Evaluation of Seasonal Heat Stress on Transcriptomic Profiles and Global DNA Methylation of Bovine Oocytes.

Heat stress affects oocyte developmental competence and is a major cause of reduced fertility in heat stressed cattle. Negative effects of heat stress on the oocyte have been observed at morphological, biochemical and developmental levels. However, the mechanisms by which heat stress affects the oocyte at the transcriptional and epigenetic levels remain to be further elucidated. Here we aimed to investigate the effect of heat stress on oocyte quality, transcriptomic profiles and DNA methylation of oocytes collected through the transition from spring to summer under Louisiana conditions. Summer season resulted in a lower number of high quality oocytes obtained compared to the spring season. There was no difference in in vitro maturation rates of oocytes collected during spring as compared to summer. RNA sequencing analysis showed that a total of 211 and 92 genes were differentially expressed as a result of heat stress in GV and MII oocytes, respectively. Five common genes (E2F8, GATAD2B, BHLHE41, FBXO44, and RAB39B) were significantly affected by heat in both GV and MII oocytes. A number of pathways were also influenced by heat stress including glucocorticoid biosynthesis, apoptosis signaling, and HIPPO signaling in GV oocytes, and Oct4 pluripotency, Wnt/beta-catenin signaling, and melatonin degradation I in MII oocytes. In addition, fluorescent immunocytochemistry analysis showed no difference in global levels of DNA methylation and DNA hydroxymethylation at either the GV or MII stage between spring and summer oocytes. The results of this study contribute to a better understanding of the effect of heat stress on the molecular mechanisms altered in bovine oocytes.

Effects of Combined Estradiol-Sulpiride Treatment and Follicle Ablation on Vernal Transition in Mares: Evaluation of Plasma and Follicular Fluid Hormones and Luteinizing Hormone Receptor Gene Expression.

This experiment assessed the hormonal production, secretory aspects, and changes in luteinizing hormone (LH) receptor gene expression of early induced ovulatory-sized follicles relative to the first ovulatory-sized follicles occurring naturally in the spring. Anovulatory mares were treated on January 21 with (1) 50 mg of estradiol cypionate (ECP, n = 8) alone or (2) with ECP followed by two 3-g sulpiride injections (n = 8), 5 and 12 days later. Half of each group also received complete follicle ablation via transvaginal aspiration before ECP treatment. Ovaries were scanned regularly until detection of a 32-35 mm follicle; follicular fluid was recovered via aspiration for analysis of hormonal concentrations. Blood was collected regularly to characterize plasma prolactin, LH, follicle stimulating hormone, progesterone, and estradiol concentrations. Mean date to first 35-mm follicle was earlier (P < .05) in sulpiride-treated mares: five of eight (63%) responded within 28 days of the first sulpiride injection. Ablation did not affect ovarian response. Plasma prolactin was stimulated (P < .0001) in ECP-sulpiride-treated mares for 16 days but did not dictate ovarian response. Estradiol stimulated plasma LH (P < .05), which was higher (P < .05) in treated mares that responded. There was no effect of treatment or ablation on follicular fluid concentrations of estradiol, progesterone, leptin, or insulin-like growth factor 1 or on LH receptor gene expression. These latter similarities indicate that ECP-sulpiride early induced follicles have apparently reached a degree of maturity equivalent to naturally occurring ovulatory-sized follicles later in the spring.

Characterization of prostate cancer cell progression in zebrafish xenograft model.

Early diagnosis of prostate cancer (PCa) is critical for the application of efficient treatment to PCa patients. However, the majority of PCas remains indolent from several months to several years before malignancy. Current diagnosis methods have limitations in their reliability and are inefficient in time cost. Thus, an efficient in vivo PCa cell xenograft model is highly desired for diagnostic studies in PCas. In the present study we present a standardized procedure to create a PCa cell xenograft model using zebrafish (Danio rerio) as the host. PC3-CTR cells, a cell line from adenocarcinoma with stable expression of calcitonin receptor (CRT), were subcutaneously injected into zebrafish larvae at 48 h post fertilization. The nursing conditions for the larvae were optimized with stable survival rates of post hatch and post PC3-CTR cell injection. In this system, the progression of PC3-CTR cells in vivo was evaluated by migration and proliferation of the cells. Massive migrations of PC3 cells in vivo were observed at post injection day (PID)3. The injected PC3-CTR cells eventually invaded the whole larval zebrafish at PID5. Quantification of PC3-CTR cell proliferation was done using quantitative PCR (qPCR) analysis targeting the expression profiles of two PCa housekeeping genes, TATA-binding protein (TBP) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) encoding genes. The excessive proliferation of PC3 cells in vivo was detected with both qPCR assays. Expression levels of one non­coding gene, prostate cancer associated 3 gene (pca3), and two other genes encoding transient receptor potential ion channel Melastatin 8 (trpm8) and prostate-specific membrane antigen (psma), showed a significantly enhanced aggressiveness of PC3-CTR cells in vivo. The model established in the present study provides an improved in vivo model for the diagnosis of PCas efficiently. This PCa cell xenograft model can also serve as a tool for high throughput anti-PCa drug screening in therapeutic treatments.

Observational study of patient and surgeon preoperative preparation in ten companion animal clinics in Ontario, Canada.

BACKGROUND: Surgical site infections (SSIs) are a recognized risk of any surgical procedure in veterinary medicine. One of the keys to prevention of SSIs is reducing exposure of the surgical site to endogenous and exogenous microbes, beginning in the preoperative period. While guidelines are available for preoperative preparation procedures, there has been no objective investigation of compliance with these recommendations in veterinary practices. The objectives of this pilot study were to describe preoperative patient and surgeon preparation practices in a sample of non-equine companion animal veterinary clinics, and to determine if there were any areas that consistently did not meet current guidelines. RESULTS: Observation of preparation practices was performed in 10 clinics over 9-14 days each using up to 3 small wireless surveillance cameras. Data were coded for 148 surgical patients, and 31 surgeons performing 190 preoperative preparations. When patient hair removal was observed, it was most commonly done using clippers (117/133, 88%), and in only one case was it performed prior to anesthetic induction. Patient contact time with soap ranged from 10-462 s (average of clinic means 75 s, average of clinic medians 67 s), and with alcohol from 3-220 s (average of clinic means 44 s, average of clinic medians 37 s). Alcohol-based hand rub (AHR) was used preoperatively in 2/10 facilities, but soap-and-water hand scrub was most commonly used at all clinics. Proximal-to-distal scrubbing was noted in 95/142 (67%) of soap-and-water scrubs. Contact time during surgeon hand preparation ranged from 7-529 s (average mean 121 s, average median 122 s) for soap-and-water and from 4-123 s (average mean 25 s, average median 19 s) for AHR. No significant changes in practices were identified over time during the observation period. Practices that did not conform to guidelines available in major companion animal surgical textbooks were commonly observed. CONCLUSIONS: Some preoperative preparation practices were relatively consistent between clinics in this study, while others were quite variable. Contact times with preoperative preparatory solutions for both patients and surgeons were often shorter than recommended. Evidence-based guidelines for these procedures in veterinary medicine should be established and implemented in order to help reduce preventable SSIs, while maintaining efficiency and cost-effectiveness.