Foetal pulmonary maturity in dogs: Estimated from bubble tests in amniotic fluid obtained via amniocentesis.

The goal of this study was to evaluate the reliability of amniocentesis during late pregnancy to assess lung maturity in puppies using a bubble test as described by Gunston and Davey (South African Medical Journal, 54, 1978, 495). Thirty-five bitches from eight different breeds were followed during late pregnancy before undergoing elective Caesarean (C)-section on days 61-62 after ovulation. Bubble tests were performed the day before the C-section (n = 11 bitches) and before the administration of aglepristone on amniotic fluid samples obtained via amniocentesis and were repeated the day of the surgery on amniotic fluid samples collected via puncture of the amniotic bags before they were opened (n = 35 bitches). No complications were observed following amniocenteses and the C-sections. The mortality rate (2.3%) was similar to the result of other studies using the same protocol for an elective C-section. Of the non-contaminated samples collected the day of the C-section, 89.6% were positive in the bubble test, which was consistent with observations of clinical maturity the day of the surgery and on the following days. In contrast, 70% of the samples collected the day before the C-section (when progesterone concentrations were still high) were negative, suggesting that the puppies were still immature at this point in the pregnancy. Additionally, we observed a significant difference in the bubble test results before and 18 hr after the administration of aglepristone, suggesting that aglepristone may act as an inducer of the final maturation of the puppies by inactivating progesterone receptors and simulating a physiological decrease in progesterone. Finally, we confirmed the need to exclude all contaminated samples, which could lead to false-negative results.

Retrospective evaluation of blood copper stable isotopes ratio 65 Cu/63 Cu as a biomarker of cancer in dogs.

Previous studies in humans with breast, colorectal or liver cancer showed that neoplasia was associated with a modification of the blood ratio between 65 Cu and 63 Cu (∂Cu). The aim of the present study was to compare the blood ∂Cu of dogs with cancer to healthy controls or dogs with non-oncologic disease. One hundred and seventeen dogs were included in the study (35 dogs with cancer, 33 dogs with non-neoplastic disease, and 49 healthy controls). The ∂Cu of dogs with cancer was significantly lower than the ratio of healthy controls (P < 0.0001) but not significantly different from dogs with non-oncologic disease. Six dogs with lymphoma were also evaluated after they achieved clinical remission and five out of six had an increase of ∂Cu. Further studies are warranted but these results suggest that ∂Cu could help in the diagnosis of cancer in a controlled clinical context, and may be a potential biomarker for the follow-up of cancer.

Prediction of Parturition Time in Queens using Radiography and Ultrasonography.

This study investigated the prediction of parturition in queens, as well as the effects of the queens' age and weight and the litter size on the accuracy of the prediction. A prospective study was performed in 24 purebred queens of 11 different breeds, all in normal body condition, using radiographic and ultrasonographic measurements of foetal femoral length (FL) and biparietal diameter (BPD) of fœtal skull that apply to different breeds. Pregnant queens with aborted or malformed foetuses were excluded. The examinations were performed up to three times during the second half of pregnancy; litter size was obtained with radiography, and FL and BPD measurements were taken of each foetus using both radiography and ultrasonography. The maximal FL and the transversal BPD were recorded for each foetus. Radiography showed the number of foetuses accurately but did not allow accurate prediction of time of parturition. However, ultrasonography did establish the best predictive model according to the data with the FL. This model corresponded to the formula Y = 37.864 - 0.193 × FL + 1.227 × W - 0.615 × LS - 0.832 × A using the FL (10(-1)  mm), litter size (LS, number of foetuses per queen) and maternal parameters (weight (W, kg) and age (A, years)). Time to parturition correlated positively with the queen's weight and negatively with her age (P < 0.01). The ultrasonographic FL associated with pregnancy and maternal factors appeared to be an accurate model to predict parturition.

Short-term pharmacokinetic study of mycophenolate mofetil in neonatal swine.

