The effects of canthaxanthin on porcine oocyte maturation and embryo development in vitro after parthenogenetic activation and somatic cell nuclear transfer

The objective of this study was to examine the effects of canthaxanthin (Cx) treatment during in vitro maturation (IVM) of porcine oocytes on embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), on intracellular glutathione (GSH) and reactive oxygen species (ROS) levels in mature oocytes, and on gene expression in both PA‐ and SCNT‐derived blastocysts. To determine the optimal effective concentration of Cx, porcine oocytes were cultured in IVM medium supplemented with various concentrations (0, 20, 40 and 80 μM) of Cx for 22 hr. Compared to other groups, supplementation with 40 μM Cx significantly improved blastocyst formation rates after PA (p < .05), but no significant differences were observed among groups in total blastocyst cell numbers. Subsequently, oocytes were cultured in IVM medium supplemented with or without 40 μM Cx. Oocytes treated with 40 μM Cx showed significantly increased cleavage and blastocyst formation rates after SCNT compared to the control group (p < .05). Moreover, significantly increased intracellular GSH and reduced ROS levels were observed in the Cx‐treated group (p < .05). In addition, both PA‐ and SCNT‐derived blastocysts from the 40 μM Cx‐treated group showed significantly increased mRNA expression of Bcl2 and Oct4 and decreased Caspase3 expression level (p < .05), when compared with the control group. PA‐derived blastocysts from the 40 μM Cx‐treated group also exhibited significantly decreased expression of Bax (p < .05). Our results demonstrated that treatment with 40 μM Cx during IVM improves the developmental competence of PA and SCNT embryos. Improvement of embryo development by Cx is most likely due to increased intracellular GSH synthesis, which reduces ROS levels in oocytes, and it may also positively regulate apoptosis‐ and development‐related genes.     

Role of bacteria in the pathogenesis of recurrent uveitis in horses from the southeastern United States

OBJECTIVE: To determine the role of intraocular bacteria in the pathogenesis of equine recurrent uveitis (ERU) in horses from the southeastern United States by evaluating affected eyes of horses with ERU for bacterial DNA and intraocular production of antibodies against Leptospira spp. SAMPLE POPULATION: Aqueous humor, vitreous humor, and serum samples of 24 clinically normal horses, 52 horses with ERU, and 17 horses with ocular inflammation not associated with ERU (ie, non-ERU inflammation). PROCEDURES: Ribosomal RNA quantitative PCR (real-time PCR) assay was used to detect bacterial DNA in aqueous humor and vitreous humor from clinically normal horses (n = 12) and horses with chronic (> 3-month) ERU (28). Aqueous humor and serum were also evaluated for anti-Leptospira antibody titers from clinically normal horses (n = 12), horses with non-ERU inflammation (17), and horses with confirmed chronic ERU (24). RESULTS: Bacterial DNA was not detected in aqueous humor or vitreous humor of horses with ERU or clinically normal horses. No significant difference was found in titers of anti-Leptospira antibodies in serum or aqueous humor among these 3 groups. Only 2 horses, 1 horse with ERU and 1 horse with non-ERU inflammation, had definitive intraocular production of antibodies against Leptospira organisms. CONCLUSIONS AND CLINICAL RELEVANCE: In horses from the southeastern United States, Leptospira organisms may have helped initiate ERU in some, but the continued presence of the organisms did not play a direct role in the pathogenesis of this recurrent disease.     

Retinal detachment in horses: 40 cases (1998-2005)

