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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.

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., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2018 Blackwell Verlag GmbH.)

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 β-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., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Puppy and kitten survival over the first weeks is particularly dependent on colostrum, a specific secretion of the mammary gland produced during the first 2 days post-partum. Colostrum is a source of nutrients and immunoglobulins. It also contributes to the digestive tract maturation. Colostrum differentiates from milk mainly based on its concentration in immunoglobulins G: 20-30 g/L in dog colostrum, 40-50 g/L in cats' vs <1 g/L in milk. IgG concentration rapidly drops after parturition (-50% in 24 hr). Immune quality of colostrum is highly variable between bitches, with no relationship with maternal blood IgG level, dam's age, breed size or litter size. In addition to systemic immune protection, colostrum also plays a major role for local digestive protection, due to IgA, lysozyme, lactoferrin, white blood cells and various cytokines. Energetic concentration of canine and feline colostrum is not superior to that of mature milk. It depends on colostrum fat concentration and is affected by breed size (higher in breeds <10 kg adult body weight). As puppies and kittens are almost agammaglobulinemic at birth, transfer of IgG from their digestive tract into their bloodstream is crucial for their survival, IgG absorption ending at 12-16 hr after birth. Energetic supply over the two first days of life, as evidenced by growth rate over the two first days of life, also affects risk of neonatal mortality. Early and sufficient suckling of colostrum is thus the very first care to be provided to newborns for their later health and survival., (© 2016 Blackwell Verlag GmbH.)

Colostrum and milk are complex mammary secretions providing the puppy with many nutritional and immunological factors, which play a crucial role for its correct development and survival. In the case of colostrum and/or milk intake deficiency, puppies are at increased risk of infectious diseases. This work reviews the various nutritional hyperimmune supplementations proposed to provide a passive immune protection and to positively impact puppies' health. Some strategies rely on canine immunoglobulins: canine colostrum banking and canine serum/plasma supplementation. Others involve heterologous sources of antibodies and other immune factors: bovine colostrum or hyperimmune egg powder. Among the different solutions evaluated from birth to weaning, canine plasma and hyperimmune egg powder showed promising beneficial effect on puppies' health. Canine plasma seems to positively impact not only growth (increased growth during the neonatal period), but also digestive health (higher species richness of intestinal microbiota) and the general health (tendency of lower morbidity). Puppies supplemented with hyperimmune egg powder presented increased neonatal growth and decreased risk of canine parvovirus infection. Nevertheless, natural canine maternal colostrum and milk ingestion remains the optimal guarantee for puppies' health and survival, as a source of immunity, energy and growth factors., (© 2016 Blackwell Verlag GmbH.)

The objective of this study was to describe efficiency of reproduction of purebred dogs in field breeding conditions, from mating to weaning in France. Data were collected between 2010 and 2014 in 5,667 French breeding kennels via a reproduction management software (Breeding Management System, Royal Canin, Aimargues, France). Effect of breed size (Mini: adult body weight <10 kg; Medium: 10-25 kg; Maxi: 25-40 kg; Giant: >40 kg), age of dam and male on pregnancy rate, abortion rate and litter size were evaluated by multivariable models. Data on 45,913 heats (all with mating), from 27,221 bitches from 248 breeds, were analysed. At mating, mean age (±SD) was 3.1 ± 1.8 years for bitches and 3.3 ± 2.0 for males. Males originated from the same kennel as the females in 88.5% of the matings. Based on breeder's evaluation of the pregnancy status, pregnancy rate (number of pregnant females based on breeders declaration/number of heats) was 87.8% and abortion rate was 6.8%. Finally, 81.9% of the mated females gave birth to a litter. On 37,946 litters (204,537 puppies), mean litter size was 5.4 ± 2.8 puppies (range 1-24), which was influenced by breed size and dam age (p < .0001). Stillbirth rate was 7.4% and puppy mortality rate (stillbirth + mortality until 2 months of age) was 13.4%. Prolificacy and puppy mortality rates were affected by breed size and within a breed size, by breed. Despite probable approximations (as data originate from breeders declaration), this large-scale analysis provides reference values on reproductive performance in dogs., (© 2016 Blackwell Verlag GmbH.)

