Fertility Assessment in Sorraia Stallions by Sperm‐Fish and Fkbp6 Genotyping

The Sorraia, a critically endangered indigenous Iberian horse breed, is characterized by low genetic variability, high rate of inbreeding, bad sperm quality and subfertility. Here, we studied 11 phenotypically normal but subfertile Sorraia stallions by karyotyping, sex chromosome sperm‐FISH and molecular analysis of FKBP6 – a susceptibility locus for impaired acrosome reaction (IAR). The stallions had normal sperm concentration (>300 million cells/ml), but the numbers of progressively motile sperm (21%) and morphologically normal sperm (28%) were invariably low. All stallions had a normal 64,XY karyotype. The majority of sperm (89%) had normal haploid sex chromosome content, although 11% of sperm carried various sex chromosome aneuploidies. No correlation was found between the percentage of sperm sex chromosome abnormalities and inbreeding, sperm morphology or stallion age. Direct sequencing of FKBP6 exon 4 for SNPs g.11040315G>A and g.11040379C>A revealed that none of the stallions had the susceptibility genotype (A/A‐A/A) for IAR. Instead, all animals had a G/G‐A/A genotype – a testimony of low genetic variability. The findings ruled out chromosomal abnormalities and genetic predisposition for IAR as contributing factors for subfertility. However, low fertility of the Sorraia stallions could be partly attributed to relatively higher rate of sex chromosome aneuploidies in the sperm.     

Effect of Dimethyl Sulfoxide on Cell Cycle Synchronization of Goldfish Caudal Fin Derived Fibroblasts Cells

Several studies have previously been conducted regarding cell cycle synchronization in mammalian somatic cells. However, limited work has been performed on the control of cell cycle stages in the somatic cells of fish. The aim of this study was to determine the cell cycle arresting effects of several dimethyl sulfoxide (DMSO) concentrations for different times on different cell cycle stages of goldfish caudal fin‐derived fibroblasts. Results demonstrated that the cycling cells or control group (68.29%) yields significantly higher (p < 0.05) arrest in G0/G1 phase compared with the group treated for 24 h with different concentrations (0.5%, 1.0% or 1.5%) of DMSO (64.88%, 65.70%, 64.22% respectively). The cell cycle synchronization in the treatment of cells with 1.0% DMSO at 48 h (81.14%) was significantly higher than that in the groups treated for 24 h (76.82%) and the control group (77.90%). Observations showed that treatment of DMSO resulted in an increase in the proportion of cells at G0/G1 phase for 48 h of culture. However, high levels of apoptotic cells can be detected after 48 h of culture treated with 1% concentration of DMSO.     

Influence of Inseminate Components on Porcine Leucocyte Migration In Vitro and In Vivo after Pre- and Post-Ovulatory Insemination

A post‐breeding migration of leucocytes (PMN) into the uterus is considered to be an important reason for sperm losses. Minimizing such effects may be necessary for successful insemination with low sperm numbers, as required with sex‐sorted spermatozoa. We examined the magnitude of PMN influx 3 h after pre‐ or post‐ovulatory insemination with various combinations of seminal plasma (SP), semen extender Androhep? (AH; Minitüb, Tiefenbach, Germany) and sperm preparations (S). Pre‐ovulatory inseminations with preparations containing 98% AH caused a massive influx of PMN, independent of whether spermatozoa were present (628 ± 189 × 10⁶ leucocytes/uterine horn) or not (580 ± 153 × 10⁶). Post‐ovulatory, 98% AH caused a comparable immigration only in the absence of sperm cells (AH: 569 ± 198 × 10⁶, AH+S: 162 ± 102 × 10⁶). The presence of SP significantly dampened the numbers of recruited uterine leucocytes. The reaction to all inseminates containing 98% SP both with and without spermatozoa, used before ovulation (SP: 14 ± 6 × 10⁶, SP+S: 73 ± 27 × 10⁶) and after ovulation (SP: 60 ± 32 × 10⁶, SP+S: 51 ± 33 × 10⁶) did not differ significantly from controls using phosphate buffered saline (PBS) (pre‐ovulatory: 1 ± 1 × 10⁶, post‐ovulatory: 11 ± 9 × 10⁶). Quantitative in vitro transmigration assays with blood‐derived PMN proved that AH‐induced leucocyte migration into the uterus to be not as a result of direct chemotaxis, because, on account of the chelator citrate, AH significantly inhibited the transmigration towards recombinant human Interleukin‐8 (rhCXCL8) (AH: 14 ± 5% migration rate vs controls: 37 ± 6%, p < 0.05). Supernatants of spermatozoa incubated in PBS for 1, 12 or 24 h showed neither chemoattractive nor chemotaxis‐inhibiting properties. SP at ≥0.1% [v/v] significantly inhibited the in vitro transmigration of PMN. With respect to in vivo migration of neutrophils, the striking difference in the results between semen extender and seminal plasma suggests that adaptation of extender composition is needed to reflect more closely the in vivo regulatory potential of natural seminal plasma.     

