Isolation of Canine Mammary Cells With Stem Cell Properties and Tumour-Initiating Potential

Recent data suggest that mammary carcinogenesis may be driven by cancer stem cells (CSCs) derived from mutated adult stem cells, which have acquired aberrant cell self-renewal or by progenitor cells that have acquired the capacity for cell self-renewal. Spontaneous mammary cancers in cats and dogs are important models for the understanding of human breast cancer and may represent alternative species model systems that can significantly contribute to the study of human oncogenesis. With the goal of identifying markers for isolating human breast CSCs, we have generated a canine model system to isolate and characterize normal and CSCs from dog mammary gland. Insight into the hierarchical organization of canine tumours may contribute to the development of universal concepts in oncogenesis by CSCs. Cells with stem cell properties were isolated from normal and tumoural canine breast tissue and propagated as mammospheres and tumourspheres in long-term non-adherent culture conditions. We showed that cells obtained from spheres that display self-renewing properties, have multi-lineage differentiation potential, could generate complex branched tubular structures in vitro and form tumours in NOD/SCID mice. We analysed these cells for the expression of human stem and CSC markers and are currently investigating the tumour-initiating properties of these cells and the hierarchical organization of normal and neoplastic canine mammary tissue.     

Equine amnionitis and fetal loss: The case definition for an unrecognised cause of abortion in mares

A series of abortions occurred in mares in New South Wales during 2004 that involved similar and unusual findings on post mortem examination of aborted fetuses and fetal membranes. The term Equine Amnionitis and Fetal Loss (EAFL) was developed to describe the condition. This form of abortion had not been previously recognised in Australia. The pathology alone is not specific for EAFL and diagnosis requires demonstration of a combination of certain pathological and bacteriological features. The purpose of this paper is to describe patterns considered consistent with EAFL cases as a working case definition for use by veterinarians and veterinary pathologists in identifying future cases of EAFL. More detailed papers are in preparation to fully describe the epidemiological, histopathological, and microbiological aspects of EAFL.     

Effects of a Simulated Agricultural Runoff Event on Sediment Toxicity in a Managed Backwater Wetland

We examined the effects of an amended mixture of three pesticides, atrazine (72.7?g), S-metolachlor (54.5?g), and permethrin (both cis and trans isomers; 11.4?g), on 10-day sediment toxicity to Hyalella azteca in a managed natural backwater wetland after a simulated agricultural runoff event. Sediment samples were collected at 10, 40, 100, 300, and 500?m from inflow 13?days prior to amendment and 1, 5, 12, 22, and 36?days post-amendment. Background pesticide concentrations ranged from <1 to 977, <1 to 119, and <1 to 2???g?kg^?1, for atrazine, S-metolachlor, and permethrin, respectively. Average post-amendment atrazine and S-metolachlor were 2,915?C3,927 and 3?C20???g?kg^?1, respectively at 10?C40?m and 538?C872 and <1???g?kg^?1, respectively at 300?C500?m. Average post-amendment permethrin was 65?C200???g?kg^?1 at 10?C40?m and 1?C10???g?kg^?1 at 300?C500?m. H. azteca 10-day survival varied spatially and temporally up to 100?m from inflow. Animal growth, independent of survival, was reduced 40 and 100?m from inflow on day?36, showing continued sediment toxicity of up to 100?m from inflow more than 1?month after amendment. Animal survival and growth were unaffected at 300 and 500?m from inflow throughout the study period. Correlations of pesticide concentrations and H. azteca responses indicated that observed sediment toxicity was primarily from permethrin with potential additional synergistic toxicity from atrazine and methyl parathion. Study results indicate that natural backwater wetlands can be managed to ameliorate pesticide mixture 10-day sediment toxicity to H. azteca within 300?m of inflow and smaller wetlands (??100?m) may require several months of effluent retention to mitigate effects.     

