Follicular Size Threshold for Ovulation Reassessed. Insights from Multiple Ovulating Dairy Cows.

In Bos. taurus cattle, follicular deviation to dominance begins when the selected ovulatory follicle reaches a mean diameter of 8.5 mm. The dominant follicle acquires the capacity to ovulate when it reaches a diameter of about 10 mm. In this study, data derived from 148 cows in estrus with one follicle of ovulatory size and another of 7-9 mm, reveal that the small follicle has the capacity to ovulate alone or with the dominant follicle; thus, giving rise to a single or twin pregnancy. This indicates that a follicle of deviation size may ovulate in the presence of a follicle of ovulatory size.

Cervix-rectum temperature differential at the time of insemination is correlated with the potential for pregnancy in dairy cows.

This study sought to establish whether temperature gradients between the cervix, vagina, and rectum at and 7 days post-artificial insemination (AI) were associated with the incidence of pregnancy in lactating dairy cows (Experiment I; n = 90 ovulating cows) and to evaluate temperature gradient dynamics from the time of insemination to 7 days post-AI under heat stress conditions (Experiment II; n = 16 ovulating and 4 non-ovulating cows). In Experiment I, 39 cows (43.3%) became pregnant. The odds ratio for pregnancy was 2.5 for each one-tenth of a degree drop in cervical temperature with reference to the control rectal temperature at the time of AI (P = 0.01), whereas the same decrease in the cervix-rectum temperature differential 7 days post-AI resulted in an odds ratio of 0.44 (P = 0.02). In Experiment II, 5 of the ovulating cows (31.3%) became pregnant. The mean values of the vagina-rectum, vagina-cervix, and cervix-rectum temperature differentials at AI (day 0), 8 h, 24 h, and 7 days post-AI changed significantly from day 0 to day 7 (within-subject effect; P < 0.02) in ovulating cows but not in non-ovulating cows. Temperature differentials on days 0 and 7 were similar between ovulating cows and cows of Experiment I. Overall, our findings support the notion that a temperature differential between the caudal cervical canal and rectum at AI may be an indicator of the likelihood of pregnancy. Possible prospects of confirming estrus at the herd-level are also suggested.

Twin Pregnancies in Dairy Cattle: Observations in a Large Herd of Holstein-Friesian Dairy Cows.

Multiple pregnancies have devastating consequences on the herd economy of dairy cattle. This observational study examines incidence patterns based on data from the ultrasonographic examination of 1130 multiple pregnancies in cows in their third lactation or more carrying twins (98.8%), triplets (1.1%), or quadruplets (0.08%), and 3160 of their peers carrying singletons. Cows became pregnant following a spontaneous estrus with no previous hormone treatments. Irrespective of a significant decrease (p < 0.0001) in the conception rate (28-34 days post-insemination) during the warm period of the year, the multiple pregnancy rate was similar for both warm (26.5%) and cool (26.3%) periods. The incidence of unilateral multiple pregnancies (all embryos in the same uterine horn) was higher than that of bilateral pregnancies (at least one embryo in each uterine horn): 54.4% versus 45.6% (p < 0.0001). This difference rose to 17% during the warm season (p = 0.03). Pregnancy was monitored in unilateral multiple pregnancies until abortion or parturition (n = 615). In the warm period, the parturition rate was 43% compared to 61% recorded in the cool period (p < 0.0001). Thus, a warm climate is the main factor compromising the fate of multiple pregnancies. Some clinical suggestions are provided.

Temperature gradients in the mammalian ovary and genital tract: A clinical perspective.

Temperature within mammalian reproductive tissues is noted to be a key component of fertility, and significant gradients in temperature can be demonstrated deep within the abdomen shortly before ovulation. Indeed, in the absence of such gradients in the ovary and genital tract, the processes of ovulation and fertilisation are severely compromised. This review aims to assess literature produced during the last five decades regarding temperature gradients in the mammalian ovary and genital tract. A large body of observations derived from rabbits, women, pigs and cattle is summarised in tabular form, ovarian follicular values being as much as 2.5 °C or more cooler than neighbouring ovarian tissues or deep rectal temperature. We highlight recent works demonstrating a positive correlation between pre-ovulatory follicular cooling and pregnancy. Understanding the significance of follicular cooling should help us (a) explain why so many potential pregnancies fail in vivo as in vitro and (b) inspire ways for improving the processes of fertilisation and establishment of a full-term pregnancy. Based on our findings in domestic animals, and most recently in cows whose Graafian follicles are comparable in size and timing of response to the LH peak with human follicles, we wish to encourage IVF and fertility preservation clinics to take advantage of this work. By so doing, the incidence of full-term pregnancies in women should be improved.

Evolutionary sequences in mammalian reproductive biology.

