Low and very low birth weight in puppies: definitions, risk factors and survival in a large-scale population.

BACKGROUND: Neonatal mortality (over the first three weeks of life) is a major concern in canine breeding facilities as an economic and welfare issue. Since low birth weight (LBW) dramatically increases the risk of neonatal death, the risk factors of occurrence need to be identified together with the chances and determinants of survival of newborns at-risk. RESULTS: Data from 4971 puppies from 10 breeds were analysed. Two birth weight thresholds regarding the risk of neonatal mortality were identified by breed, using respectively Receiver Operating Characteristics and Classification and Regression Tree method. Puppies were qualified as LBW and very low birth weight (VLBW) when their birth weight value was respectively between the two thresholds and lower than the two thresholds. Mortality rates were 4.2, 8.8 and 55.3%, in the normal, LBW and VLBW groups, accounting for 48.7, 47.9 and 3.4% of the included puppies, respectively. A separate binary logistic regression approach allowed to identify breed, gender and litter size as determinants of LBW. The increase in litter size and being a female were associated with a higher risk for LBW. Survival for LBW puppies was reduced in litters with at least one stillborn, compared to litters with no stillborn, and was also reduced when the dam was more than 6 years old. Concerning VLBW puppies, occurrence and survival were influenced by litter size. Surprisingly, the decrease in litter size was a risk factor for VLBW and also reduced their survival. The results of this study suggest that VLBW and LBW puppies are two distinct populations. Moreover, it indicates that events and factors affecting intrauterine growth (leading to birth weight reduction) also affect their ability to adapt to extrauterine life. CONCLUSION: These findings could help veterinarians and breeders to improve the management of their facility and more specifically of LBW puppies. Possible recommendations would be to only select for reproduction dams of optimal age and to pay particular attention to LBW puppies born in small litters. Further studies are required to understand the origin of LBW in dogs.

Birth weight as a risk factor for neonatal mortality: Breed-specific approach to identify at-risk puppies.

In numerous species, low birth weight is a risk factor for neonatal mortality. In the canine species, definition of a low birth weight is complex due to the huge interbreed variability in size. To identify puppies at higher risk of neonatal death, data from 6,694 puppies were analysed. The data were collected from 75 French breeding kennels, examining 27 breeds and totaling 1,202 litters of puppies. Generalised linear mixed models allowed to identify birth weight, birth weight heterogeneity within the litter, and size of the breeding kennel as significant risk factors for neonatal mortality. Receiver Operating Characteristics (ROC) and classification and regression tree (CART) analyses were combined to define breed specific thresholds for birth weight allowing the identification of puppies at higher risk of neonatal mortality. Due to differences in birth weights between breeds, including when belonging to the same breed size, analyses were conducted at the breed level. First, ROC analysis thresholds were successfully established for 12 breeds (area under the ROC ≥ 0.70; sensitivity ≥ 75%; specificity: 45-68%) and they ranged from 162 g in the Maltese to 480 g in the Bernese Mountain dog. Secondly, CART analysis thresholds from 22 breeds ranged from 105 g in the Maltese and 436 g in the Boxer. Puppies were grouped into three categories according to birth weight: low, moderate and high risk of neonatal mortality (higher than the ROC threshold, between ROC and CART thresholds, and lower than the CART threshold respectively). In the current study, 44% of the puppies were classified as at moderate risk and 5.3% for a high risk of neonatal mortality. Thresholds defined by CART analysis (and not ROC analysis) were used to define low birth weight puppies and were sometimes quite different between breeds with similar birth weight distributions suggesting a variable relationship between birth weight reduction and neonatal death. These results allow the identification of puppies at an increased risk of neonatal death, thus requiring specific nursing to improve their chances of survival. With these high risk puppies identified, both animal welfare and kennel productivity is predicted to improve.

General and type 2 parvovirus-specific passive immune transfer in puppies - Evaluation by early growth.

