by artificial insemination : An overview

Artificial insemination (AI) of swine is widely practiced in countries with an intensive pig production. It is a very useful tool to introduce superior genes into sow herds, with minimal risk for disease transmission. However, the impact of semen that is contaminated with pathogens can be enormous. Most of the micro-organisms that have been detected in boar semen are considered non-pathogenic, but some are known pathogens (e.g. porcine reproductive and respiratory syndrome virus) that can cause major economic losses. Microbial contamination of semen can be due to systemic and/or urogenital tract infections of the boar, or can occur during collection, processing and storage. It can result in reduced semen quality, embryonic or fetal death, endometritis and systemic infection and/or disease in the recipient female. Conventional techniques for isolation of bacteria and viruses from the semen do not always provide optimal results for various reasons, including lack of sensitivity and speed of testing, and difficult interpretation of the outcome. More recently, PCR tests are commonly used; they have a high sensitivity, the outcome is quickly obtained, and they are suitable for monitoring a large number of samples. The best strategy to prevent AI-transmitted diseases is to use boars that are free of specific pathogens, to monitor the animals and semen regularly, and to maintain very high biosecurity. Additional measures should be directed at treating semen with appropriate antimicrobials, and at reducing contamination during semen collection, processing, and storage.

Assessment of mammalian embryo quality: what can we learn from embryo morphology?

Embryo morphology assessment, however imperfect it may be, is at present the most popular method for embryo selection prior to transfer, both in human and bovine assisted reproduction. A major difference between human and bovine embryos is the fact that in the latter, assessment of morphology is jeopardized by the opacity of the blastomeres, which is caused by lipid droplet accumulation. This opacity makes it difficult to assess nuclear and nucleolar morphology, aspects which can easily be evaluated in human zygotes or early cleaving embryos. However, recent research which focused on correlation between bovine embryo morphology and embryonic ultrastructure, gene expression and cryoresistance, has provided evidence that much more can be deduced from mere embryo morphology than previously thought. Morphological features such as colour of the blastomeres, the extent of compaction, timing of blastocyst formation and expansion and diameter of the embryo at hatching can be linked with embryo quality. On the other hand, cattle embryos of deviant chromosomal constitution or with aberrant genetic make-up cannot be selected against by means of the current morphological techniques. Possible solutions include the visualization of bovine pronuclei at the zygote stage by means of ultracentrifugation or multiphoton laser scanning microscopy, and adjustment of genetic analysis in order to reconstruct embryo genetic make-up starting from the biopsy material.

Temporal detection of caspase-3 and-7 in bovine in vitro produced embryos of different developmental capacity

Embryo quality is most frequently evaluated at the blastocyst stage, although quality parameters further back along the developmental axis, such as early developmental kinetics or oocyte quality, can be equally valuable. Despite the fact that previous studies in bovine have linked oocyte diameter and early developmental kinetics with blastocyst formation and viability, their relation with the incidence of apoptosis during embryo development remains relatively unexplored. Therefore, we related non-invasive parameters of oocyte and embryo quality, such as embryo kinetics, embryo morphology, and oocyte diameter, to the incidence of apoptosis throughout embryo development using fluorescent detection of active caspase-3 and -7. First, bovine in vitro embryos were selected according to developmental kinetics and morphology at four set times during culture and subjected to fluorescent detection of active caspase-3 and -7. Caspase activity was significantly higher in slow developing embryos in comparison with fast cleavers (P < 0.05), but was not related to embryo morphology. Second, bovine oocytes were divided into three groups on the basis of oocyte diameter and the resulting embryos were used for staining at the same four set times. Caspase activity was significantly higher in embryos derived from growing oocytes compared with those of fully grown oocytes at 45, 80, and 117 hours post-insemination (hpi; P < 0.05), but not at 168 hpi.

Preovulatory follicular fluid during in vitro maturation decreases polyspermic fertilization of cumulus-intact porcine oocytes: In vitro maturation of porcine oocytes

