Assessment of the age for a preventive ultrasonographic examination of the prostate in the dog.

The prostate commonly develops benign prostatic hyperplasia (BPH) in dogs over 5 years, while in aged dogs other pathological findings might be revealed by ultrasonographic exam. The aim of the present study was to estimate the most suitable age for a preventive ultrasonographic examination of the prostate in the dog. The prostate of 1003 intact male dogs of 64 different breeds, of different ages (1-18 years) and bodyweights (2-55 kg) was evaluated with ultrasound, irrespective of the reason for clinical examination. The age of each dog was expressed as the ratio between the actual age and the maximum longevity expected for the breed. Dogs were divided in two groups based on breeds' life expectancy as short life (SL) and long life (LL). The size of the prostate (normal, enlarged or small) and the presence of abnormal sonographic findings were recorded for each dog. The results of the present study indicate that the most suitable age for a preventive ultrasonographic exam of the prostate in the dog is approximately at 40% of its expected longevity, both in short and long life breeds, because at this age there is a strong possibility to be able to detect abnormal prostatic findings. In 47.5% of the dogs at least one abnormal finding of the prostate was revealed by ultrasonographic exam, while dogs with long life expectancy showed a significantly higher prevalence of abnormalities, than dogs with short life expectancy. The most frequent findings were the increase of prostatic size (33.5%) and the presence of at least one cyst (33.6%), with no difference between SL and LL dogs. In conclusion, a preventive examination of the prostate starting at 40% of expected longevity in dogs of short and long life breeds is strongly recommended for early detection of abnormalities, for scheduling specific follow up and for suggesting effective therapeutic protocols.

Viability and growth of feline preantral follicles in vitro cultured with insulin growth factor and epidermal growth factor supplemented medium.

In vitro culture of ovarian preantral follicles has emerged as a reproductive technology aimed at obtaining large amount of oocytes for in vitro embryo production. The addition of growth factors (GF) in the in vitro culture of preantral follicles of different species has provided superior results of follicular development, antrum formation and proliferation of granulosa cells. However, there are only few reports regarding the use of these factors on feline preantral follicle in vitro culture. Thus, the aim of this study was to investigate the effect of a combination of IGF-1 and EGF on in vitro viability and growth of preantral follicles and enclosed oocytes collected from domestic cats. A total of 64 follicles characterized by multilayer granulosa cells were isolated and individually cultured for 6 days (T6) in minimum essential medium supplemented with IGF-1+ EGF (100 ng/ml each) or without (control). A higher percentage of follicles were viable after culture with GF than without, and an increase in size when IGF-1+ EGF were added to the medium (170 ± 32.4 µm (T0) vs. 201 ± 22.3 µm (T6); p < .05) was observed. An increase in the diameter was also observed in follicles cultured without GF, but this increase was only 8.3% compared to 15.4% of those cultured with GF (p < .05). No differences were found in the diameter of oocytes contained in follicles cultured in the non-supplemented or supplemented media (107.9 ± 11.8 µm (T0) vs. 113.2 ± 15.6 µm (T6); p > .05). These data suggest that the addition of IGF-1 and EGF to the culture medium promotes the in vitro development of preantral follicles of cats.

Matrix-assisted laser desorption/ionization imaging mass spectrometry for the spatial location of feline oviductal proteins.

With the purpose of identifying factors involved in early stages of embryo development in the domestic cat, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was used for the first time to describe the spatial localization of proteins in the oviducts of queens. Oviducts were obtained from two 2 and 4 years old cross-bred queens, divided into three segments, snap-frozen in liquid nitrogen and then stored at -80°C until use. Next, they were sectioned in a cryostat, fixed on ITO (indium tin oxide) conductive glass slides for MALDI-IMS and serial sections were collected on microscope slides for histology. As confirmed by histology, MALDI-IMS was able to show contrasting protein distributions in the oviductal infundibulum, ampulla and isthmus. Mass spectra were characterized by abundant ions of m/z 1,259, 4,939, 4,960 and 10,626, which have been tentatively attributed to keratin, thymosin ß10, thymosin ß4 and S100, respectively. Keratin and thymosins are involved in the biological response to tissue damage. S100 proteins are calcium-modulated proteins implicated in a variety of cellular activities, including cell differentiation and regulation of cell motility. These results suggest that protein composition differs between segments of the cat oviduct, which corresponds to morphological changes within these sections. Further functional studies could elucidate the effects of these proteins on feline reproductive physiology.

