No peri- and postnatal effects on calves born after transfer of in vitro produced embryos vitrified by the open pulled straw (OPS) method

The general objective of this study was to perform follow-up studies including selected peri- and postnatal characteristics on calves born after transfer of in vitro produced (IVP) embryos vitrified by the 'Open Pulled Straw' (OPS) method. An overall pregnancy rate of 16% after transfer of the OPS-vitrified IVP embryos was achieved and resulted in birth of 9 calves, with 11 AI calves serving as controls. There were no immediate or long-term effects on these calves with respect to birth weight, gestation length, perinatal mortality, growth rate, disease susceptibility and reproductive performance.     

The effect of artificial insemination prior to transfer of a limited number of vitrified and warmed porcine embryos by open pulled straw (OPS) method on their survival ability for farrowing

Embryo transfer (ET) of 20 porcine expanded blastocysts (ExBs) vitrified and warmed (VW) by open pulled straw (OPS) to a recipient allows stable piglet production. The efficiency of artificial insemination (AI) prior to ET of 10 VW ExBs for piglet production was investigated. For one trial, 10–15 VW ExBs from single donor were assigned, 10 were used for ET and the remains were assessed for their in vitro viability. In the non‐AI/ET group, 10 were transferred to each of five recipients. As AI/ET group, 10 were transferred to each of five recipients after AI. In AI/non‐ET group, only AI was performed to seven gilts. In the non‐AI/ET group, the pregnancy rate was 40%, but none of them farrowed. In the AI/ET group, all recipients produced piglets. Four (80.0%) delivered piglets from transferred VW ExBs. The survival rate of VW ExBs to term was 20.0% (10/50). In the AI/non‐ET group, six of the seven gilts farrowed. There was no difference in in vitro viability between the non‐AI/ET and AI/ ET groups (62.5% and 68.3%, respectively). AI prior to ET can be an appropriate way to maintain pregnancy and assist the development of a low number of VW ExBs to term.     

Effect of open pulled straw (OPS) vitrification on the fertilisation rate and developmental competence of porcine oocytes

Freezing technologies are very important to preserve gametes and embryos of animals with a good pedigree or those having high genetic value. The aim of this work was to compare immature and in vitro matured porcine oocytes regarding their morphology and ability to be fertilised after vitrification by the open pulled straw (OPS) method. In four experiments 830 oocytes were examined. To investigate the effect of cumulus cells on oocyte survival after OPS vitrification, both denuded and cumulus-enclosed oocytes were vitrified at the germinal vesicle (GV) stage, then after vitrification they were matured in vitro. Besides, in vitro matured oocytes surrounded with a cumulus and those without a cumulus were also vitrified. The survival of oocytes was evaluated by their morphology. After in vitro fertilisation the rates of oocytes penetrated by spermatozoa were compared. Our results suggest that the vitrification/warming procedure is the most effective in cumulus-enclosed oocytes (22.35 +/- 1.75%). There was no difference between the order of maturation and vitrification in cumulus-enclosed oocytes, which suggests the importance of cumulus cells in protecting the viability of oocytes during cryopreservation.     

Non‐surgical transfer of vitrified porcine embryos using a catheter designed for a proximal site of the uterus

This study aimed to compare the efficiency of non‐surgical embryo transfer (ET) using a newly developed catheter, which enables transferring embryos into a proximal site of the uterus (mostly uterine body), and surgical ET of vitrified porcine embryos. In Experiment 1, the catheter was inserted into 12 gilts, with each half of the group allocated to skilled or novice operators. The time required for insertion into the uterus did not differ between skilled and novice operators (4 min 9 s and 4 min 6 s, respectively). In Experiment 2, 12 gilts were used as recipients for non‐surgical and surgical ET with vitrified embryos (n = 6, each). There was no significant difference in the rate of piglet production based on the number of transferred embryos between surgical and non‐surgical ET (25.8% vs. 15.4%, p = .098). The results suggest that non‐surgical ET catheter allowed for easy insertion and transfer of embryos without special training. Although the catheter is effective for deposition of embryos into the proximal site of uterus, the efficiency of piglet production is not enhanced compared with surgical ET. The ET method using this catheter, being labor‐saving and less‐invasive, may contribute to the improvement of ET in pigs.     

Detection of turkey coronaviral enteritis (bluecomb) in field epiornithics, using the direct and indirect fluorescent antibody tests.

