The Comet assay for detection of DNA damage in canine sperm.

Sperm DNA integrity is a fundamental prerequisite in fertilization and embryo development. Among DNA integrity tests, the Comet assay is an accurate and sensitive test for the detection of sperm oxidative damage. The aim of this work was to evaluate sperm oxidative damage using the Comet assay and to study the correlation between Comet and routine assays for the evaluation of semen quality. Dogs were divided in two groups: group A (n = 6), comprising dogs with abnormal spermiogram, that is astheno-, terato- or oligoasthenoteratozoospermic (OAT); and group B (n = 8), comprising normospermic dogs. The distribution of sperm oxidative damage was significantly different between the two groups (p = .001): group A-median: 31.55%, interquartile range (IQR): 30.18-38.01; group B-median: 0.90%, IQR: 0.65-1.96. The correlation between oxidative damage and abnormal morphology was high (r = .846; p < .001). There was a negative correlation between progressive motility and oxidative damage (r = -.792; p = .001). Basal and oxidative DNA damage of spermatozoa are increased in dogs with non-normospermic semen. In conclusion, and considering the elevated correlation with classical tests of sperm quality, the Comet assay has ample potential for clinical and research purposes in dogs.

Presence of membranous vesicles in cat seminal plasma: ultrastructural characteristics, protein profile and enzymatic activity.

This study sought to verify the presence of membranous vesicles in cat seminal plasma by means of transmission electron microscopy and to identify protein profile and some of the enzymatic activities associated with these particles. The transmission electron microscopy observations showed the existence of different sized vesicular membranous structures of more or less spherical shape. These vesicles were surrounded by single-, double- or multiple-layered laminar membranes. The vesicle diameters ranged from 16.3 to 387.4 nm, with a mean of 116.5 ± 70.7 nm. Enzyme activity determinations showed the presence of dipeptilpeptidase IV, aminopeptidase, alkaline and acid phosphatase. To our knowledge, this is the first report that identifies and characterizes the membranous vesicles in cat seminal plasma. However, further studies are necessary to identify the exact site of production of these membranous vesicles in the cat male genital tract and to determine their specific roles in the reproductive events of this species.

Fertility in Adult Bitches Previously Treated with a 4.7 mg Subcutaneous Deslorelin Implant.

The absence of fertility problems in male dogs after a single treatment with deslorelin acetate (Suprelorin(®)) is well acknowledged. However, reports on the application of deslorelin in the bitch and information concerning fertility after implant treatment are still limited. In this retrospective study, data concerning induced and spontaneous oestruses of 39 bitches from 17 breeds, treated with deslorelin acetate implants (4.7 mg Suprelorin(®), Virbac, France), were retrieved to assess post-treatment fertility (ovulation rate, pregnancy rate and litter size). Animals were grouped according to treatment characteristics: group 1 (Gr1) - females submitted to oestrus induction, showing natural oestruses afterwards (n = 19); group 2 (Gr2) - females re-implanted with 4.7 mg deslorelin acetate to re-induce oestrus, showing subsequent spontaneous post-implant oestruses (n = 7); and group 3 (Gr3) - females submitted to a 4.7 mg deslorelin acetate implant for oestrus suppression, evaluated at subsequent spontaneous post-implant oestruses (n = 13). Comparison of fertility traits between induced and post-treatment spontaneous oestruses in Gr1 and Gr2 (short treatments), or between spontaneous oestruses after long-treatment schedules (Gr 3) revealed a slightly better performance in spontaneous cycles compared with induced cycles: ovulation rate post-treatment was 97.1%, 94.1% and 94.4% and the pregnancy rate post-treatment was 91.2%, 88.9% and 84.6% for groups 1, 2 and 3, respectively. Nevertheless, fertility in induced and post-treatment oestruses was considered normal. Moreover, the individual litter size did not differ within groups between induced and spontaneous cycles. From these findings, we concluded that treatment with 4.7 mg deslorelin implants did not compromise the bitches' fertility in subsequent oestruses.

Suppression of fertility in adult dogs.

