There are still major differences between in vitro production (IVP)-derived and in vivo-derived bovine blastocysts. Therefore, intrafollicular oocyte transfer (IFOT) was used in the present study to allow early embryonic development within the physiological oviductal environment, in order to avoid subsequent harmful effects of the in vitro culture environment. Using modified ovum pickup equipment, in vitro-matured oocytes were transferred into the preovulatory follicle of synchronized heifers (follicular recipients), enabling subsequent ovulation, in vivo fertilization, and in vivo development. When 1646 in vitro-matured oocytes were transferred to 28 follicular recipients, a total of 583 embryos (35.2%) were recovered in excess after uterine flushing at Day 7. Although numbers of generated extra embryos were highly variable, preovulatory follicles with a diameter of 13-14 mm delivered significantly (P < 0.05) larger amounts of extra embryos (34.3 vs. 7.3), as well as extra morulae and blastocysts (8.3 vs. 0.8), compared with follicles with a diameter of 9-10 mm. Nevertheless, the developmental rate to the blastocyst stage was lower in IFOT compared with in vitro-derived control (Vitro) embryos at Day 7 (8.0% vs. 36.5%). Likewise, cumulative developmental rates to the morula or blastocyst stage until Day 7 were lower in IFOT-derived embryos when related to the number of transferred (8.4% vs. 51.7%) or flushed (22.8% vs. 51.7%) embryos. Of the latter, IFOT-derived embryos yielded significantly lower cleavage rates compared with the Vitro controls (63.2% vs. 88.8%), and developmental rate to the morula or blastocyst stage were lower even when related to the proportion of cleaved embryos (36.8% vs. 58.2%). In contrast, lipid content and cryotolerance did not differ between IFOT and fully IVP embryos; but IFOT-derived embryos showed significantly lower lipid content (P < 0.05) and significantly higher cryotolerance compared with IVP-derived embryos cultured in CR1aa medium supplemented with estrus cow serum (ECS), but not when cultured in SOFaa medium supplemented with fatty acid-free BSA (BSA-FFA). Finally, transfer of 19 frozen-thawed IFOT-derived blastocysts to synchronized recipients (uterine recipients) resulted in pregnancy rates comparable with those obtained after transfer of fully in vivo-derived embryos or IVP-derived embryos cultured in SOFaa + BSA-FFA, whereas pregnancy rate following transfer of IVP-derived blastocysts was significantly lower when they were cultured in CR1aa + ECS (42.1% vs. 13.8%). All in all, seven pregnancies presumed to be IFOT derived went to term, and microsatellite analysis confirmed that five calves were indeed derived from IFOT. To our knowledge, these are the first calves born after IFOT in cattle. Interestingly, the average birth weight of IFOT-derived calves was lower than that of IVP-derived calves, even when embryos were cultured in SOFaa + BSA-FFA, indicating that the environment during early embryo development might cause fetal overgrowth. Taken together, for the first ti...

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This study was conducted to determine if the main components of the somatotropic axis change during the early phase of pregnancy in the maternal blood system and whether differences exist on day 18 after pregnancy recognition by the maternal organism. Blood samples of pregnant heifers (Holstein Friesian; n = 10 after embryo transfer) were obtained on the day of ovulation (day 0), as well as on days 7, 14, 16 and 18 and during pregnant, non-pregnant and negative control cycles. The oncentrations of progesterone (P4), oestrogen, growth hormone (GH), insulin-like growth factor-1 and -2 (IGF1, -2) and IGF-binding protein-2, -3 and -4 (IGFBP2, -3, -4) were measured. The mRNA expressions of growth hormone receptor 1A, IGF1, IGF2, IGFBP2, IGFBP3 and IGFBP4 were detected using RT-qPCR in liver biopsy specimens (day 18). In all groups, total serum IGF1 decreased from day 0 to 16. Notably, IGFBP4 maternal blood concentrations were lower during pregnancy than during non-pregnant cycles and synchronized control cycles. It can be speculated that the lower IGFBP4 in maternal blood may result in an increase of free IGF1 for local action. Further studies regarding IGFBP4 concentration and healthy early pregnancy are warranted.

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Summary: This study aims to review current knowledge on equine fetal sexing with special reference to the authors field experience using the non-invasive transabdominal approach. Early gender determination performed transrectally by identifying the genital tubercle is specific with a small time frame; it is best performed from day 59 until 68. The examiner has to be experienced to scan the right planes for finding the genital tubercle in either sex. Advanced sex determination can be performed transrectally from 90 up to 140 days of gestation, afterwards reaching the fetus hindquarters transrectally is almost impossible and the transabdominal alternative has to be chosen. External genitalia and gonads have to be found and good knowledge of fetal anatomy is required. For more advanced gestation, the importance of representing the gonads and especially outlining their B-Mode echotexture and vascularisation by Doppler sonography increases. For the transrectal approach for gender determination, mares have to be calm and relaxed, so the use of sedation and smooth muscle relaxants might be necessary and the risk of perforating the rectum has to be taken into consideration. Transabdominal examination shows high acceptance in mares and bears a low risk, sedation is not necessary and movement of mare and fetus can be compensated easily by moving the ultrasound probe along the abdomen. Equine fetal sex determination is a tool in equine reproductive medicine which becomes more important and interesting for breeders in order to plan the mare s future breeding prospects. With enough time, patience, practice and good equipment it can be performed by practitioners easily as an additional service for clients. The method to be selected depends on the stage of gestation and on fetal presentation. If movements of the fetus prevent the accomplishment of sex determination, the examination should be repeated later. Advantages of gender determination during advanced gestation by transabdominal ultrasound other than economical reasons are the accomplishment of a check up on pregnancy and fetal well-being, great acceptance by the clients and a good way to perform gestation control in ponies and small horses, although this may take more time for preparation and examination.Keywords: mare / fetal gender determination / transrectal ultrasonography / transabdominal sonography / reproduction Citation: Tönissen A., Martinsson G., Otzen H., Schürmann K., Schütze S., Ertmer F., Kassens A., Sielhorst J., Brehm R., Sieme H. (2015) To perform fetal gender determination in the mare by ultrasound during early and advanced gestation. Pferdeheilkunde 31,[153][154][155][156][157][158]