Background: Fecal calprotectin and immunoglobulin A (IgA) are markers of intestinal inflammation and immunity in adult dogs., Hypothesis: Fecal calprotectin and IgA concentrations in puppies are not influenced by fecal moisture in puppies but by enteropathogen shedding., Animals: Three hundred and twenty-four puppies., Methods: Fecal consistency was assessed by gross examination. Fecal moisture was evaluated before and after lyophilization. Canine parvovirus and coronavirus were detected in feces by qPCR and qRT-PCR respectively. Giardia intestinalis antigen was quantified by ELISA. The standard McMaster flotation technique was used to detect eggs and oocysts in feces. Fecal calprotectin and IgA concentrations were quantified by in-house radioimmunoassays., Results: For each marker (IgA and calprotectin), a strong positive correlation was observed between concentration in fresh feces and concentration in fecal dry matter. 75.6% of the puppies were found to be infected by at ≥1 of the enteropathogens evaluated. Fecal calprotectin concentration was significantly influenced by age (P = .001), with higher concentrations in younger puppies, but not by viral (P = .863) or parasitic infection (P = .791). Fecal IgA concentration was significantly influenced by enteropathogen shedding (P = .01), with a lower fecal IgA concentration in puppies shedding at ≥1 enteropathogen compared to puppies without any enteropathogen shedding, but not by age., Conclusions: Fecal calprotectin and IgA are of no diagnostic value to detect presence of enteropathogens in clinically healthy puppies or puppies with abnormal feces, but could help to better understand the maturation of digestive tract., (Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Breeding kennels face a high rate of neonatal mortality, on which the impact of nutrition remains to be determined. This study was designed to evaluate the impact of birth weight (reflecting intrauterine growth) and early growth rate (reflecting colostrum intake) on risk of neonatal mortality in puppies and to determine the critical thresholds of both parameters. Puppies from various breeds were weighed at birth ( = 514) and at 2 d of age, and the growth rate over that period (early growth rate) was calculated for all survivors ( = 477). Linear mixed models evaluated the effect of birth weight on mortality between birth and 2 d of age and the effect of both birth weight and early growth rate on mortality between 2 and 21 d of age. Birth weight was influenced by litter size ( = 0.003), with more low-birth-weight puppies (the lightest 25% within a breed size) in large litters compared with smaller litters. Mortality over the first 2 d after birth was associated with birth weight ( < 0.001), with 81.1% of dying puppies characterized by a low birth weight. Mortality between 2 and 21 d of age was not related to birth weight but was found to be associated with early growth rate ( < 0.001), with higher risk of death in puppies with growth rate at or below -4% after the first 2 d of life. This study demonstrates the differential effect of intrauterine nutrition impacting mortality during the first 2 d of life and that of colostrum intake impacting mortality until 21 d of life. Birth weight and early growth rate thresholds provided in this study allow identification of puppies at risk, whereby provision can be made for adequate nursing to increase their chances to survive.

Assay of blood progesterone (P4) is commonly practiced to determine the time of ovulation, diagnose luteal insufficiency, and predict time of parturition in bitches. Because of practical constraints, most blood samples cannot be assayed on site immediately after collection. The aim of this work was to evaluate the effects of various sampling and storage conditions on concentrations of P4 as determined by chemiluminescence immunoassay. The blood of 5 Beagle bitches was collected from the jugular vein to study the effect of the type of collection tube (silicone, lithium heparin, EDTA), the storage time of unseparated or separated plasma (2 h to 14 d), and the number of freeze-thaw cycles (1-10) on P4. The effect of each factor was tested within one assay session. None of the factors significantly affected P4. Thus, P4 appears to remain relatively stable in canine blood samples exposed to various processing and storage conditions., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Important reproductive events take place in the canine oviduct in the presence of increasing concentrations of progesterone (P4). To investigate the potential effects of P4 on the canine oviduct, the expression of nuclear (PR) and membrane (PGRMC1 and 2, mPRα, β and γ) P4 receptors was studied by quantitative RT-PCR and immunohistochemistry. Oviducts were collected from Beagle bitches after the onset of pro-oestrus and before the LH peak (Pre-LH), after the LH peak and before ovulation (Pre-ov) and on Days 1, 4 and 7 post-ovulation (n=6 bitches/stage). PR mRNA concentrations decreased from Pre-LH to Day 7 in the ampulla and isthmus, whereas both PGRMC1 and 2 mRNA levels increased over the same period. The main change in mPR expression was an increase in mPRβ and γ mRNAs at Day 7 in the isthmus. Furthermore, PR proteins were expressed in the nuclei of luminal epithelial, stromal and muscular cells, whereas the expression of PGRMCs and mPRs was primarily cytoplasmic and localised in the luminal epithelium. The immunostaining for PR decreased at Day 4 in the stroma and muscle, whereas it remained strong in the epithelium from Pre-LH to Day 7. PGRMC1 staining was strong at Days 4 and 7 whereas PGRMC2 was highly expressed from Pre-ov to Day 7. The most intense immunostaining signals for all three mPRs were observed at Day 7. Our results strongly support the hypothesis that P4 is an important regulator of oviductal functions in the bitch through complementary classical and non-classical P4 pathways.