Failure of passive immune transfer put puppies at a higher risk of neonatal and weaning mortality due to low immune protection against infectious agents. The aim of this study was to investigate the evolution of the general via serum IgG concentration (IgG) and the specific via serum maternally derived canine parvovirus type 2-specific antibody titer (CPV2 MDA) passive immune transfer within the first 4 weeks of age. Furthermore, the relationship between general and specific immune transfer and the possibility of non-invasive evaluation was assessed. Puppies (169) were weighed systematically between birth and Day 28. IgG and CPV2 MDA were assayed in serum samples at 2 and at 28 days of age. At Day 2, there was a positive correlation between IgG and CPV2 MDA (ρ = 0.71; p < 0.001). At Day 2, 17.9% (27/151) of puppies presented a deficit of passive immune transfer according to IgG result (defined as IgG < 2.3 g/L) and 25.8% (39/151) of puppies were under the minimal protective CPV2 MDA titer (defined as <1:160). No correlation between IgG and CPV2 MDA was observed at Day 28 (ρ = 0.14; p = 0.11). Growth rate within the first 48 hours <-2.7% allowed to distinguish puppies at high risk of the general and specific passive immune failure (Youden's index = 0.79 and 0.75, respectively). The threshold value of early growth rate, although applicable only in puppies non-supplemented with milk replacer, allows identifying via non-invasive way individuals requiring a special care. Further investigation of the mechanism of passive immune transfer in dogs is necessary to understand the relationship between the general and specific immunoglobulin levels.

Effect of selected gastrointestinal parasites and viral agents on fecal S100A12 concentrations in puppies as a potential comparative model.

BACKGROUND: Previous data suggest that fecal S100A12 has clinical utility as a biomarker of chronic gastrointestinal inflammation (idiopathic inflammatory bowel disease) in both people and dogs, but the effect of gastrointestinal pathogens on fecal S100A12 concentrations is largely unknown. The role of S100A12 in parasite and viral infections is also difficult to study in traditional animal models due to the lack of S100A12 expression in rodents. Thus, the aim of this study was to evaluate fecal S100A12 concentrations in a cohort of puppies with intestinal parasites (Cystoisospora spp., Toxocara canis, Giardia sp.) and viral agents that are frequently encountered and known to cause gastrointestinal signs in dogs (coronavirus, parvovirus) as a comparative model. METHODS: Spot fecal samples were collected from 307 puppies [median age (range): 7 (4-13) weeks; 29 different breeds] in French breeding kennels, and fecal scores (semiquantitative system; scores 1-13) were assigned. Fecal samples were tested for Cystoisospora spp. (C. canis and C. ohioensis), Toxocara canis, Giardia sp., as well as canine coronavirus (CCV) and parvovirus (CPV). S100A12 concentrations were measured in all fecal samples using an in-house radioimmunoassay. Statistical analyses were performed using non-parametric 2-group or multiple-group comparisons, non-parametric correlation analysis, association testing between nominal variables, and construction of a multivariate mixed model. RESULTS: Fecal S100A12 concentrations ranged from < 24-14,363 ng/g. Univariate analysis only showed increased fecal S100A12 concentrations in dogs shedding Cystoisospora spp. (P = 0.0384) and in dogs infected with parvovirus (P = 0.0277), whereas dogs infected with coronavirus had decreased fecal S100A12 concentrations (P = 0.0345). However, shedding of any single enteropathogen did not affect fecal S100A12 concentrations in multivariate analysis (all P > 0.05) in this study. Only fecal score and breed size had an effect on fecal S100A12 concentrations in multivariate analysis (P < 0.0001). CONCLUSIONS: An infection with any single enteropathogen tested in this study is unlikely to alter fecal S100A12 concentrations, and these preliminary data are important for further studies evaluating fecal S100A12 concentrations in dogs or when using fecal S100A12 concentrations as a biomarker in patients with chronic idiopathic gastrointestinal inflammation.

Potential of connected devices to optimize cattle reproduction.

Estrus and calving are two major events of reproduction that benefit from connected devices because of their crucial importance in herd economics and the amount of time required for their detection. The objectives of this review are to: 1) provide an update on performances reached by sensor systems to detect estrus and calving time; 2) discuss current economic issues related to connected devices for the management of cattle reproduction; 3) propose perspectives for these devices. The main physiological parameters monitored separately or in combination by connected devices are the cow activity, body temperature and rumination or eating behavior. The combination of several indicators in one sensor may maximize the performances of estrus and calving detection. An effort remains to be made for the prediction of calvings that will require human assistance (dystocia). The main reasons to invest in connected devices are to optimize herd reproductive performances and reduce labor on farm. The economic benefit was evaluated for estrus detection and depends on the initial herd performances, herd size, labor cost and price of the equipment. Major issues associated with the use of automated sensor systems are the weight of financial investment, the lack of economic analysis and limited skills of the users to manage associated technologies. In the near future, connected devices may allow a precise phenotyping of reproductive and health traits on animals and could help to improve animal welfare and public perception of animal production.

