Generation of LIF-independent induced pluripotent stem cells from canine fetal fibroblasts.

Takahashi and Yamanaka established the first technique in which transcription factors related to pluripotency are incorporated into the genome of somatic cells to enable reprogramming of these cells. The expression of these transcription factors enables a differentiated somatic cell to reverse its phenotype to an embryonic state, generating induced pluripotent stem cells (iPSCs). iPSCs from canine fetal fibroblasts were produced through lentiviral polycistronic human and mouse vectors (hOSKM/mOSKM), aiming to obtain pluripotent stem cells with similar features to embryonic stem cells (ESC) in this animal model. The cell lines obtained in this study were independent of LIF or any other supplemental inhibitors, resistant to enzymatic procedure (TrypLE Express Enzyme), and dependent on bFGF. Clonal lines were obtained from slightly different protocols with maximum reprogramming efficiency of 0.001%. All colonies were positive for alkaline phosphatase, embryoid body formation, and spontaneous differentiation and expressed high levels of endogenous OCT4 and SOX2. Canine iPSCs developed tumors at 120 days post-injection in vivo. Preliminary chromosomal evaluations were performed by FISH hybridization, revealing no chromosomal abnormality. To the best of our knowledge, this report is the first to describe the ability to reprogram canine somatic cells via lentiviral vectors without supplementation and with resistance to enzymatic action, thereby demonstrating the pluripotency of these cell lines.

Uterine artery blood flow remains unchanged in pregnant mares in response to short-term administration of pentoxifylline.

The objective of this study was to use Doppler ultrasound technology to determine whether pentoxifylline administration increased uterine blood flow in normal pregnant pony mares. Thirteen pregnant pony mares between 18 and 190 d of gestation (mean ± SEM, 101 ± 55) were utilized for the study during two trial periods. In each trial, pentoxifylline (17 mg/kg by mouth every 12h, diluted in syrup) was administered to half of the mares for 3 d, while the other mares were treated with syrup only. Doppler measurements were obtained from the right and left uterine arteries from each mare for 2 d prior to treatment and throughout the treatment period. The mean Resistivity Index (RI), Pulsatility Index (PI), Uterine Artery Diameter (D), and Total Arterial Blood Flow (TABF) from each day were compared over time and between groups. Administration of pentoxifylline did not alter uterine blood flow parameters compared with controls (values for all treatment days combined were RI: 0.517 ± 0.014 vs 0.543 ± 0.016; PI: 0.876 ± 0.048 vs 0.927 ± 0.057; D: 0.388 ± 0.018 vs 0.379 ± 0.023 cm; and TABF: 35.26 ± 7.38 vs 30.73 ± 5.29 mL/min). Uterine blood flow increased over the course of the 5 d study, irrespective of treatment, and was higher in mares of greater gestational age than in early gestational mares (RI: r(2) = 0.35; PI: r(2) = 0.37; D: r(2) = 0.66; and TABF: r(2) = 0.67 - P < 0.00001). We concluded that any immediate benefits of pentoxifylline administration in the pregnant mare were not mediated through enhanced uterine artery blood flow.

Evaluation of cytokine expression by blood monocytes of lactating Holstein cows with or without postpartum uterine disease.

Whereas neutrophils are the main phagocytic leukocytes, monocytes and macrophages are actively involved in immunomodulation after infection. Recent studies have demonstrated that neutrophil function is impaired by the state of negative energy balance around parturition, and that cows that develop uterine disease have a greater degree of negative energy balance than healthy cows. The objectives of this study were to compare monocyte gene expression and protein secretion of selected cytokines from calving to 42 d after calving in Holstein cows that did or did not develop uterine disease. Real time quantitative RT-PCR (Tumor necrosis factor-α (TNFα), Interleukin (IL)-1ß, IL-6, IL-8 and IL-10) and ELISA (TNFα, IL-1ß and IL-8) were used to evaluate cytokine response following in vitro stimulation of blood-derived monocytes with irradiated E. coli. Relative to unstimulated cells, E. coli-stimulated monocytes from cows with metritis had lower gene expression of key pro-inflammatory cytokines than healthy cows from calving to 14 d after calving (TNFα at 0, 7, and 14 d after calving, IL-1ß and IL-6 at 7 and 14 d after calving; P < 0.05). There were no significant differences between groups for expression of IL-8 or the anti-inflammatory cytokine IL-10. This was due, in part, to higher gene expression in unstimulated monocytes (TNFα, IL-1ß, IL-6 and IL-10) in early lactation from cows with metritis. Expression of mRNA in stimulated cells (relative to housekeeping genes) was lower for TNFα (7 and 14 d postpartum) and for IL-10 (7 and 14 d postpartum) in cows with metritis. Concentration of TNFα was lower in the culture medium of E. coli-stimulated monocytes from cows with metritis than healthy cows at calving and 7 and 21 d after calving (P < 0.05). Circulating cytokine concentrations were not different between groups for IL-8 and were below the limits of detection for TNFα and IL-1ß. Cytokine gene expression and production were similar between healthy cows and cows that developed endometritis, diagnosed cytologically at 42 d after calving. We concluded that altered levels of expression and production of pro-inflammatory cytokines postpartum could contribute to impaired inflammatory response and predispose cows to development of metritis.