Broad biological aspects and accepted evolutionary sequences may offer useful guidance towards a comprehensive explanation of the function of mammalian ovaries and oviducts and their vital contribution to the events of fertilization. Cooling of the preovulatory follicles before ovulation may well have its roots in the primitive stages of external fertilization in which aggregates of oocytes are shed into freshwater.

Luteal activity following follicular drainage of subordinate follicles for twin pregnancy prevention in bi-ovular dairy cows.

Twin pregnancy is undesirable in dairy cattle. This study examines luteal activity following ultrasound-guided puncture and drainage of the smaller pre-ovulatory follicle at timed AI in cows with a pre-ovulatory follicle in each ovary. Luteal activity was determined through Doppler ultrasonography and plasma progesterone (P4) concentrations. The effects of GnRH treatment on Day 7 post-AI on subsequent luteal activity were also assessed. Two study groups were established: a control group of 29 cows and a follicular drainage (FD) group of 28 cows. After drainage, all cows developed a corpus luteum (CL) in the drained ovary. On Day 21 post-AI, drainage-induced CL and fellow CL were similar in terms of size and vascularization. According to a GLM repeated measures analysis of variance (P < 0.001), non-treated drained cows had lower P4 concentrations on Day 21 post-AI than non-treated non-drained cows, whereas GnRH treated cows, both drained and non-drained, showed the highest P4 concentrations at this time point. Twin pregnancy was recorded in 3 of the 8 pregnant control cows, whereas no twins were observed in the FD group. Our results indicate that luteal structures following follicular drainage were functional. As for the presence of an additional CL, this could suggest a reduced risk of pregnancy loss. In addition, luteal activity was significantly increased following GnRH treatment on Day 7 post-AI in drained cows.

Pre-ovulatory follicular cooling correlates positively with the potential for pregnancy in dairy cows: Implications for human IVF.

INTRODUCTION: In rabbits, pigs, cows and humans, pre-ovulatory Graafian follicles may be more than 1.0 °C cooler than ovarian stroma and both these ovarian compartments are cooler than deep rectal temperatures. This study examines the effect of follicular cooling on the incidence of pregnancy in dairy cows. MATERIAL AND METHODS: Follicular measurements were compiled for cows with one ovulatory follicle (monovular) and cows with one ovulatory follicle per ovary (bi-ovular) and their corresponding uterine horn contents. The study sample consisted of 80 pre-ovulatory follicles in which antral temperatures were measured using a fine thermistor probe. RESULTS: Mean (± S.D.) follicular fluid temperature of the ovulating follicles was 1.12 ± 0.86 °C significantly cooler (P < 0.0001) than rectal temperatures. No significant differences in temperatures were found for non-ovulating follicles. In follicles undergoing cooling (n = 58), a one-tenth of a degree drop in temperature with reference to control rectal temperature gave rise to a 3.6-fold increase (odds ratio) in the pregnancy rate (P = 0.003). The follicle-rectum temperature differential giving rise to pregnancy (n = 18; 1.51 ± 1.15 °C) was significantly greater (P = 0.004) than the differential recorded in cooling follicles at that did not result in a subsequent pregnancy (n = 40; 0.83 ± 0.57 °C). CONCLUSION: Follicular cooling is needed to trigger ovulation and correlates positively with the potential for pregnancy in cows. This finding has interesting implications for human reproductive medicine.

Pre-ovulatory follicular temperature in bi-ovular cows.

In a previous study on monovular cows, follicles revealed a mean antral (follicular fluid) temperature 1.54°C cooler than rectal temperatures in ovulating cows, whereas no such temperature differences were detected in non-ovulating cows. The present study adds to our previous work, this time considering 24 bi-ovular cows (one follicle per ovary). In order to increase the number of pre-ovulatory follicles failing to ovulate, this study was performed under heat-stress conditions. Follicular temperatures of the ovulating follicles (n = 31) were 0.93°C significantly cooler (P < 0.0001) than rectal temperatures, whereas no significant differences in temperature were found in non-ovulating follicles (n = 17). Eight cows became pregnant. The results of the present study indicate that, similar to those in monovular cows, pre-ovulatory follicles in bi-ovular cows were cooler than deep rectal temperatures and those temperature gradients were not found in follicles showing ovulation failure.

Seminal plasma modulates the immune-cytokine network in the porcine uterine tissue and pre-ovulatory follicles.