Monitoring of the newborn dog and prediction of neonatal mortality.

Despite the high neonatal mortality rate in puppies, pertinent criteria for health evaluation of the newborns are not defined. This study was thus designed to measure and to characterize factors of variation of six health parameters in dog neonates, and to evaluate their value as predictors of neonatal mortality. A total of 347 purebred puppies under identical conditions of housing and management were examined within the first 8h after birth and then at Day 1. The first health evaluation included Apgar score, weight, blood glucose, lactate and ß-hydroxybutyrate concentration, rectal temperature and urine specific gravity (SG). The second evaluation at Day 1 included the same parameters, excluding Apgar score and weight. The mortality rate over the first 24h and over 21days of age was recorded. The early predictors of neonatal mortality in the dog were determined with generalized linear mixed models and receiver operating characteristic curves analyses. An Apgar score at or below 6 evaluated within the first 8h after birth was found associated with a higher risk of death during the first 24h. A reduced glucose concentration (≤92mg/dl) at Day 1 was found to be associated with higher mortality between 1 and 21days of age. Low-birth-weight puppies were characterized by both low viability (low Apgar score) and low blood glucose concentration, and thus were found indirectly at higher risk of neonatal mortality. This study promotes two low cost easy-to-use tests for health evaluation in puppies, i.e. Apgar scoring and blood glucose assay. Further investigation is necessary to establish if the strong relationship between blood glucose and neonatal survival reflects high energy requirements or other benefits from colostrum intake.

Characterization of the fecal microbiome during neonatal and early pediatric development in puppies.

Limited information is available describing the development of the neonatal fecal microbiome in dogs. Feces from puppies were collected at 2, 21, 42, and 56 days after birth. Feces were also collected from the puppies' mothers at a single time point within 24 hours after parturition. DNA was extracted from fecal samples and 454-pyrosequencing was used to profile 16S rRNA genes. Species richness continued to increase significantly from 2 days of age until 42 days of age in puppies. Furthermore, microbial communities clustered separately from each other at 2, 21, and 42 days of age. The microbial communities belonging to dams clustered separately from that of puppies at any given time point. Major phylogenetic changes were noted at all taxonomic levels with the most profound changes being a shift from primarily Firmicutes in puppies at 2 days of age to a co-dominance of Bacteroidetes, Fusobacteria, and Firmicutes by 21 days of age. Further studies are needed to elucidate the relationship between puppy microbiota development, physiological growth, neonatal survival, and morbidity.

Progesterone plays a critical role in canine oocyte maturation and fertilization.

Canine oocyte maturation and fertilization take place within the oviducts under increasing plasma levels of progesterone (P4). In order to investigate the role of P4 in these processes, 51 beagle bitches were treated with the P4 receptor antagonist aglepristone at the end of proestrus and 32 females were kept untreated. Fifteen treated and 13 control bitches were inseminated at Days +1 and +2 after ovulation (Day 0). Stages of oocyte maturation and embryo development were determined after ovariectomy at different time points after ovulation. Aglepristone did not prevent ovulation but delayed the resumption of oocyte meiosis and inhibited its progression: first metaphase I (MI) stage was observed at 173 h postovulation and 39% of oocytes reached MII as late as 335 h postovulation in treated females whereas first MI occurred at 76 h and 100% of oocytes were in MII at 109 h postovulation in controls. Aglepristone extended the stay of morphologically normal oocytes within the oviducts: first signs of oocyte degeneration were observed at 335 h in treated versus 100- to 110-h postovulation in control bitches. In inseminated females, aglepristone prevented sperm progression toward the oviducts and fertilization, although motile spermatozoa were observed in the uterine tip flush and within the cranial uterine glands. A proteomic analysis of the tubal fluid from treated and control noninseminated bitches at Day +4 found evidence of 79 differential proteins potentially involved in the oocyte phenotype. In conclusion, P4 plays key roles in postovulatory canine oocyte maturation, aging, and in fertilization.

Immunoglobulin G concentration in canine colostrum: Evaluation and variability.