A three-dimensional alginate system for in vitro culture of cumulus-denuded feline oocytes.

In the case of high valuable individuals with very precious genetic material, widening the genetic pool including gametes with poor morphological characteristics, as cumulus-denuded oocytes (CDOs), could be an option. To improve the in vitro culture of low-competence feline CDOs, an enriched three-dimensional (3D) system in association with competent cumulus-oocyte complexes (COCs) was developed. For this purpose, domestic cat CDOs were cultured with or without companion COCs in the 3D barium alginate microcapsules. The overall viability and the meiotic progression of feline CDOs cocultured with COCs or cultured separately in 3D or in 2D (traditional microdrops) system were compared. The 3D system was able to support viability and meiotic resumption of the feline oocytes, as well as the 2D microdrops. In 3D microcapsules, the presence of COCs resulted in a higher viability of CDOs (91.1%, p < .05), than that obtained without COCs or in 2D microdrops (71.2% and 67.3%, respectively), but the percentages of meiotic resumption were similar of those of CDOs cultured separately (55.4% vs. 40.4%, p > .05). It is notable that the presence of CDOs seemed to enhance the meiotic progression of the associated COCs. In conclusion, the 3D barium alginate microcapsules are a suitable system for feline oocytes in vitro culture, but more specific enriched conditions should be developed to improve the CDOs full competence in vitro.

Canine epididymal spermatozoa: A hidden treasure with great potential.

The hidden treasure represented by epididymal spermatozoa has great potential in the current reproductive technologies in dogs. In case of azoospermia or when a donor male accidentally dies or undergoes orchiectomy, the retrieval of epididymal spermatozoa opens new possibilities to generate progeny. Spermatozoa can be collected by different techniques from ex vivo or in vivo testicles and can be cryopreserved for a future use. Freeze tolerance of canine epididymal spermatozoa seems lower than that of ejaculated spermatozoa; however, puppies were born after artificial insemination with frozen epididymal semen, other than with fresh and chilled. Even though several aspects need to be further investigated, advances have been recently made in the use of epididymal spermatozoa in assisted reproduction in dogs.

The nuclear and developmental competence of cumulus-oocyte complexes is enhanced by three-dimensional coculture with conspecific denuded oocytes during in vitro maturation in the domestic cat model.

The objective of the study was to assess the efficacy of coculture with conspecific cumulus-denuded oocytes (CDOs) during in vitro maturation in a three-dimensional system of barium alginate microcapsules on the in vitro embryo development of domestic cat cumulus-oocyte complexes (COCs). In Experiment I, COCs were cocultured with conspecific CDOs or cultured separately in a 3D system for 24 hr of in vitro maturation, before assessing the meiotic progression. In Experiment II, the in vitro fertilization of COCs and CDOs was carried out with chilled epididymal spermatozoa and the presumptive zygotes were cultured in vitro separately for 7 days in 3D microcapsules before assesment of embryonic development. The results showed that the viability was maintained and that meiosis was resumed in the 3D culture system. The presence of CDOs during in vitro maturation improved the meiotic competence of the COCs, since the proportions of telophase I/metaphase II were higher than that in the groups cultured separately. The enrichment of the maturation system by companion oocytes also enhanced the ability of COCs to develop into embryos, and increased the percentages of morula and blastoycst stages. The COCs cocultured with CDOs developed at higher rates than the COCs cultured separately and the CDOs themselves. The beneficial effects of coculture with conspecific CDOs were presumably due to the paracrine action of some secreted factors that enhanced many molecular patterns related to the complex of cumulus oophorous cells. Further investigations to understand how the 3D microenvironment can influence the features of oocytes and embryos are required.