In Minnesota, efforts have been made over the past 10 years to eliminate turkey coronaviral enteritis (TCE, bluecomb) by controlled depopulation and decontamination with a rest period before restocking. In 1973, clinical observations indicated that bluecomb was restricted to one limited area in Minnesota. Five epiornithics occurred during late 1973 and 1974, involving 5 different farms in this limited geographic area. During 1975, 3 epiornithics of TCE were investigated, involving 185,000 turkeys in 17 flocks, of which approximately 17,000 died. Naturally infected turkeys representing 7 operations between 1973 and 1976 were examined by both the direct fluorescent antibody test and indirect fluorescent antibody test (IFAT). The direct fluorescent antibody test detected coronaviral antigen in intestinal tissues during the acute phase of the disease, and the IFAT was highly useful in detecting TCE serum antibodies of turkey flocks that had recovered and were potential carriers. Therefore, an IFAT surveillance program was instituted for replacement flocks on farms where clinical epiornithics of TCE had occurred in 1974 through 1976. Operation 5 involved TCE epiornithics over a 2-year period and illustrate the importance of complete depopulation with an intensive decontamination program.     

Antitumour effects of Liporaxel (oral paclitaxel) for canine melanoma in a mouse xenograft model

Paclitaxel, a member of the taxane family, exhibits antitumour effects by targeting the microtubules in cancer cells. Recently, oral paclitaxel has been developed to overcome the side effects of intravenous paclitaxel administration in human patients. The objective of this study was to investigate the antitumour effects of oral paclitaxel in vitro and in vivo. Three weeks after inoculation, oral paclitaxel (25 and 50 mg/kg) or saline was administered every week for three consecutive weeks. To explore the underlying mechanism, tumour angiogenesis was examined by immunohistochemistry with an anti‐CD31 antibody. Tumour cell apoptosis was detected by Terminal deoxynucleotidyl transferase dUTP Nick‐End Labeling assay, and cell cycle arrest was confirmed by western blot analysis. Oral paclitaxel treatment of canine melanoma cells exerted mediated antiproliferative effects and mediated cell cycle arrest in vitro. In animal experiments, after oral paclitaxel administration, the average tumour size decreased to approximately 30% of that in the control. Histologically, oral paclitaxel showed anti‐angiogenic effects and induced the apoptosis in tumour tissues. Oral paclitaxel also downregulated the intratumoural expression of cyclin D1 and inhibited cell proliferation. The study findings support potential application of oral paclitaxel as a novel chemotherapeutic strategy to treat canine melanoma. This is the first study to investigate the potential of oral paclitaxel as a therapeutic drug against canine tumours.     

Therapeutic potential of canine bone marrow stromal cells (BMSCs) in the carbon tetrachloride (CCl4) induced chronic liver dysfunction mouse model

Regenerative medicine using bone marrow cells is an attractive therapy for the cure of patients with severe liver disease. Here, we show the therapeutic potential of canine bone marrow stromal cells (BMSCs) in mouse models of CCl(4)-induced chronic liver dysfunction. We used two different models for xenotransplantation, nude mice and cyclosporine A (CSA) immunosuppressed mice. Serum parameters from a standard liver panel were not improved following transplantation. However, fibrotic liver lesions with severe inflammation were decreased in CCl(4)-treated CSA mice following BMSC transplantation. Effective migration of transplanted canine BMSCs was limited to persistently injured liver in CCl(4)-treated CSA mice, where they may be effective in resolving inflammatory fibrotic lesions. These results suggest that canine BMSCs are an effective cell source for liver regeneration.     

Evaluating the potential of (epi) genotype-by-low pass nanopore sequencing in dairy cattle:a study on direct genomic value and methylation analysis

Background Genotype-by-sequencing has been proposed as an alternative to SNP genotyping arrays in genomic selection to obtain a high density of markers along the genome. It requires a low sequencing depth to be cost effec-tive, which may increase the error at the genotype assigment. Third generation nanopore sequencing technology offers low cost sequencing and the possibility to detect genome methylation, which provides added value to gen-otype-by-sequencing. The aim of this study was to evaluat...     