Canine neonates are born hypogammaglobulinemic, and colostrum is their main source of immunoglobulins. The purpose of this study was to evaluate the immune quality of canine colostrum and its variability both among bitches and among mammary glands. The immune quality was estimated from immunoglobulin G (IgG) concentration (ELISA test). The correlation of IgG concentration with refractometry was evaluated. From a total of 44 bitches from 13 different breeds from a single breeding kennel, samples of colostrum and blood were collected one day after the parturition onset. Colostrum was collected separately from each pair of mammary glands (180 pairs). The mean colostrum IgG concentration in our population was 20.8 ± 8.1g/L (ranging from 8.0 to 41.7 g/L) with no influence of breed size, litter size, age of dam or serum IgG concentration. Colostrum IgG concentration varied widely among pairs of mammary glands within one bitch (variation coefficient: 42 ± 32.1%). Nevertheless, no single pair of mammary glands was found to produce regularly a secretion of higher quality. No difference in IgG concentration was recorded between anterior and posterior pairs either. The BRIX index and the refractive index were significantly, but moderately correlated with colostrum IgG concentration (r=0.53 and 0.42, respectively). This study demonstrates a great variability in immune quality of colostrum among bitches and among mammary glands within one bitch. Further studies on the suckling behavior of puppies and on determination of the minimal immune quality of colostrum are required to evaluate their impact of this high variability on neonatal mortality in dogs.

Methods and on-farm devices to predict calving time in cattle.

In livestock farming, accurate prediction of calving time is a key factor for profitability and animal welfare. The most accurate and sensitive methods to date for prediction of calving within 24 h are the measurement of pelvic ligament relaxation and assays for circulating progesterone and oestradiol-17ß. Conversely, the absence of calving within the next 12-24 h can be accurately predicted by the measurement of incremental daily decrease in vaginal temperature and by the combination of pelvic ligament relaxation and teat filling estimates. Continuous monitoring systems can detect behavioural changes occurring on the actual day of calving, some of them being accentuated in the last few hours before delivery; standing/lying transitions, tail raising, feeding time, and dry matter and water intakes differ between cows with dystocia and those with eutocia. Use of these behavioural changes has the potential to improve the management of calving. Currently, four types of devices for calving detection are on the market: inclinometers and accelerometers detecting tail raising and overactivity, abdominal belts monitoring uterine contractions, vaginal probes detecting a decrease in vaginal temperature and expulsion of the allantochorion, and devices placed in the vagina or on the vulvar lips that detect calf expulsion. The performance of these devices under field conditions and their capacity to predict dystocia require further investigation.

OVGP1 is expressed in the canine oviduct at the time and place of oocyte maturation and fertilization.

In the dog, oocyte maturation, fertilization, and early embryo development take place within the oviduct in the presence of increasing circulating progesterone (P4) levels. Expression of the oviduct-specific glycoprotein 1 (OVGP1), known in other species to be estrogen-dependent, was explored by real-time quantitative reverse-transcriptase PCR, Western blotting, and immunohistochemistry in oviducts from adult Beagle bitches during anestrus and at five specific time periods around ovulation: during pro-estrus before the luteinizing hormone (LH) peak (Pre-LH); after the LH peak and before ovulation (Pre-ov); and at Days 1, 4, and 7 after ovulation (n = 6 bitches per stage). Plasma estradiol-17ß (E2) and P4 were assayed at all stages. The expression of canine OVGP1 (cOVGP1) was undetectable during anestrus, increased significantly from Pre-LH to Day 1 in parallel with a decrease in plasma E2-to-P4 levels, remained high at Day 4, then decreased at Day 7 in parallel with an increase in plasma P4 levels. In contrast to other mammals, the expression of cOVGP1 was higher in the isthmus than in the ampulla at all stages. In order to explore the potential regulation of cOVGP1 expression by steroids, the 5'-flanking region of the corresponding gene was analyzed for the presence of estrogen- (ERE) and P4-response-element (PRE). An imperfect ERE and three half-ERE were found in this region, but no PREs. In conclusion, cOVGP1 is highly expressed at the time and site of oocyte maturation and fertilization, and is probably under E2 regulation. Further studies are needed to identify the potential roles of cOVGP1 in each process.

Risk factors of weaning diarrhea in puppies housed in breeding kennels.

