Epicardial adipose tissue volume in patients with coronary artery disease or non-ischaemic dilated cardiomyopathy: evaluation with cardiac magnetic resonance imaging.

AIM: To compare the amount of epicardial adipose tissue (EAT) in patients with coronary artery disease (CAD) or non-ischaemic dilated cardiomyopathy (NIDCM) with that in patients with negative cardiac magnetic resonance imaging (CMR). MATERIALS AND METHODS: One hundred and fifty patients (median age 57 years, interquartile range [IQR] 46-66 years) who underwent CMR were evaluated retrospectively: 50 with CAD, 50 with NIDCM, and 50 with negative CMR. For each patient, the EAT mass index (EATMI) to body surface area, end-diastolic volume index (EDVI), end-systolic volume index (ESVI), stroke volume (SV), ejection fraction (EF) for both ventricles, and left ventricle (LV) mass index were estimated. Intra and inter-reader reproducibility was tested in a random subset of 30 patients, 10 for each group. Mann-Whitney U test, Kruskal-Wallis test, Spearman's correlation, and Bland-Altman statistics were used. RESULTS: The EATMI in CAD patients (median 15.7 g/m2, IQR 8.3-25.7) or in NIDCM patients (15.9 g/m2, 11.5-18.1) was significantly higher than that in negative CMR patients (9.1 g/m2, 6-12; p<0.001 both). No significant difference was found between CAD and NIDCM patients (p=1.000). A correlation between EATMI and LV mass index was found in NIDCM patients (r=0.455, p=0.002). Intra- and inter-reader reproducibility were up to 80% and 72%, respectively. CONCLUSION: Patients with NIDCM or CAD exhibited an increased EATMI in comparison to negative CMR patients. CMR can be used to estimate EAT with good reproducibility.

Time-dependent changes and prognostic value of lactatemia during the first 24 h of life in brachycephalic newborn dogs.

Blood lactate concentration is known to be a good prognostic indicator associated with the severity of illness and the patient's outcome both in human and veterinary medicine. It also plays a significant role in the assessment of the newborn, being a good indicator of fetal hypoxia and the ideal predictor of morbidity at term in babies. In veterinary neonatal medicine, hyperlactatemia is considered a valid prognostic marker in critically ill foals; moreover, blood lactate measurement has been proposed for the evaluation of newborn viability and the assessment of fetal distress during delivery in dogs. Unfortunately, only a few studies have been published concerning the canine species. The present work examines 67 brachycephalic newborn dogs and their mothers, with the aim to evaluate the time-dependent changes of blood lactate and glucose concentration during the first 24 h after vaginal or caesarean delivery both in puppies and bitches. To our knowledge, this is the first published study examining the time-dependent changes of these parameters in the bitch after parturition. Within the studied population of puppies, non-surviving was significantly associated with a higher lactatemia and a lower APGAR score. Blood lactate was high at birth then progressively decreased during the first 24 h of life and a lack of normalization of blood lactate levels within this time interval was suggestive for a poor prognosis for the newborn dogs; moreover, the decrease appeared to be slower after vaginal delivery. Lactatemia also showed a positive correlation with glycemia at birth. Concerning the bitches examined, blood lactate was found to be significantly higher after vaginal delivery than after caesarean section; the normalization occurred within 24 h after parturition. Blood glucose level was significantly higher at 2 h from delivery both in the group of bitches submitted to caesarean section and in those undergoing natural whelping but no statistical correlation was found between maternal glycemia and lactatemia. The results of the present study highlighted that the monitoring of lactatemia during the first 24 h of life, in association with the assessment of the APGAR score at birth, can be an useful prognostic tool helping to identify the most severely distressed puppies and to provide them an adequate support.

Influence of Different Protocols of Urethral Catheterization after Pharmacological Induction (Ur.Ca.P.I.) on Semen Quality in the Domestic Cat.

