Expression of CD13 and CD26 on extracellular vesicles in canine seminal plasma: preliminary results.

Canine seminal plasma is a complex fluid containing proteins, peptides, enzymes, hormones as well as extracellular vesicles that are involved in many physiological and pathological processes including reproduction. We examined the expression of the extracellular vesicles surface antigens Aminopeptidase-N (CD13) and Dipeptidyl peptidase IV (CD26) by flow cytometry. For this study, third fraction of the ejaculate, from fertile adult male German Shepherd dogs, was manually collected twice, two days apart. FACS analyses revealed that CD13 and CD26 are co-expressed on the 69.3 ± 3.7% of extracellular vesicles and only a 2.0 ± 0.5% of extracellular vesicles express CD26 alone. On the other hand, 28.6 ± 3.6% of seminal EVs express CD13 alone. Our results agree with the hypothesis that CD26 needs to be co-expressed with other signal-transducing molecules, while CD13, can perform functions independently of the presence or co-expression of CD26. The results obtained in normal fertile dogs could represent physiological expression of these enzymes. Therefore, it would be interesting to carry out further studies to evaluate the expression of CD13 and CD26 on extracellular vesicles as biomarker for prostate pathological condition in dogs.

Serum concentrations of leptin in pregnant and non-pregnant bitches.

Leptin regulates body weight and several physiological processes including reproduction. We evaluated the circulating levels of leptin in pregnant and non-pregnant bitches as well as their correlation with body weight, food intake and number of foetuses. Nineteen healthy German shepherd bitches were used and divided in two groups (pregnant n = 12 and non-pregnant n = 7). Blood samples were collected every 15 days starting from ovulation (Day 0) throughout pregnancy (pregnant group, P) or throughout luteal phase (non-pregnant group, NP) In pregnant bitches, leptin concentrations increased from the day of ovulation (1.32 ± 0.06 ng/ml) up to day 45 (1.51 ± 0.06 ng/ml; p < .01) and returned to baseline values from day 60 post-ovulation. In non-pregnant bitches, leptin concentrations remained constant throughout the whole observation period (estimated marginal mean ± SE=1.33 ± 0.38 ng/ml). Pairwise comparisons showed significant differences between P and NP at day 45 post-ovulation (p < .05). Multivariable models indicated that, controlling for time and litter size, there was a positive relationship between leptin concentration and BW (p < .05) although Pearson coefficients showed that the correlation between BW and leptin was only significant in NP animals at day 45 (r = 0.76, p < .05). The multivariable approach also suggested that, holding BW and time constant, leptin concentrations tend to increase as the number of puppies increased (p = .06). Our study supports indirectly the contribution of the feto-placental unit to the circulating maternal leptin concentration.

Invader probes: Harnessing the energy of intercalation to facilitate recognition of chromosomal DNA for diagnostic applications.

Development of probes capable of recognizing specific regions of chromosomal DNA has been a long-standing goal for chemical biologists. Current strategies such as PNA, triplex-forming oligonucleotides, and polyamides are subject to target choice limitations and/or necessitate non-physiological conditions, leaving a need for alternative approaches. Toward this end, we have recently introduced double-stranded oligonucleotide probes that are energetically activated for DNA recognition through modification with +1 interstrand zippers of intercalator-functionalized nucleotide monomers. Here, probes with different chemistries and architectures - varying in the position, number, and distance between the intercalator zippers - are studied with respect to hybridization energetics and DNA-targeting properties. Experiments with model DNA targets demonstrate that optimized probes enable efficient (C50 < 1 µM), fast (t50 < 3h), kinetically stable (> 24h), and single nucleotide specific recognition of DNA targets at physiologically relevant ionic strengths. Optimized probes were used in non-denaturing fluorescence in situ hybridization experiments for detection of gender-specific mixed-sequence chromosomal DNA target regions. These probes present themselves as a promising strategy for recognition of chromosomal DNA, which will enable development of new tools for applications in molecular biology, genomic engineering and nanotechnology.

Invaders: Recognition of Double-Stranded DNA by Using Duplexes Modified with Interstrand Zippers of 2'-O-(Pyren-1-yl)methyl-ribonucleotides.