Diarrhea represents one of the most frequent disorders in dogs. In puppies, degradation of feces quality is associated with a reduced daily weight gain and an increased risk of death. Prevention of diarrhea in puppies requires a global approach encompassing enteropathogens, environment and management practices especially when housed in groups. The purpose of this study was to determine prevalence of enteropathogens in puppies in breeding kennels and to identify risk factors of diarrhea. Two hundred and sixty six puppies (between 5 and 14 weeks of age) from 29 French breeding kennels were included. For each kennel, data about environment, management of the kennel and puppies' characteristics (age, sex and breed) were collected. For each puppy, fecal consistency and fecal excretion of enteropathogens (viruses and parasites) was evaluated. At least one enteropathogen was identified in 77.1% of puppies and 24.8% of puppies presented abnormal feces. The main risk factor of weaning diarrhea was fecal excretion of canine parvovirus type 2 (odds ratio=5; confidence interval 95%: 1.7-14.7). A targeted sanitary and medical prophylaxis against canine parvovirus type 2 should be implemented to decrease risk of weaning diarrhea.

Partial urorectal septum malformation sequence in a kitten with disorder of sexual development.

A 2-month-old kitten exhibited simultaneously an imperforate anus, hypospadias, rectourethral fistula and genital dysgenesis (penis restricted to the glans, absence of prepuce and bifid scrotum). Surgical correction consisted of separation of the urinary and digestive tracts, perineal urethrostomy and connection of the rectum to the newly made anal opening. Pathological examination of the testes, conventionally removed at 9 months of age, showed no mature spermatozoa and underdevelopment of germ and Leydig cells. In humans, the absence of an anal opening in association with abnormal sexual development defines the urorectal septum malformation sequence. Here, we describe the first case of this syndrome in a kitten with a normal male karyotype (38,XY) and a normal coding sequence for the SRY gene. Both the rectourethral fistula and observed genital abnormalities might have been induced by a disturbance in the hedgehog signalling pathway. However, although four polymorphic sites were identified by DHH gene sequencing, none cosegregated with the malformation.

Effect of age, gestation and lactation on faecal IgA and calprotectin concentrations in dogs.

Faecal calprotectin and IgA have been suggested as non-invasive markers of gut health. Faecal calprotectin is a marker of intestinal inflammation in adults, whereas IgA has been suggested as a marker of intestinal immunity. The purpose of the present study was to evaluate the effect of gestation, lactation and age on faecal concentrations of these biomarkers. Thirty puppies, nineteen pregnant or lactating bitches and eighty-nine healthy control adult dogs were included in the study. Faeces were collected from the fourth week of gestation until the eighth week of lactation in pregnant and lactating bitches, and between 4 and 9 weeks of age in puppies. Faeces from the eighty-nine healthy control adult dogs were also collected. Faecal calprotectin and IgA concentrations were measured. Faecal calprotectin concentrations in control dogs were significantly lower than faecal calprotectin concentrations in puppies between 4 and 6 weeks of age (P < 0·001) or between 7 and 9 weeks of age (P = 0·004). Puppies between 4 and 6 weeks of age had significantly higher faecal IgA concentrations compared with puppies between 7 and 9 weeks of age (P = 0·001). Bitches during their second month of lactation had significantly lower faecal IgA concentrations compared with their first month of lactation (P = 0·049). Faecal calprotectin and IgA have been suggested as non-invasive and easily measured biomarkers of gut health in adults. However, the present study underlines that faecal IgA and calprotectin concentrations vary markedly depending of physiologic factors such as gestation, lactation and age. These factors need to be considered when these faecal biomarkers are used for evaluation of intestinal immunity or inflammation.

Protection against canine parvovirus type 2 infection in puppies by colostrum-derived antibodies.

During the first weeks of life puppies remain protected against canine parvovirus type 2 (CPV2) infection thanks to maternally derived antibodies (MDA) absorbed with colostrum after birth. The objective of the present study was to present the variability in CPV2-specific passive immune transfer and its consequences in puppies naturally exposed to the parvovirus. Seventy-nine puppies from one breeding kennel were included in the study at birth and followed until 56 d of age. Once per week the MDA titre for CPV2 specific antibodies was determined in blood. Viral excretion was also evaluated on a rectal swab by CPV2 PCR assay and puppies were weighed to determine growth rate. At 2 d of age, thirty-four out of seventy-nine puppies (43 %) had MDA ≤1:160 (designed group A) and forty-five puppies (57 %) had greater MDA titres (designed group B). The level of absorbed maternal antibodies was shown to be associated with breed size and growth rate during the first 48 h of life. The MDA level declined with age in all cases; however, the proportion of puppies with the antibody level considered as protective against CPV2 infection was significantly higher in group B compared with A from day 2 until 42. Among all puppies surviving until 56 d of age, sixty-seven out of seventy (95·7 %) underwent CPV2 infection. However, puppies from group A excreted CPV2 significantly earlier than puppies from group B. The present study demonstrates the link between passive immune transfer, in terms of level of specific MDA absorbed, and length of the protection period against parvovirus infection in weaning puppies.

