Clinical safety of intratesticular transplantation of allogeneic bone marrow multipotent stromal cells in stallions

Although stem cell therapy is a promising alternative for treatment of degenerative diseases, there are just few reports on the use of stem cells therapy in horse's reproductive system. This study aims to evaluate the effect of intratesticular injection of bone marrow mesenchymal stromal/stem cells (MSCs) in healthy stallions, and its outcome on seminal parameters and fertility. In Experiment 1, 24 stallions were divided into treatment group (TG) and control group (CG). In the TG, an intratesticular application of MSC was performed, and in the CG, only PBS was used. Measurements of testicular volume, surface temperature and Doppler ultrasonography were performed 24 and 48 hr after treatments. Fifteen days after application, the testicles were removed and submitted to histological analysis. In Experiment 2, 3 fertile stallions received similarly treatment with MSCs. Physical examination and sperm analysis were performed weekly during 60 days after treatment, and at the end, semen from one of them was used for artificial inseminations of 6 healthy mares. In Experiment 1, clinical examinations showed no signals of acute inflammation on both groups according to the analysed variables (p > .05). Also, no signal of chronic inflammation was observed on histological evaluation. In Experiment 2, stallions presented no physical alterations or changes in sperm parameters, and a satisfactory fertility rate (83%; 5/6) was observed after AI. The results support the hypothesis that intratesticular application of bone marrow MSCs is a safe procedure, and this could be a promising alternative to treat testicular degenerative conditions.     

Unidentified microsporidian in western pygmy perch Edelia vittata (Nannopercidae) from Australia

Specimens of western pygmy perch, Edelia vittata Castelnau 1873, were submitted for examination because of the presence of black masses in the body wall in the abdominal area. The owner reported that approximately 10% of mature fish in one dam were affected. The masses had no apparent effect on the health of affected fish, but they were unsuitable for sale for aesthetic reasons. After necropsy and histological examination, the masses were found to be microsporidial xenomas within the abdominal cavity. Transmission electron microscopy results suggest that this microsporidian is a previously unreported species, because the melanised xenoma wall and internal structure of the spores differ from previously described species. The epidemiology of the parasite remains largely unknown and highlights the need for further surveys of the disease status and parasites of Australian native fish, especially those of interest to aquaculturists.     

Genotyping of Toxoplasma gondii isolates from free range chickens in the Pantanal area of Brazil

The aim of this paper was to genetically characterize Toxoplasma gondii isolates from free range chickens in regions of Brazilian territory in the state of Mato Grosso do Sul (MS) where T. gondii strains have never been studied. In total, T. gondii isolates from 22 free range chickens were included in this study. Fifty chickens from Eldorado, thirty from Rio Verde and ten from Aquidauana were sampled between January and April 2007. In relation to the genetic diversity of T. gondii isolates from chickens in MS, the magnitude of the diversity in the isolates sampled in this study was comparable to the overall diversity in a composite data set. These 22 isolates in MS revealed 11 genotypes, whereas the 321 isolates ever genotyped in Brazil have revealed 95 genotypes. The values of Simpson's Diversity Index for the whole population of T. gondii isolates in Brazil, the whole population of T. gondii isolates from chickens in Brazil and the population surveyed in this study were 0.97, 0.95 and 0.90, respectively. Seven of the 11 genotypes revealed from chicken isolates from MS are newly described genotypes and six of them each have a single isolate. In conclusion, the results obtained from isolates in MS corroborate previous studies on T. gondii isolates in Brazil, thus confirming their diversity and atypicality. Nonetheless, the applicability of PCR-RFLP markers for epidemiological inferences remains controversial.     