Evidence is emerging that the interaction between male seminal fluid and female tissues promotes fertility, pregnancy, and health of offspring. This includes the acceleration of ovulation in a species known as a spontaneous ovulator, the domestic pig. Earlier studies revealed that seminal plasma acts by a local mechanism in the female pig. The aim of the present study was to examine local short-term and mid-term effects of seminal plasma (SP) on mRNA expression of immunoregulatory genes and transcripts associated with follicle- and oocyte maturation. In the porcine animal model, effects on mRNA expression in the female tract and preovulatory follicles were examined. SP suppressed mRNA expression of Prostaglandin-Endoperoxide Synthase 2 (PTGS2) ipsilateral to the infused uterine horn which was associated with a lower presence of immune cells in the uterine epithelium and lower PTGS2 immunoreaction. Depending on the sampling time (2 h vs. 17 h) and hormonal status, SP altered significant correlative relations of mRNA expression between PTGS2 and the transcripts Tumor Necrosis Factor Alpha, Tumor Necrosis Factor Alpha-Induced Protein 6 and Pentraxin 3 in uterus, granulosa and cumulus cells. A modulatory effect of SP on the oocyte gene network comprising eight oocyte transcripts was observed: uterine exposure to SP induced positive correlations (r >0.08, p<0.05) of maturation promoting factors among each other and with cumulus cells on the side of the treated horn. In conclusion, SP orchestrates the gene network regulating the bidirectional communication between oocytes and surrounding somatic cells. The modulation of the immune-cytokine network of the female reproductive system could contribute to the previously reported SP-induced acceleration of ovulation in the porcine species.

Whither human IVF? Fertilisable oocytes selected on the basis of follicular temperature.

Bearing in mind specific parallels between cow and human ovarian physiology, as noted in the manuscript, we have measured whether the temperature in a pre-ovulatory follicle is cooler than that in adjacent tissues. Using a novel approach not requiring anaesthetics or surgical procedures, we found that follicular fluid bathing cow oocytes shortly before ovulation is cooler than the neighbouring uterine surface and cooler than deep rectal temperature (the reference body temperature in cattle). By contrast, Graafian follicles of comparable size and ultrasonic image that do not subsequently ovulate do not have a reduced antral temperature. Human pre-ovulatory follicles have previously been reported to be cooler than other ovarian tissues, so the divergence between ovulatory and non-ovulatory follicle temperature suggests a valuable addition to selection procedures currently used in human in vitro fertilisation (IVF) clinics. In future, oocytes to be subjected to IVF might best be those taken from cooler follicles. Follicular antral temperature could become a more sensitive indicator of oocyte potential that a purely morphological assessment.

The oviductal transcriptome is influenced by a local ovarian effect in the sow.

BACKGROUND: Oviducts participate in fertilization and early embryo development, and they are influenced by systemic and local circulation. Local functional interplay between ovary, oviduct and uterus is important, as deduced from the previously observed differences in hormone concentrations, presence of sperm, or patterns of motility in the oviduct after unilateral ovariectomy (UO). However, the consequences of unilateral ovariectomy on the oviductal transcriptome remain unexplored. In this study, we have investigated the consequences of UO in a higher animal model as the pig. METHODS: The influence of UO was analyzed on the number of ovulations on the contra ovary, which was increased, and on the ipsilateral oviductal transcriptome. Microarray analysis was performed and the results were validated by PCR. Differentially expressed genes (DEGs) with a fold change ≥ 2 and a false discovery rate of 10 % were analyzed by Ingenuity Pathway Analysis (IPA) to identify the main biofunctions affected by UO. RESULTS: Data revealed two principal effects in the ipsilateral oviduct after UO: i) down-regulation of genes involved in the survival of sperm in the oviduct and early embryonic development, and ii) up-regulation of genes involved in others functions as protection against external agents and tumors. CONCLUSIONS: Results showed that unilateral ovariectomy results in an increased number of ovulation points on the contra ovary and changes in the transcriptome of the ipsilateral oviduct with consequences on key biological process that could affect fertility output.

Oviductal Transcriptome Is Modified after Insemination during Spontaneous Ovulation in the Sow.

Gene Expression Microarray technology was used to compare oviduct transcriptome between inseminated and non-inseminated pigs during spontaneous oestrus. We used an in vivo model approaching the study from a physiological point of view in which no hormonal treatment (animals were in natural oestrus) and no artificial sperm selection (selection was performed within the female genital) were imposed. It is therefore emphasised that no surgical introduction of spermatozoa and no insemination at a site other than the physiological one were used. This approach revealed 17 genes that were two-fold or more up-regulated in oviducts exposed to spermatozoa and/or developing embryos and 9 genes that were two-fold or more down-regulated. Functional analysis of the genes revealed that the top canonical pathways affected by insemination were related to the inflammatory response and immune system (Network 1) to molecular transport, protein trafficking and developmental disorder (Network 2) and to cell-to-cell signalling and interaction (Network 3). Some of the genes in network 1 had been previously detected in the oviduct of human and animals, where they were over-expressed in the presence of spermatozoa or pre-implantation embryos (C3, IGHG1, ITIH4, TNF and SERPINE1) whereas others were not previously reported (SAA2, ALOX12, CD1D and SPP1). Genes in Network 2 included RAB1B and TOR3A, the latter being described for the first time in the oviduct and clearly expressed in the epithelial cells of the mucosa layer. Network 3 integrated the genes with the highest down-regulation level (CYP51, PTH1R and TMOD3). Data in the present study indicate a change in gene expression during gamete encounter at the site of fertilization after a natural sperm selection within the female genital tract. These changes would indicate a modification of the environment preparing the oviduct for a successful fertilization and for an adequate embryo early development.