Unfortunately, the overpopulation of dogs is still a problem in the majority of countries and even though surgical methods of sterilization, the most traditional and commonly used technique, have been intensively performed, the impact on the dog population is negligible. The neutering of companion animals as ovariohysterectomy (spaying) or orchidectomy (castration) has its limitations because of the cost, the need of a surgical environment and the risk of surgical and/or anaesthetical complications (ACCD 2009). In fact, surgical castration has been banished in some northern European countries and has limited acceptance in other countries. In a survey performed in Sao Paulo, Brazil, 56.5% of the owners of adopted shelter dogs were against the surgical procedure for different reasons (Soto et al. 2005). Currently, the options for contraception, defined as suppression of fertility are based on hormonal treatment. The treatments can be divided into analogues of gonadotropin releasing hormone (GnRH), progestins and androgens. Other possibilities of contraception are via the immunological system with vaccinations against GnRH, the luteinizing hormone (LH) receptor and the zona pellucida proteins. Finally, there is also the intra-epididymal or intratesticular injection of sclerosing substances in dogs. Mechanical devices to disrupt fertility are not used anymore due to the side effects. Suppression of fertility in adult dogs will be reviewed in order of use and possible impact on the dog population.

Results of GnRH agonist implants in oestrous induction and oestrous suppression in bitches and queens.

Over the last 10-15 years, long-acting GnRH agonists have become widely available. In the field of small animal reproduction, most recent studies have focused on the use of two compounds developed under the form of subcutaneous implants: azagly-nafarelin and deslorelin. Only the latter has been commercially available for use in male dogs, first in Australia and New Zealand, then in several countries of the European Union since 2008. Although officially marketed for male dogs, this compound has also been studied in bitches and more recently in queens. Some published papers or recent presentations at congresses--still unpublished--have focused on the use of GnRH agonists implants in females.

Folliculogenesis, ovulation and endocrine control of oocytes and embryos in the dog.

Reproductive physiology in dogs is quite unusual compared with that in other mammalian species. The peculiarities include the presence of numerous polyoocyte follicles, the ovulation of an immature oocyte (GV stage, non-fertilizable) and a peri-ovulatory period during which concentrations of circulating progesterone are particularly high. The aim of this review is to examine the unusual aspects of the reproductive physiology of dogs and how this relates to the clinical biology of this species.

Clinical use of GnRH agonists in canine and feline species.

GnRH (gonadotrophin releasing hormone) is a key hormone of reproductive function in mammals; agonist forms have been largely developed, and data concerning their use in small animal reproduction are now abundant. GnRH agonists act by a two-step mechanism. First, their agonist properties on the pituitary will cause marked LH (luteinizing hormone) and FSH (follicle-stimulating hormone) secretion into the bloodstream, accompanied by an increase in the concentrations of sex steroid hormones. Then, in case of constant administration, GnRH agonists will lead to pituitary desensitization, and FSH and LH levels will collapse. These two effects have been widely documented, and these compounds have many potential benefits in a clinical context, capitalizing both on their stimulating and sterilizing effects.

Estimated pregnancy length from ovulation to parturition in the bitch and its influencing factors: a retrospective study in 162 pregnancies.

An accurate timing of parturition is very useful for managing canine parturition. It is generally accepted that parturition in bitches occurs between 64 and 66 days after the luteinizing hormone peak. In this retrospective study, we determined pregnancy length in different breeds and its influencing factors dating it from the estimated day of ovulation (EDO), defined as the day when peripheral plasma level of progesterone (P4) reaches 6 ng/ml. From January 2001 to December 2006, 162 pregnancies in 151 bitches of 53 different breeds were followed. Different parameters concerning the bitch, the litter, the type of semen and the type of reproduction were studied. The mean estimated pregnancy length in the bitch from EDO to parturition was 63.1±2.1 days. The main influencing factors for the pregnancies studied were the breed, the size of the bitch and the number of puppies within the litter.

A review of maternal behaviour in dogs and potential areas for further research.

Here we review information on maternal behaviour in dogs, defined as a combination of all the acts of the mother towards her offspring, which begins before parturition and continues until weaning. In dogs, maternal care is measured using the most commonly observed behaviours, such as the time spent in contact, licking/grooming and nursing of the puppies. Since newborn puppies have a very limited capacity for movement, maternal interaction is essential to their survival, nourishment and protection. It is also an important element of the bonding process between puppies and the bitch and is thought to play a role in the social development of the puppies. Nevertheless, some questions still need to be clarified, such as the best way to quantify factors that may interfere with maternal behaviour. In recent studies, maternal care, or maternal style, was measured using a scoring system and found to be influenced by factors such as litter size, breed and parity, or even human interaction. However, the impact of the emotional state of the bitch and the quality of maternal behaviour on puppy survival and development remain unclear. The long-lasting effects of mother-puppy interactions on puppy behaviour during their adult life are still poorly understood, despite their importance for breeders who wish to prevent future problem behaviours.

Triplex doppler ultrasonography to describe the uterine arteries during diestrus and progesterone profile in pregnant and non-pregnant bitches of different sizes.