Urethane anesthesia in adult female rats: preliminary observations

Urethane is widely used as a rodent anesthetic in the laboratory setting, and is characterized as producing long‐lasting anesthesia. The purpose of this study was to evaluate the quality of anesthesia provided by a single dose of urethane based on the response to a noxious stimulus. If the quality of anesthesia was insufficient to prevent gross purposeful movement (GPM), isoflurane was also administered until no response to noxious stimulation occurred. Five adult Harlan Sprague Dawley rats (6 months of age, 250–300 g) were given urethane (1.4 g kg^?1 IP) and evaluated for 120 minutes post‐injection. If the rats became laterally recumbent by 20 minutes post‐injection, a large hemostat was positioned around the tail and the response to tail clamping was assessed. If no GPM occurred, an additional 20 minutes was allowed to elapse. If the rats were not laterally recumbent or GPM was present, they were placed in a chamber and isoflurane in oxygen was administered. Inspired isoflurane concentrations (ISO) were measured using a S/5 anesthetic gas analyzer (Datex‐Ohmeda Division, Helsinki, Finland) calibrated before each experiment with a standardized calibration gas mixture (DOT‐34 NRC 300/375 m 1014, Datex‐Ohmeda Division, Helsinki, Finland). A period of 20 minutes was allowed for equilibration to inspired ISO. The tail‐clamp stimulus was then re‐applied and the animal's response recorded. If GPM was absent, ISO was lowered by 10–20% and an additional 20 minute interval elapsed. In contrast, if GPM was present, ISO was increased by 10–20%. This procedure was repeated until the ISO required to prevent GPM was determined in duplicate. The position within the estrus cycle influenced pain thresholds in the rats. As such, a vaginal smear was prepared from each rat and the position in the estrus cycle was determined based on vaginal cytology. Rats were euthanatized at the end of the study period. All values were mean ± SD. Four rats became recumbent after urethane injection (time to recumbency: 45 ± 17 seconds). Of these, two rats (one estrus, one metestrus) did not require isoflurane supplementation for the duration of the study. The three remaining rats (two metestrus, one estrus) required isoflurane supplementation. The mean ISO required to prevent GPM was 0.26 ± 0.16%. Position within the estrus cycle did not appear to affect the animal's response to urethane. These results indicate that urethane anesthesia is not long lasting in all rats and provides variable quality of anesthesia. This is of particular concern in the laboratory setting where muscle relaxants are often administered to rats shortly after urethane injection.     

Pregnancy Diagnosis in Cats Using a Rapid, Bench-top Kit to Detect Relaxin in Urine

Relaxin is a pregnancy-specific hormone in the queen and is produced by the placenta. Both serum and urinary relaxin levels can be used to diagnose and monitor pregnancy in the cat; however, only serum levels are commonly measured in practice. The present study aimed to assess whether urine could be used for the rapid diagnosis of pregnancy at an early stage in domestic cats using a bench-top kit to detect relaxin. Paired serum and urine samples were collected during the first month of gestation in six cats. The samples were tested by applying neat serum, urine or urine diluted in non-pregnant cat serum to the Witness Relaxin kit. Relaxin concentrations in the paired samples were also measured by radioimmunoassay. All undiluted urine samples from pregnant cats tested negative using the bench-top kit; however, the kit was able to detect relaxin in urine after dilution with non-pregnant cat serum. Using this as the test sample, the kit was accurate at diagnosing pregnancy from 28 days after mating and some samples tested positive at 21 days after mating. This preliminary work could lead to the development of a home pregnancy test for cats.     

Comparison of two injectable anesthetic regimes in feral cats at a large-volume spay clinic