Considerations of viscosity in the preliminaries to mammalian fertilisation.

Migration of spermatozoa in the female genital tract will be strongly influenced by the viscosity of the fluids encountered, yet little systematic analysis has been given to such a consideration. This essay reviews the series of milieux confronting a fertilising sperm during its progression to the oviduct ampulla. Two groups are discussed, first those in which ejaculation is into the vagina, second those in which semen enters the uterus during a protracted mating. Viscous glycoprotein secretions that accumulate in the oviduct isthmus of both groups before ovulation are highlighted, as is the environment generated in the ampulla by the post-ovulatory suspension of oocyte(s), cumulus cells and spermatozoa; follicular and peritoneal fluids may also be present. The viscosity of all female tract fluids responds to cyclical variations in temperature, and these exist within the oviduct near the time of ovulation. Gradations in viscosity influence the pattern and strength of sperm flagellar activity and the rate of forward movement. Measurements of sperm motility are currently made in a physiological medium of constant viscosity and temperature, thereby overlooking changes in the female genital tract. A more sophisticated approach might reveal an adequate fertilising potential in a proportion of putatively poor semen samples.

Peritoneal fluid concentrations of progesterone in women are higher close to the corpus luteum compared with elsewhere in the abdominal cavity.

OBJECTIVE: To investigate the potential role of peritoneal fluid activity for the local delivery of gonadal steroid hormones. DESIGN: Prospective clinical study. SETTING: University hospital. PATIENT(S): Nine women undergoing tubal sterilization during laparoscopic surgery. INTERVENTION(S): Peritoneal fluid was collected during laparoscopic surgery by means of sterile cotton swabs on the surface of the peritoneum facing the ovary with an active corpus luteum and on the contralateral side, at the right and left paracolic gutter, and at the pouch of Douglas; simultaneous systemic blood sampling was performed. MAIN OUTCOME MEASURE(S): P was quantified by competitive immunochemical assay. Final steroid concentrations were calculated relative to the standard recovery rate. RESULT(S): P concentrations in the peritoneum close to the corpus luteum were 4 times (range, 1.4-9.2) higher than in the other peritoneal samples and about 5 times (range, 3.9-9.5) higher than in the systemic blood. CONCLUSION(S): The results indicate that P enters the peritoneal cavity locally and may therefore influence the function of other nearby tissues, especially those of the ipsilateral fallopian tube and proximal region of the uterus. Indeed, the peritoneal cavity and its fluid contents may be a powerful yet overlooked component of the regulatory systems within the body.

Physiological routes from intra-uterine seminal contents to advancement of ovulation.

Whole boar semen or seminal plasma has been demonstrated to advance the time of ovulation in gilts. As a means of clarifying this influence, the contribution of uterine lymphatics and their white cell populations has been examined. After duct visualisation with Evan's blue, lymph was sampled from a mesometrial vessel in eight pre-ovulatory gilts whose uterine lumen was infused simultaneously with whole semen in one ligated horn and saline in the contralateral ligated horn. Lymph was collected from cannulated vessels for periods of up to four hours under general anaesthesia. Thereafter, mesometrial lymph nodes, utero-tubal junction and uterine wall tissues were sampled. The proportion of nucleated cells in the sampled lymph increased towards the end of the collection period, but erythrocytes were found in all instances preventing a meaningful differentiation and identification of leukocytes. Prominent uterine lymph nodes were present in the mesometrium on both sides of the reproductive tract in 7 of 10 gilts. Differences in cellular contents were demonstrated between the side of the tract infused with semen and that infused with saline control. Two of 4 gilts had lower values for CD4 (Cluster Differentiation) and 3 of 6 gilts higher values for MHC II (Major Histocompatibility Complex) markers on the side challenged with semen. In contrast, values remained constant for CD8 but ranged widely for CD18. Immunohistochemical analysis of uterine tissue samples for MHC II+ cells revealed significant differences (P < 0.05) between the control and semen-treated ligated portions of the horns, as well as between the tissue sample of uterine wall and that from the utero-tubal junction, but there were no significant differences for CD4+ cells. It therefore remains plausible that semen-induced cytokines in the uterine lymph undergo counter-current transfer to the ipsilateral ovary and accelerate the final maturation of pre-ovulatory Graafian follicles.