As puppies are born with very low immunoglobulin concentrations, they rely on passive immune transfer from ingested colostrum to acquire a protective immunity during the first few weeks of life. The purpose of this study was to describe the timing of gut closure in canine neonates. Twenty-two Beagle puppies received 3 ml of standardized canine colostrum at 0, 4, 8, 12, 16 or 24 h after birth using a feeding tube. Blood immunoglobulins G (IgG, M and A) were assayed 0, 4 and 48 h after colostrum ingestion. IgG absorption rate was significantly affected by the time of colostrum administration, and the IgG concentrations in puppies serum 48 h after administration were significantly higher when colostrum was ingested at 0-4 h of age than at 8-12 h or 16-24 h (1.68 ± 0.4, 0.79 ± 0.07 and 0.35 ± 0.08 g/l, respectively; p < 0.001). In the canine species, gut closure seems thus to begin as early as 4-8 h after birth and to be complete at 16-24 h. Consequently, this phenomenon appears to occur earlier in puppies than in most other species., (© 2012 Blackwell Verlag GmbH.)

In canine species, in vitro maturation (IVM) rates of oocytes collected from anoestrous ovaries are low (<20%). Several IVM media have been tested without significant improvements. A critical step in the evaluation of culture conditions is the observation of the meiotic stage reached by the oocytes. The present study was designed to investigate the chromatin patterns of in vitro matured oocytes by visualizing Germinal Vesicle (GV) and Germinal Vesicle Breakdown (GVBD) structures at 72 h of IVM. Nuclear stages of 1678 oocytes were evaluated by confocal microscopy after IVM. 1204 oocytes were non-degenerated, and 94.4% were still immature and at GV stage. Five different patterns of chromatin configuration were observed. Higher percentages of oocytes with unmodified GV and with diffuse (58%; Type A) and filamentous chromatin (19%; Type B) were observed in comparison with those with modifications in the GV such as patched chromatin (12.5%; Type C), surrounded-nucleolus (3%; Type D) and in vivo type chromatin/fully grouped chromatin (2.5%; Type E). These results indicate that GVBD (absence of nucleolus, nucleus breakdown) is rarely observed in vitro. The percentage of type C-D-E GVs and MI (meiotic resumption) and of MII (completion of meiosis) can be used to evaluate meiotic resumption after IVM. Our results indicate that although a low number of in vitro matured oocytes exhibit the chromatin configurations observed in in vivo collected oocytes, chromatin changes in the GV can be induced during IVM., (© 2012 Blackwell Verlag GmbH.)

In the bitch, oocyte meiosis resumption takes place in the oviduct. Using oocytes from anestrous bitches, in vitro maturation (IVM) generally gives very poor results. To investigate the contribution of oocyte competence to the low IVM yield, we compared in vivo maturation in an optimal environment with conventional IVM. A total of 418 grade 1 cumulus-oocyte complexes (COCs) from 10 anestrous bitches were transferred into the oviducts of recipient bitches either on Day -1 (n = 3 recipients), Day 0 (n = 2) or on Day +1 (n = 2) relative to ovulation. For each donor bitch, 20 grade 1 COCs were also cultured in vitro. After 72 h of in vivo or IVM, the nuclear stage of oocytes was determined after DNA and tubulin staining. Of the 154 oocytes recovered and examined after intratubal transfer, only 2% reached the metaphase I or II stage and 38.3% were degenerated. Oocytes cultured in vitro displayed a higher metaphase rate (7.6%, n = 170) and lower degeneration rate (12.9%) compared with transferred oocytes (p < 0.001). These results clearly demonstrate that the oocyte competence is the major limiting factor of IVM efficiency in the dog. Mimicking the tubal environment may thus not be sufficient to increase IVM yield in this species., (© 2012 Blackwell Verlag GmbH.)