Characteristics of methicillin resistant Staphylococcus aureus isolated from chicken meat and hospitalized dogs in Korea and their epidemiological relatedness

Methicillin resistant Staphylococcus aureus (MRSA) is one of the most important pathogens in human and veterinary hospitals. The isolation of MRSA from animals and foodstuffs has been reported with an increased incidence. However, methicillin (oxacillin) is not used in animal husbandry or in animal hospitals in Korea. In this study, three pre-MRSA and one silent mecA-carrying methicillin susceptible S. aureus (smMSSA) were isolated from retail chicken meat, and three MRSA were isolated from hospitalized dogs in Korea. The three pre-MRSA isolates were determined to have a staphylococcal cassette chromosome mec (SCCmec) type III, and the smMSSA isolate was not classified. The animal hospital isolates were found to contain SCCmec type II. Seven and 15 S. aureus isolated from hospitalized humans and bovine milk, respectively, were also examined in this study in order to determine the epidemiological origins of MRSA. Multilocus sequencing typing (MLST) revealed that the chicken meat and bovine milk isolates were closely related to the animal hospital isolates. The SCCmec characteristics and MLST analyses indicated the possibility of the human to animal transmission of MRSA. These results highlight the importance of identifying MRSA carriers as well as intercepting MRSA transmission because MRSA is becoming increasingly widespread without any plausible relationship with the use of methicillin (oxacillin).     

Comparison of orbital prosthesis motility following enucleation or evisceration with sclerotomy with or without a motility coupling post in dogs

Objective  To evaluate motility of silicone orbital implants and corneoscleral prostheses, with and without use of a motility coupling post (MCP) in dogs.
Animals studied  Eighteen mixed-breed dogs.
Procedures  The motility of an orbital silicone implant and corneoscleral prosthesis after enucleation ( n  = 6), evisceration ( n  = 6), or use of a MCP with evisceration ( n  = 6) in dogs were compared. One eye from each dog had surgery whereas the opposite eye was used as a control. Clinical evaluations were performed three times a week. Histopathology of the orbital tissues was performed 8 and 12 weeks after surgery.
Results  Implant motility in dogs with evisceration (vertical movement [VM] 8.04 ± 2.13; horizontal movement [HM] 11 ± 3.05) and evisceration with MCP (VM 9.61 ± 1.59); HM was significantly greater than the enucleation group (VM 0.51 ± 0.5; HM 1.22 ± 0.68) ( P <  0.01). Prosthetic motility in dogs with evisceration with MCP was significantly greater than in dogs with evisceration; dogs with evisceration had significantly greater motility than dogs with enucleation ( P <  0.01). No postoperative complications were observed in any of the groups. No significant abnormalities were noted on histopathology.
Conclusions  MCP placement in silicone orbital implants significantly enhanced the prosthetic motility in dogs. This study supports the use of MCP in silicone orbital implants to enhance corneoscleral prosthesis motility and cosmetics in dogs.     

Rat allogeneic mesenchymal stem cells did not prolong the survival of corneal xenograft in a pig-to-rat model

Objective  Recent reports have demonstrated that mesenchymal stem cells (MSCs) can modulate/suppress immunologic responses through interactions with different immune cells. We performed this study in order to investigate the immunomodulatory effects of MSCs in corneal xenotransplantation.
Animals studied  Pig and rat.
Procedures  We orthotopically transplanted pig corneas into rats and topically applied allogeneic rat MSCs to the corneas for 2 h immediately after transplantation. Graft survival was clinically assessed using slit-lamp biomicroscopy and the median survival time (MST) was calculated. The rejected grafts were histologically examined using antibodies against CD4, CD8, CD161, and CD68. The expression of IL-2, IL-6, IL-10, and IFN-γ was also evaluated in the rejected grafts using an enzyme-linked immunosorbent assay.
Results  The survival of corneal xenografts was not significantly prolonged by MSC application (MST 10.5 days) compared with the controls (MST 9.67 days) ( P  = 0.4189). Histologically, the rejected grafts in both groups were massively infiltrated with neutrophils and macrophages. Some CD8⁺ T cells and rare NK cells were found in the rejected grafts. The levels of IL-6 and IL-10 were significantly increased in the rejected grafts from MSC-treated rats compared with the grafts from MSC-untreated rats. However, the levels of IL-2 and IFN-γ were not different between the two groups.
Conclusions  Topical application of allogeneic rat MSCs was ineffective in prolonging corneal xenograft survival in a pig-to-rat model.     

