Rate Of Embryonic Loss Research Articles

Effect of post-mating GnRH or TAK-683 (Kisspeptin analog) treatment on the reproductive performance and serum progesterone concentration in ewes during the non-breeding season

This study was designed to evaluate the effect of post-mating GnRH or TAK-683 (kisspeptin analog) treatment on serum progesterone (P4) concentrations and fertility parameters of ewes during the non-breeding season. One hundred and twenty multiparous, lactating Awassi ewes were used. For estrus synchronization, vaginal sponges containing 60 mg of medroxyprogesterone acetate (MAP) were inserted into the vagina and kept there for nine days. On the day of sponge removal (day 0), 500 IU of eCG and 75 µg of d-cloprostenol were given intramuscularly. Rams were introduced in the flock 24 h after sponge removal and kept with the ewes for 26 days, allowing a second mating period after synchronized estrus. On the fourth day after mating (day 6) the ewes mated by the rams between the 24th and 72nd hours (n = 105) were randomly divided into three groups. Control group (n = 36) received 1 ml of saline solution subcutaneously; GnRH group (n = 34) received 4 μg of buserelin acetate intramuscularly; TAK-683 group (n = 35) received 5 μg of TAK-683 acetate subcutaneously. Blood samples were taken to measure serum P4 concentrations (ng/ml) at sponge insertion and on the 6th, 9th, 12th, 15th, 19th, 35th, and 50th days after sponge removal. Pregnancy diagnosis was conducted via serum P4 concentration measurement on the 19th day and transrectal ultrasonography on days 35 and 50. The results showed that GnRH treatment (4.823 ± 0.238; 2.151 ± 0.272) increased (P<0.05) the mean P4 concentration (ng/ml) in pregnant and non-pregnant ewes during the first mating period compared to Control (3.708 ± 0.207; 1.515 ± 0.164), while TAK-683 treatment (4.457 ± 0.181; 1.967 ± 0.213) was not different between groups. GnRH (58.82 %) and TAK-683 (42.10 %) treatments increased the pregnancy rate in the second mating period in comparison to the Control group (17.39 %) (P<0.05). Cumulative pregnancy and fecundity rates were higher in GnRH (79.41 %; 120.58 %) and TAK-683 (68.57 %; 100.0 %) groups than in the Control group (17.39 %; 58.33 %) (P<0.05). There was no difference in late embryonic loss rate among the groups (P>0.05), whereas early fetal loss rate tended to be lower in the TAK-683 (0 %) and GnRH (0 %) groups than in the Control group (15.38 %) (P=0.07). The total pregnancy loss rate (from day 19 to day 50 post-mating) was lower in GnRH (0 %) and TAK-683 (0 %) groups relative to the Control group (21.42 %) (P<0.05). There were no statistical differences among the groups in terms of the first mating period pregnancy rate, twinning rate, and litter size (P>0.05). As a result, treatment with GnRH and TAK-683 on the fourth day after mating increased fecundity in lactating ewes outside the breeding season by preventing total pregnancy losses (from day 19 to day 50) and increasing second mating pregnancy rates.

Jiawei Shoutai Pill promotes decidualization by regulating the SGK1/ENaC pathway in recurrent spontaneous abortion

Ethnopharmacological relevanceJiawei Shoutai Pill (JWSTW) is a traditional herbal formula for recurrent spontaneous abortion (RSA). Although JWSTW significantly improves the clinical symptoms of RSA patients, its molecular mechanism remains unclear. Aim of studyThis study evaluated the expression and function of the serum/glucocorticoid regulated kinase 1/epithelial sodium channel (SGK1/ENaC) pathway and decidualization level in RSA patients and mice. It also investigated the therapeutic effects and potential mechanisms of JWSTW. Materials and methods30 early RSA patients and 30 normal pregnant women undergoing induced abortion during the same period were included in the study. Decidual tissues were collected, and HE staining, immunohistochemistry, Western blot, and RT-PCR were used to detect protein and mRNA expression levels of SGK1, ENaC-a, estrogen Rreceptor β (ERβ), and progesterone receptor (PR) in patients’ decidual tissues. Protein expression levels of prolactin receptor (PRLR) and insulin-like growth factor binding protein 1 (IGFBP-1) were also detected. A classical RSA mouse model was constructed, and the mice were randomly divided into four groups: normal, model, dydrogesterone (DQYT) (0.33 g/kg/d), and JWSTW (1.66 g/kg/d). The normal and model groups received the same volume of distilled water by gavage for 8 and 14 days after pregnancy. On the 14th day of pregnancy, the embryonic loss rate of each group, the number of offspring born to naturally delivered mice, and the protein or mRNA expression levels of key factors of the SGK1/ENaC pathway (SGK1, ENaC-a, ERβ, and PR), decidual proliferation marker (Ki67), mesenchymal-epithelial transition (E-cadherin and Vimentin), and decidualization markers (PRLR and IGFBP-1) in mouse decidual tissue on the eighth day of pregnancy were observed. ResultsThe decidual tissue structure of RSA patients was abnormal. Immunohistochemical analysis revealed significantly reduced positive expression of SGK1, ENaC-a, ERβ, and PR proteins in the decidual tissue of RSA patients (P < 0.001). Western blot and RT-PCR analyses demonstrated significantly decreased protein and mRNA expression of SGK1, ENaC-a, ERβ, and PR in the decidual tissue of RSA patients (all P < 0.05). Additionally, protein expression of PRLR and IGFBP-1 was significantly reduced (both P < 0.001). The RSA mouse model exhibited a significant increase in embryo loss rate and decreased litter size (both P < 0.001). Treatment with DQYT and JWSTW rescued the embryo loss rate and litter size to varying extents (all P < 0.05). The protein or mRNA expression levels of SGK1, ENaC-a, ERβ, PR, Ki67, E-cadherin, vimentin, PRLR, and IGFBP-1 in RSA mice were improved to different degrees after treatment with DQYT and JWSTW (all P < 0.05). ConclusionsAbnormal SGK1/ENaC signaling pathway regulation is closely associated with early endometrial damage in RSA patients. JWSTW promotes endometrial proliferation and mesenchymal-epithelial transition through the SGK1/ENaC signaling pathway, improving endometrial shedding. Consequently, JWSTW is a potential treatment for RSA.