The aim of this study was to evaluate the influence that different protocols of urethral catheterization after pharmacological induction (Ur.Ca.P.I.) may have on the semen quality of the domestic cat. The study has been divided into two experiments: one in which different dosages of medetomidine administrated are evaluated and the second one in which the timing of the catheterization after pharmacological induction is tested. In the first experiment, 18 cats were sedated with the recommended dosage of medetomidine (130 µg/kg i.m.) while the other 18 were sedated with a lower dose of the same drug (50 µg/kg i.m.). In the second experiment, three groups were implemented, each containing 25 subjects. In group 1, the semen collection was performed immediately once the pharmacological effect of the drug was reached; in group 2, the semen collection was performed three times every 5 min after the pharmacological effect was reached; finally, in group 3, Ur.Ca.P.I. was performed 20 min after the pharmacological effect was reached. All the different protocols permitted sperm collection, nevertheless the first experiment showed a better quality in terms of volume, concentration, total number of spermatozoa (p < 0.01) and quality of the movement (motility p < 0.05 and forward progressive motility p < 0.01), using a high medetomidine dosage rather than 50 µg/kg i.m. In the second experiment, forward motility was statistically higher (p < 0.01) in the first group and total volume was higher (p < 0.01) in the second and third group, while other parameters were statistically not different. Results suggest that a single catheterization immediately after the onset of the pharmacological effect leads to a good-quality semen with the lowest possibility of damaging the urethra and that a sedation with 130 µg/kg of medetomidine leads to a better quality sperm collection than 50 µg/kg does.

Endoscopic transcervical catheterization in the domestic cat.

Feline-assisted reproduction is still not routinely performed in veterinary practice, although there is an increasing interest on the subject by cat breeders. In recent years, many techniques for artificial insemination in the domestic cat have been developed with regard to the intrauterine deposition of sperm through the catheterization of the cervix. Transcervical catheterization has been described also for diagnostic and therapeutic purposes. This article provides the first description of a new method for cervical catheterization, under the direct visualization of the cervix, using a rigid endoscope and a new specially designed transcervical catheter. The procedure was performed on 14 queens with a success rate of 85.71%.

Validation of a model to develop a symptom index for benign prostatic hyperplasia in dogs.

Benign prostatic hyperplasia (BPH) is a spontaneous and age-related condition in humans and intact male dogs. A symptom index for BPH in men was created by the American Urological Association. In this study, it has been developed and statistically validated as a model to assign an objective score to canine BPH severity based on clinical signs observed and/or subjectively reported to the veterinarian by dog owners. The medical records of the Animal Reproduction Unit of University of Bologna (Italy) were used to select dogs with a clinical diagnosis of BPH. A data set was built up, and the animals were included in the statistical analysis as dependent variables. A score of 1-3 was assigned to the disease severity of each case based on signs annotated, graded using a scale ranging from 1 to 4. Signs of BHP were entered as predictors while disease severity as dependent variable to generate the predictive model. The model was finally used to re-classify each case of the data set, and the percentage of corrected predictions calculated. Overall, 373 subjects were entered in the model. Between them, 243, 107 and 23 animals have been represented based on medical records with a BPH severity score of 1, 2 and 3, respectively. The model correctly predicted the response variable in 97.3% of the cases. In this study, a BPH symptom index was created for the first time in dogs, which may be useful to standardize BPH severity with an objective score and to evaluate the necessity, the kind and the effectiveness of treatment.

In vitro evaluation of fresh sperm quality in tomcats: a comparison of two collection techniques.

The collection of semen from tomcats by urethral catheterization (CT) after medetomidine administration offers a novel and easy approach to obtain good quality sperm for in vitro fertilization. This study was designed to compare the sperm quality parameters and in vitro fertilizing capacity of CT spermatozoa with those of spermatozoa retrieved after epididymal slicing (EP). Semen was collected in seventeen adult cats by urethral catheterization, after which the cat was orchiectomized. Motility, morphology, plasma membrane integrity, acrosomal status, and in vitro fertilizing capacity of both fresh CT and EP samples were evaluated. The results showed that both total and progressive motility, as well as the percentage of normal spermatozoa, were higher for EP sperm than for CT sperm (P<0.01). Epididymal sperm had a lower percentage of spermatozoa with an intact acrosome (P<0.01), while CT sperm contained more spermatozoa with tail abnormalities (P<0.01). Other morphological parameters, as well as plasma membrane integrity, did not differ (P>0.05) between CT and EP sperm. Nevertheless, no difference (P>0.05) in in vitro fertilizing capacity between spermatozoa collected by means of the two different methods was found. In conclusion, semen collection by means of urethral catheterization after medetomidine administration yields fertilization results similar to epididymal slicing, despite the fact that several sperm variables were different. Since this novel catheterization technique is repeatable, is easy to perform and facilitates semen preparation protocols, it may be preferable for routine IVF experiments with fresh spermatozoa.