Same‐day mass sterilization of feral cats requires rapid onset, short‐duration anesthesia. The purpose of this study was to compare our current anesthetic protocol, Telazol–ketamine–xylazine (TKX) with medetomidine–ketamine–buprenorphine (MKB). Feral female cats received either IM TKX (n = 68; 0.25 mL cat^?1; tiletamine 12.5 mg, zolazepam 12.5 mg, K 20 mg, and X 5 mg per 0.25 mL) or MKB (n = 17; M 40 µg kg^?1, K 15 mg kg^?1, and B 10 µg kg^?1). Intervals measured included time from injection to recumbency, time to surgery, duration of surgery, and time from reversal of anesthesia (TKX: yohimbine 0.50 mg cat^?1 IV; MKB: atipamezole 0.50 mg cat^?1 IM) to sternal recumbency. Following instrumentation (Vet/Ox 4403 and Vet/BP Plus 6500), physiological measurements were recorded at 5‐minute intervals, and included rectal temperature, heart rate (HR), respiratory rate (RR), SpO₂ (lingual or rectal probes), and indirect mean arterial blood pressure (MAP) (oscillometric method). Nonparametric means were compared using Mann–Whitney U‐tests. Parametric means were compared using a two‐factorial anova with Bonferroni's t‐tests. The alpha‐priori significance level was p < 0.05. Values were mean ± SD. Body weight (TKX: 2.9 ± 0.5 kg, MKB: 2.7 ± 0.7 kg), time to recumbency (TKX: 4 ± 1 minutes, MKB: 3 ± 1 minutes), time to surgery (TKX: 28 ± 7 minutes, MKB: 28 ± 5 minutes), and duration of surgery (TKX: 11 ± 7 minutes, MKB: 8 ± 5 minutes) did not differ between groups. In contrast, MKB cats required less time from reversal to sternal recumbency (TKX: 68 ± 41 minutes, MKB: 7 ± 2 minutes) and were recumbent for shorter duration (TKX: 114 ± 39 minutes, MKB: 53 ± 6 minutes). Temperature decreased during the study in both groups, but overall temperature was higher in MKB cats (38.0 ± 0.95 °C) than in TKX cats (37.5 ± 0.95 °C). RR, HR, and SpO₂ did not change during the study in either group. However, overall HR and RR were higher in TKX cats (RR: 18 ± 8 breaths minute^?1, HR: 153 ± 30 beats minute^?1) compared to MKB cats (RR: 15 ± 7 breaths minute^?1, HR: 128 ± 19 beats minute^?1). In contrast, overall SpO₂ was lower in the TKX group (90 ± 6%) compared to the MKB group (94 ± 4%). MAP was also lower in the TKX group (112 ± 29 mm Hg) compared to that in the MKB group (122 ± 20 mm Hg). However, MAP increased in the TKX group during surgery compared to pre‐surgical values, but did not change in the MKB group. The results of this study suggested that MKB might be more suitable as an anesthetic for the purpose of mass sterilization of feral female cats.     

Above-ground biomass dynamics after reduced-impact logging in the Eastern Amazon

Changes in above-ground biomass (AGB) of 17 1 ha logged plots of terra firme rain forest in the eastern Amazon (Brazil, Paragominas) were monitored for four years (2004–2008) after reduced-impact logging. Over the same time period, we also monitored two 0.5 ha plots in adjacent unlogged forest. While AGB in the control plots changed little over the observation period (increased on average 1.4 Mg ha⁻¹), logging resulted in immediate reductions in ABG that averaged 94.5 Mg ha⁻¹ (±42.0), which represented 23% of the 410 Mg ha⁻¹ (±64.9) present just prior to harvesting. Felled trees (dbh > 55 cm) accounted for 73% (±15) of these immediate losses but only 18.9 Mg ha⁻¹ (±8.1) of biomass was removed in the extracted logs. During the first year after logging, the annual AGB balance (annual AGB gain by recruitment and growth − annual AGB loss by mortality) remained negative (−31.1 Mg ha⁻¹ year⁻¹; ±16.7), mainly due to continued high mortality rates of damaged trees. During the following three years (2005–2008), average net AGB accumulation in the logged plots was 2.6 Mg ha⁻¹ year⁻¹ (±4.6). Post-logging biomass recovery was mostly through growth (4.3 ± 1.5 Mg ha⁻¹ year⁻¹ for 2004–2005 and 6.8 ± 0.9 Mg ha⁻¹ year⁻¹ for 2005–2008), particularly of large trees. In contrast, tree recruitment contributed little to the observed increases in AGB (1.1 ± 0.6 Mg ha⁻¹ year⁻¹ for 2004–2005 and 3.1 ± 1.3 Mg ha⁻¹ year⁻¹ for 2005–2008). Plots with the lowest residual basal area after logging generally continued to lose more large trees (dbh ≥70 cm), and consequently showed the greatest AGB losses and the slowest overall AGB gains. If 100% AGB recovery is desired and the 30-year minimum cutting cycle defined by Brazilian law is adhered to, current logging intensities (6 trees ha⁻¹) need to be reduced by 40–50%. Such a reduction in logging intensity will reduce financial incomes to loggers, but might be compensated for by the payment of environmental services through the proposed REDD (reduced emissions from deforestation and forest degradation) mechanism of the United Nations Framework Convention on Climate Change.     