Determination of gestational time and prediction of parturition in dogs and cats: an update.

Accurate prediction of delivery date in canine and feline allows a better management of parturition, reducing the loss of neonates. This review evaluates the most common methods adopted to accurately predict the day of delivery: determination of ovulation and hormonal assays, first appearance of embryonic/foetal structures using ultrasound or radiography, echographic measurement of extra-foetal and foetal structures, or evaluation of foetal flux and heart rate. Determination of ovulation and hormonal assays at the time of breeding and close to pregnancy term is widely used to predict parturition in dogs (Concannon et al. American Journal of Veterinary Research 44, 1983, 1819; Hayer et al. Journal of Reproduction and Fertility, Suppl. 47, 1993, 93; Hase et al. Journal of Veterinary Medical Science, 62, 2000, 243; Kutzler et al. Theriogenology, 60, 2003a, 1187). In cats, some studies have been carried out, but no hormonal parameters for accurate prediction of parturition have been described so far (Buff et al. Journal of Reproduction and Fertility, Suppl. 57, 2001, 187; De Haas van Dorsser et al. Biology of Reproduction, 74, 2006, 1090; DiGangi et al. Journal of the American Veterinary Medical Association, 237, 2010, 1267; Dehnhard et al. Theriogenology, 77, 2012, 1088). Many studies suggested that gestational timing can be obtained by observation using ultrasound or radiography of specific structures in relation to the time of appearance during gestation (Concannon and Rendano American Journal of Veterinary Research, 44, 1983, 1506; Rendano et al. Veterinary Radiology, 25, 1984, 132; Shille and Gontarek Journal of the American Veterinary Medical Association, 187, 1985, 1021; Davidson et al. Veterinary Radiology, 27, 1986, 109; England et al. Journal of Small Animal Practice, 31, 1990, 324; Yeager et al. American Journal of Veterinary Research, 53, 1992, 342; Zambelli et al. Theriogenology, 57, 2002a, 1981; Zambelli et al. Journal of Feline Medicine and Surgery, 4, 2002b, 95; Zambelli and Prati 2006; Lopate Theriogenology, 70, 2008, 397; Davidson and Baker Topics in Companion Animal Medicine, 24, 2009, 55). Ultrasonographic measurement of extra-foetal and foetal structures is a common and accurate method for the prediction of parturition day during pregnancy, when specific formulae are used depending on the ultrasonographic parameter, the species and, in canines, the size of the bitch (Shille and Gontarek Journal of the American Veterinary Medical Association, 187, 1985, 1021; England et al. Journal of Small Animal Practice, 31, 1990, 324; Luvoni and Grioni Journal of Small Animal Practice, 41, 2000, 292; Luvoni and Beccaglia Reproduction in Domestic Animals, 41, 2006, 27; Lopate Theriogenology, 70, 2008, 397; Michel et al. Reproduction in Domestic Animals, 46, 2011, 926; Beccaglia and Luvoni Reproduction in Domestic Animals, 47, 194, 2012). Recent studies demonstrated that in dogs, the imminence of parturition could be predicted by evaluating foetal flux and foetal heart rate by ultrasound (Gil et al. Theriogenology, 82, 2014, 933; Giannico et al., Animal Reproduction Science, 154, 2015, 105). For an accurate prediction of parturition date, the combination of different methods is desirable.

Prediction of whelping date in large and giant canine breeds by ultrasonography foetal biometry.