Quality and fertilizing ability of electroejaculated cat spermatozoa frozen with or without Equex STM Paste.

An optimal protocol for cat semen cryopreservation has not yet been defined. Addition of Equex STM Paste has been tested for epididymal cat spermatozoa but not for ejaculated cat spermatozoa. Furthermore, the effect of Equex STM Paste on fertilizing ability of cryopreserved semen has never been evaluated in that species. Therefore, the aims of the current study were to investigate if addition of Equex STM Paste to a freezing extender for electroejaculated cat (Felis catus) semen would improve postthaw sperm quality and if sperm fertilizing ability after cryopreservation with or without Equex STM Paste was preserved. Semen was collected by electroejaculation and frozen in a Tris-glucose-citrate egg yolk extender supplemented with (0.5% vol/vol) or without Equex STM Paste. In Experiment 1, sperm motility, membrane integrity, and acrosomal status were determined immediately after collection and at 0, 3, and 6h postthaw. In Experiment 2, frozen semen from the two groups was used for in vitro fertilization (IVF) of in vitro-matured cat oocytes. Cleavage rate was recorded 30h after IVF, and embryo development was evaluated on Days 6 and 7 of culture. In Experiment 1, the rate of motile spermatozoa after freezing-thawing was higher when Equex STM Paste was added to the freezing extender, but progressive motility score was not influenced (P>0.05). Sperm membrane integrity was positively affected (P<0.05) by the addition of the detergent. Intact acrosomes after thawing were similar (P>0.05) between groups. Even if the decreasing rates of motility and membrane integrity were more rapid in presence of Equex than those in controls, total motility and sperm viability were similar at 3 and 6h after thawing (P>0.05). In Experiment 2, there was no difference in fertilizing ability and embryo development between the two groups (P>0.05). The results of this study demonstrate that the addition of Equex STM Paste in the freezing extender avoids the loss of motile spermatozoa and maintains fertilizing ability of frozen-thawed spermatozoa.

Comparative immunolocalization of GLUTs 1, 2, 3 and 5 in boar, stallion and dog spermatozoa.

Spermatozoa, as other eukaryotic cells, need hexoses to produce energy to maintain membrane homeostasis, to move along the female genital tract and to carry the male genome to the female gamete. GLUTs are a family of proteins that permit and improve the passive transport of hexoses inside cells. This study was aimed at investigating the presence and localization of GLUTs 1, 2, 3 and 5 in boar, stallion and dog spermatozoa by both immunofluorescence and western blotting. GLUTs exhibited a peculiar distribution along the sperm cell depending on the isoforms considered, the hexose they transport and the different species. The localization of GLUTs after capacitation and acrosome reaction highlighted the possible changes in their distribution because of the different functional moment. Only in dog spermatozoa changes in GLUTs distribution were demonstrated; these changes could be related to the different metabolic needs and modifications occurring in the sperm cell.

Cat blastocysts produced in vitro from oocytes vitrified using the cryoloop technique and cryopreserved electroejaculated semen.

Oocyte preservation is still a challenge in the cat. The aim of this study was to evaluate the efficiency of oocyte vitrification in cryoloop in the domestic cat and to assess the embryonic development after IVF with cryopreserved semen. In vitro matured cat oocytes were vitrified in cryoloop after exposure to 10% ethylene glycol (EG, 0.9 M) in hepes synthetic oviductal fluid (HSOF) for 1 min, 20% EG (1.8M) in HSOF for 1 min, and 40% EG (3.6M), 10mg/ml Ficoll 70 and 0.3M sucrose in HSOF for 20s. Warmed oocytes were fertilized in vitro with frozen-thawed semen collected by electroejaculation and presumptive zygote were cultured in vitro for 10 days. Results showed that percentage of degenerated oocytes was higher (P<0.01), while cleavage rate and morulae blastocysts rate on day 6 were significantly lower (P<0.01) for vitrified oocytes than control. Blastocyst rate on day 8 was higher (P<0.01) for control oocytes than vitrified counterparts, and also developmental ability was higher (P<0.05) for non-vitrified oocytes, while the hatched blastocyst rate on day 10 was higher (P<0.05) for vitrified oocytes than control. In conclusion cat oocytes can be vitrified in cryoloop with a fairly good survival rate, cleavage rate and embryo development until pre-implantation stage.