Reliability of Hoechst 33342 staining under wide-field microscopy for evaluation of the nuclear status of living dog oocytes.

Due to the marked cytoplasmic opacity of canine oocytes, the diagnosis of their nuclear status is difficult. The objective of the present study was to evaluate the accuracy of Hoechst staining observed under epifluorescence wide-field microscopy [living oocyte observation (LivOO)] by comparison to a reference technique [DNA staining with ethidium homodimer-2 under confocal microscopy; fixed oocyte observation (FixOO)]. Four Hoechst 33342 concentrations (200 ng, 500 ng, 1 µg, 2 µg/mL) were tested and 1 µg/mL was the lowest one with the lowest proportion of oocytes in which DNA was missed. At this concentration, LivOO procedure did not affect the degeneration rate. On 379 oocytes observed individually with the two techniques successively, diagnosis of meiosis resumption by LivOO was exact in 87.3% of the cases, but the meiosis resumption rate was underestimated (23.5% versus 34.3% with FixOO; p < 0.001). Diagnosis for metaphase II was exact in 80% of the cases, but LivOO detected only 72.7% of the metaphase II oocytes present. Metaphase rates did not differ between LivOO and FixOO. This study contributes to a better interpretation of in vitro maturation results. The developmental potential of metaphase II canine oocytes sorted after Hoechst staining is to be evaluated.

Validation of a fecal scoring scale in puppies during the weaning period.

In puppies weaning is a high risk period. Fecal changes are frequent and can be signs of infection by digestive pathogens (bacteria, viruses, parasites) and indicators of nutritional and environmental stress. The aim of this study was to define a pathological fecal score for weaning puppies, and to study the impact on that score of two intestinal viruses (canine parvovirus type 2 and canine coronavirus). For this, the quality of stools was evaluated on 154 puppies between 4 and 8 weeks of age (100 from small breeds and 54 from large breeds). The scoring was performed immediately after a spontaneous defecation based on a 13-point scale (from 1; liquid to 13; dry and hard feces). Fecal samples were frozen for further viral analysis. Each puppy was weighed once a week during the study period. The fecal score regarded as pathological was the highest score associated with a significant reduction in average daily gain (ADG). Fecal samples were checked by semi-quantitative PCR or RT-PCR for canine parvovirus type 2 and canine coronavirus identification, respectively. The quality of feces was affected by both age and breed size. In small breeds, the ADG was significantly reduced under a fecal score of 6 and 7 for puppies at 4-5 and 6-8 weeks of age, respectively. In large breeds, the ADG was significantly reduced under a fecal score of 5 whatever the age of the puppy. Whereas a high viral load of canine parvovirus type 2 significantly impacted feces quality, no effect was recorded for canine coronavirus. This study provides an objective threshold for evaluation of fecal quality in weaning puppies. It also emphasizes the importance to be given to age and breed size in that evaluation.

The canine oocyte: uncommon features of in vivo and in vitro maturation.

The biology of the canine oocyte is unusual compared with that of other mammalian females. The present paper reviews both in vivo and in vitro specificities of canine oocytes. Final follicular growth in the bitch is characterised by an early appearance of LH binding sites in the granulosa, a high proportion of polyovular follicles and a preovulatory luteinisation, starting at the time of the LH surge. Through follicular fluid, preovulatory oocytes are thus exposed to high levels of progesterone, as high as 1000-fold plasma concentrations. The composition of the follicular fluid is affected by the size of the female. The more specific aspect of oocyte biology in the bitch is ovulation: oocytes are expelled immature, at the Prophase I stage. Ovulatory follicles are 6-8 mm in diameter, releasing oocytes from 110 µm, with dark cytoplasm. Resumption of meiosis occurs from 48 h postovulation, MII stages appearing 48-54 h after ovulation. The mechanisms controlling such a late meiotic resumption are still unknown. Granulosa cells seem to play a central role as in other mammalian species, but not with cAMP as the principal mediator. The importance of a transient reactivation of oocyte transcription a few hours before meiotic resumption is to be explored. These specific features may contribute to the low efficiency of IVM. Only 10-20% oocytes reach the metaphase stage and suffer from a poor cytoplasmic maturation. Moreover, in vitro culture of canine oocytes is associated with a high proportion of degeneration. To date, IVM of the oocytes is the main limiting factor for the development of assisted reproductive techniques in the canine. A better knowledge of the basic physiology of folliculogenesis and the molecular mechanisms controlling oocyte meiosis resumption in this species may allow us to overcome this obstacle.