BACKGROUND: Mycophenolate mofetil (MMF) is an effective immunosuppressive agent that has been frequently used in laboratory animals including swine; however, the pharmacokinetic properties of MMF in swine have not been studied. This short-term study was designed to evaluate the feasibility and the pharmacokinetic profiles of MMF therapy in neonatal swine. MATERIALS AND METHODS: Twelve neonatal pigs were randomized into four groups including one control and three treated groups with oral MMF administered at 0.5, 1, and 2 g/m(2)/d for 4 days, divided by 2 half-doses at 9:00 and 17:00 (except day 4 during which MMF was not administered at 17:00). Blood samples were collected at 9:00 on days 0, 2, 3 and 4 for complete blood count and hepatic/renal function examination; the trough concentration of plasma mycophenolic acid (MPA) was also determined. On days 2 and 4, blood was collected to determine the area under the curve (AUC) of plasma MPA concentration. Animal body-weight growth and manifestations of MMF side-effects such as anorexia, vomiting, and diarrhea were also observed. RESULTS: MMF has no acute hepatic/renal toxicity in newborn pigs; however, less body-weight growth was observed in treated groups. In the control group, a spontaneous increase of lymphocyte count was observed; in contrast, MMF therapy with doses of 1 and 2 g/m(2)/d reduced both lymphocyte and monocyte counts of piglets. Oral MMF had high bioavailability in neonatal swine. MPA-AUC0-12h of doses 0.5, 1, and 2 g/m(2)/d was 22.00 ± 3.32, 57.57 ± 34.30, and 140.00 ± 19.70 µg × h/mL, respectively. Neither MPA trough concentration (MPA-C0), nor MPA maximum concentration (MPA-Cmax) or MPA-AUC0-6h had high correlation with MMF-dose. For surveillance of MPA exposure, MPA-C0 had significant correlation with MPA-AUC0-12h (Spearman's ρ = 0.933, AUC0-12h = 17.882 × C0 + 14.479, r(2) = 0.966). CONCLUSION: To reach adequate drug exposure and to reduce dose-dependent side effects, an MMF dose of 1 g/m(2)/d is recommended to be used as an initial dose for immunosuppressive therapy in piglets, and MPA-C0 monitoring is the most practical strategy for experimental transplantation study.

First description of scrotal testicles in a dog affected by 78, XX testicular disorder of sex development.

An eight-month-old female dog presented with ambiguous external genitalia. A thorough clinical examination together with various imaging techniques and a histology examination showed the presence of two testicles linked to both the Mullerian and Wolffian ducts. The discovery of the 78,XX SRY-negative karyotype led to the diagnosis of incoherence between the chromosomal and gonadal sex, which is typical for a 78,XX testicular disorder of sex development. Our case was unique because the testicles were still located in their normal scrotal position, whereas the literature contains reports of the presence of cryptorchid testicles in this karyotype setting. To our knowledge, this is the first case that describes an SRY-negative 78,XX testicular disorder of sex development with bilateral scrotal testicles.

Improved cryopreservation of in vitro-produced bovine embryos using a chemically defined freezing medium.

