Association between prepartum vaginal temperature changes and placenta expulsion time in Holstein dairy cattle.

Retained placenta (RP) adversely affects postpartum productivity and reproduction in dairy cattle. Thus, methods to predict the occurrence of RP before calving would be desirable. Herein, we assessed whether vaginal temperature measurements (which have already been applied to detect calving) could be used to predict the occurrence of RP in cattle. A vaginal temperature recording device was inserted into the vagina of 49 pregnant Holstein-Friesian heifers (n = 16) and cows (n = 33); this device recorded the vaginal temperature every 5 min until the device dropped out at calving. Serum was collected 10 days before the expected calving date. The time points of calving and placental expulsion were identified via video recordings. We further calculated calving duration (temperature decrease to calving) and placenta expulsion time (PE time = calving to placenta expulsion). The PE times were divided into four categories (0-4 h, 4-8 h, 8-12 h, and RP at >12 h), while subsequent analysis revealed that an extension of the PE time dependent on the shortening of the calving duration (P < 0.05). The vaginal temperature patterns also differed in a PE time-dependent manner, and cows with RP did not show any re-elevation of vaginal temperature. Serum analyses indicated an energy deficiency in RP cattle. These results suggest that RP may be detected early as a specific change in the vaginal temperature associated with reproductive hormone secretion.

Deterioration of mitochondrial biogenesis and degradation in the endometrium is a cause of subfertility in cows.

To investigate possible causes of reproductive failure, we conducted global endometrial gene expression analyses in fertile and subfertile cows. Ingenuity pathway analysis showed that RICTOR and SIRT3 are significant upstream regulators for highly expressed genes in fertile cows, and are predicted to be activated upstream regulators of normal mitochondrial respiration. Canonical pathway analysis revealed that these highly expressed genes are involved in the activation of mitochondrial oxidative phosphorylation. Therefore, in subfertile cows, the inactivation of RICTOR and SIRT3 may correlate with decreased capacity of mitochondrial respiration. Furthermore, the expression levels of most mitochondrial DNA genes and nuclear genes encoding mitochondrial proteins were higher in subfertile cows. The mitochondrial DNA copy number was significantly higher in the endometrium of subfertile cows, whereas the ATP content did not differ between fertile and subfertile cows. Quantitative reverse transcription-PCR analysis demonstrated that the expression of PGC1a, TFAM, MFN1, FIS1, and BCL2L13 were significantly lower in subfertile cows. In addition, transmission electron microscopy images showed mitochondrial swelling in the endometrial cells of the subfertile cow. These results suggest that poor-quality mitochondria accumulate in the endometrium owing to a reduced capacity for mitochondrial biogenesis, fusion, fission, and degradation in subfertile cows, and may contribute to infertility.

Changes in immunoglobulin A and immunoglobulin G concentrations in the saliva of Japanese Black beef cows during calving: A pilot study.

Immunoglobulin A (IgA) in saliva, mostly consisting of secretory IgA, plays an important role in the mucosal immune mechanism. This study evaluated changes in salivary IgA and Immunoglobulin G (IgG) concentrations in Japanese Black cows (n = 16) during calving. Individual saliva samples were collected -2, 0, and 2 weeks postpartum. Immunoglobulin concentrations differed significantly among weeks (P < 0.05), but the effect of parity and week × parity was insignificant. Salivary IgA concentrations decreased drastically (P < 0.05) after calving compared with those at -2 weeks postpartum and remained low until 2 weeks postpartum. The salivary IgG concentrations decreased gradually during peripartum and differed at -2 and 2 weeks postpartum (P < 0.05). Considering the immunoglobulin concentrations at -2 weeks postpartum as the reference standard for 100%, the rates of decrease in IgA concentrations (36.7 ± 6.9%) were significantly lower (P < 0.05) than those of IgG (70.3 ± 10.1%) at calving day. To our knowledge, this is the first report indicating that salivary IgA concentrations decreased drastically after calving in Japanese Black cows. Further studies monitoring the secretory functions of IgA in the salivary gland are essential for understanding maternal immunity in cattle.