A comparison of culture and cooling for the short term preservation of in vivo derived dromedary camel embryos of varying morphological quality

Despite recent advancements in the cryopreservation of dromedary camel embryos, widespread application of the technique is still limited by the need for specialised vitrification equipment and supplies. Temporary, liquid-phase embryo storage methods provide a useful tool for short-term preservation of camel embryos.In the current study, we compared the use of in vitro embryo culture with cold liquid storage in order to maintain both high- (Grade 1- Excellent and 2-Good) and low- (Grade 3- Moderate and 4-Poor) morphological grade Day-7 dromedary camel embryos in vitro for up to 3 days. Embryos were either cooled and placed in Hams-F10 medium supplemented with HEPES and 10% FBS and then kept at 4 °C; or placed in Hams-F10 supplemented with sodium bicarbonate and 10% FBS and then cultured in a humidified atmosphere of 6% CO2 at 37 °C before being assessed for viability at 24 h. In high-morphological grade embryos, both cold storage and culture supported 100% viability (maintenance of normal morphology) over this period (Cooled n = 22, Cultured n = 20). In low-morphological grade embryos, culture supported higher viability (16/18, 88.9%) than did cooling (4/18, 22.2%). We then evaluated the effect of up to 3 days of cold storage or culture on embryo morphological grade, diameter, and developmental competence following embryo transfer. High-grade embryos were divided between culture and cold storage; low-grade embryos were evaluated only after culture. Over 3 days of culture, both high- and low-grade embryos tended to either maintain or improve upon their initial morphological score (P < 0.05) and increased in diameter (P < 0.001). Embryos subjected to cooling tended to have reduced morphological scores by 48 h of storage and decreased in diameter by 72 h (P < 0.05). No significant influence of storage method (cooling vs. culture), duration (24–72 h), or embryo grade (high vs low) was observed on pregnancy establishment at Day-60 (22.2%–57.2% pregnancy rates for all treatments). Overall, rates of pregnancy establishment were similar for transferred cultured (n = 45) and cooled (n = 45) embryos (pregnancy rates at Day 18, 48% vs 51.1%; at Day 60, 37.7% vs 37.7%). Rates of embryonic loss also were similar (22.7% vs 26%).In conclusion, whilst similar rates of pregnancy and pregnancy loss were observed following the transfer of both cooled and cultured embryos held in vitro for up to 3 days, amongst the two methods, only embryo culture appears to provide a means of effectively preserving Day- 7 dromedary camel embryos with reduced morphological values in vitro. Considering these embryos appear to show poor tolerance to the cooling procedure and are unlikely candidates for vitrification, embryo culture may provide an effective method for deriving pregnancies from low-morphological grade embryos when immediate transfer is not possible on the day of flushing.

