Oocyte and Embryo Quality: Effect of Origin, Culture Conditions and Gene Expression Patterns

In general, the majority of immature bovine oocytes fail to develop to the blastocyst stage following maturation, fertilization and culture in vitro. The evidence suggests that while culture conditions during in vitro embryo production can impact on the developmental potential of the early embryo, the intrinsic quality of the oocyte is the key factor determining the proportion of oocytes developing to the blastocyst stage. In addition, evidence suggests that the period of post‐fertilization embryo culture is the most critical in determining blastocyst quality. This paper reviews the current literature, with emphasis on the bovine model, demonstrating evidence for an effect of oocyte origin and/or in vitro maturation conditions on the developmental capacity and gene expression patterns in the oocyte. Furthermore, the well‐documented effects of post‐fertilization culture environment on embryo gene expression and quality are highlighted.     

Protection of pigs from swine dysentery by vaccination with recombinant BmpB, a 29.7 kDa outer-membrane lipoprotein of Brachyspira hyodysenteriae

Swine dysentery (SD) is an important endemic infection in many piggeries, and control can be problematic. In this study the efficacy of BmpB, a 29.7 kDa outer-membrane lipoprotein of Brachyspira hyodysenteriae, was evaluated as an SD vaccine. Non-lipidated BmpB was expressed in Escherichia coli as a histidine-tagged protein (His6-BmpB), or as an 8 kDa carboxy-terminal portion fused to maltose-binding protein (MBP-BmpB-F604). The purified proteins were emulsified with oil-based adjuvants for intramuscular (im) administrations. In experiment 1, 20 weaner pigs were vaccinated im with 1 mg of His6-BmpB. After 3 weeks, 10 received 1 mg of the protein orally (im/oral), and 10 received 1 mg im (im/im). Ten acted as unvaccinated controls. In experiment 2, 12 pigs were vaccinated im with 1 mg of His6-BmpB, and 12 with 1 mg of MBP-BmpB-F604. Three weeks later, each was given 1 mg of the same protein orally. Twelve pigs acted as unvaccinated controls. All pigs were challenged orally with B. hyodysenteriae 2 weeks after their second vaccination. In both experiments, all pigs vaccinated with His6-BmpB developed serum antibodies to BmpB, and oral administration provided boosting of im-induced serum antibody titres. In experiment 1, seven non-vaccinated control pigs developed dysentery and severe colitis. Three pigs vaccinated im/oral developed diarrhoea; two had severe colitis and one had mild lesions. Four pigs vaccinated im/im developed diarrhoea; one had severe colitis and the others had mild lesions. In experiment 2, six control pigs developed SD with severe colitis. Two His6-BmpB vaccinated pigs developed SD with mild colitis. Nine pigs vaccinated with MBP-BmpB-F604 developed SD and severe colitis. Overall, 50-70% of controls and 17-40% of His6-BmpB vaccinated pigs developed disease. Vaccination with MBP-BmpB-F604 did not induce serum titres against BmpB, nor confer protection. The incidence of disease for the three His6-BmpB vaccinated groups was significantly less (P = 0.047) than for the control groups, with a approximately 50% reduction. BmpB appears to have potential as an SD vaccine component.     

Expression of interleukin 4, interleukin 4 splice variants and interferon gamma mRNA in calves experimentally infected with Fasciola hepatica

Interleukin 4 (IL-4) is expected to play a dominant role in the development of T helper (Th) 2 cells. Th2 immune responses with expression of relatively large amounts of interleukin 4 (IL-4) but little interferon gamma (IFN-gamma) are characteristic for chronic helminth infections. But no information is available about IL4 expression during early Fasciola hepatica (F. hepatica) infections in cattle. Therefore, we investigated F. hepatica specific IL-4 and IFN-gamma mRNA expression in peripheral blood mononuclear cells (PBMCs) from calves experimentally infected with F. hepatica. Cells were collected prior to infection and on post-inoculation days (PIDs) 10, 28 and 70. Interestingly, PBMCs responded to stimulation with F. hepatica secretory-excretory products (FhSEP) already on PID 10 and expressed high amounts of IL-4 but not of IFN-gamma mRNA suggesting that F. hepatica induced a Th2 biased early immune response which was not restricted to the site of infection. Later in infection IL-4 mRNA expression decreased whereas IFN-gamma mRNA expression increased slightly. Isolated lymph node cells (LNCs) stimulated with FhSEP and, even more importantly, non-stimulated LN tissue samples indicated highly polarized Th2 type immune responses in the draining (hepatic) lymph node, but not in the retropharyngeal lymph node. During preliminary experiments, two splice variants of bovine IL-4 mRNA, boIL-4delta2 and boIL-4delta3, were detected. Since a human IL-4delta2 was assumed to act as competitive inhibitor of IL-4, it was important to know whether expression of these splice variants of bovine IL-4 have a regulatory function during an immune response to infection with F. hepatica. Indeed, IL-4 splice variants could be detected in a number of samples, but quantitative analysis did not yield any clue to their function. Therefore, the significance of bovine IL-4 splice variants remains to be determined.     