Effect of E-64 Supplementation during In Vitro Maturation on the Developmental Competence of Bovine OPU-Derived Oocytes.

Recovery of bovine oocytes using the ovum pick-up (OPU) technique offers the advantage of rapid genetic improvement through propagation of desired genes from animals with high genetic qualities. However, the developmental competence of OPU-derived immature oocytes remains relatively poor. We previously found that cathepsin B gene expression and activity are increased in poor quality oocytes and embryos compared to good quality ones. In this study, we investigated the effect of E-64 (cathepsin B inhibitor) supplementation during in vitro maturation (IVM) on the developmental competence of OPU-derived immature oocytes and the quality of the produced blastocysts. Our results showed that supplementation of IVM medium with E-64 significantly improved the developmental competence of OPU-derived immature oocytes as evidenced by the significant increase of the blastocyst rate. Importantly, the presence of E-64 during IVM also significantly improved blastocyst quality by increasing the total cell number and decreasing the percentage of TUNEL positive cells. These results indicate that E-64 supplementation during IVM is a promising tool to improve the efficiency of OPU-IVF program by improving the developmental competence of OPU-derived immature oocytes.

[The role of reproductive biology in SDGs] Global warming and cattle reproduction: Will increase in cattle numbers progress to global warming?

The livestock industry produces a large amount of greenhouse gases (GHG) that cause global warming. A high percentage of GHG emissions are derived from cattle and has been suggested to be a factor in global warming. With the global increase in the consumption of livestock products, the number of farm animals has increased. In addition, the reduction in productivity and reproductive capacity of cattle has resulted in accelerated GHG emissions. In a high-temperature environment, the pregnancy rate decreases, leading to an increase in animals that do not contribute to production. Consequently, GHG emission per unit product increases, thereby accelerating global warming. To reduce this environmental impact, it is important to improve the breeding efficiency of cattle by the use of reproductive technology and, thus, reduce the number of non-productive animals. Thus, reproductive biology plays a major role in mitigating global warming related to the livestock industry.

Steroidal but not embryonic regulation of mucin 1 expression in bovine endometrium.

In cow herd management, inadequate embryo implantation leads to pregnancy loss and causes severe economic losses. Thus, it is crucial to understand the molecular mechanisms underlying endometrial receptivity and subsequent embryo implantation. Transmembrane glycocalyx mucin 1 (MUC1) has a large and highly glycosylated extracellular domain known to inhibit embryo implantation via steric hindrance. The role of MUC1 in the bovine endometrium remains to be explored. Herein, we used simple but reliable in vivo and in vitro experiments to investigate the expression and regulation of MUC1 in the bovine endometrium. MUC1 gene expression was analyzed in endometrial epithelial cells collected by the cytobrush technique using reverse transcription-quantitative polymerase chain reaction. MUC1 protein expression was evaluated by immunohistochemical analysis of endometrial samples collected from slaughtered cows. We used an in vitro cell culture model to study the regulation of MUC1 expression by treating cells with sex steroidal hormones or co-culturing cells with a blastocyst. The results revealed that MUC1 was expressed and localized to the apical surface of luminal epithelial cells in the bovine endometrium. MUC1 expression disappeared during the luteal phase of the estrous cycle and during pregnancy. 17ß-estradiol induced MUC1 expression, whereas progesterone inhibited its increase and co-culturing with blastocysts did not affect the expression. A long postpartum interval is a known risk factor for reduced fertility, and MUC1 expression was higher in this compromised condition. Our results demonstrated the MUC1 regulation by steroid hormones in bovine endometrium for embryo implantation, and we observed a negative correlation between MUC1 expression and fertility.

Vaginal temperature before calving assessed with wireless vaginal temperature sensor in dairy and beef cattle.