Embryo transfer technique affects pregnancy rates in recipient mares

Embryo transfer in horses started in the 1970′s and has evolved since then in several technical and biological areas that may influence the overall outcome. The transfer method evolved from surgical to non-surgical (NSET), with pregnancy rates for NSET related closely to operator skills. Recently, high pregnancy rates have been reported by non-specialized operators using the cervical forceps method (Cuervo Arango et al., Veterinary Record. 2018,183;10,1-5). The aim of this retrospective study was to compare pregnancy and early embryonic loss rates between conventional and cervical forceps transfer techniques (Wilsher and Allen, Equine Vet. Educ. 2004. 16; 1, 39-44), operated by two veterinarians with different degrees of experience. During the last 12 breeding seasons in a commercial equine embryo transfer program in Chile, 345 embryos were transferred by two operators with low / moderate (n=86) or high experience (n=259), using a conventional manual technique and 228 embryos were transferred by the same two operators (n=81) and (n=147) respectively, using a bovine cervix forceps/Polansky speculum technique. Embryo flushes were performed at 8 or 9 days after ovulation on 102 donor mares aged 4 to 24 years old and transferred to recipient mares (n=387) aged 3 to 12 years old. Ovulation synchrony ranged from -1 to +4 days when comparing the recipient to the donor. No medications were used during embryo transfer and procedures were done at the samefacilities. Data were analyzed by a binary logistic regression model in which pregnancy and embryo loss were treated as dependent variables, and the experience of the operator and the ET technique as independent variables. Pregnancy rates at first diagnosis (12 to 14 days post ovulation) and embryo loss rate until 60 days of gestation were compared between and within operator groups. Overall pregnancy rates were higher (P<0.001) by the cervical forceps / speculum embryo transfer technique (195/228; 85%) than with the conventional technique (236/345; 65%) in both groups combined. Early embryonic loss rate did not differ (P>0.1) between the groups (6.0 vs. 4.6%, respectively). For the conventional technique, there was a significant operator effect (P=0.015) on the pregnancy rates at the first diagnosis (57 vs. 72%); no operator effect (P>0.1) was observed when using cervical forceps (84 vs. 86 %). These results agree with the report of Cuervo-Arango et al. (2018). We also confirmed that simple bovine cervical forceps work as well as the equine specific forceps reported by Wilsher and Allen (2004). In conclusion, the cervical forceps technique yielded higher pregnancy rates, with no effect of the operator's experience compared to the conventional technique in which the skill and experience of the operator is crucial and needs time and losses to achieve.

NAD+ precursor concentrations are elevated in the follicular fluid of mares following oral supplementation of nicotinic acid

Niacin deficiency has recently been associated with congenital malformations in humans and embryonic death and resorption in mice (Shi et al. New England Journal of Medicine. 2017; 377(6):544-552). Mares suffer from high rates of early embryonic loss (Newcombe. Equine Veterinary Education. 12(2):88-101), the aetiology of which remains unknown. Increasing the levels of nicotinamide adenine dinucleotide (NAD+) through supplementing NAD+ precursors has resulted in an improvement in oocyte quality in mice (Bertoldo et al. Cell Rep. 2020; 30: 1670-1681). However, little is known regarding the requirement of niacin and NAD+ in reproductive processes in the mare. The aim of this study was to determine the effect of oral nicotinic acid (NA) supplements, a form of niacin, on the composition of nicotinamide adenine dinucleotide (NAD+) metabolites in the blood and follicular fluid of mares. Vehicle alone or NA (3 g per os) was administered to 7 mares over a minimum of 3 consecutive days during the oestrous cycle. Blood samples were collected immediately prior tosupplemental feeding and at follicular fluid aspiration. Follicular fluid was collected from the dominant follicle through transvaginal ultrasound guided aspiration. Blood and follicular fluid samples were processed and analysed by mass spectrometry. The concentration of nicotinamide mononucleotide (NMN) in the follicular fluid of NA-fed mares was greater than that in the corresponding plasma (15.0 ± 6.5 vs. 3.3 ± 0.9 ng/µL; p < 0.05) and in the follicular fluid of vehicle-fed mares (1.5 ± 0.5 ng/µL; p < 0.001). Meanwhile, the concentration of NA, nicotinamide (NAM) and nicotinuric acid (NUR) tended to be greater in the follicular fluid of NA supplemented mares (1043.9 ± 953.5, 1641.7 ± 256.1 and 5.5 ± 5.1 ng/µL) than in the corresponding plasma (70.8 ± 15.4, 875.6 ± 361.1 and 0.6 ± 0.2, respectively; p < 0.1). The results show that NA supplementation increased the bioavailability of NAD+ precursors in the follicular fluid of the dominant follicle, which is proposed to better promote the maturation of good quality oocytes, especially in older mares.

Early embryonic death in equines and camelids.

This paper includes the study of early embryonic death (EED), predisposing factors of EED and treatment. EED refers to the fetal mortality which varies in mare and camelids but most probably not later than 50 days of gestation. This duration may be divided into very early mortality, early mortality and late embryonic mortality. This also varies in mare and camelids. There are different embryonic, maternal, environmental/external, and infectious and noninfectious factors which lead to early embryonic loss. Diagnosis is very difficult as in most of the cases resorption of fetus occurs but it is done by the use of ultrasound. Unfortunately, there is no treatment to avoid early embryonic mortality. However, new reproductive technologies have increased the service rate in a herd, and efforts are still being made to determine the rate and frequency of camel embryonic loss.

Adiponectin Reduces Embryonic Loss Rate and Ameliorates Trophoblast Apoptosis in Early Pregnancy of Mice with Polycystic Ovary Syndrome by Affecting the AMPK/PI3K/Akt/FoxO3a Signaling Pathway

