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.

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.

Effect of a porcine circovirus type 2 infection on embryos during early pregnancy.

The aim of the present study was to assess the effects of porcine circovirus type 2 (PCV2) on porcine embryos and their receptor sows during the first 21 days of pregnancy. Hatched blastocysts exposed to 10(5.0) TCID50 PCV2 per ml (strain 1121, fifth passage PK15) and negative control embryos were transferred to PCV2-immune receptor sows at the 7th day of the cycle. Two weeks after transfer (D21), the receptor sows were euthanized and embryos were recovered. They were assessed macroscopically for viability and examined for viral antigen-positive cells by immunoperoxidase staining. The embryonic survival rate of the PCV2-exposed embryos (6.4%, 7 viable embryos out of 110 transferred) was significantly lower than the survival rate of the negative control embryos (65.4%, 34 viable embryos out of 52 transferred). All of the non-viable PCV2-exposed embryos (n=9) displayed immunohistochemical positive signals for PCV2-antigen in degenerated tissues. In the PCV2-exposed embryos that were categorized as viable at D21, small clusters (n=4) or no PCV2-positive cells (n=3) were detected. The pregnancy results of the receptor sows that received PCV2-exposed embryos (1/5) were considerably different from the negative control receptors (2/2), with 3 out of 5 sows displaying a regular return to oestrus. In conclusion, it can be stated that PCV2 can replicate in embryos and might lead to embryonic death. In a small proportion of embryos, PCV2 exposure does not have a detrimental effect on embryo development before D21.

Developmental competence of bovine oocytes is not related to apoptosis incidence in oocytes, cumulus cells and blastocysts.

The number of follicles undergoing atresia in an ovary is very high, and isolation of cumulus-oocyte complexes (COCs) from such atretic follicles may impair subsequent embryo development in vitro. Our aim was to study if stringent selection by morphological assessment of COCs can improve embryo development, and to evaluate whether oocyte diameter is related with apoptotic ratio in oocytes and blastocysts. COCs from slaughtered cattle were recovered by follicle aspiration and classified depending on oocyte diameter: (A) <110 microm; (B) 110-120 microm; (C) >120 microm. COCs were matured, fertilized and cultured in vitro. Early and late stages of apoptosis were detected by Annexin-V and TUNEL staining, respectively, in denuded oocytes, COCs and blastocysts. Immature oocytes from Group A showed higher apoptotic ratio assessed by TUNEL assay, and the COCs corresponding to this group also showed a higher proportion of apoptotic cumulus cells. After maturation, no differences were present in the incidence of apoptosis among oocytes from different groups, but COCs corresponding to the largest diameter showed less apoptotic cumulus cells. In addition, the percentage of apoptotic oocytes decreased during in vitro maturation in all groups. Apoptotic cell ratio (ACR) in blastocysts was not related to oocyte diameter. In conclusion, oocyte selection and oocyte morphological evaluation prior to maturation was not sufficient to select non-atretic oocytes. When oocyte diameter was used as an additional selection the embryonic developmental potential increased together with oocyte diameter, but this improvement was not related to a lower incidence of apoptosis in the largest oocytes.

Receptor-determined susceptibility of preimplantation embryos to pseudorabies virus and porcine reproductive and respiratory syndrome virus.

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.

Non-esterified fatty acids in follicular fluid of dairy cows and their effect on developmental capacity of bovine oocytes in vitro.

In this study concentration and composition of non-esterified fatty acids (NEFA) in follicular fluid (FF) of high-yielding dairy cows were determined during the period of negative energy balance (NEB) early post partum. NEFA were then added during in vitro maturation at concentrations measured previously in FF to evaluate their effect on the oocyte's developmental competence. At 16 and 44 days post partum, FF of the dominant follicle and blood were collected from nine high-yielding dairy cows. Samples were analysed for NEFA concentration and composition. NEFA concentrations in FF (0.2-0.6 mmol/l) during NEB remained +/- 40% lower compared with serum (0.4-1.2 mmol/l). The NEFA composition differed significantly between serum and FF with oleic acid (OA), palmitic acid (PA) and stearic acid (SA) being the predominant fatty acids in FF. Based on these results, 5115 oocytes were matured for 24 h in serum-free media with or without (negative control) the addition of 0.200 mmol/l OA, 0.133 mmol/l PA or 0.067 mmol/l SA dissolved in ethanol or ethanol alone (positive control). Matured oocytes were fertilized and cultured for 7 days in SOF medium. Addition of PA or SA during oocyte maturation had negative effects on maturation, fertilization and cleavage rate and blastocyst yield. More (late) apoptotic cumulus cells were observed in cumulus-oocyte complexes matured in the presence of SA or PA. Ethanol or OA had no effect. These in vitro results suggest that NEB may hamper fertility of high-yielding dairy cows through increased NEFA concentrations in FF affecting oocyte quality.

Susceptibility of pig embryos to porcine circovirus type 2 infection.