We evaluated the daily and hourly vaginal temperature changes and the relationships between the dams' breed and parity by using a commercially available vaginal temperature sensor in 72 Holstein (Hol) calvings and 101 Japanese Black (JB) calvings. Vaginal temperature sensors inserted 7-10 days before the expected calving day sounded two alerts: when the temperature fell below the threshold (Alert 1), and when the sensor reached the ambient temperature after falling out of the dam's vagina with the rupture of the allantoic sac (Alert 2). The durations from Alert 1 to Alert 2 (Time 1) and from Alert 2 to delivery (Time 2) were calculated. Only Time 1 in the Hol group tended to be affected by parity and parity × calf body weight. In the JB group, none of the factors examined affected Time 1 or Time 2. The alert detection rates did not differ by parity in either breed or by the temperature threshold in Hol. However, the Hol group's alert detection rate was significantly lower than the JB group's (p < 0.05). The daily average temperature was higher in the Hol group and the primiparous dams than those in the JB and multiparous dams; it increased slightly from Day -7 to -3 (Day 0 = the day of calving) and then dropped dramatically on Days -1 and 0. The hourly vaginal temperature difference from -48 h of calving showed a typical pattern, i.e., a decrease from -30 h of Alert 1 and an increase at -6 h of Alert 1. The decrease and increase might be the regression of the pregnant corpus luteum and the beginning of the contractions, respectively. The temperature differences were significantly affected by parity and calving ease (p < 0.01). The primiparous dams showed wider temperature differences compared to the multiparous dams in both breeds (p < 0.001). No typical temperature difference pattern was observed in assisted calving or dystocia. The alert detection rate, the Time durations, and the vaginal temperature differences were affected by the dams' breed and parity. However, measuring vaginal temperatures proved useful for predicting the calving regardless of the breed and parity. The effect of calving ease remains unclear due to the low number of assisted calvings herein.

Prepartum change in ventral tail base surface temperature in beef cattle: comparison with vaginal temperature and behavior indices, and effect of ambient temperature.

Prediction of parturition is essential for sustainable production in beef and dairy cattle, yet the present methods are limited by their high invasiveness and low utility. Here we compared prepartum changes in ventral tail base surface temperature (ST) with changes in vaginal temperature (VT) and behavioral indices. We analyzed 22 parturitions from 22 beef cows. Changes in daily values of ST, VT, and behavioral indices over the 7 days before parturition were investigated. Hourly values were calculated as the actual values minus the mean values for the same hour over a 3-day period, and the changes in hourly values over the 48 h before parturition were investigated. To test the effect of ambient temperature, tested cows were assigned to two season-groups based on the ambient temperature to which they were exposed (warm: n = 13; cool: n = 9), and the daily and hourly values of the indices were compared between seasons. A decrease in ST occurred approximately 30 h before parturition, which was similar to the time of the decrease in VT and earlier than the increase of behavioral indices. In addition, a unique fluctuation of ST observed in the last few hours before parturition indicates that ST could provide a sign for parturition not only in the long-term like VT, but also in the short-term like behavioral indices. Although ST was more sensitive to ambient temperature than VT or the behavioral indices, the day of parturition could be predicted from ST in both the warm and cool seasons.

Production of lipid mediators in mastitic milk of cow.

Bovine mastitis is one of the most prevalent and costly diseases in the dairy industry. Lipid mediators are signaling molecules which coordinately and intricately modulate inflammation. They are produced from polyunsaturated fatty acids (PUFAs) in the cellular membrane via several enzymes including cyclooxygenase (COX) and lipoxygenase (LOX). In the present study, we performed comprehensive analysis of lipid production in milk obtained from clinical or subclinical mastitic cows using liquid chromatography/mass spectrometry. We detected 26, 24, and 40 kinds of lipid constantly in healthy, subclinical, and clinical mastitic milk, respectively. In clinical mastitic milk, the amount of a major n-6 PUFA, arachidonic acid (AA), tended to increase, whereas amounts of major n-3 PUFAs, eicosapentaenoic acid and docosahexaenoic acid, tended to decrease. The amounts of several AA-derived lipids including COX-catalyzed prostaglandin (PG) D2 and PGE2 , and LOX-catalyzed leukotriene (LT) B4 were increased in clinical mastitic milk. Although subclinical mastitic milk represented similar trend of lipid production to healthy milk, amounts of several lipids such as LTD4 , 14,15-dihydroxyeicosatrienoic acid, and 14-epoxyeicosatrienoic acid changed. These findings would be helpful for better understanding of mastitis pathology and give us some insights to develop a new diagnostic and therapeutic strategy.