Reports in recent years have suggested that adiponectin (APN) improves insulin resistance and inhibits apoptosis by activating the AMP-activated protein kinase (AMPK) pathway and the PI3K/Akt signaling pathway after binding to its receptor. This study aims to explore the mechanism by which APN reduces embryo loss rate and trophoblast apoptosis in early pregnancy of mice with polycystic ovary syndrome (PCOS). PCOS mice were subcutaneously injected with APN (10 μg mg kg−1 day−1) on 11 consecutive days from the 3rd day of pregnancy onwards to observe the change of the embryo loss rate of PCOS mice induced by APN. Quantitative real-time PCR and Western blot were used to determine the relative expressions of mRNA and the proteins AMPK, PI3K, and Akt in mouse uterine tissue. At the same time, primary cultured mouse villous trophoblast cells were used to further explore the underlying mechanisms in vitro. APN significantly reduces the pregnancy loss rate of PCOS mice. At the same time, APN increases phosphorylation and mRNA expression levels of AMPK, PI3K, and Akt in PCOS mouse uterine tissue. In addition, trophoblast cells of model mice were treated with APN and inhibitors, and APN was found to reduce trophoblast cell apoptosis by affecting the phosphorylation levels of AMPK, PI3K, Akt, and FoxO3a proteins. APN reduces the embryo loss rate and ameliorates trophoblast apoptosis in PCOS mice by affecting the AMPK/PI3K/AKT/FoxO3a signaling pathway.

170 Embryonic loss and pregnancy rate in response to resynchronisation using oestradiol benzoate or injectable progesterone at 14 days after timed AI in Bos taurus×Bos indicus beef heifers

The effect of the exposure of pregnant cattle to oestradiol benzoate (EB) or injectable long-acting progesterone (iP4), as well as their use for early resynchronisation of non-pregnant (NP) females within the second week after timed AI (TAI), is yet to be assessed. This study aimed to assess the pregnancy rate and embryonic loss of cross-bred Bos taurus×Bos indicus beef heifers in response to an EB- or iP4-based resynchronisation strategy implemented 14 days after the first TAI. A total of 677 cyclic heifers (Braford and Brangus; 24 months old) were subjected to an EB/P4 synchronisation protocol for the first TAI (Day 0=TAI). On Day 14, all heifers received a new P4 intravaginal device (1g, Sincrogest, Ourofino Saúde Animal) and were randomly assigned to two groups: EB (1mg intramuscularly, Sincrodiol; n=339) or iP4 (75 mg intramuscularly, Sincrogest Injetável; n=338). On Day 22, P4 devices were removed and heifers underwent colour Doppler ultrasonography evaluation. Non-pregnant females were identified by the assessment of structural regression of the corpus luteum, as previously reported by our group. On Day 22, NP heifers received 1mg of oestradiol cipionate (SincroCP), 250µg of sodium cloprostenol (Sincrocio), and 200IU of equine chorionic gonadotrophin (SincroeCG), and they were inseminated on Day 24. On Day 22, the diameter of the preovulatory follicle (POF) was also recorded. Pregnancy was diagnosed via B-mode ultrasonography on Days 35-40 and 54. Statistical analyses were performed using SAS software (ver. 9.2, SAS Institute Inc.). The MIXED procedure was used to evaluate the POF diameter, and the GLIMMIX procedure was used to develop a multivariate model including treatment, breed, age, farm (1-7), and possible interactions of treatment with farm, breed, and age to evaluate the binomial data (pregnancy rates and embryonic loss). In NP heifers, the diameter of the POF on Day 22 did not differ (P&amp;gt;0.05) between the EB (9.77±0.25mm) and iP4 (9.92±0.22 mm) groups. Exposure to EB or iP4 on Day 14 post-TAI did not influence pregnancy rates as assessed on Day 22 (56.3% (191/339) vs. 60.1% (203/338)) and on Days 35-40 (49.6% (168/339) vs. 53.3% (180/338)). In addition, embryonic loss between Days 22 and 35-40 was not different between the EB (12%, 23/191) and iP4 (11.3%, 23/203) groups. On the other hand, pregnancy rates in heifers submitted to the second TAI were greater (P=0.01) in the EB group (45.95%, 68/148) than in the iP4 group (31.85%, 43/135). In conclusion, improved pregnancy rates can be achieved using 1mg of EB associated with a P4 intravaginal device on Day 14 of the resynchronisation protocol without affecting the first TAI pregnancy rates and embryonic loss. However, further investigation is warranted to assess the lower pregnancy rates obtained in response to the association of iP4 and P4 device. This research was supported by FAPESP (2015/10606-9; 2019/15514-6), Graduate Studies Support Program (PAPG-UNIPAMPA), and Ourofino Saúde Animal.

Protein expression profiles in Meishan and Duroc sows during mid-gestation reveal differences affecting uterine capacity, endometrial receptivity, and the maternal\u2013fetal Interface

BackgroundEmbryonic mortality is a major concern in the commercial swine industry and primarily occurs early in gestation, but also during mid-gestation (~ days 50–70). Previous reports demonstrated that the embryonic loss rate was significant lower in Meishan than in commercial breeds (including Duroc). Most studies have focused on embryonic mortality in early gestation, but little is known about embryonic loss during mid-gestation.ResultsIn this study, protein expression patterns in endometrial tissue from Meishan and Duroc sows were examined during mid-gestation. A total of 2170 proteins were identified in both breeds. After statistical analysis, 70 and 114 differentially expressed proteins (DEPs) were identified in Meishan and Duroc sows, respectively. Between Meishan and Duroc sows, 114 DEPs were detected at day 49, and 98 DEPs were detected at day 72. Functional enrichment analysis revealed differences in protein expression patterns in the two breeds. Around half of DEPs were more highly expressed in Duroc at day 49 (DUD49), relative to DUD72 and Meishan at day 49 (MSD49). Many DEPs appear to be involved in metabolic process such as arginine metabolism. Our results suggest that the differences in expression affect uterine capacity, endometrial matrix remodeling, and maternal-embryo cross-talk, and may be major factors influencing the differences in embryonic loss between Meishan and Duroc sows during mid-gestation.ConclusionsOur data showed differential protein expression pattern in endometrium between Meishan and Duroc sows and provides insight into the development process of endometrium. These findings could help us further uncover the molecular mechanism involved in prolificacy.