Hemodynamics of uterine vascularization is modified throughout pregnancy to meet the increasing demand of the growing fetuses and triplex doppler ultrasonography is widely used in human medicine to study the uterine arteries and assess the fetal and placental conditions. The aim of our study was to confirm this observation in the bitch, to evaluate differences between bitches of different sizes and to study abnormal pregnancies. Forty-four bitches were monitored during the estrous period to determine ovulation and every 10 days from ovulation to 50 days post-ovulation: the resistivity (RI) and pulsatility (PI) indexes of the right uterine artery were measured as well as usual assessment of fetal development and follow up of the luteal function. Thirty-three out of forty-four bitches were pregnant, including 6 abnormal pregnancies (resorption of more than 10% of the embryos). We also divided them in four weight categories: 8 were small (<10 kg), 13 medium (10-25 kg), 13 large (>25-40 kg) and 10 were giant breeds (>40 kg). We observed that RI and PI decreased over time and were significantly lower for pregnant bitches compared to non-pregnant ones from 30 days post-ovulation. In contrast, RI and PI did not significantly vary with the size of the bitches and we could not determine a significant impact of abnormal pregnancies either. In conclusion, we found no significant difference related to the size of bitches in the RI and PI. The only significant difference between pregnant and non-pregnant bitches was observed from 30 days post-ovulation.

Elective caesarean operation in the bitch using aglepristone before the pre-partum decline in peripheral progesterone concentration.

The aim of this study was to investigate caesarean operation (CO) undertaken before the pre-partum decrease of progesterone but following administration of a progesterone receptor antagonist and to evaluate the innocuity of this procedure for the dam and pups. Thirty seven bitches of 15 different breeds, received an injection of 15 mg/kg aglepristone 59 or 60 days after the estimated day of ovulation, determined by progesterone quantitative assays, and caesarean section (CS) was performed between 20 and 24 h after administration. Progesterone remained above 6 nmol/l at the time of CS (mean = 15.75, SD = 3.84). No post-operative clinical complications were reported in any of the bitches. All bitches were able to nurse and feed their puppies in the first 24 h following surgery. No pups showed any signs of prematurity and 5 out of 188 pups (2.6) died in the first 2 weeks after delivery. This small study demonstrates that a CS may be safely and successfully performed an average of 2 days before the expected date of parturition following the administration of aglepristone, without any harmful consequence for the dam and her neonates.

Thyroid function and infertility in the dog: a survey in five breeds.

In our study, a clinical and reproductive examination was undertaken in 204 dogs of five different breeds. Thyroxine and thyroid-stimulating hormone plasma concentrations were assayed in order to assess the real incidence of hypothyroidism associated with reproductive disease. Among these animals, only two breeds were affected by hypothyroidism (4.5% of the Dogue de Bordeaux and 2.4% of the Leonbergers). Moreover, these animals did not suffer from any reproductive disease. Our study showed also that 70% of the male Dogue de Bordeaux were hypothyroxinemic compared with Great Danes, English Mastiff and Leonbergers whose male population was 100% normothyroidic. We found the same tendency in bitches. In our data, there was no difference in thyroxin plasma concentrations between normo- and hypofertile dogs, but our results were not statistically significant.

Induction of parturition with aglepristone in various sized bitches of different breeds.

The objective of this study was to confirm in various breeds of dogs the efficacy and safety of a parturition induction treatment described to be successful in Beagle dogs. Parturition was induced in seven various sized pregnant bitches of different breeds, with 15 mg aglepristone per kg at day 59-61 post-estimated ovulation day, followed 24 h later by 0.15 IU oxytocin per kg subcutaneous injections every 2 h. Two bitches were small-sized bitches (<10 kg), three bitches were large-sized bitches (30-40 kg) and two bitches were giant bitches (>40 kg). The results were compared to a control group (n = 6), in which bitches underwent a natural delivery in the same environmental conditions as the induced group. In the induced group, parturition was successfully induced in 7/7 bitches. The first pup in a litter was born on average 25.9 +/- 3.29 h after aglepristone administration (21-30 h). Two of seven bitches from the small-sized group delivered some of their pups before the first administration of oxytocin. The mean duration of parturition was 9.6 +/- 5.4 h vs 8.0 +/- 4.8 h in the control group. The mean interval between two successive pups being delivered was 115.6 +/- 82.8 min (34-265) vs 68.8 +/- 24.5 min in the control group (p < 0.03). The mean weight at parturition did not differ significantly between the two groups. One litter of four Yorkshire Terrier pups in the induced group were premature at the time of birth and died between 19 and 29 h post-delivery. This study, although on a very limited number of dogs, confirms the efficacy of the aglepristone/oxytocin protocol to induce parturition in dogs.