The invasion has begun: Invaders are shown to recognize DNA hairpins in cell-free assays and chromosomal DNA during non-denaturing fluorescence in situ hybridization (nd-FISH) experiments. As Invaders are devoid of inherent sequence limitations, many previously inaccessible DNA targets could become accessible to exogenous control with important ramifications for karyotyping, in vivo imaging, and gene regulation.

Evaluation of adrenocortical function in Florida manatees (Trichechus manatus latirostris).

The study objectives were to determine the predominant manatee glucocorticoid; validate assays to measure this glucocorticoid and adrenocorticotropic hormone (ACTH); determine diagnostic thresholds to distinguish physiological vs. pathological concentrations; identify differences associated with sex, age class, female reproductive status, capture time, and lactate; and determine the best methods for manatee biologists and clinicians to diagnose stress. Cortisol is the predominant manatee glucocorticoid. IMMULITE 1000 assays for cortisol and ACTH were validated. Precision yielded intra- and inter-assay coefficients of variation for serum cortisol: ≤23.5 and ≤16.7%; and ACTH: ≤6.9 and ≤8.5%. Accuracy resulted in a mean adjusted R(2)≥0.87 for serum cortisol and ≥0.96 for ACTH. Assay analytical sensitivities for cortisol (0.1 µg/dl) and ACTH (10.0 pg/ml) were verified. Methods were highly correlated with another IMMULITE 1000 for serum cortisol (r=0.97) and ACTH (r=0.98). There was no significant variation in cortisol or ACTH with sex or age class and no correlation with female progesterone concentrations. Cortisol concentrations were highest in unhealthy manatees, chronically stressed by disease or injury. ACTH was greatest in healthy free-ranging or short-term rehabilitating individuals, peracutely stressed by capture and handling. Cortisol concentrations ≥1.0 µg/dl were diagnostic of chronic stress; ACTH concentrations ≥87.5 pg/ml were diagnostic of peracute stress. In healthy long-term captive manatees, cortisol (0.4±0.2 µg/dl) and ACTH (47.7±15.9 pg/ml) concentrations were lower than healthy free-ranging, short-term rehabilitated or unhealthy manatees. Capture time was not significantly correlated with cortisol; ACTH correlation was borderline significant. Cortisol and ACTH were positively correlated with lactate.

Preliminary evaluation of deslorelin, a GnRH agonist for contraception of the captive variable flying fox Pteropus hypomelanus.

BACKGROUND: This study was conducted to study the effects of a gonadotropin-releasing hormone (GnRH) agonist, deslorelin, on luteinizing hormone (LH), testosterone (males), semen characteristics and pregnancy in the variable flying fox Pteropus hypomelanus. STUDY DESIGN: Male (n = 3) and female (n = 5) bats received a 4.7-mg implant and were housed with untreated bats (eight females and three males, respectively). Plasma was collected twice monthly and analyzed for hormone concentrations, and semen was collected from untreated and treated males 1 month preimplantation, 3 months postimplantation and 4 months postimplantation. RESULTS: Administration of a GnRH challenge 1 month postimplantation showed an attenuated response in treated (n = 4), but not in untreated (n = 4), male and female bats. Plasma LH was lower in treated versus untreated males (p = .04), but not in females. Testosterone was lower in treated versus untreated males (p < .001). Spermic ejaculates were obtained from treated males, although no untreated females became pregnant during the 8-month study. One treated female became pregnant 6 months after implantation. CONCLUSION: Deslorelin is a useful and reversible contraceptive for P. hypomelanus.

Urodynamic and morphologic changes in the lower portion of the urogenital tract after administration of estriol alone and in combination with phenylpropanolamine in sexually intact and spayed female dogs.