Impact of days in milk at the initiation of ovulation synchronization protocols on the efficiency of first AI in multiparous Holstein cows

The objective of this study was to evaluate the impact of days in milk (DIM) at the initiation of ovulation synchronization protocols [spontaneous estrus (n=899), Presynch (n=1277), and CIDRsynch (n=978)] on fertility indices of the first AI in multiparous Holstein cows. The days from calving to the initiation of timed-AI (TAI) were categorized into: <50days (D1), 51–65days (D2) and 66–80days (D3). Pregnancy rate (P/AI at day 75) of cows at D2 period was significantly greater than that in the D1 period [OR (odds ratio)=1.26; P=0.050). Additionally, embryonic loss rate was significantly reduced in D2 and D3 groups compared to the reference group (OR=0.68 and 0.63; P=0.012 and 0.001, respectively). Conception (P/AI at day 28) and pregnancy rates in the Presynch group were significantly increased from 26.5 and 19.3% at D1 period to 35.9 and 28.9% at D2 period, respectively (OR=1.59 and 1.72; P=0.009 and 0.001, respectively). In contrast, conception and pregnancy rates using the CIDRsynch protocol did not differ at the different DIM at the initiation of TAI. The results of the Cox regression model reported significant associations for parity and season of calving with the hazard of embryonic loss (P=0.029 and 0.036, respectively). The current results suggest that using the Presynch regimen may achieve satisfactory conception and pregnancy rates at 51–65days in milk; however, the use of CIDRsynch regimen may provide a stable and homogenous conception and pregnancy rates at the different DIM at initiation of TAI.

CA125, progesterone & β-hCG in prediction of first trimester abortion

IntroductionPregnancy loss is a common medical problem in reproductive-age as more than fifty percent of human pregnancies are aborted before term. The majority are unrecognized occurring before or with the expected next menses. About 10–12 percent of all clinically diagnosed pregnancies are lost as first-trimester or early second trimester. The rate of fetal death after 14 weeks’ gestation is much lower than the rate of pre-embryonic and embryonic loss. CA125 is a member of the mucin family glycoproteins. CA125 has found application as a tumor marker or biomarker that its level may be increased in the serum of some patients with specific types of cancers. Some studies detected that the abortion risk is increased in pregnant women with higher CA125 levels. Progesterone belongs to the C21 group of progestogen. Its main source in humans is the corpus luteum.Human chorionic gonadotropin (HCG) is a glycoprotein produced by syncytiotrophoblast. Aim of the workThe aim of this study was to determine the effectiveness of measuring maternal serum β-HCG, progesterone, CA125 in prediction of first trimester abortion. PatientsThe study included 90 pregnant women attending the ANC clinic in El-Shatby Maternity University Hospital. Patients were divided equally into two groups: Group I: 45 women with threatened abortion, subdivided into 2 subgroups: Subgroup A – Cases ended in abortion; Subgroup B – Cases continued as normal pregnancies. Group II: 45 pregnant women with normal pregnancy and were further subdivided into two subgroups: Subgroup C – Cases ended in abortion; Subgroup D – Cases continued as normal pregnancies. Exclusion criteria(1) Multiple pregnancies; (2) Anembryonic pregnancy; (3) Pregnant women with prior treatment with progesterone; (4) History of endometriosis; (5) Fibromyoma with pregnancy. MethodsAfter clinical and sonographic examination, 3mL venous blood have been taken once for estimation of serum level of β-hCG, progesterone and CA125 by quantitative ELISA. ResultsThis is a case–control study. Out of the 90 pregnancies, 15 cases (16.6%) had aborted during follow-up, 9 cases (60%) of them had history of threatened abortion while 6 cases (40%) had no history of threatened abortion. Regarding Serum Progesterone level between studied groups, the calculated p value was <0.001. For Serum β HCG, the calculated p value was <0.001. In Serum CA125 the calculated p value was <0.001.

Altered expression of hormone receptor, integrin β3 and pinopode in the endometrium of luteal phase defect women

Objective: To invistigate estrogen receptor (ER), progesterone receptor (PR), integrin β3, and pinopode expression in luteal phase deficiency (LPD) women.Methods: There were 52 nulligravidas consecutive infertile patients undergoing a routine assistant reproduction consultation included in this study. An endometrial biopsy sample was randomly obtained between days 4 and 10 of the luteal phase. Endometrial morphology was examined with scanning electron microscopy. Expressions of ER, PR, integrin β3 were determined in the endometrium of LPD patients with immunohistochemistry.Results: The incidence of LPD was 15.3% (8/52) in this study. On day luteinizing hormone (LH) surge + 9∼LH + 10, noted regressing pinopodes resembling a day LH + 7∼LH + 8 in the endometrium of the control group. The expressions of ER and PR in glandular epithelium were significantly increased in endometrium of LPD than that in the control group (p < 0.05). In contrast, there was a statistically significant decrease expression of the integrin β3 in women from the group of LPD (p < 0.05).Conclusion: The altered expression of ER and PR may be associated with the expression variation of integrin and pinopode formation in endometrium of LPD women. This alteration may imply the association of low rates of cycle fecundity and high rates of embryonic loss in LPD women.