Normal DNA methylation status in sperm from a somatic cell cloned bull and their fertilized embryos.

Epigenetic reprogramming confers totipotency even during somatic cell nuclear transfer (SCNT), which has been used to clone various animal species. However, as even apparently healthy cloned animals sometimes have aberrant epigenetic status, the harmful effects of these defects could be passed onto their offspring. This is one of the biggest obstacles for the application of cloned animals for livestock production. Here, we investigated the DNA methylation status of four developmentally regulated genes (PEG3, XIST, OCT4, and NANOG) in sperms from a cloned and a non-cloned bull, and blastocysts obtained by in vitro fertilization using those sperms and SCNT. We found no differences in the methylation status of the above genes between cloned and non-cloned bull sperms. Moreover, the methylation status was also similar in blastocysts obtained with cloned and non-cloned bull sperms. In contrast, the methylation status was compromised in the SCNT blastocysts. These results indicate that sperm from cloned bulls would be adequately reprogrammed during spermatogenesis and, thus, could be used to produce epigenetically normal embryos. This study highlights the normality of cloned bull offspring and supports the application of cloned cattle for calf production.

Sericin enhances the developmental competence of heat-stressed bovine embryos.

We investigated the effects of sericin on the developmental competence of bovine embryos exposed to heat stress (HS). Putative zygotes were cultured with sericin and subjected to HS (40.5°C for 6 hr) on Day 2 or 7 followed by continuous culture at 38.5°C until Day 8. Day 2 HS significantly decreased blastocyst development on Day 8 as well as mitochondrial activity, and significantly increased the amount of intracellular reactive oxygen species and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL)-positive cells, whereas Day 7 HS only significantly decreased mitochondrial activity and increased the number of TUNEL-positive cells in Day 8 blastocysts. These detrimental effects were neutralized by sericin supplementation. Next, to investigate the potential production of blastocysts with high viability in terms of thermotolerance, embryos were cultured with sericin until Day 7, and then exposed to HS in the sericin-free medium. TUNEL-positive cell numbers were significantly lower in blastocysts produced by sericin culture than in control blastocysts. Transcript abundance for HSPA1A and BAX was significantly decreased but IFNT2 levels were increased in blastocysts produced by sericin culture. In conclusion, these findings demonstrate the anti-oxidative and anti-apoptotic activities of sericin, and the potential use of sericin to produce embryos with high viability in vitro.

Efficiency of a pedometer device for detecting estrus in standing heat and silent heat in Japanese Black cattle.

The usefulness of a radiotelemetric pedometer for estrus detection in standing (ST) heat, or in silent heat without ST events, but in which ovulation is observed, in Japanese Black cattle was investigated. The duration of an increase in steps in ST heat was 11.8 ± 1.3 hr, and it was similar to that of ST events (duration: 10.1 ± 0.8 hr). Even in silent heat, the change pattern and the duration (11.6 ± 0.2 hr) of the period with an increase in steps during estrus were not different compared with ST heat. When artificial insemination (AI) was performed at 15.5 ± 0.6 hr from the onset of estrus detected by the pedometer in ST heat cases, the conception rate was 57.1% (8/14). Furthermore, fertility in cattle that underwent silent heat was evaluated. When AI was performed at 14.4 ± 2.0 hr from the onset of estrus detected by the pedometer, the conception rate was 60% (3/5) in silent heat cases. The overall results suggest that the radiotelemetric pedometer is a valid device for detecting estrus and it can even detect silent heat in Japanese Black cattle. Moreover, even silent heat cattle are fertile when AI is performed at the appropriate time.