OBJECTIVE: To compare the urodynamic and morphologic effects of the administration of estriol alone and in combination with phenylpropanolamine on the lower portion of the urogenital tract in female dogs. ANIMALS: 3 sexually intact and 3 spayed female Beagles without urinary incontinence. PROCEDURE: Dogs received estriol (2 mg, PO) once daily for 7 days followed by estriol (2 mg, PO) and phenylpropanolamine (1.5 mg/kg, PO) once daily for 7 days. Urethral pressure profilometry, diuresis cystometry, and vaginourethrography were performed before treatment (day 0) and at days 7 and 14. The maximum urethral pressure (MUP) and closure pressure (MUCP), urethral functional and anatomic profile lengths, integrated pressure (IP), plateau, distance before MUP, maximum meatus pressure, threshold pressure, threshold volume, compliance, urethral length, and vaginal length and width were measured. RESULTS: Before treatment, no urodynamic differences were observed between the 2 groups; however, vaginal length and width were significantly shorter in spayed dogs. Compared with day 0 values, estriol treatment significantly increased MUP, MUCP, and IP values at day 7, but at day 14, this effect decreased despite phenylpropanolamine administration. No morphologic changes from baseline were detected after either treatment in any dog. CONCLUSIONS AND CLINICAL RELEVANCE: Data suggest that estriol mainly acts on the urethral sphincter mechanism by increasing urethral resistance in sexually intact and spayed female dogs without urinary incontinence. Administration of estriol and phenylpropanolamine did not increase the urethral resistance more than estriol alone. The urodynamic effects of estriol in female dogs with urinary incontinence remain to be elucidated.

Evaluation of the urodynamic and hemodynamic effects of orally administered phenylpropanolamine and ephedrine in female dogs.

OBJECTIVE: To compare the urodynamic and hemodynamic effects of different dosages of phenylpropanolamine and ephedrine and determine effective dosages in increasing urethral resistance in female dogs. ANIMALS: 20 sexually intact female Beagles. PROCEDURE: Dogs were allocated into 4 groups and received phenylpropanolamine once, twice, or 3 times daily, or ephedrine twice daily, for 14 days. On days 0, 7, and 14, urethral pressure profiles were performed while dogs were anesthetized with propofol. Variables recorded included maximum urethral pressure, maximum urethral closure pressure, integrated pressure, functional profile length, anatomic profile length, plateau distance, distance before maximum urethral pressure, and maximum meatus pressure. Arterial and central venous pressures were measured before anesthetic induction and 10 and 35 minutes after induction. RESULTS: Administration of phenylpropanolamine once daily or ephedrine twice daily significantly increased maximum urethral pressure and maximum urethral closure pressure. Values for integrated pressure were significantly increased after 14 days of once-daily administration of phenylpropanolamine. Variables did not change significantly from day 7 to day 14. Diastolic and mean arterial blood pressures increased significantly during the treatment periods, and arterial pressure decreased during propofol infusion. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of phenylpropanolamine once daily or ephedrine twice daily increased urethral resistance in clinically normal dogs and may be recommended for management of urethral sphincter mechanism incompetence. Treatment efficacy may be assessed after 1 week. Dogs with concurrent cardiovascular disease should be monitored for blood pressure while receiving alpha-adrenergic agents because of the effects on diastolic and mean arterial pressure.

Influence of the estrous cycle on urodynamic and morphometric measurements of the lower portion of the urogenital tract in dogs.

OBJECTIVES: To compare the values of the urodynamic parameters of the lower portion of the urinary tract and vaginourethral measurements obtained during the phases of the estrous cycle in dogs and determine possible functional or anatomic modifications of the lower portion of the urinary tract associated with those phases. ANIMALS: 7 adult female Beagles. PROCEDURE: Urethral pressure profilometry, diuresis cystometry, and vaginourethrography were performed in each dog during proestrus; estrus; early, mid, and late diestrus; and early and late anestrus. The maximum urethral pressure (MUP), maximum urethral closure pressure (MUCP), urethral functional and anatomic profile lengths (UFPL and UAPL, respectively), integrated pressure, threshold pressure, threshold volume, compliance, urethral length, and vaginal length and width were measured. RESULTS: For all measurements, significant interindividual variation was detected. Integrated and threshold pressures, APL, and each morphometric value significantly increased from late anestrus to proestrus. Compared with other phases, MUP, MUCP, and integrated pressure values were significantly lower in estrus and early diestrus; UAPL and UFPL values were significantly lower in late diestrus. At each cycle phase in old dogs, MUP, MUCP, threshold pressure, and vaginal length and width were significantly lower (except in proestrus for vaginal measurements) and threshold volume and compliance values were significantly higher, compared with middle-aged dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Urodynamic and morphometric measurements of the lower portion of the urogenital tract are affected by the changes in hormonal balance that occur during the estrous cycle. In sexually intact female dogs, estrous phase determination is important for the interpretation of urodynamic data.