OBJECTIVES: To derive the growth curves of the inner chorionic cavity and the biparietal diameter in large and giant dog breeds by ultrasonographic foetal biometry. To evaluate their accuracy in the prediction of whelping date and the effect of litter size and foetal sex ratio. METHODS: Foetal biometry parameters were obtained using serial ultrasonographic examinations in eight large (26 to 40 kg) and nine giant (>40 kg) pregnant bitches with known breeding dates and concentrations of serum progesterone during oestrus. The relationship between inner chorionic cavity or biparietal diameter growth and days to parturition were analysed by linear regression and the equations derived from the growth curves were applied to predict the whelping day. The accuracy of the prediction (whelping day ±1 day and ±2 days) and the litter size and sex ratio were recorded. RESULTS: The results showed a significant relationship between days before parturition and inner chorionic cavity or biparietal diameter. The overall accuracy at ±2 days was greater, than that at ±1 day. In giant breed bitches, the accuracy of the prediction by biparietal diameter was significantly lower in small, than normal litter size. No effect of foetal sex ratio was observed. CLINICAL SIGNIFICANCE: Foetal biometry parameters obtained by ultrasonography can be used to predict whelping dates in large and giant dog breeds.

Feto-maternal heart rate ratio in pregnant bitches: effect of gestational age and maternal size.

Few information is available on parameters that can be used to objectively assess the foetal health during canine pregnancy. To identify a reliable parameter for the evaluation of foetal well-being, the effect of pre-gestational maternal bodyweight and gestational age on foetal heart rate (FHR) and on feto-maternal heart rate ratio (FHR/MHR) was investigated. Seventeen client-owned pregnant bitches of different pre-gestational maternal bodyweight were examined by serial echo colour Doppler. Only data from 11 uncomplicated pregnancies were included in the statistical analysis. The relationship between FHR, and FHR/MHR, and independent variables was analysed by polynomial regression (p ≤ .05). The FHR and the FHR/MHR significantly fitted a multiple quadratic regression for all independent variables. They both increased from 35 to 20 days before parturition and then a decreasing pattern followed. Higher values of both parameters were observed in bitches of lowest and highest bodyweight. Patterns of FHR and FHR/MHR were similar, but the ratio better describes the effect of the independent variables on the data. Thus, the highest significance of FHR/MHR compared to FHR alone encourages the application of this ratio to evaluate foetal well-being. The equation derived by the regression analysis of FHR/MHR could be applied in clinical practice to obtain its expected values in healthy pregnancies.

Congenital abnormality of the vagina complicated by haemato-pyocolpos in a 1-year labrador retriever.

A 1-year-old female Labrador retriever was referred with a few days history of haematic-like vulvar discharge. Physical examination, vaginal inspection and palpation did not reveal any remarkable finding. Transabdominal ultrasound showed echogenic fluid accumulation in the vagina suggesting haemato-pyocolpos. An exploratory laparotomy was performed: a well-delimited ectasic vagina was identified. Ovariohysterectomy and partial vaginectomy and vaginoplasty were performed to spay the bitch and to remove the ectasic vagina. Post-operative recovery and 12-month follow-up were uneventful. Clinical, morphological and histological findings were consistent with a congenital abnormality of the muscular layer of the vagina complicated by haemato-pyocolpos. The disorganization of the vaginal tunica muscularis may have acted as locus minoris resistentiae in the vaginal wall. The organ was dilated and atonic due to the gradual accumulation of physiological fluids complicated by an overgrowth of genital bacteria. This congenital disorder has to be taken into account as differential diagnosis of haemato-pyocolpos with vaginal discharge in young bitches.

Quality of canine spermatozoa retrieved by percutaneous epididymal sperm aspiration.