OBJECTIVE: To determine clinical features, ophthalmic examination findings, etiology, treatment, and outcome of horses diagnosed with retinal detachment (RD). ANIMALS STUDIED: Forty horses, presented to the North Carolina State University and The Ohio State University Veterinary Teaching Hospitals from 1998 to 2005 that were diagnosed with RD. PROCEDURE(S): Horses with documented RD, confirmed either on ophthalmic examination or by ultrasonography, and with a complete medical record were included. Information retrieved from the medical records included signalment, presenting complaint, duration of clinical signs, ophthalmologic examination findings, diagnostics performed, identified cause of the retinal detachment, treatment given, and outcome. RESULTS: Forty horses (46 eyes) were diagnosed with RD. Mean +/- SD duration of clinical signs of ocular disease was 10.5 +/- 14.7 months. Thirty-four horses presented with unilateral involvement, 6 with bilateral, 14 with partial and 32 with complete RD. Ultrasonography was used to make the diagnosis in 26 eyes, while RD was diagnosed on routine ocular examination in 20 eyes. Bullous RD was the only type of RD observed, although small vitreal traction bands were considered secondary to the underlying inflammation or trauma. RD caused by equine recurrent uveitis (ERU) was diagnosed in 27 of 40 (67.5%) horses. Trauma-induced RD involved 10 of the 40 horses (25%). Presenting problems included known ERU (n = 16), acute or progressive vision loss (n = 9), known ocular trauma (n = 6), cataract (n = 6), and a cloudy cornea (n = 3). No horses regained vision after RD despite therapy. Many eyes were enucleated or eviscerated, or the horses were euthanized. Seven eyes with complete RD were noted to be unchanged and comfortable with medical therapy. CONCLUSIONS: The visual prognosis of RD in horses is grave; however, horses with nontraumatic RD (most commonly ERU) may be able to maintain a comfortable but blind globe with anti-inflammatory medical therapy.     

Surface Deformation and Lower Crustal Flow in Eastern Tibet

Field observations and satellite geodesy indicate that little crustal shortening has occurred along the central to southern margin of the eastern Tibetan plateau since about 4 million years ago. Instead, central eastern Tibet has been nearly stationary relative to southeastern China, southeastern Tibet has rotated clockwise without major crustal shortening, and the crust along portions of the eastern plateau margin has been extended. Modeling suggests that these phenomena are the result of continental convergence where the lower crust is so weak that upper crustal deformation is decoupled from the motion of the underlying mantle. This model also predicts east-west extension on the high plateau without convective removal of Tibetan lithosphere and without eastward movement of the crust east of the plateau.     

Effect of gentamicin on CD3+ T-lymphocyte proliferation for treatment of equine recurrent uveitis: An in vitro study

Objective

The objective of the study was to determine the effect of gentamicin on CD3+ T-lymphocyte proliferation and cell viability using an in vitro cell culture model as a means of investigating the mechanism of action of low-dose intravitreal gentamicin injection.

Animals Studied

Three adult horses with no evidence of ophthalmic or systemic disease.

Procedure

Peripheral blood lymphocytes were treated with gentamicin at concentrations 37.5 μg/mL, 112.5 μg/mL, 187 μg/mL, 375 μg/mL, or 750 μg/mL then stimulated to proliferate with concanavalin A (ConA). 4′,6-diamidino-2-phenylindole (DAPI) and carboxyfluoroscein succinimidyl ester (CSFE) were used as markers of cell viability and cell proliferation, respectively. Following 5-day culture, live cell counts and CSFE fluorescent intensity data were collected via automated cell count and flow cytometry. The experimental design was duplicated using preservative-free gentamicin and a proprietary brand formulation. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test.

Results

No statistically significant comparisons in CD3+ T-lymphocyte live cell counts and geometric mean fluorescent intensity of CSFE were identified between gentamicin concentrations or formulations.

Conclusions

Gentamicin had no effect on equine peripheral blood CD3+ T-lymphocyte cell viability and proliferation in concentrations ranging from “safe” to “retinotoxic” in relation to intravitreal injection volumes. Low-dose intravitreal gentamicin may not suppress the Th1- and Th17-mediated immune response.     

A retrospective comparison of surgical removal and subsequent CO2 laser ablation versus topical administration of mitomycin C as therapy for equine corneolimbal squamous cell carcinoma

Objective To compare the complications and nonrecurrence rate following topical mitomycin C (MMC) therapy vs. CO₂ laser ablation for treating equine corneolimbal squamous cell carcinoma (SCC). Study design Retrospective study. Sample population Twenty‐five horses with corneolimbal SCC. Procedures Medical records of horses undergoing surgical tumor resection followed by either topical MMC therapy (0.04%) or CO₂ laser ablation between the years of 2004 and 2010 were reviewed. Recurrence and complications were compared between groups and within MMC subgroups defined by the time at which treatment was initiated relative to surgery. Results Therapy with topical MMC resulted in a nonrecurrence rate comparable to that achieved with CO₂ laser ablation (82.4% vs. 85.7%, respectively). Initiation of MMC following epithelialization of the surgical site a mean of 15 days postoperatively did not result in increased recurrence rates relative to treatment in the immediate postoperative period. Vision‐ or globe‐threatening complications tended to occur with greater frequency in horses receiving topical MMC in the immediate postoperative period (5 of 6 major complications) relative to following epithelialization of the surgical site (1 of 6 major complications). Conclusions Horses receiving adjunctive topical MMC therapy were no more likely to experience tumor recurrence than were horses undergoing CO₂ laser ablation in the horses in this study. Initiation of two to three rounds of MMC following epithelialization of the surgical site results in fewer major complications and achieves comparable disease resolution relative to treatment in the immediate postoperative period.     