The aim of the present study was to determine if porcine circovirus type 2 (PCV2) is able to infect embryonic cells of in vivo produced porcine embryos with and without zona pellucida (ZP). ZP-intact and ZP-free morulae (6-day post-insemination) and early blastocysts (7-day post-insemination), and hatched blastocysts (8-day post-insemination) were exposed to 10(5.0) TCID50 PCV2 per ml (strain 1121, fifth passage PK15). At 48 h post-incubation, the percentage of infected embryos and the percentage of viral antigen-positive cells per embryo were determined by indirect immunofluorescence (IF). Significantly different percentages of infected embryos were detected: 15% for ZP-free morulae, 50% for ZP-free early blastocysts and 100% for hatched blastocysts. The percentage of cells that expressed viral antigens was similar for the three stages of development. PCV2 exposure did not affect the in vitro development of the embryos during the 48 h study period. All ZP-intact embryos remained negative for viral antigens. In an additional experiment the diameter of the channels in the porcine ZP was determined. After incubation of early blastocysts with fluorescent microspheres of three different sizes, beads with a diameter of 20 nm and beads with a diameter of 26 nm crossed the zona whereas beads with a diameter of 200 nm did not. In conclusion, it can be stated that PCV2 is able to replicate in in vivo produced ZP-free morulae and blastocysts and that the susceptibility increases during development. The ZP forms a barrier to PCV2 infection, but based on the size of the channels in the ZP the possibility that PCV2 particles cross the ZP cannot be excluded.

Motility characteristics of boar spermatozoa after addition of prostaglandin F2alpha.

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.

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.

Efficacy of tilmicosin phosphate (Pulmotil premix) in feed for the treatment of a clinical outbreak of Actinobacillus pleuropneumoniae infection in growing-finishing pigs.

A double-blind randomized clinical trial was carried out to investigate the efficacy of tilmicosin (Pulmotil premix) for the treatment of a clinical outbreak of Actinobacillus pleuropneumoniae infection in growing-finishing pigs. The effects of tilmicosin administration in the feed at 400 mg/kg and an injection therapy of clinically diseased pigs with long-acting oxytetracycline (Terramycine LA) at 20 mg/kg bodyweight were compared. Both groups, totalling 147 pigs, were compared during a medication period of 15 days and a post-medication period of 11 days by means of different clinical and performance parameters. During the medication period, the tilmicosin group showed a significant advantage with respect to the number of new disease cases (P < 0.01), and a non-significant advantage regarding the number of removed pigs (P = 0.16), the number of sick pigs that recovered (P = 0.27) and the time to recovery (P = 0.42). During the post-medication period, the pigs of the tilmicosin group showed numerical non-significant benefits (P > 0.05) with respect to the clinical parameters. During the overall study period (26 days), the average daily gain and the feed conversion ratio were both significantly (P < 0.01) better in pigs from the tilmicosin group compared with pigs from the oxytetracycline group. This study demonstrated that in-feed medication of tilmicosin at a dosage of 400 mg/kg is efficacious for the treatment of a clinical respiratory disease outbreak of A. pleuropneumoniae infection in growing-finishing pigs. Compared with oxytetracycline injection of clinically diseased pigs, the tilmicosin treatment is particularly beneficial in the prevention of new disease cases while increasing or maintaining the performance of the pigs.

A comparative study of the preventive use of tilmicosin phosphate (Pulmotil premix) and Mycoplasma hyopneumoniae vaccination in a pig herd with chronic respiratory disease.

This study was conducted to compare the effects of a preventive in-feed medication programme using tilmicosin (Pulmotil 200 premix, Elanco Animal Health) at 200 p.p.m. with those of Mycoplasma hyopneumoniae (Mh) vaccination programme (Stellamune Mycoplasma, Pfizer Animal Health). A pig herd with chronic respiratory disease in which infection with Mh played an important role was selected, and a total of 204 piglets were randomly allocated to either the medication (P) or the vaccination (V) group. Pigs in the P group received medicated feed for 3 weeks after weaning (days 34-55), and for 2 weeks late in the nursery period (days 77-98). The piglets in the V group were vaccinated twice intramuscularly, at 4 and 22 days of age. The two groups were compared on the basis of average daily gain (ADG), feed conversion rate (FCR), additional curative medication days (CMD), overall mortality (major variables), a coughing index, pneumonia lesions, and serology against Mh, influenza H1N1 and influenza H3N2 viruses, Actinobacillus pleuropneumoniae (App) and porcine reproductive and respirator, syndrome virus (PRRSV) (minor variables). No significant differences (P > 0.05) were observed for ADG (555 g/day in P group; 567 g/day in V group), FCR (2.64 in P group; 2.41 in V group) and mortality rate (11% in P group; 7% in V group). The average number of additional curative medication days (CMD) per pig was significantly higher (P < 0.01) in the P group (1.5) than in the V group (0.58). At slaughter age, the serological results and the prevalence of macroscopic lung lesions were comparable in the two groups (P > 0.05). With the exception of CMD, the preventive use of tilmicosin at this swine farm was found to confer similar beneficial effects to Mh vaccination.