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., (© 2012 Blackwell Verlag GmbH.)

In the bitch, oocyte maturation, sperm storage, fertilization and early embryo development take place within the oviducts under high and increasing circulating progesterone concentrations. To investigate the potential effects of progesterone on the canine oviduct, nuclear progesterone receptors (PR) were localized. Oviducts were collected by ovariectomy from adult Beagle bitches during anestrus, after the onset of proestrus but prior to the Luteinizing Hormone (LH) peak (Pre-LH), after the LH peak but before ovulation (Pre-ov) and on Days 1, 4 and 7 after ovulation (n = 3 bitches per stage). The cellular distribution of PR was studied by immunohistochemistry (IHC) in the ampulla, isthmus and tubal part of the utero-tubal junction (UTJ). Plasma progesterone and 17β-oestradiol were assayed on the day of surgery. PR were specifically expressed in the nuclei of epithelial, stromal and muscular cells in the ampulla, isthmus and UTJ. The IHC scores did not vary from one oviductal region to another. However, the epithelium displayed higher scores than the stroma at anestrus, Pre-ov, Days 4 and 7, and also higher scores than muscle at Days 4 and 7 (p < 0.05). Immunohistochemistry scores in the stroma and muscle decreased at Days 4 and 7 compared with previous stages (p < 0.05). Furthermore, muscular IHC scores were positively correlated with circulating 17β-oestradiol concentrations and negatively correlated with circulating progesterone concentrations (p < 0.05). In conclusion, PR were identified in the canine oviduct, with differences in expression between tissues and times around ovulation, suggesting that progesterone may regulate tubal functions and reproductive events in this species., (© 2012 Blackwell Verlag GmbH.)

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.

The IGF system plays a crucial role in ovarian folliculogenesis, and changes in IGF-binding protein (IGFBP) levels modulate IGF bioavailability. Data from various mammalian models suggest a link between body size, IGF1 in serum and female reproduction parameters. Among the vertebrate species, the dog exhibits the widest span in body height. Height is known to be positively correlated with the concentration of serum IGF1. In this work, the ovarian physiology of 40 bitches exhibiting a wide span of height, and breed type was investigated. IGF1, IGF2, IGFBP3, estradiol (E(2)), and progesterone concentrations in plasma and preovulatory follicular fluid were quantified. A total of 455 follicles, 2-8 mm in diameter, were recovered at the preovulatory stage, measured, and punctured. Intrafollicular levels of IGF1 were positively correlated with plasma levels, and plasma IGF1 levels were positively correlated with both bitch height and weight. The concentrations were threefold higher in large dogs compared with small dogs. A positive correlation between intrafollicular and plasmatic IGFBP3 levels and a positive correlation between plasmatic IGFBP3 levels, and both height and weight of the bitches were observed. The number of preovulatory follicles and the diameter of the three largest follicles were positively correlated with bitch height. E(2) intrafollicular concentrations were higher in preovulatory follicles from small animals than in those from large animals. In conclusion, the strong variability in height between dogs appeared to be associated with dramatic differences in IGF1, and IGFBP3 levels, in both plasma and follicular fluid. These differences were associated with significant differences in some functional aspects of ovarian follicles.

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.