Heat-shock-induced cathepsin B activity during IVF and culture compromises the developmental competence of bovine embryos.

Heat stress can cause significant reproductive dysfunction in mammals and previous studies report that expression and activity of cathepsin B (CTSB), a lysosomal cysteine protease, is negatively correlated with the developmental competence of bovine oocytes and embryos. However, the relationship between heat shock (HS) and CTSB remains largely unknown. Here, we investigated the effects of HS during IVF and early embryonic stages of IVC on CTSB activity and developmental competence in bovine embryos. HS (40 °C for 6 h during IVF and 20 h during IVC) caused a significant increase in CTSB activity irrespective of the developmental stage or duration of HS. The developmental rate to the blastocyst stage was also significantly decreased by HS. Additionally, HS during IVC significantly increased the number of apoptotic cells in blastocysts. Notably, these HS-induced changes in blastocyst development and quality were significantly improved by inhibition of CTSB activity, indicating a key role for CTSB. These results showed that CTSB activity plays an essential role in HS-induced dysfunction in bovine embryo development, and that inhibition of this activity could enhance the developmental competence of heat-shocked embryos.

Live births from artificial insemination of microfluidic-sorted bovine spermatozoa characterized by trajectories correlated with fertility.

Selection of functional spermatozoa plays a crucial role in assisted reproduction. Passage of spermatozoa through the female reproductive tract requires progressive motility to locate the oocyte. This preferential ability to reach the fertilization site confers fertility advantage to spermatozoa. Current routine sperm selection techniques are inadequate and fail to provide conclusive evidence on the sperm characteristics that may affect fertilization. We therefore developed a selection strategy for functional and progressively motile bovine spermatozoa with high DNA integrity based on the ability to cross laminar flow streamlines in a diffuser-type microfluidic sperm sorter (DMSS). The fluid dynamics, with respect to microchannel geometry and design, are relevant in the propulsion of spermatozoa and, consequently, ultrahigh-throughput sorting. Sorted spermatozoa were assessed for kinematic parameters, acrosome reaction, mitochondrial membrane potential, and DNA integrity. Kinematic and trajectory patterns were used to identify fertility-related subpopulations: the rapid, straighter, progressive, nonsinuous pattern (PN) and the transitional, sinuous pattern (TS). In contrast to the conventional notion that the fertilizing spermatozoon is always vigorously motile and more linear, our results demonstrate that sinuous patterns are associated with fertility and correspond to truly functional spermatozoa as supported by more live births produced from predominant TS than PN subpopulation in the inseminate. Our findings ascertain the true practical application significance of microfluidic sorting of functional sperm characterized by sinuous trajectories that can serve as a behavioral sperm phenotype marker for fertility potential. More broadly, we foresee the clinical application of this sorting technology to assisted reproduction in humans.

Vaginal temperature measurement by a wireless sensor for predicting the onset of calving in Japanese Black cows.

We evaluated the utility of the continuous measurement of vaginal temperature by a wireless sensor and wireless connection for predicting the onset of calving and for clarifying the relationships among dystocia, calf conditions, and temperature changes at a commercial beef cattle farm in Japan. A total of 625 effective delivery data was collected. The temperature sensor inserted to the vagina on 7 days before the expected due date and collected the vaginal temperature every 5 min. The sensor detected two alerts according to the temperature change, one was the vaginal temperature of 4 h moving average compared to the same time temperature of last two days decreased more than 0.4 °C (Alert 1) and the other was the rupture of the allantoic sac and the dropped sensor temperature reached to the ambient temperature (Alert 2). The detection rates of Alert 1 and Alert 2 were 88.3% and 99.4%, respectively. The average time between Alert 1 and Alert 2 (Time 1) was 22 h, and that between Alert 2 and delivery (Time 2) was 2 h. These results indicated that the continuous measurement of vaginal temperature is effective for predicting the calving time. The necessity of assistance was correlated with dystocia, calf birth weight (BW), sex, and gestation periods. Interestingly, the durations of Times 1 and 2 were also associated with dystocia. The calf BW, sex, and gestation periods affected the length of Time 2. Our findings indicate that the BW of the calf is the most important factor for dystocia risk, and that the continuous measurement of vaginal temperature could become a good indicator for predicting not only the onset of calving, but also the necessity of assistance.

