Lack of transplacental transmission of Bartonella bovis.

Transplacental transmission of Bartonella spp. has been reported for rodents, but not for cats and has never been investigated in cattle. The objective of this study was to assess vertical transmission of Bartonella in cattle. Fifty-six cow-calf pairs were tested before (cows) and after (calves) caesarean section for Bartonella bacteremia and/or serology, and the cotyledons were checked for gross lesions and presence of the bacteria. None of the 29 (52%) bacteremic cows gave birth to bacteremic calves, and all calves were seronegative at birth. Neither placentitis nor vasculitis were observed in all collected cotyledons. Bartonella bovis was not detected in placental cotyledons. Therefore, transplacental transmission of B. bovis and multiplication of the bacteria in the placenta do not seem likely. The lack of transplacental transmission may be associated with the particular structure of the placenta in ruminants or to a poor affinity/agressiveness of B. bovis for this tissue.

Effect of blood handling conditions on progesterone assay results obtained by chemiluminescence in the bitch.

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.

Expression of nuclear and membrane progesterone receptors in the canine oviduct during the periovulatory period.

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, ß 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 mPRß 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.

Effect of endocervical inflammation on days to conception in dairy cows.

In contrast to endometritis, now diagnosed by cytological examination, the effect of endocervical inflammation on reproductive performance has been inadequately investigated. In this study, endocervical and endometrial cytological specimens were collected from 168 Holstein cows between 21 and 60 days in milk (DIM) to investigate the prevalence of endocervical inflammation and effect on days to conception. Statistical analyses were stratified based on DIM at examination (<35 vs. ≥35 DIM). Endocervical inflammation with ≥5% neutrophils before 35 DIM (disregarding the level of endometrial inflammation) was associated with decreased hazard of pregnancy within 300 DIM (adjusted hazard ratio, 0.4; 95% confidence interval: 0.3-0.8). A decrease in hazard of pregnancy was observed when >6% neutrophils were counted in endometrial smears (adjusted hazard ratio, 0.4; 95% confidence interval: 0.2-0.7). The study suggested an additive effect of combined endocervical and endometrial inflammation on the hazard of pregnancy within 300 DIM. Using the thresholds of 5% neutrophils for the cervix and 6% neutrophils for the uterus, 11% of the cows examined before 35 DIM presented cervicitis only, 13% were affected by endometritis only, and 32% suffered from both endometrial and endocervical inflammation. The presence (absence) of cervicitis was not indicative of the presence (absence) of endometritis. This study showed that in addition to uterine inflammation, endocervical inflammation in early lactation affects conception. Thus, the global evaluation of genital tract health may be more beneficial for reproductive performance than that of endometrial inflammation.

Immunolocalization of progesterone receptors in the canine oviduct around ovulation.

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 17ß-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 17ß-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.

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.

Are oocytes from the anestrous bitch competent for meiosis?

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.

Timing of the intestinal barrier closure in puppies.

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.

[The mammalian oviduct revisited]. / L'oviducte de mammifère : un organe revisité

The oviducts, or uterine tubes, support the transport and final maturation of gametes, and harbour fertilization and early embryo development. The oviduct environment is finely regulated by ovarian steroids as well as by gametes and embryos that interact with it. Previously regarded as a simple transit zone, the oviduct is now regarded as a complex organ with multiple functions in these various processes. The tubal fluid, now better characterized, is to be regarded as the first interface between the mother and the embryo. It may play a major role in the quality of the conceptus.

[Polyovular follicles]. / Les follicules polyovocytaires.

Folliculogenesis covers the sequential steps in the development of a follicle, from primordial to preovulatory. Most of the time, one follicle contains a single oocyte, but some follicles are polyovular in that they contain several. These follicles were considered earlier as pathological, but they are, actually, fairly common in several species, from mice to humans. The frequency of polyovular follicles (number of polyovular/total number of follicles) varies among species, <0.1% to 14% in the dog, and with age (more polyovular follicles during the prepubertal period). More than 20 oocytes (and even more than 100 in the marsupials like the opossum) may be present in a single follicle. These follicles may form during the earliest stages of follicle formation, due to an imbalance between somatic and germinal cells, which induces an incomplete germ cell cyst breakdown. In polyovular follicles, the quality (size and maturity) of the various oocytes is often heterogeneous. Numerous authors reported that polyovular follicles are able to reach ovulation and ovulate. Polyovular follicles, naturally found in several species, may also be induced by exposure to therapy or agents in the environment, especially with estrogen activity such as pesticides or diethylstilbestrol/DES. Polyovular follicles are also observed in the ovaries of mutated rodents.