This study investigates the mitochondrial (mt) distribution in canine ovarian oocytes examined at recovery time, as related to the reproductive cycle stage, and in oviductal oocytes. Ovarian Germinal Vesicle (GV) stage oocytes were recovered from bitches in anestrous (A, n=2), follicular phase (F, n=4), ovulation (O, n=2), early luteal (EL, n=7) and mid/late luteal phase (MLL, n=2). Oviductal GV, metaphase I (MI) or MII stage oocytes were recovered from six bitches between 56 and 110 h after ovulation. Mitochondria were revealed by using MitoTracker Orange CMTM Ros and confocal microscopy. In ovarian oocytes, three mt distribution patterns were found: (I) small aggregates diffused throughout the cytoplasm; (II) diffused tubular networks; (III) pericortical tubular networks. Significantly higher rates of oocytes showing heterogeneous mt patterns (II+III) were obtained from bitches in F (75%) and in O (96%) compared with bitches in A (31%; F vs. A: P<0.05; O vs. A: P<0.001), in EL (61%; O vs. EL: P<0.01), or in MLL (0%; F vs. MLL: P<0.05; O vs. MLL: P<0.001). Fluorescence intensity did not vary according to mt distribution pattern except that it was lower in oocytes recovered in EL phase and showing small mt aggregations (P<0.001). The majority of ovulated MII stage oocytes (79%) showed diffused tubular mt network. We conclude that mt distribution pattern of canine ovarian immature oocytes changes in relation to reproductive cycle stage and that patterns observed in oocytes recovered from bitches in periovulatory phases are heterogeneous and similar to those of in vivo matured oocytes.

In vitro maturation (IVM) of bitch oocytes is, to date, a very inefficient process, with common metaphase rates approximately 0-20% and mean degeneration rates approximately 20-30%. In other mammals, meiotic resumption is controlled in the cumulus-oocyte complex by the disappearance of the coupling between granulosa cells and the oocyte. The first aim of this study was to evaluate the influence on meiotic resumption of a mechanical denudation of the oocytes before maturation. The nuclear stage was determined by DNA staining with ethidium-homodimer-2 under confocal microscopy. Denuded (n = 318) and control (n = 378; no mechanical denudation) oocytes had similar degeneration rates (respectively 32.1 vs 28.6%). However, meiosis resumption rates were significantly higher for denuded oocytes (DO; 26.9 vs 17.8%). Secondly, we aimed to evaluate oocytes experiencing spontaneous denudation during the 72 h IVM period. Denuded oocytes, having lost cumulus cells on at least 75% of their perimeter (n = 440), were compared with surrounded oocytes (SO), with 100% of their perimeter surrounded by granulosa cells (n = 860). As above, the nuclear stage was determined by confocal microscopy, but cytoplasmic maturation was also evaluated through transmission electron microscopy. Degeneration rates but also meiosis resumption and metaphase rates were significantly higher for denuded than for SO (9.6 vs 2.8% for metaphase rate). Nevertheless, ultrastructurally, metaphase DO have scarcer organelles unevenly distributed, with smooth endoplasmic reticulum concentrated in aggregates in the cortical zone. Denudation, whether mechanical or spontaneous, is thus an inefficient mean to obtain metaphase II oocytes suitable for in vitro fertilization.

The aim of the present study was to describe the canine oocyte ultrastructural modifications during in vivo maturation, with precise reference to the timing of the LH surge and of ovulation. Twenty-five bitches were ovariectomized at specific stages between the onset of proestrus and the fifth day post-ovulation: 65 oocytes were observed by transmission electron microscopy (TEM), either before the LH surge (n = 10), between the LH surge and ovulation (n = 12) or after ovulation (n = 43). Prior to the LH surge, the oocyte nucleus had already begun its displacement to the vicinity of the oolemma and reticulated nucleoli were infrequent. The cytoplasm showed signs of immaturity (few organelles preferentially located in the cortical zone, "mitochondrial cloud", scarce cortical granules). The LH surge was immediately followed by cumulus expansion but the ovulation occurred 2 days later. Retraction of the transzonal projections and the meiotic resumption occurred after another 3 days (5 days after the LH peak). The ovulation was then followed by gradual cytoplasmic modifications. Nucleoli re-assumed a reticulated aspect around 24 hr post-ovulation. From 48 hr post-ovulation mitochondria and SER were very numerous and evenly distributed. In conclusion canine oocyte maturation began prior to the LH surge and no cytoplasmic or nuclear modifications followed immediately the LH surge and ovulation. This study suggests that two distinct signals are needed for the final in vivo maturation: one prior to the LH surge (to induce maturation) and another one, around 3 days post-ovulation (to induce meiotic resumption)., ((c) 2007 Wiley-Liss, Inc.)