Observations on the potential for natural transfer of Psoroptes cuniculi and Chorioptes bovis (Acari: Psoroptidae) between goats and sheep

Goats and sheep were pastured together and held in close contact in yards twice a month for 21 months. The goats had a high prevalence of both the rabbit ear canker mite, Psoroptes cuniculi and the chorioptic mange mite, Chorioptes bovis. The sheep were not infested with P. cuniculi at the beginning of the study and viable C. bovis could not be detected. At no time during the study did P. cuniculi establish in the ears of sheep, despite a maximum prevalence of >95% P. cuniculi in goats. This may have been related to the viscous nature of sheep cerumen. Levels of cerumen in goats' ears increased with increasing prevalence of the mite in the flock. The prevalence of C. bovis in the sheep flock did not exceed 10%, despite a maximum prevalence of >90% infested goats. The difficulty of detecting light infestations of mites means that it was not clear whether the C. bovis infestation of sheep was derived from goats or was present when the study began. Whatever the source, close contact with goats with an active and extensive C. bovis infestation seems unlikely to seriously influence populations of the ectoparasite in sheep. Psoroptes cuniculi was found to he established in goats as young as five days and post-mortem examination of goats' ears was more successful at revealing P. cuniculi than was use of an otoscope during life.     

Embryo transfer: a discussion on its potential for infectious disease control based on a review of studies on infection of gametes and early embryos by various agents

Studies on laboratory animals have shown that viruses vary as to whether or not they are transmissible by the gametes or are capable of passing through the zona pellucida and infecting the embryo.

Methods of studying early embryos for the presence of infectious agents include electron microscopy, immunocytochemistry and cell cultivation.

Determination that early bovine embryos do not become infected by certain agents might allow for easing of restrictions in the current import and export regulations for cattle embryos.

Embryo transfer could be used as a means of controlling or eliminating disease in a herd or flock if the causal agent does not infect the early embryo via the gametes or by penetrating the zona pellucida.

     

Comparison of direct colony count methods and the MPN-method for quantitative detection of Listeria in model and field conditions]

In order to compare the plate count method for quantitating Listeria, as published in the "Official Collection of Testing Methods" in section 35 LMBG (L. 00.00-22), to an MPN-method for Listeria based on the same mediums, these two detection methods for Listeria were tested in three sets of experiments and a routine sample status evaluation. A pure broth culture of L. monocytogenes, artificially with L. monocytogenes contaminated ground meat, artificially contaminated and cold stored ground meat as well as 77 ground beef samples from Berlin retail food stores were used in the four trials. The detection limit of the MPN-method is about 66% lower than the plate count method allowing detection of a clearly greater number of Listeria-positive samples from naturally contaminated ground meat. The MPN-method yielded more Listeria spp.-positive samples (rel. 43%) and more L. monocytogenes-positive samples (rel. 21%) versus the colony count method based on the results from the field trial using ground beef samples from retail food stores in Berlin. Nevertheless the standardized colony count method is preferred over the MPN-method for routine use because of its slightly higher productivity and much smaller variation in the results. However, the MPN-method is preferable for epidemiological studies because of the significance of the lower detection level. The random sampling evaluation of ground beef from retail stores indicated that 39% of the samples were Listeria spp.-positive and 31% were L. monocytogenes-positive when using the colony count method. A total of 56% of the meat samples were found to be Listeria spp.-positive and 38% L. monocytogenes-positive when the MPN-method was used. Population levels ranged from 10 to 580 cfu/g (Listeria spp.-positive samples) and from 10 to 270 cfu/g (L. monocytogenes-positive samples) for the colony count method. The MPN-method yielded population levels of 3.6 to 930 MPN/g for Listeria spp.-positive samples and 3.6 to 150 MPN/g for L. monocytogenes-positive samples. L. monocytogenes strains isolated using the colony count method belonged to the following serovars: 1/2a (46%), 1/2b (13%), 1/2c (33%), 3b (4%) and 4c (4%). A similar serovar isolation pattern was found for L. monocytogenes-positive MPN-tubes. The most common serotype was 1/2a (43%), followed by 1/2c (32%) and 1/2b (14%). The serotypes 3c, 4b and 4c were all isolated 4% of the time.     

Feline head and neck squamous cell carcinoma: a natural model for the human disease and development of a mouse model

Head and neck squamous cell carcinoma (H/N SCC) is a devastating disease in humans and cats, and shares similar features between the two species. The large population of pet cats inthe United States, along with the high incidence of oral SCC in the cat, makes the cat an attractive candidate as a natural model for the human disease. There are similarities in pathology, progression, outcome, resistance to treatment, possible aetiologies and p53 expression, and we discuss the benefits of the cat as a natural model. We describe the development of a nude mouse xenograft model of feline oral SCC using the SCCF1 cell line transfected with a luciferase expression construct. In vivo tumour growth and metastasis were measured using serial bioluminescent imaging, and tumours grew best in the subcutis. The cat and nude mouse models will be useful to investigate the pathogenesis and the molecular basis of H/N SCC, and for preclinical drug screening.     