Comparative immunolocalization of heat shock proteins (Hsp)-60, -70, -90 in boar, stallion, dog and cat spermatozoa.

Heat shock proteins (Hsp)-60, -70 and -90 are important testis chaperones that fulfil several functions during sperm cell maturation. In post-meiotic cells, their expression may change or may be undetectable and in some species it may be evident in mature spermatozoa. The aims of this study were to verify whether Hsp60, -70 and -90 are present in the sperm, and to compare their localization in boar, stallion, cat and dog spermatozoa by immunofluorescence. Hsp-60 immunoreactivity was detected in sperm midpiece in all the species examined. In stallion sperm, Hsp70 signal was localized in the sub-equatorial band, whereas immunoreactivity was evident on the neck of dog spermatozoa and on both neck and sub-equatorial region of cat spermatozoa. In agreement with our previous observations, a triangular fluorescent signal in the equatorial segment of fresh boar sperm was detected. Hsp90 immunoreactivity was present in different portions of sperm tail: in the midpiece of both boar and cat spermatozoa and in the neck and throughout the tail in dog and stallion spermatozoa, respectively. When capacitation and acrosome reaction were induced in boar, stallion and dog spermatozoa, no changes in both Hsp60 and -90 were recorded by either Western blot or immunofluorescence. After induction of acrosome reaction, a Hsp70 redistribution in boar spermatozoa and an increased percentage of stallion spermatozoa showing the post-acrosomal signal were observed although no changes were recorded by Western blot; in dog spermatozoa, no changes in Hsp70 were found by Western blot and immunofluorescence after capacitation and acrosome reaction.

Quality and in vitro fertilizing ability of cryopreserved cat spermatozoa obtained by urethral catheterization after medetomidine administration.

Quality and in vitro fertilizing ability of frozen-thawed cat semen collected by urethral catheterization (CT) or electroejaculation (EE) after medetomidine administration were compared. Sperm collection was performed by an urinary tomcat catheter and, 4 days apart, by electroejaculation from each of eight tomcats. Results showed that semen collected by CT was characterized by lower volume (10.5+/-5.3 microL, P<0.05), higher sperm concentration (1868.4+/-999.8 x 10(6)/mL, P<0.05) and lower pH (7.0+/-0.4, P<0.05) than that collected by EE (67.1+/-25.9 microL, 542.9+/-577.9 x 10(6)/mL, and 7.9+/-0.4, respectively). Spermatozoa characteristics after thawing at 0, 3 and 6h did not differ between the two methods of collection. Also cleavage rate and embryo production from oocytes fertilized with frozen-thawed spermatozoa collected by CT or EE showed no significant differences (P>0.05). In conclusion, the results obtained in the present study indicate that good quality freezable semen can be collected from cats by urethral catheterization after medetomidine administration. This new method of semen collection appears very useful in practice and, compared with the electroejaculation protocol, permits to obtain semen samples characterized by a higher concentration of spermatozoa, lower total volume and lower pH.

Effects of ketamine or medetomidine administration on quality of electroejaculated sperm and on sperm flow in the domestic cat.

The effects of two commonly used drugs for anaesthesia in the domestic cat, ketamine and medetomidine, on features of electroejaculated semen and on sperm flow in this species were evaluated performing three experiments. This is the first study about these topics in the domestic cat. In Experiment 1, ketamine or medetomidine effects on cat sperm quality after collection by electroejaculation (E.E.) have been assessed in nine animals. Results showed that mean sperm concentration was significantly higher (p<0.01) after medetomidine than after ketamine administration. In Experiment 2, ketamine or medetomidine effects on sperm flow in 12 electroejaculated cats were studied. Mean sperm concentration and mean total number of spermatozoa resulted significantly higher (p<0.01) in medetomidine than in ketamine treated animals. The number of spermatozoa displaced in urethra was significantly higher (p<0.01) using medetomidine. No significant differences were observed in percentages of retrograde flow. In Experiment 3, ketamine or medetomidine effects on urethral sperm flow, without any stimulation for sperm collection, were evaluated. Data obtained showed a significantly higher (p<0.05) number of spermatozoa displaced in urethra after medetomidine than after ketamine injection. In conclusion, E.E. in the cat after medetomidine administration determined a higher number of spermatozoa per ejaculate than after ketamine administration, with a good pharmacological restriction and without increasing sperm retrograde flow.