B-mode and Doppler ultrasonography in pony mares with experimentally induced ascending placentitis.

REASONS FOR PERFORMING STUDY: Early, accurate diagnosis of ascending placentitis in mares remains a key challenge for successful treatment of the disease. Doppler ultrasonography has shown promise as a tool to diagnose pregnancy abnormalities and is becoming more available to equine clinicians. However, to date, no studies have prospectively compared this technique to standard B-mode measurement of the combined thickness of the uterus and placenta (CTUP). OBJECTIVES: The objective of the current study was to compare Doppler and B-mode ultrasonography for the detection of experimentally-induced ascending placentitis in mares. METHODS: Eleven healthy pony mares in late gestation were used in this study. Placentitis was induced in 6 mares between Days 280 and 295, while 5 mares served as negative controls. All mares were intensively monitored until delivery. Fetal heart rate, CTUP, uterine artery blood flow (resistance index, pulsatility index, arterial diameter and total arterial blood flow) and physical examination findings were recorded at each examination. Mares with an increased CTUP above published values were treated in accordance with published recommendations. Foals and fetal membranes were examined at birth. Ultrasonographic parameters were compared between groups using ANOVA. Foal viability and histological presence of placentitis were compared using a Fisher's exact test. RESULTS: The CTUP was increased above normal in 5 of 6 inoculated mares within 3 days after inoculation (P = 0.05). The sixth inoculated mare was excluded from subsequent data analysis. Uterine artery blood flow, physical examination findings and fetal heart rate were not different between groups. Gradual increases in CTUP, arterial diameter and total arterial blood flow were detected with increasing gestational age in the control mares (P = 0.02, P = 0.00001 and P = 0.00001, respectively). CONCLUSION: The CTUP, but not uterine blood flow, was different between groups (P = 0.00001). Recorded CTUP values for control pony mares were similar to previously published values for light breed horses.

Use of reagent test strips for diagnosis of endometritis in dairy cows.

The use of leukocyte esterase (LE), protein, and pH tests were evaluated on widely available urinary test strips (Multistix 10 SG; Bayer Corporation, Elkart, IN, USA) on uterine lavage samples as a potential cow-side test for the diagnosis of cytologic endometritis. Uterine lavage samples of 563 lactating Holstein cows between 40 and 60 days postpartum from 28 herds were evaluated. Endometrial cytology was used as the reference for endometritis, with a cutoff point of ≥10% neutrophils. All three (LE, protein, and pH) were increased in cows with cytologic endometritis and the associations were highly significant. Optimal cutoff points determined by receiver operating characteristic analysis for LE, protein, and pH were ≥++, ≥300 mg/dL, and ≥7.0, respectively. Combining the results for LE and pH improved the performance of the test strip, but this resulted in a group of cows (20.6% of cows) which were approximately equally likely (46% with endometritis and 54% without endometritis) to have cytologic endometritis or not, and therefore could not be accurately classified. The direct relationship between reagent strip test and reproductive performance was also evaluated. Reproductive impairment due to endometritis was restricted to multiparous cows; significantly decreased reproductive performance was observed for multiparous cows with lavage fluid LE ≥+++ (154 vs. 115 median days not-pregnant), as well as cows with pH ≥ 7.0 (150.5 vs. 111.5 median days not-pregnant), but not in cows with high protein, even at the highest cutoff point. In conclusion, reagent strip test results were strongly associated with cytologic endometritis and reproductive impairment; however, in comparison with conventional cytology, the performance of reagent strip as an alternative test was relatively poor and may require further refinement.

Association between uterine disease and indicators of neutrophil and systemic energy status in lactating Holstein cows.

The objective of this study was to evaluate the association between uterine disease and indicators of neutrophil (PMN) and systemic energy status in dairy cows. Peripheral blood (120 mL) was collected weekly from 84 Holstein cows for PMN isolation and plasma collection from calving until 42 d in milk (DIM). The final analysis included 80 cows. Of those, 20 cows were classified as having metritis (fetid uterine discharge and fever), 15 as having subclinical endometritis (SCE; >or=10% PMN on uterine cytology), and 45 as healthy controls. Plasma haptoglobin concentration was increased only in cows that developed metritis. Neutrophil glycogen content was reduced in cows developing metritis compared with healthy cows on the day of calving and at 7 and 42 DIM. Cows with SCE had lower PMN glycogen content than healthy cows at 7, 28, and 42 DIM. Blood glucose was affected by disease status within parity. Primiparous metritis cows had greater blood glucose concentrations than healthy primiparous cows. Multiparous metritis cows tended to have lower blood glucose concentration than multiparous SCE cows. Cows that developed metritis and SCE had or tended to have greater NEFA and BHBA than healthy cows, mainly around calving. At calving, cows that developed metritis had higher plasma estradiol concentration than healthy cows and greater plasma cortisol than cows that had SCE. Plasma insulin was not affected. Plasma glucagon was increased for SCE cows. Cows that developed uterine disease experienced a greater degree of negative energy balance and had decreased lower intracellular PMN glycogen levels, which could be a major predisposing factor for disease because of decreased availability of oxidative fuels.