This study evaluates a new synthetic substitute (CRYO3, Ref. 5617, Stem Alpha, France) for animal-based products in bovine embryo cryopreservation solutions. During the experiment, fetal calf serum (FCS) and bovine serum albumin (BSA) were used as references. A combination of a thermodynamic approach using differential scanning calorimetry and a biological approach using in vitro-produced bovine embryo slow-freezing was used to characterize cryopreservation solutions containing CRYO3, FCS and BSA. The CRYO3 and fetal calf serum (FCS) slow-freezing solutions were made from Dulbecco's phosphate-buffered saline containing 1.5 m ethylene glycol, 0.1 m sucrose and 20% (v.v(-1)) of CRYO3 or FCS. The bovine serum albumin (BSA) solution was made by adding 0.1 m sucrose to a commercial solution containing 1.5 m ethylene glycol and 4 g L(-1) BSA. These solutions were evaluated using three characteristics: the end of melting temperature, the enthalpy of crystallization (thermodynamic approach) and the embryo survival and hatching rates after in vitro culture (biological approach). The CRYO3 and FCS solutions had similar thermodynamic properties. In contrast, the thermodynamic characteristics of the BSA solution were different from those of the FCS and CRYO3 solutions. Nevertheless, the embryo survival and hatching rates obtained with the BSA and FCS solutions were not different. Similar biological properties can thus be obtained with slow freezing solutions that have different physical properties within a defined range. The embryo survival rate after 48 h of in vitro culture obtained with the CRYO3 solution (81.5%) was higher than that obtained with the BSA (42.2%, P = 0.000 12) and FCS solutions (58%, P = 0.016). Similarly, the embryo hatching rate after 72 h of in vitro culture was higher with the CRYO3 solution (61.1%) than with the BSA (31.1%, P = 0.0055) and FCS solutions (36%, P = 0.018). We conclude that CRYO3 can be used as a chemically defined substitute for animal-based products in in vitro-produced bovine embryo cryopreservation solutions.

Control of the estrous cycle in guinea-pig (Cavia porcellus).

The aim of this work was to look for a simple method to obtain synchronized ovulation in guinea pigs under farming conditions while respecting animal welfare. The luteolytic activity of three different prostaglandins F2alpha (PGF2α) analogs (D-cloprostenol, D,L-cloprostenol and luprostiol) and a daily treatment with oral progestagen (altrenogest) was tested successively at different stages of the estrous cycle on the same group of females during a period of 8 mo. The estrous cycle length was not modified by the administration of PGF2α analogs, whatever the stage of the estrous cycle when the treatment was initiated. Our results led us to reject the use of PGF2α analog to induce practical synchronization of the estrus in this species. In females (n = 29), given 15 days with altrenogest (0.1 mL po once a day), ovulation occurred 4.43 ± 0.13 days after the end of the treatment. Altrenogest treatment was followed by mating. No negative impacts of the treatment on the pregnancy rates, delivery rates and litter sizes were observed. This standard method of guinea-pig estrus synchronization is less stressful for the animals compared to techniques using progesterone tubing.

Follicle development in cryopreserved bitch ovarian tissue grafted to immunodeficient mouse.

The present study evaluated: (1) in vivo follicular development in canine ovarian tissue after slow freezing and xenotransplantation; and (2) the use of erythropoietin (EPO) as an angiogenic factor to optimise the transplantation procedure. Frozen-thawed ovarian tissue from five bitches was grafted into severe combined immunodeficient (SCID) mice (n=47) treated with or without EPO (500 IU kg(-1), once daily for 3 days) (Groups A and B, respectively) and analysed after 0, 1, 8 or 16 weeks. Follicle grade, follicle density, follicle morphology and stromal cells density were assessed by histological analysis, whereas vascularisation of the graft was quantified by immunohistochemistry with anti-α-smooth muscle actin antibody. Despite a massive loss of follicles after grafting, secondary follicle density was higher at 8 and 16 weeks than at 1 week regardless of EPO treatment. EPO significantly improved early follicle morphology and stromal cell density after 8 weeks and blood vessel density at 16 weeks after transplantation (P<0.05). Intact secondary follicles with more than three granulosa cells layers were observed 16 weeks after transplantation. The results suggest that canine ovarian tissue can be successfully preserved by our slow-freezing protocol because the tissue showed follicular growth after xenotransplantation. EPO treatment did not lessen the massive loss of follicles after transplantation.

Amino acids in cat fallopian tube and follicular fluids.