In the female dog, in contrast with most mammals, the growing follicle starts to luteinise several days before ovulation. Little is known about the physiological control of the final follicular growth in this species. In order to better understand the pituitary regulation of follicular growth, specific binding sites for FSH and LH were localised and quantified by autoradiography using [(125)I]-porcine (p) gonadotrophins on ovarian sections (7 microm) from adult Beagle bitches during the follicular phase. Follicles were analysed either before the LH surge (n = 4 bitches; n = 117 follicles) or after the LH surge and before ovulation (n = 5 bitches; n = 110 follicles). FSH binding sites were specifically and homogeneously expressed at high levels on granulosa cells of all healthy follicles from the preantral stage onwards. In contrast, LH binding sites were detected homogeneously and at high levels only on granulosa cells of follicles larger than 1 mm in diameter, including luteinised follicles. Theca binding of LH (but not FSH) was also observed, but only when using high concentrations of [(125)I]-pLH. The overall incidence of atresia was 45.8% and was dependent upon follicular diameter. Quantitative analysis of labelling showed that atretic follicles had reduced levels of both FSH and LH binding sites compared with healthy follicles. In healthy follicles, levels of both FSH and LH binding sites changed with follicle diameter. Compared with other mammals, the acquisition of LH binding on canine granulosa cells occurs in smaller sized follicles relative to the size of ovulation.

Cumulus-oocyte complexes (COCs) were recovered from ovaries of bitches during anoestrus. The ultrastructural organisation of COCs was determined before and after 72 h in vitro maturation (IVM) by transmission electron microscopy. The aim of the study was to determine the quality of oocytes used for IVM and to assess cytoplasmic maturation of IVM metaphase (M) II oocytes. In addition, we examined whether the oocytes that did not reach MII were engaged in an erratic maturation process or whether they were blocked during their progression through a normal maturation process. Before IVM, there were two populations of oocytes: (1) oocytes with a centrally located germinal vesicle, a transcriptionally active aspect and an immature cytoplasm; and (2) oocytes with an eccentric nucleus, a transcriptionally inactive aspect and a more mature cytoplasm. After IVM, most oocytes were still at the germinal vesicle stage with three different patterns and all showing a good synchronisation between nuclear and cytoplasmic maturation. MI oocytes had a similar cytoplasmic maturation to that observed in vivo, but failed to complete meiosis; however, IVM MII oocytes had a very poor cytoplasmic maturation. Ultrastructural analysis demonstrated that even when nuclear maturation is achieved, cytoplasmic maturation may not be obtained in vitro. Thus, all IVM systems should be evaluated on both criteria.

This review reports existing and original data concerning the biology of the canine oocyte and early embryo. It describes specific aspects of intra- and extra-follicular maturation of the oocyte during the peri-ovulatory period, methods to detect ovulation, sperm survival and fertilization and timing of preimplantation embryo development.

Early development in canine species follows a very specific pattern. Oocytes are ovulated at the germinal vesicle stage and meiotic resumption occurs in the oviduct. However, because of difficulties in the accurate determination of ovulation time and in the observation of oocyte nuclear stage by light microscopy, these early events have not been fully described. Moreover, the oocyte stage at which sperm penetration occurs is still uncertain since fertilization of immature oocytes has been reported in vivo and in vitro. The aim of this study was to establish the exact timing of in vivo meiotic resumption, fertilization and early embryo development in the bitch with reference to ovulation. Ovulation was first determined by ultrasonography, artificial inseminations were performed daily and oocytes/embryos were collected between 17 and 138 h after ovulation. After fixation and DNA/tubulin staining, the nuclear stage was observed by confocal microscopy. Of the 195 oocytes/embryos collected from 50 bitches, the germinal vesicle stage was the only one present until 44 h post-ovulation, and the first metaphase II stage was observed for the first time at 54 h. Sperm penetration of immature oocytes appeared to be exceptional (three out of 112 immature oocytes). In most cases, fertilization occurred from 90 h post-ovulation in metaphase II oocytes. Embryonic development was observed up to the eight-cell stage. No significant influence of bitch breed and age on ovulation rate, maturation and developmental kinetics was observed. However, some heterogeneity in the maturation/development process was observed within the cohort of oocytes/embryos collected from one bitch. In conclusion, the most peculiar aspect of the canine species remains oocyte meiotic maturation whereas fertilization follows the same pattern as in other mammals.