Factors Affecting Pregnancy Rate in Artificial Insemination with Frozen Semen During Non-Breeding Season in Murciano–Granadina Goats: a Field Assay

An artificial insemination programme was carried out to study the effect of factors such as depth of semen deposition, inseminator skill, farm, sire and expression of oestrus on pregnancy rate in Murciano-Granadina (MG) goats during non-breeding season and using frozen semen. Frozen-thawed semen from six males was applied by three technicians to inseminate a total of 551 goats in 17 farms distributed throughout the Mediterranean area of Spain. Pregnancy rate was determined at 6 weeks after insemination by transabdominal ecography. Overall pregnancy rate was 57%. Farm and depth of semen deposition affected pregnancy rate, whereas the sire and the technician had no effect. The deeper the semen was deposited in the genital tract, the higher was the rate of pregnancy obtained, being greater when the catheter reached the uterus. In spite of the relevant difference observed (48.2% vs 59.0%), pregnancy rate of females not coming into oestrus until 30 h after sponge removal was not significantly different, compared with those showing oestrus during the OD procedure. In conclusion, our field assay data on AI in MG goat with frozen-thawed semen showed that post-cervical insemination presented significantly greater pregnancy rate in comparison to when semen is deposited in the vagina or in the caudal part of the cervix.     

Seminal Plasma Composition from Ejaculates Collected by Artificial Vagina and Electroejaculation in Guirra Ram

This study was conducted to evaluate changes in ram seminal plasma composition from ejaculates obtained using artificial vagina (AV) and electroejaculation (EE). To address this question, we assessed the effect of semen collection method on volume, sperm concentration, sodium concentration, potassium concentration, sodium/potassium ratio, total protein content and protein profile using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and 2-D polyacrylamide gel electrophoresis. The main findings from this study were: (i) similar volume was obtained, while sperm concentration was significantly lower for EE method; (ii) potassium and sodium/potassium concentration ratio were not influenced by recovery method, while sodium concentration increased significantly when semen was recovered using EE; (iii) approximately 80% of the total relative seminal plasma protein is represented by four protein fractions of molecular weights around 15, 21, 24 and 50 kDa and there were not differences and (iv) focussing the two-dimensional SDS-PAGE gel on the 10–25 kDa rank, the image analysis software detected around 22 spots with isoelectric points ranging from 5.1 to 6.1. Two protein spots (15 kDa and 5.5 and 22 kDa and 5.2 for molecular weight and isoelectric point respectively) increased significantly when semen was recovered using EE. One spot protein with molecular weight around 25 kDa and isoelectric point of 5.2 were only found in the seminal plasma from the semen recovery by AV. As it was demonstrated, ejaculates obtained with EE modify the sodium concentration, alter two proteins concentration and induced the loss of one protein in seminal plasma.     

Serology of a Neospora abortion outbreak on a dairy farm in New Zealand: a case study

AIM: To describe the kinetics of serological titres after an abortion outbreak in April-May 1995 due to Neospora caninum affected 17 dairy cows in a herd of 320. METHODS: Thirty-five cows, that had either aborted, carried mummified calves, were not pregnant or calved normally were: bled several times at regular intervals and the sera tested for Neospora antibodies in the indirect fluorescence antibody test (IFAT). RESULTS: Maximal IFAT titres of up to 1:4000 occurred within 6 weeks of the abortion outbreak, decreased over the next 2 months to < or = 1:200 and remained at this level until the next scheduled bleed a further 2 months later. A rise in titres was subsequently observed in the cows that had aborted or were not pregnant (at the time of the abortions) or had carried mummified foetuses. Seroconversion was also observed in some of the control cows, which had, up until then, remained seronegative. A dog and cat in contact with the cows in the herd investigated were, however, negative in the IFAT. CONCLUSIONS: Maximal serological titres in Neospora abortions are observed within weeks of the abortion event and then quickly return to very low levels. Subsequently, a recrudescence of titres can be observed in infected cows during the next pregnancy, without it being associated with repeat abortions.     

Evaluation of electrophoretic immunoblotting for Brucella ovis infection in deer using ram and deer serum

AIMS: Recently the first case of natural infection of deer with Brucella ovis was discovered. The aim of this study was to develop and evaluate an electrophoretic immunoblotting method for testing deer serum for specific B. ovis antibodies. METHODS: An existing immunoblotting method for sheep serum was altered by using a recombinant protein G-alkaline phosphatase conjugate and Tris-buffered saline containing 3% non-fat dry milk powder for the blocking step and the serum and conjugate dilutions. The method was evaluated using 106 sheep sera from B. ovis - negative, accredited flocks, 69 sera from chronically infected rams shedding B. ovis in their semen, 110 sera from a B. ovis-infected flock, 18 sera from stags from which B. ovis was isolated, and 48 sera from deer flocks free from B. ovis infections. The immunoblotting method was applied to another 85 deer sera. RESULTS: The sensitivity of the new immunoblotting method was 98.6% for sheep and 94.4% for deer, and the specificity 99.1% for sheep and 100% for deer. Sixty-nine out of 97 deer sera, originating from the property from which the first B. ovis deer case had been reported, tested positive or suspicious in the complement fixation test. Of these, 53 sera exhibited staining patterns in blots typical for B. ovis infections and also one serum which was negative in the CFT. Only six out of 1498 deer sera. from throughout New Zealand had positive or suspicious reactions in the B. ovis complement fixation test. Of these, one exhibited a staining pattern in the blot suggestive of a B. ovis infection, while four showed patterns of suspicious reactions. CONCLUSION: The new immunoblotting technique is useful as a confirmatory serological test method for B. ovis infections in deer.