Aminograms of tubal and follicular fluids were obtained using fluids collected by aspiratory puncture from six cats. The amino acids were separated and quantified by high-performance liquid chromatography analysis. The serum of the cats was used as control. The three most prevalent amino acids quantified in cat tubal fluid were glycine, glutamic acid, and taurine. Their mean concentrations were 840 µmol/l (µm), 808 µm and 596 µm, respectively. The three most prevalent amino acids quantified in cat follicular fluid were alanine, glutamine, and taurine. Their mean concentrations were 359 µm, 351 µm, and 258 µm, respectively. This result is consistent with aminograms of tubal fluid previously determined in other mammals. As previously observed in other species and humans, glycine was quantitatively the most abundant and most prevalent free amino acid in cat tubal fluid. The total quantity of amino acids in tubal fluid was similar in cats and other species. However, in contrast with other species studied, hypotaurine was not detected in tubal and follicular fluids of female cats.

Ex vivo recovery of preimplantatory embryos in bitches.

The present study was conducted to investigate the timing of preimplantatory development in the dog and to evaluate the efficiency of flushing oviducts and uterine horns to collect embryos. Among the embryonic structures collected between day 8 and day 12 after ovulation, 43 % were at the 1-16 cells stage, 23% were at the morula stage and 34% at the blastocyst stage. Our collection method yielded to a recovery rate of 61.3 %, and 7.1 ± 0.7 embryos were harvested per bitch. In addition, the ovulation rate reached 11.6 ± 0.8 per bitch. The first morulae were observed from day 9 post-ovulation, while the first blastocyst appeared from day 10. Two-thirds of the collected morulae-blastocysts were obtained between the 11th and the 12th day after ovulation. To the moment, we suggest this is the best period to harvest canine embryo for cryopreservation.

Can video cameras replace visual estrus detection in dairy cows?

A 6-mo experiment was conducted in a dairy herd to evaluate a video system for estrus detection. From October 2007 to April 2008, 35 dairy cows of three breeds that ranged in age from 2 to 6 yr were included in the study. Four daylight cameras were set up in two free stalls with straw litter and connected to a computer equipped with specific software to detect movement. This system allowed the continuous observation of the cows as well as video storage. An observation method related to the functionality of the video management software ("Camera-Icons" method) was used to detect the standing mount position and was compared to direct visual observation (direct visual method). Both methods were based on the visualization of standing mount position. A group of profile photos consisting of the full face, left side, right side, and back of each cow was used to identify animals on the videos. Milk progesterone profiles allowed the determination of ovulatory periods (reference method), and a total of 84 ovulatory periods were used. Data obtained by direct visual estrus detection were used as a control. Excluding the first postpartum ovulatory periods, the "Camera-Icons" method allowed the detection of 80% of the ovulatory periods versus 68.6% with the direct visual method (control) (P = 0.07). Consequently, the "Camera-Icons" method gave at least similar results to the direct visual method. When combining the two methods, the detection rate was 88.6%, which was significantly higher than the detection rate allowed by the direct visual method (P < 0.0005). Eight to 32 min (mean 20 min) were used daily to analyze stored images. When compared with the 40 min (four periods of 10 min) dedicated to the direct visual method, we conclude that the video survey system not only saved time but also can replace direct visual estrus detection.

Relationships between fetal biometry, maternal factors and birth weight of purebred domestic cat kittens.

The goal of this study was to evaluate the relation between kittens' birth weights and biometrical factors from the kittens and the mother during pregnancy. Knowing fetal birth weight could help in detecting abnormalities before parturition. A Caesarean-section or a postnatal management plan could be scheduled. Consequently, the neonatal mortality rate should be decreased. We used ultrasonographic measurements of femur length (FL) or fetal biparietal diameter (BPD), pregnancies, and maternal factors to obtain a model of prediction. For this purpose, linear mixed-effects models were used because of random effects (several fetuses for one queen and a few paired measurements) and fixed effects (litter size, pregnancy rank, weight, wither height, and age of the queen). This study was performed in 24 purebred queens with normal pregnancies and normal body conditions. Queens were scanned in the second half of pregnancy, using a micro-convex probe. They gave birth to 140 healthy kittens whose mean birth weight was 104 g (ranged 65 to 165 g). No correlation between the birth weight and the age of the queen, as a maternal factor alone, was observed. But the birth weight was found to be inversely proportional to the pregnancy rank and the litter size. Moreover, birth weight increased when the weight and wither height of queen increased. BPD and FL increased linearly during pregnancy so a model was used to estimate mean birth weight. Using this model, we found a correlation between mean birth weights and an association of parameters: maternal factors (wither height and age), and litter size.

