The induction of a secondary corpus luteum on day 12 post-ovulation can delay the time of luteolysis in high-producing Holstein cows.

Luteolysis before the time of maternal recognition of pregnancy is one cause of low fertility in high-producing dairy cows. The objective of this study was to assess whether induction of a secondary corpus luteum (CL) late in the luteal phase would delay the time of luteolysis. Twenty high-producing Holstein cows were synchronized to ovulation (Day 0) with the Ovsynch protocol and received hCG (1500 IU im) on Day 12. Corpora lutea formation (as evaluated by ultrasonography) and plasma P4 concentrations were monitored from Days 4 to 36. hCG treatment induced the formation of one secondary CL (CL2) in 11 of 20 cows (55%) from the dominant follicle (mean diameter: 14.2 ± 0.9 mm) of two-wave (3/11) and three-wave (8/11) cycles. The maximal diameter of the CL2 (23.3 ± 1.9 mm) was reached approximately 6 days after hCG treatment and was correlated with its structural lifespan (p < 0.01). Cows that formed a CL2 after hCG had higher mean plasma P4 concentrations on Day 14 (+4.5 ng/ml) and Day 18 (+3.0 ng/ml) compared with cows without CL2 (p < 0.05). The structural regression of CL2 begun approximately 8 days after that of the CL1, and the median time at which the first drop in circulating P4 levels occurred was later in cows that formed a CL2 than in those that did not (Day 26 vs Day 18; p < 0.01). Thus, the induction of a CL2 by hCG on Day 12 might reduce the risk of premature luteolysis in high-producing dairy cows after insemination.

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.

Towards an automated detection of oestrus in dairy cattle.

Heat detection is a key factor in the profitability of dairy herds. However, this detection demands a significant part of the breeder's working time and is made difficult by the short duration and the discrete behavioural changes associated with oestrus in modern dairy cows. Progress has been made in monitoring cow with electronics, biosensors and computer. As a result, automated heat detection systems have been developed. Currently available tools are automated detectors of standing heat, activity-metres and automated in-line systems measuring milk progesterone. Camera-software systems and monitoring of body temperature are being developed and may also be used as heat detection tools. The heat detection rate of most systems is above 80% with a specificity of detection generally higher than 90%. The accuracy, however, may vary considerably depending on the tool and model developed. The initial investment of several thousands of euros required for these automated systems becomes a source of profit in large herds, provided the recorded data are properly managed.

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.

[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.

Induction of final maturation by sperm penetration in canine oocytes.

In contrast to oocytes of most mammals, the canine oocyte is at the germinal vesicle stage at ovulation. Moreover, the bitch is receptive to mating while immature oocytes are present in the oviducts. The aims of this study were to examine the influence of fertilization in immature oocytes on the resumption of meiosis, and the modification of both male and female chromatin in fertilized oocytes. Canine cumulus-oocyte complexes collected from routine ovariectomies were cultured in medium 199 with 20% fetal calf serum for 24 h, incubated in the same medium with fresh semen for 24 h, washed, cultured for a further 24 h and fixed. Control oocytes were cultured in the same medium but without spermatozoa for 24, 48 or 72 h. After fixation, chromatin was stained with propidium iodide and examined using laser scanning confocal microscopy. The data indicate that sperm penetration can occur in immature canine oocytes and that it induces resumption of meiosis. After 72 h of culture, the percentage of oocytes at the germinal vesicle stage was significantly lower in fertilized oocytes (40% versus 60.3% for control oocytes; P < 0.05) and the percentage of oocytes beyond metaphase I was significantly greater in fertilized oocytes (28.3% metaphase I and II, and two pronuclei versus 10.2% metaphase I and II for control oocytes; P < 0.01). Observation and measurement of the area of chromatin in fertilized oocytes showed an overall parallel condensation-decondensation of both female and male chromatin from the germinal vesicle stage to the pronuclear stage.

In vitro maturation of bitch oocytes: effect of sperm penetration.

In contrast to most mammals, bitches ovulate immature oocytes at the germinal vesicle stage. Spermatozoa can be present in the oviduct of bitches at ovulation. The aim of this study was to investigate the influence of premature fertilization on the resumption of meiosis. In addition, the relative behaviour of male and female chromatin was observed. Canine cumulus-oocyte complexes cultured in medium 199 supplemented with 20% fetal calf serum for 24 h were incubated in vitro in the presence or absence of fresh dog semen for 24 h, and then rinsed and cultured in the same medium for a further 24 h. Chromatin was stained by propidium iodide and all oocytes were examined under laser scanning confocal microscopy. Results show that sperm penetration can occur in vitro in immature oocytes and that this induces a resumption of meiosis: at 72 h, the percentage of oocytes at the germinal vesicle stage decreased significantly and the percentage of oocytes beyond metaphase I increased. However, premature fertilization was ineffective for 40% of oocytes, which remained at the germinal vesicle stage. Measurement of chromatin areas in fertilized oocytes showed an overall parallel condensation-decondensation of both female and male chromatin from the germinal vesicle stage to the pronuclear stage, indicating that male and female chromatins are sensitive to the same cell cycle regulators.