Diagnosis of endometritis in the bitch: a new approach.

There are a few investigations into endometritis in the bitch and its relationship with failure to conceive remains unclear. This may be because of the difficulty in collecting uterine samples for further investigations. Recently, transcervical catheterization by vaginal endoscopy has been introduced allowing the evaluation of the endometrium. In this study, uterine cytology and bacteriology were evaluated in 26 infertile bitches. Endometritis was bacterial in origin in most cases (70% of affected bitches), but these results may be underestimated, as some other pathogens (anaerobic bacteria, mycoplasms and fungi) were not investigated. Endometritis, in our opinion, should be investigated in each case of unexplained infertility in bitches. The method used here seems reliable although defining more accurate classification criteria will improve the efficiency of this non-invasive technique.

Evaluation of a structured educational programme for type 2 diabetes patients seen in private practice.

AIM: Structured education is necessary in the management of a chronic disease such as diabetes and should be readily offered to patients in different settings. Our aim was to demonstrate the feasibility and advantages of a group education programme for type 2 diabetic patients in a private setting in France. METHODS: A programme of group education for patients with type 2 diabetes was initiated by a multidisciplinary group of volunteer healthcare providers, including general practitioners, specialists in diabetology and non-medical members. All volunteers received one day of training, and physicians were instructed to organize several sessions of group education for the type 2 diabetic patients who regularly attended their practice. The first 427 patients entering the programme were included in the study, and asked to fill in a questionnaire to assess their knowledge, beliefs and behaviours with regard to diabetes. Their physician filled in a medical form. Six months later, the same questionnaire and form were sent for follow-up information. RESULTS: At six months versus baseline, patients exhibited small, but consistent, improvements: (i) fasting glucose 142+/-42 mg/dL (P<0.04) vs 146+/-44 mg/dL (P<0.04); (ii) HbA(1c) 7.41+/-1.26% vs 7.57+/-1.33% (P<0.01); and (iii) all of the main parameters of diabetes self-management recorded in the study. The percentage of patients who inspected their feet at least once a week increased from 67 to 77% (P<0.001). Patients improved their knowledge of the disease and developed a more positive attitude towards their diabetes. CONCLUSION: Our study demonstrates that it is possible to organize educational sessions for diabetic patients in a private-practice setting. At six months, patients receiving these sessions showed benefits in terms of blood glucose control and other important markers of self-management of their disease.

A retrospective study of canine prostatic diseases from 2002 to 2009 at the Alfort Veterinary College in France.

A retrospective study was used to investigate the incidence of prostatic diseases in a large population of dogs at Alfort Veterinary Hospital and to clarify epidemiologic features, which might be of a great help to veterinarians in managing and discriminating prostatic disorders. During the investigation period, a total of 72,300 male dogs (coming mainly from the Ile-de-France region) were registered in the Alfort Veterinary College database, and 481 of them (0.7%) were found to have prostatic disorder. The diagnosis was carried out on the basis of clinical signs and ultrasound findings. Among dogs experiencing a prostatic disorder, most frequently recorded diseases were benign prostatic hyperplasia (45.9%) and prostatitis (38.5%), followed by abscesses (7.7%), cysts (5.0%), neoplasia (2.6%), and squamous metaplasia (0.2%). Our study revealed an incidence of 0.3% of prostatic disorders observed in intact male dogs, except in the case of prostatic neoplasia. The mean age of the dogs experiencing prostatic disorders was 8.6 ± 3.2 years. This was significantly different (P < 0.001). Large dogs were significantly more affected by prostatic disorders (P < 0.05), except for prostatic neoplasia. A breed predisposition was suspected in German Shepherd (odds ratio [OR] = 2.1; 95% confidence interval [CI]: 1.5-2.9), Rottweiler (OR = 1.8; 95% CI: 1.2-2.7), American Staffordshire Terrier (OR = 3.8; 95% CI: 2.5-5.8), Berger de Beauce (OR = 3.7; 95% CI: 2.2-6.1), and Bernese Mountain Dog (OR = 2.5; 95% CI: 1.3-4.7).

Risk factors for NIDDM in white population. Paris prospective study.