OBJECTIVES: To investigate the feasibility of percutaneous epididymal sperm aspiration in dogs and whether it might provide a population of epididymal spermatozoa similar to the population that can be obtained by processing isolated epididymis caudae. METHODS: Concentration and total sperm number, motility, morphology and acrosomal integrity of spermatozoa retrieved by percutaneous epididymal sperm aspiration, in vitro aspiration and mincing of the cauda of the epididymis were compared. RESULTS: Percutaneous epididymal sperm aspiration is a feasible procedure to retrieve a population of spermatozoa in dogs. Quality is similar to that of spermatozoa collected in vitro, although a wide variation amongst animals was observed. CLINICAL SIGNIFICANCE: In case of ejaculation failure due to pathological conditions in dogs, the collection of spermatozoa from the cauda of the epididymis could be an option for providing gametes for assisted reproductive technologies. Percutaneous epididymal sperm aspiration can be used in dogs with compromised reproductive performance, in which orchiectomy cannot be performed for medical or owner reasons. Further studies aimed to investigate whether the percutaneous epididymal sperm aspiration technique might be feasible for repeated semen collection and to accurately evaluate side effects are required.

Vitrification of immature feline oocytes with a commercial kit for bovine embryo vitrification.

The aim of this study was to evaluate the suitability of a commercial kit for bovine embryo vitrification for cryopreserving cat oocytes and to evaluate comparatively the effects of its use with slow freezing procedure on cryotolerance in terms of morphology and oocyte resumption of meiosis. Germinal vesicle stage oocytes isolated from cat ovaries were either vitrified (n = 72) using a vitrification kit for bovine embryo or slow frozen (n = 69) by exposing oocyte to ethylene glycol solution before being transferred to a programmable embryo freezer. After thawing and warming, oocytes were cultured for 48 h and then were examined for meiosis resumption using bisbenzimide fluorescent staining (Hoechst 33342). Fresh immature oocytes (n = 92) were used as the control group. The proportion of oocytes recovered in a morphologically normal state after thawing/warming was significantly higher in frozen oocytes (94.5%) than in the vitrified ones (75%, p < 0.01). Morphological integrity after culture was similar in vitrified (73.6%) and slow frozen oocytes (76.8%); however, only 37.5% of the morphologically normal oocytes resumed meiosis after vitrification compared to 60.9% of those submitted to slow freezing procedure (p < 0.01). Fresh oocytes showed higher morphological integrity (91.3%) and meiosis resumption rates (82.6%, p < 0.002) than cryopreserved oocytes, irrespective of the procedure used. These results suggest that immature cat oocytes vitrified with a kit for bovine embryos retain their capacity to resume meiosis after warming and culture, albeit at lower rates than slow frozen oocytes. Vitrification and slow freezing methods show similar proportions of oocytes with normal morphology after culture, which demonstrate that thawed and warmed oocytes that resist to cryodamage have the same chances to maintain their integrity after 48 h of culture.

Vitrification with DAP 213 and cryotop of ex situ and in situ feline cumulus-oocyte complexes.

This study was undertaken to compare cryotolerance, in terms of viability and resumption of meiosis after warming and culture (24 and 48 h), of ex situ (isolated) and in situ (enclosed in the ovarian tissue) feline cumulus-oocyte complexes (COCs) vitrified with DAP 213 (2 m DMSO, 1 m acetamide, 3 m propylene glycol) in cryotubes or Cryotop method. Ovaries were harvested from 49 pubertal queens. Of each pair of ovaries, one was dissected to release COCs randomly divided into three groups: fresh COCs (control), ex situ COCs vitrified with DAP 213 and Cryotop. The cortex of the other ovary was sectioned into small fragments (approximately 1.5 mm(3)) and randomly assigned to be vitrified by DAP 213 or Cryotop. After warming, ex situ and in situ (retrieved form vitrified ovarian tissue) COCs were matured in vitro. Viability of oocytes was highly preserved after warming and culture in all treatments. Proportions of oocytes surrounded by complete layers of viable cumulus cells were remarkably decreased (p < 0.00001) in both vitrification procedures compared to fresh oocytes. Resumption of meiosis occurred in all treatments. After 24 h of culture, results were similar in ex situ and in situ vitrified oocytes regardless of the vitrification protocol used (range 29-40%), albeit lower (p < 0.05) than those of fresh oocytes (65.8%). After 48 h of culture, ex situ oocytes vitrified with Cryotop achieved the rates of meiosis resumption similar to fresh oocytes (53.8% vs 67.5%; p > 0.05) and ex situ and in situ oocytes vitrified with DAP 213 showed similar rates of resumption of meiosis. These findings demonstrated that DAP 213 and Cryotop preserve the viability of ex situ and in situ oocytes, but cumulus cells are highly susceptible to vitrification. However, the capability to resume meiosis evidences that feline immature oocytes vitrified as isolated or enclosed in the ovarian cortex have comparable cryotolerance.