Follicle population, cumulus mucification, and oocyte chromatin configuration during the periovulatory period in the female dog.

This study was designed to describe the follicular population present on the canine ovary (Canis familiaris) during the preovulatory period and essentially the changes in oocyte size, mucification, and chromatin configuration occurring from before the luteinizing hormone (LH) surge up to postovulation. In a first experiment, ovaries of beagle bitches were collected before (n=21) or after LH surge but before ovulation (post-LH surge/preovulation stage, n=24) as determined using hormone (LH, estradiol, progesterone) assays and ultrasonography. All large (>2mm) follicles were measured and punctured. The numbers of oocytes collected per follicle and the degree of cumulus mucification were recorded. In a second experiment, ovaries were similarly collected before (n=13) and after the LH surge but before ovulation (n=11) as well as after ovulation as determined by ultrasonography (n=9). Chromatin configuration of the oocytes was observed by DNA staining and confocal microscopy. In Experiment 1, before the LH peak, an average of 13.5+/-0.7 follicles per bitch (total 284 follicles) were detected, and the maximal follicle diameter reached 6.5mm. Large follicles were observed already in this period of the cycle and as early as when progesterone was still below 0.5 ng/mL. After the LH peak but before ovulation, 11.0+/-0.7 follicles were present (total 264 follicles). Fully mucified cumulus cells were observed only in follicles larger than 4mm. Multi-oocytic follicles represented 7% (before LH peak) and 4% (after LH peak) of the follicular population. In Experiment 2, all the oocytes were at the germinal vesicle (GV) stage, but three chromatin configurations could be distinguished: diffuse, partly grouped, and fully grouped chromatin. The proportion of oocytes with fully grouped chromatin increased with the follicular diameter and the time in estrus, the maximum being observed after the LH peak. These results suggest that (1) before LH peak, follicles are already of large diameter, similar to the ones at ovulation; (2) the ability for cumulus mucification is acquired during the late steps of follicular growth; (3) three GV patterns may be observed during the periovulatory period.

Folliculogenesis and morphometry of oocyte and follicle growth in the feline ovary.

This study was designed to describe, both quantitatively (morphometry) and qualitatively (histological differentiation), follicle and oocyte growth in the feline ovary. The ovaries of 43 cats were collected and processed for histology. The diameters of 832 follicle/oocyte pairs were measured, with and without zona pellucida (ZP), and a special emphasis was placed on the study of early folliculogenesis. Primordial, primary, secondary, pre-antral and early antral follicles were measured at 44.3, 86.2, 126.0, 155.6 and 223.8 microm in diameter respectively. A biphasic pattern of follicle and oocyte growth was observed. Before antrum formation, follicle (x) and oocyte (y) size were positively and linearly correlated (y = 0.500x + 20.01, r(2) = 0.89). Antrum formation occurred when the follicle reached 160-200 microm in diameter (when oocyte was at 102 microm). After antrum formation, a decoupling was observed, a minimal increase in oocyte size contrasting with a significant follicle development (y = 0.001x + 114.39, r(2) = 0.01). The pre-ovulatory follicle diameter was approximately 3500 microm and the maximal oocyte diameter was 115 microm. The ZP, absent in primordial and primary follicles, appeared at the secondary stage and reached almost 6 microm at the pre-ovulatory stage. These results suggest that (i) in feline ovary, follicle and oocyte growth pattern is similar to that observed in other mammals; (ii) the antrum forms in 160-200 microm follicles, which represents 5% of the pre-ovulatory diameter and (iii) the oocyte had achieved more than 90% of its maximal growth at the stage of antrum formation.

In vivo meiotic resumption, fertilization and early embryonic development in the bitch.

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.