Dimethylformamide Improves the In vitro Characteristics of Thawed Stallion Spermatozoa Reducing Sublethal Damage

A total of 42 ejaculates were used in the experiment; six ejaculates per stallion, obtained from seven Pure Spanish stallions (PRE), were split and frozen in freezing media with different concentrations and combinations of cryoprotectant (CPA): (i) Cáceres (skim milk based extender) containing 2.5% glycerol (2.5GL), (ii) Cáceres containing 1.5% glycerol and 1.5% dimethylformamide (1.5%GL–1.5%DMFA), (iii) Cáceres extender supplemented with 1.5% glycerol and 2.5% dimethylformamide (1.5%GL–2.5%DMFA) and (iv) Cáceres extender supplemented with 4% dimethylformamide (4%DMFA). After at least 4 weeks of storage in liquid nitrogen (LN), straws were thawed and semen analysed by computer‐assisted sperm analysis and flow cytometry (membrane lipid architecture (Merocyanine 540), integrity and sublethal damage (YoPro‐1) and mitochondrial membrane potential (JC‐1)). After thawing, better results were observed in samples frozen in 4%DMFA or in combinations of 1.5%GL–2.5%DMFA, in fact total motility increased by 16% in the 4%DMFA group compared to 2.5%GL (P < 0.05). Also, there was an increment in the percentage of progressive motile sperm in the 1.5%GL–2.5%DMFA group (9.8% 2.5GL vs 19% in the 1.5%GL–2.5%DMFA group p < 0.05); also, samples frozen in the 4%DMFA group had more intact (YoPro‐1 negative) sperm post‐thawing, 29.3% in 2.5%GL vs 36.7% in 4%DMFA group (p < 0.05). Membrane lipid architecture was not affected by any of the cryoprotectants tested, while samples frozen in 4%DFMA had a lower percentage of mitochondria with lower membrane potential. It is concluded that DMFA improves the outcome of cryopreservation of stallion spermatozoa mainly reducing sublethal cryodamage.     

In vitro Evaluation of the Quality and Fertilizing Capacity of Boar Semen Frozen in 0.25 ml straws

Twenty-two boar ejaculates were frozen in 0.25 ml straws using a controlled cooling rate, then evaluated in vitro in order to assess: (i) the extent to which a range of semen evaluation parameters accurately characterize sperm quality, (ii) the value of quality assessment in the characterization of long-term sperm survival and fertility and (iii) the suitability of the cryopreservation protocol used for yielding semen with good quality and fertilizing capacity. Motility with or without caffeine, plasma membrane integrity (PMI) evaluated with both propidium iodide (PI) and Hoechst 33258, and acrosome morphology were studied, the ejaculates being then classified into five quality groups. A thermoresistance test and a homologous in vitro fertilization test were applied to selected ejaculates of these groups. Caffeine-stimulated motility and PMI evaluated with PI provided better estimations of semen quality than the other tests of motility, PMI, or acrosome morphology, but this quality assessment could not reveal differences in fertilizing capacity or thermoresistance among ejaculates. Over 43% spermatozoa survived cryopreservation in 19 of the 22 ejaculates, with inter-boar and inter-ejaculate variability in the freezing success being observed. The fertilizing capacity, however, was seriously affected by the process regardless of the semen quality. It is concluded that caffeine-stimulated motility and PMI evaluated with PI give accurate information on sperm quality, but important aspects to the valuation of semen such as thermoresistance and fertilizing capacity are not revealed by this quality study. Moreover, the approach of selecting suitable protocols of cryopreservation does not appear to be sufficient for guaranteeing systematically good quality and fertilizing capacity in the frozen-thawed semen.     

Two Unusual Cases of Canine Prostatitis: Prostatitis in a Castrated Dog and Preputial Oedema in an Intact Male

In this study, two unusual presentations of canine prostatitis are described; in the first case a 10‐years‐old neutered Boxer dog was presented to the Veterinary Teaching Hospital of the University of Extremadura with a complaint of anorexia, apathy and preputial discharge. In the second case, a local veterinarian referred an 8‐years‐old male Labrador to the Veterinary Teaching Hospital of the University of Extremadura. The dog had a history of pain in the caudal abdomen and preputial oedema. The final diagnosis in both cases was acute prostatitis. It is concluded that although canine prostatitis is a common disease, sometimes can have presentations that may differ from those classically described in the literature.