Exposure of bovine oocytes and embryos to elevated non-esterified fatty acid concentrations: integration of epigenetic and transcriptomic signatures in resultant blastocysts.

BackgroundMetabolic stress associated with negative energy balance in high producing dairy cattle and obesity in women is a risk factor for decreased fertility. Non-esterified fatty acids (NEFA) are involved in this pathogenesis as they jeopardize oocyte and embryo development. Growing evidence indicates that maternal metabolic disorders can disturb epigenetic programming, such as DNA methylation, in the offspring. Oocyte maturation and early embryo development coincide with methylation changes and both are sensitive to adverse environments. Therefore, we investigated whether elevated NEFA concentrations affect establishment and maintenance of DNA methylation in oocytes and embryos, subsequently altering transcriptomic profiles and developmental competence of resultant blastocysts.ResultsBovine oocytes and embryos were exposed to different NEFA concentrations in separate experiments. In the first experiment, oocytes were matured in vitro for 24 h in medium containing: 1) physiological (“BASAL”) concentrations of oleic (OA), palmitic (PA) and stearic (SA) acid or 2) pathophysiological (“HIGH COMBI”) concentrations of OA, PA and SA. In the second experiment, zygotes were cultivated in vitro for 6.5 days under BASAL or HIGH COMBI conditions. Developmental competence was evaluated by assessing cleavage and blastocyst rate. Overall gene expression and DNA methylation of resultant blastocysts were analyzed using microarray. DNA methylation data were re-evaluated by pyrosequencing. HIGH COMBI-exposed oocytes and embryos displayed a lower competence to develop into blastocysts compared to BASAL-exposed counterparts (19.3% compared to 23.2% and 18.2% compared to 25.3%, respectively) (P < 0.05). HIGH COMBI-exposed oocytes and embryos resulted in blastocysts with altered DNA methylation and transcriptomic fingerprints, compared to BASAL-exposed counterparts. Differences in gene expression and methylation were more pronounced after exposure during culture compared to maturation suggesting that zygotes are more susceptible to adverse environments. Main gene networks affected were related to lipid and carbohydrate metabolism, cell death, immune response and metabolic disorders.ConclusionsOverall, high variation in methylation between blastocysts made it difficult to draw conclusions concerning methylation of individual genes, although a clear overview of affected pathways was obtained. This may offer clues regarding the high rate of embryonic loss and metabolic diseases during later life observed in offspring from mothers displaying lipolytic disorders.

The efficiency of new CIDR and once-used CIDR to synchronize ovulation in primiparous and multiparous Holstein cows

The objective of this study was to elucidate the efficiency of Ovsynch protocol, new (N-CIDRsynch) and once-used (U-CIDRsynch) controlled internal drug-release (CIDR) devices to synchronize the ovulation time of primiparous and multiparous Holstein cows. The current study was performed on a total of 2207 cows using three different synchronized groups. Conception [pregnancy per AI (P/AI) at day 28] and pregnancy (P/AI at day 75) rates using the N-CIDRsynch and U-CIDRsynch regimens (35.9% and 29.6%; 29.2% and 25.1%, respectively) were significantly greater than those in the Ovsynch group [Odds ratio (OR)=1.88 and 1.44; 1.92 and 1.52, respectively]. Embryonic loss rate did not differ significantly (P>0.05) among the Ovsynch, N-CIDRsynch and U-CIDRsynch regimens. In primiparous cows, no significant differences were found in conception and pregnancy rates between the U-CIDRsynch and N-CIDRsynch groups (OR=0.95 and 1.12; p=0.693 and 0.451, respectively). In multiparous cows, conception and pregnancy rates in the N-CIDRsynch group were significantly greater than those observed in the U-CIDRsynch group (OR=0.66 and 0.71; p=0.016 and 0.027, respectively) and embryonic loss rate in multiparous cows was lower for the U-CIDRsynch treatment than for the N-CIDRsynch treated cows (COR=0.58; p=0.048). The present study indicates that the use of a progesterone device in a synchronization protocol may improve fertility in dairy cows compared to the Ovsynch treatment. The use of U-CIDRsynch regimen may achieve satisfactory fertility results compared to those reported with N-CIDRsynch in primiparous Holstein cows.