Antiproliferative activity of CCN3: involvement of the C-terminal module and post-translational regulation.

Previous work had suggested that recombinant CCN3 was partially inhibiting cell proliferation. Here we show that native CCN3 protein secreted into the conditioned medium of glioma transfected cells indeed induces a reduction in cell proliferation. Large amounts of CCN3 are shown to accumulate both cytoplasmically and extracellularly as cells reach high density, therefore highlighting new aspects on how cell growth may be regulated by CCN proteins. Evidence is presented establishing that the amount of CCN3 secreted into cell culture medium is regulated by post-translational proteolysis. As a consequence, the production of CCN3 varies throughout the cell cycle and CCN3 accumulates at the G2/M transition of the cycle. We also show that CCN3-induced inhibition of cell growth can be partially reversed by specific antibodies raised against a C-terminal peptide of CCN3. The use of several clones expressing various portions of CCN3 established that the CT module of CCN3 is sufficient to induce cell growth inhibition.

Ultrasonography for pregnancy diagnosis and evaluation in queens.

In the present paper, we describe the clinical utility of ultrasonography for diagnosing and evaluating pregnancy in domestic cats. Ultrasonography is a non-invasive technique that permits an accurate diagnosis of pregnancy and allows serial evaluation of the developing embryo/fetus and the extrafetal structures. The first ultrasonographic indication of pregnancy is a gestational chamber seen on day 10 after mating as a small circular anechoic structure. From day 30, it is possible to recognize different fetal organs, and between 38 and 43 days, the gender of the fetus can be determined. Measurements obtained during the second half of gestation can be used to determine fetal age and calculations can then be made that may more accurately predict the time of parturition. Further studies are needed in the queen to determine the applicability of the echo-Doppler technique used routinely in human obstetric medicine. This type of ultrasonography could potentially provide useful information about fetal health and the maturity of the placenta.

Fertilizing ability of electro-ejaculated cryopreserved semen in the domestic cat.

Semen collection and AI in the cat are still not routine procedures. The correlation between semen quality and fertility under natural conditions is a relatively unknown field in the cat. In the present study, functional in vitro tests, such as the ability to bind and penetrate the zona pellucida or to fertilize in vitro, were used to determine fertilizing ability of sperm cryopreserved with a practical and efficient freezing protocol previously developed in our laboratory. Semen was collected by electroejaculation, evaluated for motility and diluted with Tris-glucose-citrate egg-yolk extender supplemented with Equex STM paste (0.5% v/v). After equilibration and loading into 0.25 ml straws, semen was frozen at 3.85 degrees C/min. Frozen-thawed semen was co-cultured with in vitro matured cat oocytes. Penetration rate was recorded 30 h after in vitro fertilization and cleaved zygotes were cultured in vitro until day 7. A correlation was found between sperm motility index (SMI) after thawing and semen fertilizing ability (p<0.05). In conclusion, it was demonstrated that the post-thaw motility quality, expressed as SMI, of spermatozoa frozen using the protocol mentioned above can be considered an index of the sperm ability to penetrate in vitro matured oocytes.

Vaginal and cervical anatomic modifications during the oestrus cycle in relation to transcervical catheterization in the domestic cat.