Effect of vitrification of feline ovarian cortex on follicular and oocyte quality and competence.

Cryopreservation of ovarian cortex has important implications in the preservation of fertility and biodiversity in animal species. Slow freezing of cat ovarian tissue resulted in the preservation of follicular morphology and in the follicular development after xenografting. Vitrification has been recently applied to ovarian tissues of different species, but no information is available on the effect of this method on feline ovarian cortex. Moreover, meiotic competence of fully grown oocytes isolated from cryopreserved tissue has not been reported. The aim of this study was to evaluate the effect of vitrification of feline ovarian cortex on follicular morphology and oocyte integrity, as well as meiotic competence. A total of 352 fragments (1.5-2 mm(3) ) were obtained from ovarian cortical tissues: 176 were vitrified and 176 were used fresh as control. Histological evaluation of fresh and vitrified fragments showed intact follicles after cryopreservation procedures with no statistically significant destructive effect from primordial to antral follicles. After IVM, oocytes collected from vitrified ovarian fragment showed a higher proportion of gametes arrested at germinal vesicle (GV) stage compared to those isolated from fresh control tissue (33.8% vs 2.9%; p < 0.001). However, oocytes isolated from vitrified tissues were able to resume meiosis, albeit at lower rate than those collected from fresh tissues (39.8% vs 85.9%; p < 0.00001). Vitrification induced changes in the organization of cytoskeletal elements (actin microfilaments and microtubules) of oocytes, but significantly only for actin network (p < 0.001). Finally, chromatin configuration within the GV was not affected by the cryopreservation procedure. Our study demonstrated that vitrification preserves the integrity of ovarian follicles and that oocytes retrieved from cryopreserved tissue maintain the capability of resuming meiosis. To our knowledge, this has not previously been reported in the cat.

In vitro survival of follicles collected from domestic cats' ovaries at different stages of oestrous cycle and cultured with IGF-1.

Optimal conditions for in vitro culture of feline ovarian follicles have not yet been defined. Follicular development is regulated by intraovarian growth factors, as insulin-like growth factor (IGF-1), and during the different stages of the oestrous cycle, follicles are exposed to specific hormonal environments. The aim of this study was to investigate the effect of IGF-1 on in vitro growth and granulosa cell (GC) viability of preantral follicles collected from domestic cats at follicular and luteal phases of the oestrous cycle. Oestrus and ovulation were induced in 12 cats. A total of 39 and 32 follicles collected at the follicular and luteal phases, respectively, were individually cultured in vitro for 6 days in minimum essential medium media supplemented with or without IGF-1 (100 ng/ml). Follicles collected during the follicular phase and cultured without IGF-1 displayed a significant increase in size and higher GC viability (46.5 ± 22.1 µm, 66.7%, respectively) than that of follicles collected at the luteal phase and cultured without IGF-1 (26.7 ± 14.4 µm, 50%, respectively; p < 0.05). In contrast, when IGF-1 was added to the culture medium, no differences were observed in size or GC viability between follicles collected at the two phases of the cycle. Nonetheless, follicles collected at the luteal phase and cultured with IGF-1 had a significant increase in their diameter and GC viability (31.9 ± 15.9 µm, 63.6%, respectively) than that cultured without IGF-1 (26.7 ± 14.4 µm, 50%, respectively; p < 0.05). These data suggest that in vitro growth and GC survival of feline preantral follicles are affected by the oestrous cycle phase, and the IGF-1 exerts a positive effect on follicles collected at the luteal phase.