Impact of parity on the efficiency of ovulation synchronization protocols in Holstein cows

The objective of this study was to elucidate the impact of parity on the efficiency of three different protocols to synchronize time of ovulation in Holstein cows. All cows enrolled in this trial were categorized into primiparous and multiparous (M1 = 2–3 and M2 ≥3 parities). Conception (P/AI at Day 28) and pregnancy (P/AI at Day 75) rates in the Presynch and CIDRsynch (31.7% and 35.5%; 26.3% and 28.4%, respectively) groups were significantly greater than that in spontaneous estrus (SE; 24.7 and 20.4%, respectively) group (P = 0.048 and 0.024; 0.041 and 0.011, respectively); however, no significant differences were detected between the SE and Ovsynch group for conception, pregnancy, and embryonic loss rates. Conception and pregnancy rates in the Presynch group decreased from 37.8% and 33.5%, respectively in primiparous cows to 29.6% and 23.1%, respectively in M1 cows (P = 0.022 and 0.007, respectively). However, conception and pregnancy rates using the CIDRsynch regimen were increased from 31.1% and 23.8% in primiparous cows to 41.4% and 34.7% in M1 cows (P = 0.017 and 0.008, respectively), and 40.9% and 33.6% in M2 cows (P = 0.021 and 0.019, respectively). Embryonic loss rate using the CIDRsynch protocol was decreased from 22.9% in primiparous cows to 15.1% and 18.2% in M1 and M2 cows (P = 0.013 and 0.130, respectively). On the contrary, embryonic loss rate using the Ovsynch protocol was increased from 11.5% in primiparous cows to 22.1% and 21.8% in M1 and M2 cows (P = 0.001 and 0.003, respectively). The Cox proportional-hazards model of embryonic loss showed significant associations for parity and season of calving with the hazard of embryonic loss (P = 0.001 and 0.016, respectively). Multiparous cows (M1 and M2) had a higher risk of embryonic loss than primiparous cows (Hazard ratio = 1.32 and 1.89, respectively). Our results indicate that use of the CIDRsynch regimen may achieve satisfactory conception and pregnancy rates in multiparous Holstein cows. However, synchronizing time of ovulation in primiparous cows with use of the Presynch treatment increases the fertility indices.

Incompatibility between Nuclear and Mitochondrial Genomes Contributes to an Interspecies Reproductive Barrier

Vertebrate cells carry two different genomes, nuclear (nDNA) and mitochondrial (mtDNA), both encoding proteins involved in oxidative phosphorylation. Because of the extensive interactions, adaptive coevolution of the two genomes must occur to ensure normal mitochondrial function. To investigate whether incompatibilities between these two genomes could contribute to interspecies reproductive barriers, weperformed reciprocal mtDNA replacement (MR) in zygotes between widely divergent Mus m. domesticus (B6) and conplastic Mus m. musculus (PWD) mice. Transfer of MR1 cybrid embryos (B6nDNA-PWDmtDNA) supported normal development of F1 offspring with reduced male fertility but unaffected reproductive fitness in females. Furthermore, donor PWD mtDNA was faithfully transmitted through the germline into F2 and F3 generations. Incontrast, reciprocal MR2 (PWDnDNA-B6mtDNA) produced high embryonic loss and stillborn rates, suggesting an association between mitochondrial function and infertility. These results strongly suggest that functional incompatibility between nuclear and mitochondrial genomes contributes to interspecies reproductive isolation in mammals.

Impact of thermal stress on the efficiency of ovulation synchronization protocols in Holstein cows

The objective of this study was to evaluate the impact of thermal stress on the efficiency of three different protocols to synchronize time of ovulation in purebred Holstein cows under subtropical Egyptian conditions. The influence of temperature humidity index (THI) on the conception, pregnancy, embryonic loss and early abortion rates were investigated. Conception and pregnancy rates using the CIDRsynch and Presynch (37.5% and 33.9%; 29.5 and 29. 6%, respectively) were significantly greater than that for cows expressing spontaneous estrus (SE) and with use of the Ovsynch (28.5% and 24.3%; 21.6% and 24.6%, respectively) treatment regimen. Conception and pregnancy rates using the Ovsynch protocol were significantly decreased from 31.6% and 26.3% at the lesser THI to 11.5% and 9.9%, respectively than at the greater THI [crude odds ratio (COR)=0.28 and 0.32; P=0.001 and 0.004, respectively]. However, conception (P/AI at 28 days) and pregnancy (P/AI at 75 days) rates using the Presynch protocol were significantly reduced at either the lesser or greater THI (COR=0.47 and 0.42; 0.48 and 0.34, respectively). Embryonic loss rate with the Presynch group was significantly increased from 11.5% at the lesser THI to 22.2% at the greater THI (COR=2.28; P=0.039). In contrast, conception, pregnancy and embryonic loss rates did not differ significantly (P>0.05) with the CIDRsynch protocol at the different THI. Results from the present study indicate that use of the CIDRsynch protocol may provide consistent and satisfactory conception and pregnancy rates in Holstein cows under subtropical environmental conditions.