IL-10 delivery by AAV5 vector attenuates inflammation in mice with Pseudomonas pneumonia.

Lung infections with Pseudomonas aeruginosa and other pathogens in cystic fibrosis (CF) cause progressive airway obstruction and tissue damage, the predominant cause of morbidity and mortality in CF. We investigated whether a recombinant adeno-associated virus type 5 (AAV5) vector expressing murine interleukin (IL)-10 (AAV5.Cbeta-mIL-10), a regulatory/anti-inflammatory cytokine, could decrease airway inflammation in IL-10 knockout mice chronically infected with mucoid P. aeruginosa. Mice that received AAV5.Cbeta-mIL10 through intratracheal inoculation produced IL-10 at an average of 25 000 pg/ml in the epithelial lining fluid (ELF) and 12 000 pg/g-lung tissue 6 weeks post-vector delivery, significantly higher levels than in placebo-treated mice. At 3 days post-infection, proinflammatory cytokines (IL-1beta, tumor necrosis factor (TNF)-alpha, macrophage inhibitory protein (MIP)-1alpha and (KC) in the ELF and lung homogenate were decreased (1-9 folds) in the AAV5.Cbeta-mIL10-treated mice accompanied by less pronounced and more localized neutrophil infiltration in lung sections, when compared with placebo-treated mice. These results suggest that AAV5.Cbeta-mIL10 induces IL-10 levels in the lungs mediating a significant anti-inflammatory response and making AAV-IL-10 gene transfer a potentially useful therapy in the treatment of CF lung disease.

Oral instillation with surfactant phospholipid: a reliable alternative to intratracheal injection in mouse studies.

The intratracheal (IT) injection technique has been widely used in the mouse studies of pulmonary diseases. Here, we describe a non-invasive technique using oral instillation challenge with the surfactant phospholipid that may advantageously replace the traditional IT technique. We performed comparative studies between oral instillation and IT injection of both vectors (adeno-associated virus, AAV vector) and bacteria (Pseudomonas aeruginosa). Our results demonstrated that the oral instillation is a reliable alternative to IT injection. The administration of a fluorophore-labelled AAV vector demonstrated a similar pattern of distribution and quantity of vector delivered by oral instillation compared with IT injection. In addition, administration of AAV5-alpha-1 antitrypsin (AAT) to the lungs by oral instillation resulted in similar levels of AAT in both the lung homogenates and sera compared with the IT injection group. In our study of P. aeruginosa delivery, oral instillation resulted in similar mouse weight loss, cytokine levels in the epithelial lining fluid [interleukin (IL)-1beta, IL-6, tumour necrosis factor-alpha, neutrophil chemokine and macrophage inflammatory protein-1alpha], lung histology/pathology and bacterial loads. Therefore, we conclude that oral instillation of materials mixed with surfactant phospholipid is an adequate and reproducible technique to replace the invasive IT injection procedure for the delivery of either vector or bacteria to the lungs. This procedure has the benefits of eliminating the discomfort, local inflammation and mortality associated with the more invasive IT surgical procedures.

[Follicular morphology after ovarian cortex cryopreservation in the rabbit doe(Oryctolagus cuniculus)]. / Morphologie folliculaire après cryoconservation du cortex ovarien chez la lapine (Oryctolagus cuniculus).