Porcine follicular fluid (pFF), as a supplement of maturation media, has been shown several times to improve the in vitro production (IVP) of porcine embryos. As a transudate of serum, pFF contains locally produced factors in addition to the ones derived from serum. The objective of this study was to determine the additional positive effects of these pFF specific factors on the nuclear and cytoplasmic maturation of porcine oocytes. Follicular fluid and autologous serum were collected from sows in the preovulatory phase of the estrous cycle. Subsequently, oocytes from prepubertal gilts were matured in NCSU23 supplemented with either 10% pFF or 10% autologous serum derived from the same sow. Oocytes were then fertilized and the putative zygotes were cultured for 7 days. Nuclear maturation and cumulus expansion were assessed after the maturation culture. For evaluation of cytoplasmic maturation, oocyte glutathione (GSH) content, fertilization parameters and embryonic development were evaluated. After in vitro maturation (IVM) of the oocytes, both cumulus expansion rate and oocyte GSH content were increased for oocytes matured in pFF (P<0.05). More monospermic penetration was found when cumulus-intact oocytes had been matured in 10% pFF but this effect was lost after fertilization of cumulus denuded oocytes indicating that the pFF was acting through the cumulus. We speculate that the increased cumulus expansion and increased glutathione content, which were prevalent after IVM in pFF, are responsible for the positive effects on fertilization and the pre-implantation development of the embryos.

Motility characteristics of boar spermatozoa after addition of prostaglandin F2α

Addition of prostaglandin F2alpha (PGF2alpha) to extended boar semen has been shown to slightly increase reproductive parameters in sows such as the conception rate and the total number of piglets born alive. The mechanisms by which PGF2alpha affect these parameters have not yet been elucidated, but it is possible that the sperm transport after insemination is increased. This study investigated whether the sperm motility from 20 Piétrain boars improved when PGF2alpha (Dinolytic; 5 mg PGF2alpha/ml) was added to diluted semen. Different amounts of PGF2alpha (0, 0.5, 1 and 2 ml/100 ml) were tested and the motility was evaluated immediately after addition of PGF2alpha, after 30 min, 2 h, and 24 h. Two computer-assisted semen analysis (CASA) systems, namely the Sperm Quality Analyzer (SQA-IIC) and the Hamilton Thorne (HTR Ceros 12.1) were used to assess the motility parameters. With the SQA-IIC, sperm motility index values of the treated groups were only slightly higher (P>0.05) compared to the negative control group. The different motility parameters measured with the HTR Ceros 12.1 were similar between the treatment groups, except for beat cross frequency, which was higher in the control group (1.5-5%; P<0.001). This study documented that the addition of 2.5, 5 or 10 mg PGF2alpha to 100 ml diluted boar sperm does not increase any sperm motility parameter. Further research is necessary to elucidate mechanisms by which PGF2alpha in diluted semen may improve the reproductive performance in swine farms.

Transmission of Mouse Minute Virus (MMV) but Not Mouse Hepatitis Virus (MHV) Following Embryo Transfer with Experimentally Exposed In Vivo-Derived Embryos

Abstract The present study investigated the presence and location of fluorescent microspheres having the size of mouse hepatitis virus (MHV) and of mouse minute virus (MMV) in the zona pellucida (ZP) of in vivo-produced murine embryos, the transmission of these viruses by embryos during embryo transfer, and the time of seroconversion of recipients and pups. To this end, fertilized oocytes and morulae were exposed to different concentrations of MMVp for 16 h, while 2-cell embryos and blastocysts were coincubated for 1 h. In addition, morulae were exposed to MHV-A59 for 16 h. One group of embryos was washed, and the remaining embryos remained unwashed before embryo transfer. Serological analyses were performed by means of ELISA to detect antibodies to MHV or MMV in recipients and in progeny on Days 14, 21, 28, 42, and 63 and on Days 42, 63, 84, 112, 133, and 154, respectively, after embryo transfer. Coincubation with a minimum of 105/ml of fluorescent microspheres showed that particles with a diameter of 20 nm but not 100 nm crossed the ZP of murine blastocysts. Washing generally led to a 10-fold to 100-fold reduction of MMVp. Washed MMV-exposed but not MHV-exposed embryos led to the production of antibodies independent of embryonic stage and time of virus exposure. Recipients receiving embryos exposed to a minimum of 107 mean tissue culture infective dose (TCID50)/ml of MHV-A59 and 102 TCID50/ml of MMVp seroconverted by Day 42 after embryo transfer. The results indicate that MMV but not MHV can be transmitted to recipients even after washing embryos 10 times before embryo transfer.

Receptor-Determined Susceptibility of Preimplantation Embryos to Pseudorabies Virus and Porcine Reproductive and Respiratory Syndrome Virus