Chemical composition of lipids present in cat and dog oocyte by matrix-assisted desorption ionization mass spectrometry (MALDI- MS).

The aim of the present study was to investigate the level of information on the chemical structures and relative abundances of lipids present in cat and dog oocytes by matrix-assisted laser desorption mass spectrometry (MALDI-MS). The MALDI-MS approach requires a simple analysis workflow (no lipid extraction) and few samples (two or three oocytes per analysis in this work) providing concomitant profiles of both intact phospholipids such as sphingomyelins (SM) and phosphatidylcholines (PC) as well as triacylglycerols (TAG). The lipids were detected in oocytes by MALDI using dihydroxybenzoic acid (DHB) as the matrix. The most abundant lipid present in the MS profiles of bitch and queen oocytes was a PC containing 34 carbons and one unsaturation [PC (34:1)]. Oocytes of these two species are characterized by differences in PC and TAG profiles detected qualitatively as well as by means of principal component analysis (PCA). Cat oocytes were mainly discriminated by more intense C52 and C54 TAG species and a higher number of unsaturations, indicating predominantly linoleic and oleic fatty acyl residues. Comparison of the lipid profile of bitch and queen oocytes with that of bovine oocytes revealed some similarities and also some species specificity: TAG species present in bovine oocytes were also present in bitches and queens; however, a more pronounced contribution of palmitic, stearic and oleic fatty acid residues was noticed in the lipid profile of bovine oocytes. MALDI-MS provides novel information on chemical lipid composition in canine and feline oocytes, offering a suitable tool to concomitantly monitor, in a nearly direct and simple fashion the composition of phospholipids and TAG. This detailed information is highly needed to the development of improved protocols for in vitro culture and cryopreservation of cat and dog oocytes.

Prediction of parturition in dogs and cats: accuracy at different gestational ages.

In bitches and queens, the ultrasonographic measurement of extrafoetal and foetal structures allows the evaluation of gestational age and the prediction of the parturition term for an extended period of time. The aim of this study was to investigate whether the accuracy of parturition date prediction is affected by the week of pregnancy when the ultrasonographic examination is performed. The results were obtained by retrospective analysis on the gestational period basis (from week 4 to week 9 of pregnancy) in 495 ultrasonographic examinations of pregnant bitches (small and medium size) and 60 of pregnant queens. They demonstrated that a similar accuracy (p > 0.05) was obtained by the measurement of inner chorionic cavity at week 4 and 5 of pregnancy (± 1 day, 81% vs 67.7%; ± 2 days, 93.1% vs 85.9%). Accuracy (± 1 day) based on biparietal (BP) measurement was similar at week 5 and 6 of pregnancy (78.6% vs 78.9%; p > 0.05), whereas a significant decrease (p < 0.05) was observed at week 7 and 8 compared to week 6. No difference in the accuracy of the prediction at ± 2 days based on BP was observed from week 5 to 8 of gestation (range from 82.3% to 95.2%). Close to term (week 9), the accuracies of the prediction based on BP were significantly lower than those obtained at week 5 (± 1 day: 50.9% vs 78.6%, p < 0.05; ± 2 days: 69.8% vs 95.2%, p < 0.005). The accuracy of the prediction in bitches and queens is influenced by the gestational period when ultrasonographic measurements are performed, particularly for BP measurement. Although a decrease in BP accuracy occurs along the pregnancy, the accuracy at ± 1 and ± 2 days of prediction of parturition term is highly consistent for 6 and 8 weeks of gestation, respectively.

Cryosurvival of ex situ and in situ feline oocytes.