In a previous study we observed that it is possible to reach the cervix in all queens with a 1 mm diameter probe only. So, we developed both a new technique and a catheter (1 mm diameter) to allow transcervical insemination [Zambelli and Castagnetti 2001]. The aims of this study were to investigate vaginal and cervical anatomic modifications during the various stages of the oestrus cycle and to test the previously described technique of transcervical catheterization during the various stages of the oestrus cycle. In experiment 1, silicon impression moulds were obtained from the reproductive tracts of 21 queens' cadavers and vaginal and cervical measures were taken. The results showed that there are some significant anatomic modifications during the various stages of the oestrus cycle in vaginal and cervical anatomy, principally related to the dorsal medial fold increase induced by the follicular phase. In experiment 2, transcervical catheterization was attempted in 95 queens at various stages of oestrus cycle both during reproductive and non-reproductive season. After catheterization, methylene blue solution was injected through the cervical catheter. Successful catheterization was assessed during surgery, when colour was observed in the uterine horns. It was possible to perform transcervical catheterization during non-reproductive season in 16 of 20 anoestrus queens and in 12 of 15 induced oestrus queens; during reproductive season in nine of 21 interoestrus queens, in eight of 13 dioestrus/pregnancy queens, in four of 18 oestrus queens and in seven of eight queens in first oestrus during lactation.

Cryopreservation of cat semen in straws: comparison of five different freezing rates.

The first pregnancies in domestic cats were obtained using semen frozen in pellets (Platz et al. 1978). Other freezing methods, vials (Lengwinant and Blottner 1994) or straws (Pope et al. 1991; Hay and Goodrowe 1993), have also been used. Pelleted freezing has often been the standard method (Howard 1986). Opinions about the freezing method are discordant; the best method for Pope et al. (1991) was using straws; in fact, the post-thaw motility and the percentage of normal acrosomes were of 44 +/- 4 and 62 +/- 3%, respectively, with straws and 11 +/- 3 and 26 +/- 4%, respectively, with pellets. According to Wood et al. (1993), there are no differences between the two methods, with a motility of 66.2% and a percentage of normal acrosomes of 28.6% for the pellet method and a motility of 67.0% and a percentage of normal acrosomes of 27.4% for the straw container. However, these two authors used two different freezing protocols. A high concentration of glycerol (i.e. 8%, vol/vol) damages cat semen (Zambelli 1994; Nelson et al. 1999), because of his toxicity to spermatozoa (Graham 1996); while a concentration of 4% is suggested (Zambelli 1994). Fast green FCF Bengal pink staining is often used to evaluate the acrosomal morphology (Wood et al. 1993; Zambelli et al. 1993). As there are no studies on the influence of freezing rate on motility and on acrosomal morphology, the aim of this study was to test five freezing rates in order to verify which is the best for the cryopreservation of cat semen in straws.

Correlation between the age of the conceptus and various ultrasonographic measurements during the first 30 days of pregnancy in domestic cats (Felis catus).

We ultrasonographically evaluated the prenatal development in cats, from the early phases to Day 30 of pregnancy, subjecting a group of pregnant cats (n = 12) to a daily ultrasonographic exam. The ultrasonographic images allowed us to measure the minor diameter of the gestational sac and the crown-rump length of the embryo/fetus. Ten subjects underwent ovariohysterectomy at specific intervals during the pregnancy, with the aim of comparing the ultrasonographic data with real data; only two subjects brought their pregnancy to term. The earliest ultrasonographic observation of the gestational sac was on Day 10 after mating, while the embryo could be measured only beginning with Day 18. This study allowed to gather useful new data in order to clinically monitor the normal course of pregnancy in cats and to date the gestational age.

Ultrasound aspects of fetal and extrafetal structures in pregnant cats.

Prenatal feline fetal growth and utero-placental development were ultrasonographically evaluated using an ultrasound scanner with a 10 MHz sector probe. Uterus, placenta, embryo, fetus and fetal membranes in 16 pregnant cats were monitored during the course of pregnancy; 13 subjects underwent an ovariectomy on specific days while three subjects went to term. Various anatomic structures, fixed in Carson-buffered formalin, were sectioned and then compared to ultrasound images. By ultrasound examination it is possible to evaluate every stage of the fetal development; the gestational chamber can be seen on the 10th and the embryo inside the chamber on the 14th day. By the 20th day it is possible to evaluate all the fetal membranes, and later it is possible to appreciate organs and structures such as the stomach, intestine, eyes (crystalline lens), kidneys and the cerebral choroid plexi, on the 30th, 40th, 50th, 39th and 40th day respectively. Based on our observations, it will be simpler to locate anomalies of development or pathologies during ultrasound examination of pregnant queens.