151 REPRODUCTIVE PARAMETERS OF MINI-HORSE MARES

The Mini-horse is a new equine breed that is on the rise in Brazil. These are animals with a maximum adult height of 98 cm for females and 93 cm for males, with a well-proportioned body. Those animals are easily handled by children and require small spaces with low maintenance costs. The Mini-horse is under constant genetic selection that is aimed mainly at decreasing its height. For this reason, mares of low stature are required, justifying their use in assisted reproduction programs. Reproductive data of these animals are not available in the literature. Only data of full-size mares are referenced, not taking into account the particularities of this breed. Therefore, the aim of this study was to describe one technique of embryo transfer (ET) for Mini-horse mares. Reproductive rates obtained during the 2012/2013 Brazilian breeding season are presented. Thirteen Mini-horse donor mares were monitored daily by transrectal ultrasonography and ovulation induction was performed with injection of deslorelin acetate or hCG, along with natural breeding using the same stallion for all mares. Eight days after detection of ovulation, flushing for embryo collection was performed. The recipient mares were also monitored daily to detect ovulation and those selected ovulated in the range of up to four days after the donor mare. In order to prevent cervical injuries, the large catheter traditionally employed in uterine washing of large mares was replaced by a Foley catheter (24 Fr in diameter). A range of 50 to 250 mL of Ringer's lactate solution was used to fill the uterus up to 5 times or until the embryo was observed in the collector filter. After the embryo collection, the donor mare received a dose of luteinizing drug (sodic cloprostenol – Sincrocio®, Ouro Fino, Brazil). The recipient mare was examined for a corpus luteum, absence of uterine fluid or endometrial oedema, and good cervical and uterine tone. Recipients were also selected by height (similar to donors) so that the newborn foal could reach the recipient mare's udder to nurse. For non-surgical transfer, the embryos were aspirated by an artificial insemination pipette with ~0.5 mL of holding medium interspersed with small columns of air and then inserted into the uterine body of the recipient mares. Daily follicular growth was 2.2 ± 1.0 mm and the size of follicles at ovulation induction was 31.6 ± 2.8 mm. In 57 embryo collections performed, 46 embryos were obtained and transferred into the uteri of recipient mares, resulting in 35 pregnancies. The embryo recovery and pregnancy rate were 80.7% and 76.1% respectively. The relationship between pregnancy and number of embryo flushes performed was 61.4%. The time between two consecutive flushes was 20 ± 4.5 days. The rate of embryonic loss between 20 and 60 days was 20%. The easy handling of the animals and the high rates of ET program success were promising. However, information regarding full-size mares should be used with caution, respecting the specific characteristics of this breed.

201 USING THE TRANSCRIPTOME SIGNATURE TO PREDICT PREGNANCY SUCCESS IN BEEF COWS AT 6 DAYS AFTER ARTIFICIAL INSEMINATION

Pregnancy success is critical to the profitability of cattle operations. Attempts to reduce high rates of early embryonic loss mainly focus on the critical phase of embryo recognition by maternal tissue. However, the molecular events driving the uterine tissue toward a favourable stage, facilitating the maternal receptivity, are poorly understood. This study aimed to characterise the endometrial transcriptome profiles of pregnant versus nonpregnant beef cows during early pregnancy and attempted to define a potential set of marker genes that can be valuable for predicting pregnancy outcome. Therefore, pluriparous, cyclic Nellore (Bos indicus) cows were synchronized (n = 51) and artificially inseminated (n = 36) at detected oestrus using semen from a single high-fertility bull. Six days after AI (Day 6), jugular blood samples and an endometrial biopsy from the uterine horn contralateral to the ovary containing the CL were collected. Pregnancy diagnosis was performed by ultrasonography on Days 22 and 30. Based on pregnancy outcome, samples were retrospectively allocated to the following groups: pregnant (P; n = 6) and nonpregnant (NP; n = 5). Both groups had similar plasma progesterone concentrations on Day 6 (less than 1 ng mL–1 between lowest and greatest concentrations). Endometrial biopsies were submitted to RNA-Seq analysis in an Illumina single flow cell line (Illumina Inc., San Diego, CA). The 272 685 768 million filtered reads were mapped to the Bos taurus UMD3.1 reference genome and 14 654 genes were effectively analysed for differential expression between groups. Transcriptome data showed that 216 genes are differently expressed when comparing P v. NP endometrial tissue (adjusted P &lt; 0.1). More specifically, 36 genes showed a significantly up-regulated expression for pregnant cows and 180 are up-regulated for non-pregant cows. Functional enrichment and pathway analyses revealed enriched expression of genes associated with extracellular matrix remodelling in NP cows and nucleotide binding, microsome, and vesicular fraction in P cows. From the 40 top-ranked genes, six that were down- and three that were up-regulated in pregnant cows were further analysed by qRT–PCR in an additional 26 cows. Subsequent quantitative expression data were evaluated using multivariate statistics. Both principal component analysis (PCA; R2 = 0.82 and Q2 = 0.40) and orthogonal projections to latent structures analysis (OPLS), using pregnancy as the dependent variable (R2Y = 0.95 and Q2 = 0.86), efficiently separated P from NP animals. In conclusion, this study characterizes a unique set of genes, expressed in the endometrium as early as 6 days after AI, that indicate a receptive state leading to pregnancy success. Furthermore, expression of such genes can be used as potential markers to efficiently predict pregnancy success.

Evidence of placental endoplasmic reticulum stress in the pathophysiology of gestational diabetes mellitus

Evidence of placental endoplasmic reticulum stress in the pathophysiology of gestational diabetes mellitus