Functional Role of Feline Zona Pellucida Protein 4 Trefoil Domain: A Sperm Receptor or Structural Component of the Domestic Cat Zona Pellucida?

The trefoil domain (TFD) is a protein structure characterized by six cysteines, which form a typical three-loop structure by three disulphide bridges. It is assumed that two of these loops generate a hydrophobic groove, which could be a binding site for carbohydrate residues or proteins. The zona pellucida (ZP) protein, ZP4, contains such a TFD. The carbohydrate-/protein-binding property of TFD allows us to assume a potential sperm receptor function of this domain. Additionally, gastrointestinal trefoil peptides are stable against proteases; therefore, a structural role of TFD within the ZP might also be possible. We were able to show that the synthesized and natural folded feline TFD (fTFD) expresses the typical protease resistance that vanished under reducing conditions and after substitution of cysteine residues within the peptide. Furthermore, an antibody directed against the first loop of fTFD was almost unable to bind to intact in vitro mature cat oocytes. Pre-incubation of oocytes in the reducing agent (DDT), however, improved antibody binding substantially. Therefore, we suggest structural masking of the fTFD domain within the intact ZP. An interaction between fTFD and feline sperm cells was examined using several methods, including immunocytochemistry, immunoelectron microscopy, co-immunoprecipitation and far western blot, but we found no indication for an involvement of TFD in the primary sperm binding to the ZP. To summarize, there is increasing evidence that the TFD of fZP4 has a structural rather than a sperm-binding function.     

Bartonella vinsonii subspecies berkhoffi as a possible cause of anterior uveitis and choroiditis in a dog

A 2-year old, neutered, female spaniel mixed breed was referred to the North Carolina State University Veterinary Teaching Hospital for evaluation of bilateral anterior uveitis. The dog was febrile and, in addition to anterior uveitis, multifocal hyporeflective lesions were present in the tapetal fundus of both eyes. The antibody titer for Bartonella vinsonii subspecies berkhoffi was positive (1 : 512). Aqueous paracentesis was performed for PCR in an attempt to detect B. vinsonii in the eye but was unsuccessful. The ocular manifestations of Bartonella infection in humans are currently expanding as more sensitive serologic and PCR techniques are being developed to identify Bartonella spp. In addition to optic neuritis and neuroretinitis, retinochoroidal lesions are one of the most common manifestations of B. henselae infection, and are frequently accompanied by vitreous or anterior segment inflammation. Diagnosis of a Bartonella infection in humans can be made on serology alone, in conjunction with ocular examination findings. The ultimate proof of B. vinsonii (berkhoffi) as a direct cause of ocular disease would be detection of the infectious agent in the eye. However, it is unknown at this time whether Bartonella causes ocular disease primarily, secondarily via an autoimmune reaction, or both. Due to the difficulties associated with culture of Bartonella spp. and the limitations of PCR, serology is currently the most useful tool for screening dogs for possible Bartonella spp. infection. In the case presented here, even though the PCR was negative, the clinical signs of anterior uveitis and choroiditis might reasonably be associated with B. vinsonii (berkhoffi) seroreactivity, which was repeatable on three separate occasions. Clinical improvement was also accompanied by a post-treatment decrease in B. vinsonii (berkhoffi) seroreactivity, potentially supporting resolution of Bartonella infection in this dog. This is the first reported case of a possible association between uveitis, choroiditis and Bartonella infection in the dog, without clinical manifestations of other organ or tissue involvement. Future studies based on PCR analysis of intraocular fluids may clarify the involvement of B. vinsonii (berkhoffi) in dogs with intraocular inflammatory disease. Furthermore, performing fluorescein angiography in dogs with elevated Bartonella titers may also prove useful in the identification and characterization of lesions.