Abstract In the present study, the in vitro interaction of embryos with pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) was investigated by viral antigen detection and by evaluating the expression of virus receptors, namely, poliovirus receptor-related 1 (PVRL1; formerly known as nectin 1) for PRV and sialoadhesin for PRRSV. Embryonic cells of zona pellucida intact embryos incubated with PRV remained negative for viral antigens. Also, no antigen-positive cells could be detected after PRV incubation of protease-treated embryos, since the protease disrupted the expression of PRVL1. However, starting from the five-cell-stage onwards, viral antigen-positive cells were detected after subzonal microinjection of PRV. At this stage, the first foci of PVRL1, also a known cell adhesion molecule, were expressed. At the expanded blastocyst stage, a lining pattern of PVRL1 in the apicolateral border of trophectoderm cells was present, whereas the expression in the inner cell mass was low. Furthermore, PVRL1-specific monoclonal antibody CK41 significantly blocked PRV infection of trophectoderm cells of hatched blastocysts, while the infection of the inner cell mass was only partly inhibited. Viral antigen-positive cells were never detected after PRRSV exposure of preimplantation embryos up to the hatched blastocyst stage. Also, expression of sialoadhesin in these embryonic stages was not detected. We conclude that the use of protease to investigate the virus embryo interaction can lead to misinterpretation of results. Results also show that blastomeres of five-cell embryos up to the hatched blastocysts can become infected with PRV, but there is no risk of a PRRSV infection.

Effectiveness of treatment with lincomycin hydrochloride and/or vaccination against Mycoplasma hyopneumoniae for controlling chronic respiratory disease in a herd of pigs

A herd of pigs infected with Mycoplasma hyopneumoniae was used in a double-blind randomised trial to assess the effectiveness of three control strategies against chronic respiratory disease in growing-finishing pigs. One group of 61 pigs received 220 ppm lincomycin hydrochloride in the feed from day 71 to day 91, a second group was vaccinated against M hyopneumoniae at four and 28 days of age, and a third group received both treatments; a fourth group was left untreated as a control. Throughout the nursery-finishing period (day 29 to slaughter) the average daily weight gain and feed conversion rate of all the treated groups were slightly better than in the controls, but there were no significant differences between them. There were no significant differences between the treated groups in terms of clinical signs, serology, pathology or mortality, which was very low throughout the trial.

Pathogenicity of Actinobacillus minor, Actinobacillus indolicus and Actinobacillus porcinus strains for gnotobiotic piglets.

The purpose of the study was to evaluate the clinical significance of Actinobacillus minor, Actinobacillus porcinus and Actinobacillus indolicus strains in gnotobiotic piglets. Twenty-two 6-h-old Caesarean-delivered and colostrum-deprived piglets were intranasally and orally inoculated with 2 x 10(6) colony-forming units of an A. minor (group 2; n = 9), A. indolicus (group 3; n = 5), or A. porcinus (group 4; n = 8) strain. Six other piglets were inoculated in the same way with phosphate-buffered saline solution and used as controls (group 1). All pigs were observed for clinical signs and rectal temperatures were taken until euthanasia 7 days after inoculation. At necropsy, conchae, tonsils, lungs, brains, liver, spleen and kidneys were macroscopically examined for lesions and samples were taken for bacteriology. None of the pigs developed fever. Mild ataxia was observed in one pig from group 3 for 2 days. Clinical signs were not observed in the other animals. In none of the animals were macroscopic lesions detected at necropsy. NAD-dependent Pasteurellaceae were not isolated from control animals (group 1). The A. minor, A. indolicus and A. porcinus strains were isolated from the tonsils of one, two and one pigs, respectively. Actinobacillus porcinus was isolated from the brains of the pig with central nervous symptoms and from the conchae of another pig. The inoculation strains were not demonstrated in the other samples. It was concluded that, using these inoculation routes and dose, the A. minor, A. indolicus and A. porcinus strains had low capacity to colonize the upper respiratory tract of gnotobiotic piglets and demonstrated low or no pathogenicity in such animals.

Ex vivo modeling of feline herpesvirus replication in ocular and respiratory mucosae, the primary targets of infection

Feline herpesvirus 1 (FeHV-1) is a major cause of rhinotracheitis and ocular diseases in cats. In the present study, the viral replication at the primary infection sites was studied using feline respiratory and ocular mucosa explants. The explants of three cats were maintained in an air-liquid culture up to 96 hours without loss of viability. After inoculation with FeHV-1 (C27), no evidence of infection was noted in corneal epithelium, while plaque-wise replication was observed in conjunctival and tracheal mucosae beginning from 24 h post inoculation (hpi). The viral plaque diameters increased over time in trachea and conjunctiva and were larger in tracheal explants than in conjunctival explants at 48 hpi. FeHV-1 penetrated the basement membrane in conjunctival and tracheal explants between 24 and 48 hpi. At 48 and 72 hpi, viral invasion was going deeper in tracheal explants than in conjunctival explants. Our study indicates that FeHV-1 has a better capacity to invade the respiratory mucosa than the conjunctival mucosa, and prefers the conjunctiva, but not the cornea as a portal of entry during ocular infection.