Risk factors for non-insulin-dependent diabetes mellitus (NIDDM) were assessed in a population of 5042 middle-aged white men, initially nondiabetic, who were followed 3 yr. The subjects were participants in the Paris Prospective Study I. Sixty-three subjects developed diabetes during the follow-up. Plasma glucose concentration in the years before the occurrence of the disease was a major risk factor. Subjects with normal glucose tolerance but elevated fasting plasma glucose exhibited a similar risk of developing NIDDM as did subjects classified as having impaired glucose tolerance on the basis of 2-h postload glucose. In a multiple logistic regression, a high fasting plasma insulin concentration and a low 2-h plasma insulin concentration after a glucose load in association with a high body mass index were independent predictors of conversion to NIDDM from impaired glucose tolerance. Previously, this result had been found only in Nauruans, Pima Indians, and Japanese. This demonstrates for the first time in a white population that a high fasting and low 2-h insulin concentration is predictive of conversion to NIDDM from impaired glucose tolerance.

Features of insulin-resistance syndrome in men from French Caribbean Islands. The Telecom Study.

The prevalence of diabetes is known to be high in the West Indies, whereas CVD seems relatively rare. This apparently contradicts the existence of the insulin-resistance syndrome, a cluster of metabolic abnormalities supposedly favoring both diabetes and cardiovascular complications. To address the question of whether this contradiction could be accounted for by specific features of this syndrome, we compared 1505 Caucasian and 181 Afro-Caribbean men, all participants in the French Telecom Study. The Afro-Caribbeans were of the same age and had the same BMI as the Caucasians; they also had significantly higher subscapular skin-fold thickness and fasting insulin level, but similar BP and blood glucose level, and significantly lower plasma triglyceride level. Thus, although some features of the insulin-resistance syndrome were present (central adiposity and high insulin levels), none of the associated metabolic abnormalities were present. However, within the Afro-Caribbean group, subjects with plasma insulin concentrations above the median (> 52 pM) had higher mean BP and glucose and triglyceride levels compared with subjects with insulin concentrations < or = 52 pM (P < 0.001). After adjustment for age and BMI, these differences, though smaller, still were statistically significant. These findings confirm that higher insulin concentrations are associated with higher levels of potentially atherogenic and diabetogenic metabolic parameters. However, depending on ethnic origin, the mean levels of these parameters seem to be different, with the consequence that, even if they are elevated with increasing insulin levels, they may not reach values high enough to determine a substantial risk of disease.

Factors associated with upper respiratory tract disease caused by feline herpesvirus, feline calicivirus, Chlamydophila felis and Bordetella bronchiseptica in cats: experience from 218 European catteries.

A full history of the management practices and the prevalence of upper respiratory tract disease (URTD) at 218 rescue shelters, breeding establishments and private households with five or more cats was recorded. Oropharyngeal and conjunctival swabs and blood samples were taken from 1748 cats. The prevalences of feline herpesvirus (FHV), feline calicivirus (FCV), Chlamydophila felis and Bordetella bronchiseptica were determined by PCR on swab samples. An ELISA was applied to determine the prevalence of antibodies to B. bronchiseptica. The rates of detection by PCR of each pathogen in the cats in catteries with and without ongoing URTD were, respectively, FHV 16 per cent and 8 per cent; FCV 47 per cent and 29 per cent; C. felis 10 per cent and 3 per cent; and B. bronchiseptica 5 per cent and 1.3 per cent; the seroprevalences of B. bronchiseptica were 61 per cent and 41 per cent, respectively. There was evidence that FHV, FCV and B. bronchiseptica played a role in URTD. The risk factors associated with the disease were less than excellent hygiene, contact with dogs with URTD, and larger numbers of cats in the cattery or household.

Insulin and cardiovascular disease. Paris Prospective Study.

The Paris Prospective Study is a long-term investigation of coronary heart disease (CHD) risk factors in a large population of working men. The baseline cohort included 7028 men, 6093 who had a 75-g oral glucose tolerance test with measurement of plasma insulin and glucose levels (0 and 2 h) and 125 who were known non-insulin-treated diabetic patients. After a mean follow-up of 11 yr, 126 deaths ascribed to CHD were reported. Major independent predictors of CHD death were blood pressure, smoking, plasma cholesterol level, and fasting and 2-h postload plasma insulin level. Impairment of glucose tolerance, including overt diabetes, did not rank as an independent predictor when other baseline variables were accounted for. In the subset of the baseline cohort who presented with impaired glucose tolerance or diabetes (n = 943), 26 died from CHD during the follow-up. The strongest independent predictor of subsequent CHD death in this subsample with abnormal glucose tolerance was plasma triglyceride level. In view of the accumulating evidence that hyperinsulinemia and hypertriglyceridemia generally occur in the same type of subjects, in relation to insulin resistance and central obesity, the epidemiological findings of the Paris Prospective Study and of other investigations support the hypothesis that a constellation of mild metabolic abnormalities may play a deleterious role with regard to cardiovascular disease risk.