Cryosurvival of feline oocytes preserved as isolated cells (ex situ) or enclosed in ovarian follicles (in situ) has been demonstrated, and significant advances have recently been achieved. However, an ideal protocol for oocyte cryopreservation has not been established to date because of extreme sensitivity of the structural complex to chilling injury. Several factors, such as stage of maturation, membrane permeability and plasticity of the cytoskeleton, affect cryosurvival of the oocyte. Also, intercellular communications between cumulus cells and oocyte are compromised after freezing or vitrification of ex situ or in situ cumulus-oocyte complexes, which has a detrimental effect on oocyte maturational competence. Despite these issues, embryo development, pregnancies and live kittens have been obtained after in vitro fertilization (by ICSI) and transfer of embryos derived from cryopreserved oocytes. It is a general belief that the efficiency of cryopreservation would increase through a better understanding of oocyte responses to cryoprotectants, cooling rates and all the physical events occurring during the exposure of feline oocytes to low temperatures. Cryobanking of feline oocytes would significantly contribute to the preservation of rare genotypes and to the maintenance of a valuable source of genetic material for research applications.

DNA fragmentation in canine oocytes after in vitro maturation in TCM-199 medium supplemented with different proteins.

In the present study, the effect of different protein supplementation on meiotic nuclear configuration, DNA fragmentation (TUNEL assay) and metabolic parameters of dog oocytes cultured in vitro for 72 h was investigated. TCM-199 medium was supplemented either with 0.3% bovine serum albumin (BSA) or with 10% bitch heat inactivated plasma (OBP) collected before the LH peak or with OBP collected between the LH peak and ovulation or OBP collected after ovulation. After culture, more than 70% of the cumulus-oocyte complexes cultured in plasma groups presented extensive cell expansion, while none of those cultured in BSA showed extensive expansion of the cumulus (P < 0.05). Glucose consumption and lactate production was lower (P < 0.05) in the BSA-supplemented medium than in plasma-supplemented groups. In all groups, high amounts of alanine were produced. A higher number of oocytes with DNA fragmentation were observed in the BSA group, while in the plasma-supplemented groups more oocytes presented undistinguishable nuclear material. Only a small percentage of the oocytes (7.4-12.7%) had intact DNA after culture and within these, no differences were observed between groups in number of oocytes at each chromatin configuration stage. No differences in the percentage of oocytes reaching metaphase II (MII) were observed between experimental groups. Still, only 2% of cultured oocytes reached MII, but 85.7% of these had intact DNA. Conversely, all other chromatin configurations presented a high proportion of fragmented DNA (germinal vesicle 79.8%; meiosis resumption 73.3%; unclassified 95.2%). In conclusion, a high percentage of canine oocytes that do not complete meiotic maturation to MII are degenerated, whereas a high proportion of MII oocytes have intact DNA, independently of the protein supplement used.

Pharmacokinetics, intraoperative effect and postoperative analgesia of tramadol in cats.

Tramadol is a synthetic codeine analogue used as an analgesic in human and veterinary medicine, but not approved for use in cats. Tramadol (2 mg/kg) was administered intravenously (IV) as preoperative analgesic in 12 cats (6 males) undergoing surgical gonadectomy. The pharmacokinetic profile of the drug and its O-desmethyl metabolite were determined in 8 animals (4 males), while intraoperative effects and postoperative analgesia, estimated by subjective pain score (0-24), were evaluated in all. Mean intraoperative isoflurane consumption was reduced, but hypoventilation was not observed. Sex-related differences were not observed, particularly in terms of postoperative analgesia: rescue analgesic was never administered. Concentrations of the active O-desmethyl metabolite were persistently high in all the animals. Considering the results obtained in this study, tramadol, at the dose of 2 mg/kg IV, did not produce any evident intraoperative cardiorespiratory side effects and with additional investigation may prove to be an appropriate intraoperative analgesic in cats undergoing gonadectomy.