OBJECTIVE: To compare the effects of two cryoprotective agents (DMSO and 1,2-PROH) used at two concentrations (1,5 and 2 M) on the morphology of small ovarian cortex follicles in doe. MATERIALS AND METHODS: Ovarian cortexes (n=40) were frozen in TCM199+10% FCS medium added to 1.5 or 2 M of DMSO or 1,2-PROH. Two controls were realized (fresh and frozen without cryoprotectant). The equilibration in cryoprotective solutions before freezing, and the elimination of the cryoprotective agents after thawing, was performed step by step. The effects induced by cryopreservation were evaluated by histological examination. RESULTS: Fresh ovarian tissue showed 68.6% of intact follicles. After freezing, only 1.5 M of 1,2-PROH preserved 48.0% of normal follicles, with no significant difference compared to the fresh control. The proportion of follicles without morphological defect observed after cryopreservation with DMSO was significantly reduced (respectively 28.8 and 34.8% for 1,5 and 2 M of DMSO). DISCUSSION AND CONCLUSIONS: Our results suggest that 1,2-PROH is a more effective cryoprotectant than DMSO, for the cryopreservation of doe ovarian cortex. These results differ from those that were obtained for other species, credibly because of a higher fragility of the ovarian tissue of the doe. Nevertheless, this species is an interesting animal model which allows rapid results after cryopreserved ovarian tissue graft.

Circulating relaxin concentrations in pregnant and nonpregnant bitches: evaluation of a new enzymeimmunoassay for determination of pregnancy.

A new kit (ReproCHEK RELAXIN) intended for the diagnosis of pregnancy in bitches is now available for veterinary use. This assay measures relaxin concentrations in plasma and whole blood samples, and the presence of significant amounts of relaxin is indicative of pregnancy. A clinical trial was carried out to evaluate the performance of the test. Serial blood samples were collected on alternate days, and relaxin concentrations were determined from day 15 to day 35 after the LH surge (estimated by progesterone concentrations). Pregnancy was confirmed using ultrasonography. At the end of pregnancy, both the day of whelping and the size of the litter were recorded. Pregnancy was established in 61 bitches. The day that pregnancy was detected using the relaxin assay ranged from day 19 to day 28 after the LH surge and had a mean (+/- SD) of 25.4 +/- 2.5 days. The day of parturition was taken as a reference point, and pregnancy was detected from -46 to -38 days (mean -40.2 +/- 2.4 days) before parturition. False positives were not observed in pseudopregnant bitches (n = 16) or in the control group (30 anoestrous and ten unmated bitches). These results demonstrate that the new assay kit is an inexpensive, user-friendly and reliable technique for determining pregnancy.

Taurine and hypotaurine in spermatozoa and epididymal fluid of cats.

Taurine and hypotaurine have been found in spermatozoa and seminal plasma of numerous species and are known to have beneficial effects on sperm characteristics in mammals. Taurine is considered an essential dietary constituent in cats. Dietary deficiency has been associated with a range of serious clinical disorders. Quantification of taurine and hypotaurine in the genital tracts of male cats has not been reported. In this study, the concentrations of taurine and its precursors were measured in serum, spermatozoa, epididymal fluid and seminal plasma from cats. The concentrations of taurine measured in serum samples confirmed that the cats were not deficient in taurine. Significant amounts of taurine and hypotaurine were found in spermatozoa, seminal plasma and epididymal flushing fluid. Hypotaurine was not detected in serum samples. These results indicate that hypotaurine may be synthesized in cat testes or epididymides. Cysteamine was not detected in any of the samples.

Candida albicans pneumonia: radiographic appearance.

A retrospective series of patients with pure Candida albicans pulmonary opportunistic infection confirmed at autopsy were examined for any characteristic radiographic pattern. Of the 20 patients examined, eight showed nonlobar, nonsegmental, bilateral disease; the others exhibited unilateral or bilateral lobar or segmental patterns. Cavitation, adenopathy, masslike opacities, or a miliary pattern were not identified. Radiographically these "negative" findings can be useful in distinguishing Candida from other fungal opportunistic infections. Histologic evidence of lung invasion by Candida is necessary for definitive confirmation. The previously described association of Candida infection with certain underlying diseases (leukemia and lymphoma) was again demonstrated.