Effect of synchronization of follicle-wave emergence with estradiol and progesterone and superstimulation with follicle-stimulating hormone on milk estrogen concentrations in dairy cattle

Very little is known about the effects of hormonal synchronization of follicle waves and superovulation on the estrogen content of a cow’s milk. The objective of this study was to determine the effect in dairy cows of synchronization with estradiol-17β (E2) and progesterone (P4) on milk E2 concentrations and to compare these levels with those achieved during superstimulation for 4 d with porcine follicle-stimulating hormone (FSH). The milk E2 concentrations were raised significantly above pretreatment levels (P < 0.05) for 2 d after synchronization, the mean peak being 40.2 ± 18.5 (standard error) pg/mL and the pretreatment mean 1.5 ± 0.5 pg/mL. The mean peak E2 concentration during ovarian stimulation was 4.4 ± 0.7 pg/mL. The mean E2 concentration was significantly higher (P < 0.05) after synchronization than during superstimulation for the 1st milking after synchronization but not subsequent milkings. The milk estrone concentrations were above pretreatment levels for 1 d after synchronization and were not different from those observed during superstimulation.     

3D-culture models as drug-testing platforms in canine lymphoma and their cross talk with lymph node-derived stromal cells

BackgroundMalignant lymphoma is the most common hematopoietic malignancy in dogs, and relapse is frequently seen despite aggressive initial treatment. In order for the treatment of these recurrent lymphomas in dogs to be effective, it is important to choose a personalized and sensitive anticancer agent. To provide a reliable tool for drug development and for personalized cancer therapy, it is critical to maintain key characteristics of the original tumor.ObjectivesIn this study, we established a model of hybrid tumor/stromal spheroids and investigated the association between canine lymphoma cell line (GL-1) and canine lymph node (LN)-derived stromal cells (SCs).MethodsA hybrid spheroid model consisting of GL-1 cells and LN-derived SC was created using ultra low attachment plate. The relationship between SCs and tumor cells (TCs) was investigated using a coculture system.ResultsTCs cocultured with SCs were found to have significantly upregulated multidrug resistance genes, such as P-qp, MRP1, and BCRP, compared with TC monocultures. Additionally, it was revealed that coculture with SCs reduced doxorubicin-induced apoptosis and G2/M cell cycle arrest of GL-1 cells.ConclusionsSCs upregulated multidrug resistance genes in TCs and influenced apoptosis and the cell cycle of TCs in the presence of anticancer drugs. This study revealed that understanding the interaction between the tumor microenvironment and TCs is essential in designing experimental approaches to personalized medicine and to predict the effect of drugs.     

Seroprevalence of Canine Herpesvirus‐1 in the Belgian Dog Population in 2000

Canine herpesvirus‐1 (CHV‐1) is known to be associated with fertility and fecundity disorders as well as neonatal mortality in puppies of less than 3 weeks of age. The virus is presumed to be enzootic in dogs all over the world and recent studies in several European countries suggest a high seroprevalence among the dog population. In the year 2000, a total of 647 Belgian canine sera from 102 privately owned patients and 545 breeding dogs were analysed with an enzyme‐linked immunosorbent assay (ELISA). Furthermore 77 of the samples were submitted to two serum neutralization (SN) tests for comparison. An overall CHV‐1 seroprevalence of 45.75% was observed in the Belgian dog population. No significant differences could be observed based on breeding status, reason for consultation or sex. The correlation between the ELISA and both SN tests appeared to be moderate with a significantly greater sensitivity of the ELISA. This study also demonstrated that the CHV‐1 seroprevalence in the Belgian dog population is similar to that in other recently investigated European countries and that the incidence in breeding units is not necessarily higher than in non‐breeding dogs.     

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.