Selected Metabolic and Hormonal Profiles during Maintenance of Spontaneous Ovarian Cysts in Dairy Cows

Information is lacking regarding the relationship between metabolic and hormonal profiles and the maintenance of spontaneous ovarian cyst disease in dairy cows. For this reason, the concentrations of non‐esterified fatty acids (NEFA), insulin‐like growth factor I (IGF‐I) and cortisol (C) were investigated during the spontaneous course of ovarian cyst disease in dairy cows (n = 6) between the 7th and 16th weeks post‐partum (PP). The control group consisted of normally cycling cows (n = 6). Blood samples were collected twice a day, and plasma was analysed using different techniques. Progesterone and 15‐ketodihydro‐PGF_2α plasma profiles were investigated to confirm the ovulatory or anovulatory conditions of the cows. Cortisol plasma levels were not significantly different among sampling times within each group or between the two groups. NEFA plasma levels were significantly higher in cycling cows compared to cystic cows at the 16th week PP (p < 0.01), but with rather low values, indicating by now sparse mobilization of fat stores. Insulin‐like growth factor I plasma concentrations were higher in cystic cows during the 8th, 10th, 11th (p < 0.01) and 16th week PP (p < 0.05), indicating that the presence of ovarian cysts coincides with increased IGF‐I levels. These results suggest no influence of cortisol and NEFA levels in cysts maintenance, while a possible involvement of IGF‐I can be suspected not only in the pathogenesis, as already known, but also in the maintenance of spontaneous cystic ovarian disease in cattle.     

Fertility and motility of sperm from Atlantic halibut (Hippoglossus hippoglossus) in relation to dose and timing of gonadotrophin-releasing hormone agonist implant

In broodstocks of Atlantic halibut, Hippoglossus hippoglossus, male and female gamete production often becomes unsynchronised towards the end of the spawning season—milt becomes very viscous and difficult to express while the females are still producing batches of good quality eggs. Gonadotrophin-releasing hormone agonist (GnRHa) has been shown to stimulate spermiation in a number of fish species. Therefore, we conducted two experiments where male halibut were implanted intramuscularly with pellets containing GnRHa. The effect of the pellets was tested at three periods: before, at the height of and at the end of spermiation. In the middle period, GnRHa was tested at two doses (5 and 25 μg/kg bodyweight). Measurements were made of milt hydration, sperm motility and fertilisation rate. Implanted males began spermiation at least 4 weeks before control males. Both doses of GnRHa increased the fluidity of the milt. This effect lasted for at least 20 days in the low dose group and for 40 days in the high dose group. When applied at the end of the season, GnRHa reversed the normal trend for the milt to become more viscous. GnRHa treatments did not affect fertilisation rates obtained with the sperm. However, towards the end of the spawning season, sperm motility was enhanced in males treated with the high dose of GnRHa (25 μg/kg) compared to controls. As described previously, plasma concentrations of the gonadal steroids, 5β-pregnane-3β,17,20β-triol 20-sulphate and 17,20-dihydroxy-4-pregnen-3-one, were significantly enhanced by GnRHa treatment. Concentrations of testosterone on the other hand decreased when spermiating males were treated with GnRHa. Our data suggest that 17,20β-dihydroxy-4-pregnen-3-one or its metabolites are involved in milt hydration, possibly through affecting ion transport.     

A comparison of the autecology of Quercus robur L. and Q. pyrenaica Wild.: present habitat in Galicia, NW Spain

The biotopes occupied by pedunculate oak (Quercus robur L.)and rebollo oak (Quercus pyrenaica Willd.) in Galicia were characterizedusing different parameters, enabling a comparative analysisof its habitats. Thirty-nine plots of pedunculate oak and 40of rebollo oak were inventoried and 25 ecological parameters(physiographic, climatic and edaphic) were measured to describethe biotopes, along with 14 stand parameters to characterizethe structure and the silviculture of these forests; some ofthem were used in previous works about the autecology of bothspecies. Quercus robur was found to occupy a wider range ofecological habitats and their distribution is oceanic; Q. pyrenaicapresents a more Mediterranean phytoclimatic position. The resultsindicated that the distribution of oak forests in Galicia ismore closely related to physiographic and climatic featuresthan to edaphic, because the nature of the substrates that theyoccupy is similar. Analysis of fragility/aggressiveness betweenboth species and chestnut shows that the aggressiveness of bothoaks, mainly pedunculate, was much higher, except at high altitudes,and in areas with a high mean temperature, where rebollo ismore aggressiveness. Comparison of the two Quercus shows formost of the parameters that Q. robur is the more aggressivespecies.     