Steroid hormones content and proteomic analysis of canine follicular fluid during the preovulatory period.

BACKGROUND: Follicular fluid contains substances involved in follicle activity, cell differentiation and oocyte maturation. Studies of its components may contribute to better understanding of the mechanisms underlying follicular development and oocyte quality. The canine species is characterized by several ovarian activity features that are not extensively described such as preovulatory luteinization, oocyte ovulated at the GV stage (prophase 1) and poly-oocytic follicles. In this study, we examined the hypothesis that the preovulatory LH surge is associated with changes in steroid and protein content of canine follicular fluid prior to ovulation. METHODS: Follicular fluid samples were collected from canine ovaries during the preovulatory phase, before (pre-LH; n = 16 bitches) and after (post-LH; n = 16) the LH surge. Blood was simultaneously collected. Steroids were assayed by radioimmunoassay and proteomic analyses were carried out by 2D-PAGE and mass spectrometry. RESULTS: The concentrations of 17beta-estradiol and progesterone at the pre-LH stage were 737.2 +/- 43.5 ng/ml and 2630.1 +/- 287.2 ng/ml in follicular fluid vs. 53 +/- 4.1 pg/ml and 3.9 +/- 0.3 ng/ml in plasma, respectively. At that stage, significant positive correlations between follicular size and intra-follicular steroid concentrations were recorded. After the LH peak, the intrafollicular concentration of 17beta-estradiol decreased significantly (48.3 +/- 4.4 ng/ml; p < 0.001), whereas that of progesterone increased (11690.2 +/- 693.6 ng/ml; p < 0.001). Plasmatic concentration of 17beta-estradiol was not modified (49 +/- 9.6 pg/ml) after the LH peak, but that of progesterone significantly increased (9.8 +/- 0.63 ng/ml).Proteomic analysis of canine follicular fluid identified 38 protein spots, corresponding to 21 proteins, some of which are known to play roles in the ovarian physiology. The comparison of 2D-PAGE patterns of follicular fluids from the pre- and post-LH stages demonstrated 3 differentially stained single spot or groups of spots. One of them was identified as complement factor B. A comparison of follicular fluid and plasma protein patterns demonstrated a group of 4 spots that were more concentrated in plasma than in follicular fluid, and a single spot specific to follicular fluid. These proteins were identified as gelsolin and clusterin, respectively. CONCLUSION: Our results provide the first demonstration of size-related changes in the steroid concentrations in canine follicular fluid associated with the LH surge. 2D protein mapping allowed identification of several proteins that may play a role in follicle physiology and ovarian activity at the preovulatory stage. This may help in the future to explain and to better understand the species specificities that are described in dogs.

Embryo biotechnology in the dog: a review.

Canine embryos are a scarce biological material because of difficulties in collecting in vivo-produced embryos and the inability, to date, to produce canine embryos in vitro. The procedure for the transfer of in vivo-produced embryos has not been developed adequately, with only six attempts reported in the literature that have resulted in the birth of 45 puppies. In vitro, the fertilisation rate is particularly low ( approximately 10%) and the incidence of polyspermy particularly high. So far, no puppy has been obtained from an in vitro-produced embryo. In contrast, cloning of somatic cells has been used successfully over the past 4 years, with the birth of 41 puppies reported in the literature, a yield that is comparable to that for other mammalian species. Over the same period, canine embryonic stem sells and transgenic cloned dogs have been obtained. Thus, the latest reproductive technologies are further advanced than in vitro embryo production. The lack of fundamental studies on the specific features of reproductive physiology and developmental biology in the canine is regrettable in view of the increasing role of dogs in our society and of the current demand for new biological models in biomedical technology.