Behavioural studies on the potential for direct transmission of tuberculosis from feral ferrets (Mustela furo) and possums (Trichosurus vulpecula) to farmed livestock

Studies were conducted to evaluate the response of cattle and deer to ferrets which were sedated so they behaved like terminally tuberculous animals, and to compare this with the response of cattle, deer and sheep to sedated possums. Six groups of deer and two groups of cattle were exposed to a sedated ferret and to a sedated possum. Both livestock species showed interest in the possum by sniffing and licking it, but they only briefly touched the ferret and no licking or extended investigation was observed. The proportion of available time spent in physical contact with the possum by cattle was 7.7 times as high as for the ferret, and for the deer was 5.7 times as high. The behavioural response of three groups of sheep to a sedated possum was investigated, and sheep showed limited interest beyond viewing the possum from a distance. The amount of time spent by sheep investigating the possum was very low and the intensity of exploration was also low. For possums, at least one deer was within 1.5 m (an estimate of the distance that tuberculosis can be transmitted by aerosol) for 50.9% of observation time, and in physical contact with the possum for 9.5% of time. The figures for cattle were 69.3% and 17.3%, while those for sheep were 6.9% and 0.3%. In interactions with ferrets, the equivalent figures were 29.8% within 1.5 m and 2.2% in physical contact for cattle, and 20.8% and 1.1% for deer. Tuberculous possums commonly and tuberculous ferrets less commonly have lung lesions and/or discharging sinuses, and may excrete Mycobacterium bovis intermittently or continuously in aerosols or discharges. The exploratory behaviour of deer and cattle in this study would provide opportunities for them to become infected with M. bovis if they had contact with infectious possums, and less probably with ferrets. The response of sheep to possums suggests that they would be much less likely to contract the disease.     

A study of the transfer of tetracycline resistance genes between Escherichia coli in the intestinal tract of a mouse and a chicken model

Experiments to demonstrate the transfer of genes within a natural environment are technically difficult because of the unknown numbers and strains of bacteria present, as well as difficulties designing adequate control experiments. The results of such studies should be viewed within the limits of the experimental design. Most experiments to date have been based on artificial models, which only give approximations of the real-life situation. The current study uses more natural models and provides information about tetracycline resistance as it occurs in wild-type bacteria within the environment of the normal intestinal tract of an animal. Tetracycline sensitive, nalidixic acid resistant Escherichia coli isolates of human origin were administered to mice and chicken animal models. They were monitored for acquisition of tetracycline resistance from indigenous or administered donor E. coli. Five sets of in vivo experiments demonstrated unequivocal transfer of tetracycline resistance to tetracycline sensitive recipients. The addition of tetracycline in the drinking water of the animals increased the probability of transfer between E. coli strains originating from the same animal species. The co-transfer of unselected antibiotic resistance in animal models was also demonstrated.     

ELISA and direct immunofluorescence test to detect equine arteritis virus (EAV) using a monoclonal antibody directed to the EAV-N protein

A monoclonal antibody (mAb) directed against the equine arteritis virus (EAV) nucleocapsid (N) protein was used for indirect enzyme-linked immunosorbent assays (ELISAs) using viral antigen from different sources. The same mAb was labelled with fluorescein isothiocyanate for direct immunofluorescence tests (DIFTs). The N-specific mAb appeared to be suitable for the detection in both ELISA and DIFT of different EAV strains and field isolates from semen and tissue samples after passage in lines of RK-13, Vero and fetal equine kidney cells. The ELISA described is an easy and fast method which can be used in most cases to replace the microneutralization test to prove the EAV specificity of the cytopathic effect of cell cultures. The DIFT, however, is more sensitive than both the ELISA and the microneutralization test because EAV antigen can be detected even in cell cultures without or with very weak cytopathic effect.     