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Progesterone (P4) is important for the developmental competence of cumulus–oocyte complexes (COC) used for in vitro maturation (IVM). In a recent study, we were able to show that circulating P4 concentrations had an effect on the molecular quality of COC recovered during repeated ovum pickup sessions, which might affect further development. (Schlüter et al. 2013 Reprod. Fertil. Dev. 25, 250). The aim of the present study was to determine the influence of different P4 concentrations during IVM on the molecular quality of bovine COC. The COC were collected from slaughterhouse ovaries and cultured as described recently (Stinshoff et al. 2011 Theriogenology 76, 1433–1441). The IVM medium was supplemented with 0, 50, 150, 300, and 450 ng mL–1 P4. Ethanol served as vehicle control. After IVF with a bull of proven fertility, the presumptive zygotes were cultured in SOF under 5% oxygen (Stinshoff et al. 2011). Cleavage and developmental rates were determined at Day 3 and Day 7/8 (Day 0: IVF). Additionally, maturation rates were assessed. For mRNA analysis, immature and matured denuded COC (n = 5) were individually frozen at –80°C to analyse the relative transcript abundance using RT-qPCR. The transcripts studied play important roles during oocyte development [growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), glucose transporter 1 (SCL2A1), hypoxia inducible factor 2α (HIF2α), nuclear progesterone receptor (PGR), progestin and adipoQ receptor 5 (PAQR5), progesterone receptor membrane component 1 and 2 (PGRMC1, PGRMC2)]. Data were tested using ANOVA followed by multiple pairwise comparisons using Tukey's test. A P-value of <0.05 was considered significant. The percentage of oocytes that reached the MII stage was similar in oocytes from all treatment groups (82.2–88.1%). Despite similar cleavage rates across all groups, the developmental rates did show significant differences. More embryos developed to the blastocyst stage stemming from oocytes cultured without any supplement or cultured only with alcohol compared to oocytes stemming from the group cultured with less than 50 ng mL–1 P4 (25.4 ± 5.7 and 27.9 ± 7.2 v. 15.8 ± 2.6). The relative abundance of SCL2A1, BMP15, and PGRMC1transcripts in single oocytes did not show differences related to the supplementation of the IVM medium, whereas GDF9, HIF2α, and PAQR5 mRNA was reduced in oocytes of all groups compared with immature ones. The PGR and PGRMC2 transcripts were increased in matured oocytes of the control group and the vehicle control group (PGRMC2). In summary, supplementation of the IVM medium with different P4 concentrations had an effect on the molecular quality of oocytes after IVM, which might affect further development. The financial support of the FBF (Förderverein Biotechnologieforschung) e.V. is gratefully acknowledged.

Summary: Sonographic parameters were studied in healthy Thoroughbred mares (n = 286) in mid-gestation (day 84 -232), and included: combined thickness of uterus and placenta (CTUP), echogenicity of the allantoic fluid (ALF), presence of free floating particles (FFP), and the fetal heart rate (FHR). In case of an abortion or stillbirth, data of the sonographic parameters were compared with results from necropsies. Abnormalities in CTUP values were seen in 18 cases (maximum value of 17.7 mm). The sonographic appearance of ALF did not change during gestation. There was a significant difference in the presence of FFP with respect to the month of gestation. Abortion rates were determined to be 6.33 times higher for mares with presence of FFP. The mean FHR decreased significantly during gestation. In 7 mares a fetal bradycardia was found, while in 14 mares a tachycardia was found. Overall, 13 abortions (4.8 %) occurred between 201 and 315 days of gestation, which was about 94 ± 46.1 days after the sonographic examination. In one case of an abortion, an abnormal CTUP (8.9 mm), and in another case an abnormal FHR (170 beats/min) was determined. In the remaining 11 cases no abnormalities were detected during sonographic examinations. Although deviations from mean values for sonographic parameters do not necessarily result in an abortion or stillbirth, such abnormalities should be taken seriously and continuously monitored. We conclude that the month of gestation affects FFP as well as FHR in Thoroughbred mares. Furthermore, sonographic evaluation of CTUP, FFP, and FHR alone is not sufficient to foresee placental failure and impending abortion.Keywords: horse / pregnancy / ultrasound / placentitis / combined thickness of uterus and placenta / abortion / reproduction Citation: Seehafer H., Oldenhof H., Kassens A., Klewitz J., Ulrich R., Martinsson G., Rohn K., Baumgärtner W., Sieme H. (2015) Sonographic parameters for assessment of fetal well-being in mid-gestation in german Thoroughbred mares. Pferdeheilkunde 31,[477][478][479][480][481][482][483][484]