Evaluating the electrical impedance and mucus-related gene expression of uterine endometrial tissues in mares.

We investigated the electrical impedance of the reproductive tracts (vagina and uterine endometrial tissues) and the expression of mucus-related genes to identify the stage of the estrous cycle in mares. We first examined vaginal impedance in native Hokkaido mares during their estrous cycle and found no significant differences. However, impedance levels tended to decrease towards ovulation. Furthermore, we investigated the estrous cycle by measuring the electrical impedance of the uterine endometrial tissues obtained from carcasses of mares. We found that impedance levels in the endometrial tissues decreased in the regressed phase of the corpus luteum (CL). Expression of mucus-related genes (ATP1A1, CFTR, AQP3, and AQP5) varied at different stages of the estrous cycle. Among them, AQP3 expression was consistent with previous reports. We concluded that electrical impedance in the uterine endometrial tissues of mares could be potentially used to verify the presence of active CL in horses for experimental purposes. However, further studies are needed to determine the reference value and to identify the day of the estrous cycle in mares.

Different thermotolerances in in vitro-produced embryos derived from different maternal and paternal genetic backgrounds.

The present study evaluated the effects of genetic backgrounds on the developmental competence and thermotolerance of bovine in vitro-produced (IVP) embryos. First, Holstein (Hol) and Japanese Black (JB) oocytes were fertilized with sperm from Hol, JB and a thermotolerant breed (Brahman), and in vitro development was evaluated when the embryos were exposed to heat shock on Day 2 (Day 0 = day of fertilization). Sperm genetic backgrounds affected the developmental competence in controls (P < 0.05). Second, the effect of sperm pre-incubation for 4 h on subsequent in vitro fertilization was assessed using different sperm genetic backgrounds. The pre-incubation of sperm did not decrease the embryonic development regardless of the breed of the sperm. A milder heat shock (40.0°C) effect on parthenotes (Hol and JB) and IVP embryos were evaluated. JB parthenotes showed developmental arrest after Day 4, and the rate of development to the blastocyst stage decreased by heat shock, but not in Hol parthenotes. Heat shock decreased developmental competence after cleavage of IVP embryos regardless of genetic background. The thermotolerance of IVP embryos would be controlled by both maternal and paternal factors but genetic involvement was still unclear. Further evaluation is needed to reveal the genetic contribution to thermotolerance.

Effects of heat stress on bovine preimplantation embryos produced in vitro.

Summer heat stress decreases the pregnancy rate in cattle and has been thought to be associated with the early embryonic death caused by the elevation of maternal body temperature. In vitro cultures have been widely used for the evaluation of effects of heat stress on oocytes, fertilization, preimplantation, and embryonic development. Susceptibility to heat stress is present in developmental stages from oocytes to cleavage-stage (before embryonic gene activation, EGA) embryos, leading to a consequent decrease in developmental competence. On the other hand, advanced-stage embryos such as morula or blastocysts have acquired thermotolerance. The mechanism for the developmental stage-dependent change in thermotolerance is considered to be the accumulation of antioxidants in embryos in response to heat-inducible production of reactive oxygen species. The supplementation of antioxidants to the culture media has been known to neutralize the detrimental effects of heat stress. Besides, EGA could be involved in acquisition of thermotolerance in later stages of embryos. Morulae or blastocysts can repair heat-induced unfolded proteins or prevent DNA damage occurring in processes such as apoptosis. Therefore, embryo transfer (ET) that can bypass the heat-sensitive stage could be a good solution to improve the pregnancy rate under heat stress. However, frozen-thawed ET could not improve the pregnancy rate as expected. Frozen-thawed blastocysts were more sensitive to heat stress and showed less proliferation upon heat exposure, compared to fresh blastocysts. Therefore, further research is required to improve the reduction in pregnancy rates due to summer heat stress.