Adjuvant and immunostimulatory effects of a D-galactose-binding lectin from Synadenium carinatum latex (ScLL) in the mouse model of vaccination against neosporosis

Vaccination is an important control measure for neosporosis that is caused by a coccidian parasite, Neospora caninum, leading to abortion and reproductive disorders in cattle and serious economic impacts worldwide. A D-galactose-binding lectin from Synadenium carinatum latex (ScLL) was recently described by our group with potential immunostimulatory and adjuvant effects in the leishmaniasis model. In this study, we evaluated the adjuvant effect of ScLL in immunization of mice against neosporosis. First, we investigated in vitro cytokine production by dendritic cells stimulated with Neospora lysate antigen (NLA), ScLL or both. Each treatment induced TNF-α, IL-6, IL-10 and IL-12 production in a dose-dependent manner, with synergistic effect of NLA plus ScLL. Next, four groups of C57BL/6 mice were immunized with NLA + ScLL, NLA, ScLL or PBS. The kinetics of antibody response showed a predominance of IgG and IgG1 for NLA + ScLL group, whereas IgG2a response was similar between NLA + ScLL and NLA groups. Ex vivo cytokine production by mouse spleen cells showed the highest IFN-γ/IL-10 ratio in the presence of NLA stimulation for mice immunized with NLA + ScLL and the lowest for those immunized with ScLL alone. After parasite challenge, mice immunized with NLA + ScLL or ScLL alone presented higher survival rates (70-80%) and lower brain parasite burden as compared to PBS group, but with no significant changes in morbidity and inflammation scores. In conclusion, ScLL combined with NLA was able to change the cytokine profile induced by the antigen or lectin alone for a Th1-biased immune response, resulting in high protection of mice challenged with the parasite, but with low degree of inflammation. Both features may be important to prevent congenital neosporosis, since protection and low inflammatory response are necessary events to guide towards a successful pregnancy.     

Effect of sex differences in donor foetal fibroblast on the early development and DNA methylation status of buffalo (Bubalus bubalis) nuclear transfer embryos

Low efficiency of somatic cell nuclear transfer (SCNT) embryos is largely attributable to imperfect reprogramming of the donor nucleus. The differences in epigenetic reprogramming between female and male buffalo cloned embryos remain unclear. We explored the effects of donor cell sex differences on the development of SCNT embryos. We and then compared the expression of DNA methylation (5‐methylcytosine‐5mC and 5‐hydroxymethylcytosine‐5hmC) and the expression level of relevant genes, and histone methylation (H3K9me2 and H3K9me3) level in SCNT‐♀ and SCNT‐♂ preimplantation embryos with in vitro fertilization (IVF) counterparts. In the study, we showed that developmental potential of SCNT‐♀ embryos was greater than that of SCNT‐♂ embryos (p < 0.05). 5mC was mainly expressed in SCNT‐♀ embryos, whereas 5hmC was majorly expressed in SCNT‐♂ embryos (p < 0.05). The levels of DNA methylation (5mC and 5hmC), Dnmt3b, TET1 and TET3 in the SCNT‐♂ embryos were higher than those of SCNT‐♀ embryos (p < 0.05). In addition, there were no significant differences in the expression of H3K9me2 at eight‐stage of the IVF, SCNT‐♀ and SCNT‐♂embryos (p < 0.05). However, H3K9me3 was upregulated in SCNT‐♂ embryos at the eight‐cell stage (p < 0.05). Thus, KDM4B ectopic expression decreased the level of H3K9me3 and significantly improved the developmental rate of two‐cell, eight‐cell and blastocysts of SCNT‐♂ embryos (p < 0.05). Overall, the lower levels of DNA methylation (5mC and 5hmC) and H3K9me3 may introduce the greater developmental potential in buffalo SCNT‐♀ embryos than that of SCNT‐♂ embryos.     

Therapeutic effect of anti-TNF-alpha antibody and levofloxacin (LVFX) in a mouse model of enterohemorrhagic Escherichia coli O157 infection

The ability of an anti-TNF-alpha antibody to confer protection against enterohaemorrhagic Escherichia coli (EHEC) O157 was investigated in germfree IQI mice. The use of an antibiotic levofloxacin (LVFX) alone or with the antibody was also studied. Protection included an increase in survival rate. Treatment with the anti-TNF-alpha antibody inhibited the histological signs associated with EHEC infection but did not prevent the colonization of EHEC or production of Shiga toxin (Stx). No clinical signs were observed and EHEC was completely eliminated in the mouse model receiving both anti-TNF-alpha antibody and LVFX. Anti-TNF-alpha antibody suppressed inflammatory cytokine response in the mouse kidney and brain by EHEC infection.