Validation and comparison of the use of diuresis cystometry and retrograde filling cystometry at various infusion rates in female Beagle dogs.

OBJECTIVES: To compare retrograde filling cystometry at infusion rates of 5, 10, and 20 mL/min with diuresis cystometry for determination of an appropriate infusion rate and to confirm the reproducibility of measurements obtained by urethral pressure profilometry (UPP) and cystometry in female Beagles. ANIMALS: Adult female Beagles. PROCEDURE: Successive UPP and cystometry were performed by use of a water perfusion catheter on dogs anesthetized with propofol. Dogs randomly underwent each of the following at 1-week intervals: retrograde filling cystometry at 5, 10, and 20 mL/min, and diuresis cystometry. The maximum urethral pressure and closure pressure, functional and anatomic profile lengths, threshold pressure, threshold volume, and compliance were measured. RESULTS: For each UPP variable, significant differences were found among dogs, but no significant differences were found in intra- or interstudy measurements for individual dogs. For retrograde filling cystometry, threshold pressure was not significantly different between a 5 and 10 mL/min infusion rate. Threshold pressure was significantly higher during retrograde filling cystometry at 20 mL/min, compared with 5 and 10 mL/min, and was associated with bladder wall damages. Threshold pressure was significantly lower during diuresis cystometry, compared with retrograde filling cystometries. Threshold volume and compliance were not significantly different among retrograde filling cystometries but were significantly higher during diuresis cystometry. CONCLUSIONS AND CLINICAL RELEVANCE: Retrograde filling cystometry at 20 mL/min leads to unacceptable sudden increase in threshold bladder pressure. Retrograde filling cystometry at 10 mL/min can be recommended in a clinical setting, shortening the anesthesia time. However, diuresis cystometry approximates physiologic bladder filling most accurately.

Effects of a medetomidine-midazolam-butorphanol combination on renal cortical, intestinal and muscle microvascular blood flow in isoflurane anaesthetized dogs: a laser Doppler study.

OBJECTIVE: To obtain renal cortical, ileal, colonic and skeletal muscle microvascular blood flow measurements in dogs using the laser Doppler technique and to characterize the effects of medetomidine-midazolam-butorphanol combination on these flows. STUDY DESIGN: Prospective randomized experimental study. ANIMALS: Fourteen clinically normal beagles (two groups of seven), aged 1-4 years and weighing 13.2 ± 1.8 kg. METHODS: All dogs were anaesthetized with 1.7% end-tidal isoflurane in oxygen. In the treatment group, after instrumentation and when anaesthesia was considered stable, medetomidine (1 mg m-2 body surface area (BSA)) was administered intramuscularly (time 0). Midazolam (1 mg kg-1) and butorphanol (0.1 mg kg-1) were administered intravenously 20 minutes later. Atipamezole (2.5 mg m-2 BSA) was administered intramuscularly 60 minutes after medetomidine. In the control group, saline (0.5, 2.5 and 0.25 mL) was administered at the corresponding times. Heart rate, systolic, diastolic and mean arterial pressures, body temperature, renal cortical, ileal, colonic and skeletal muscle microvascular blood flows were measured before time 0, and 5, 15, 25, 40, 60, 65, 70 and 90 minutes thereafter. RESULTS: Heart rate, ileal and skeletal muscle blood flows decreased in the treatment group, while no changes were observed in the control group. CONCLUSIONS: Laser Doppler flowmetry allowed the measurement of microvascular blood flow in different organs. The medetomidine-midazolam-butorphanol combination decreases intestinal and skeletal muscle microvascular blood flows, while renal cortical blood flow is maintained. CLINICAL RELEVANCE: Medetomidine-midazolam-butorphanol combination can be used to induce a short duration anaesthesia in dogs, but it will induce cardiovascular depression. This depression appears to be accompanied by a redistribution of blood flow.