Regulation of gene expression in the bovine blastocyst by colony stimulating factor 2.

BACKGROUND: Colony stimulating factor 2 can have multiple effects on the function of the preimplantation embryo that include increased potential to develop to the blastocyst stage, reduced apoptosis, and enhanced ability of inner cell mass (ICM) to remain pluripotent after culture. The objective of the current experiment was to identify genes regulated by CSF2 in the ICM and trophectoderm (TE) of the bovine blastocyst with the goal of identifying possible molecular pathways by which CSF2 increases developmental competence for survival. Embryos were produced in vitro and cultured from Day 6 to 8 in serum-free medium containing 10 ng/ml recombinant bovine CSF2 or vehicle. Blastocysts were harvested at Day 8 and ICM separated from TE by magnetic-activated cell sorting. RNA was purified and used to prepare amplified cDNA, which was then subjected to high-throughput sequencing using the SOLiD 4.0 system. Three pools of amplified cDNA were analyzed per treatment. RESULTS: The number of genes whose expression was regulated by CSF2, using P < 0.05 and >1.5-fold difference as cut-offs, was 945 in the ICM (242 upregulated by CSF2 and 703 downregulated) and 886 in the TE (401 upregulated by CSF2 and 485 downregulated). Only 49 genes were regulated in a similar manner by CSF2 in both cell types. The three significant annotation clusters in which genes regulated by ICM were overrepresented were related to membrane signaling. Genes downregulated by CSF2 in ICM were overrepresented in several pathways including those for ERK and AKT signaling. The only significant annotation cluster containing an overrepresentation of genes regulated by CSF2 in TE was for secreted or extracellular proteins. In addition, genes downregulated in TE were overrepresented in TGFß and Nanog pathways. CONCLUSIONS: Differentiation of the blastocyst is such that, by Day 8 after fertilization, the ICM and TE respond differently to CSF2. Analysis of the genes regulated by CSF2 in ICM and TE are suggestive that CSF2 reinforces developmental fate and function of both cell lineages.

Evidence for a PGF2α auto-amplification system in the endometrium in mares.

In mares, prostaglandin F2α (PGF2α) secreted from the endometrium is a major luteolysin. Some domestic animals have an auto-amplification system in which PGF2α can stimulate its own production. Here, we investigated whether this is also the case in mares. In an in vivo study, mares at the mid-luteal phase (days 6-8 of estrous cycle) were injected i.m. with cloprostenol (250 µg) and blood samples were collected at fixed intervals until 72 h after treatment. Progesterone (P4) concentrations started decreasing 45 min after the injection and continued to decrease up to 24 h (P < 0.05). In turn, 13,14-dihydro-15-keto-PGF2α (PGFM) metabolite started to increase 4h after an injection and continued to increase up to 72 h (P < 0.05). PGF receptor (PTGFR) mRNA expression in the endometrium was significantly higher in the late luteal phase than in the early and regressed luteal phases (P < 0.05). In vitro, PGF2α significantly stimulated (P < 0.05) PGF2α production by endometrial tissues and endometrial epithelial and stromal cells and significantly increased (P < 0.05) the mRNA expression of prostaglandin-endoperoxide synthase-2 (PTGS2), an enzyme involved in PGF2α synthesis in endometrial cell. These findings strongly suggest the existence of an endometrial PGF2α auto-amplification system in mares.