Monoclonal antibodies for coagglutination of Streptococcus suis type 1

Monoclonal antibodies (MAbs) were sought that would identify Streptococcus suis. Spleen cells from Balb/c mice immunized with formalin-killed S. suis were fused with the murine cell-line SP-2/0. Hybridomas positive for S. suis were identified by a solid-phase radioimmunoassay with a mixture of all available serotypes of S. suis. On further screening with individual serotypes of S. suis (Types 1,2,1/2,3,4,5,7,8,9 and 12) and other related and unrelated bacteria, a hybridoma specific for Type 1 was identified. Another hybridoma was positive for all serotypes of S. suis. S. suis Type 1-specific MAbs in the form of ascites fluid was used in a coagglutination test. Agglutination with S. suis Type 1 was observed within 45 s, while there was no reaction with other serotypes of S. suis or other bacteria, during the 5-min observation period. A library of type-specific MAbs should help in rapid and accurate serotyping of S. suis isolates.     

Influence of Post‐Mortem Sperm Recovery Method and Extender on Unstored and Refrigerated Rooster Sperm Variables

Many post‐mortem sperm collection techniques have been described for mammalian species, but their use in birds is scarce. This paper compares the efficacy of two post‐mortem sperm retrieval techniques ‐ the flushing and float‐out methods ‐ in the collection of rooster sperm, in conjunction with the use of two extenders, i.e., L&R‐84 medium and Lake 7.1 medium. To determine whether the protective effects of these extenders against refrigeration are different for post‐mortem and ejaculated sperm, pooled ejaculated samples (procured via the massage technique) were also diluted in the above extenders. Post‐mortem and ejaculated sperm variables were assessed immediately at room temperature (0 h), and after refrigeration at 5°C for 24 and 48 h. The flushing method retrieved more sperm than the float‐out method (596.5 ± 75.4 million sperm vs 341.0 ± 87.6 million sperm; p < 0.05); indeed, the number retrieved by the former method was similar to that obtained by massage‐induced ejaculation (630.3 ± 78.2 million sperm). For sperm collected by all methods, the L&R‐84 medium provided an advantage in terms of sperm motility variables at 0 h. In the refrigerated sperm samples, however, the Lake 7.1 medium was associated with higher percentages of viable sperm, and had a greater protective effect (p < 0.05) with respect to most motility variables. In conclusion, the flushing method is recommended for collecting sperm from dead birds. If this sperm needs to be refrigerated at 5°C until analysis, Lake 7.1 medium is recommended as an extender.     

Experience and confidence of final year veterinary students in performing desexing surgeries

AIMS: To describe the level of experience and confidence of veterinary students in performing canine and feline desexing procedures at the end of their final clinical year.

METHOD: A cross-sectional survey was conducted with veterinary students at Massey University in November 2017 after completion of their final clinical year. The questions included career plans after graduation, number of assisted and unassisted desexing procedures performed, approximate time to complete desexing surgeries, level of confidence with different aspects of desexing surgeries, what aspects of their desexing surgery training were most helpful, and what could be done to improve training in desexing surgical skills in veterinary school.

RESULTS: The survey was completed by 70/95 (74%) students in their final clinical year. Among respondents, 55/70 (70%) had performed >2 unassisted feline neuters before graduation. However 38/70 (54%) students had never performed an unassisted feline spay, 31/70 (44%) had never performed an unassisted canine neuter, and 44/70 (63%) students had never performed an unassisted canine spay. The median reported times to complete a feline neuter, feline spay, canine neuter, and canine spay were 9, 40, 30 and 60 minutes, respectively. The median level of confidence for these procedures were 9, 6, 7 and 5 (on a scale from 1=least confident to 10=most confident), respectively. The reported time to complete procedures and the confidence in performing procedures did not change markedly with increasing total number of procedures performed. Students were most concerned about their ability to perform the desexing procedures in a reasonable amount of time and to prevent post-operative bleeding from occurring. Students were least concerned with their ability to manage post-operative pain in patients and to select the appropriate suture material. Free-text comments revealed that 62/70 (89%) students wanted more hands-on surgical experience prior to graduation.

CONCLUSION AND CLINICAL RELEVENCE: Many students are currently completing veterinary school with limited experience and low confidence with performing routine canine and feline desexing procedures. Further research is needed to identify the most effective ways for addressing this issue within the constraints of the veterinary curriculum and teaching hospital resources.