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We recently demonstrated that conceptus-derived interferon tau (IFNT), responsible for maternal recognition in cattle, acts on the uterus in a dose- and time-dependent manner by upregulating key interferon-stimulated genes (ISGs) in the endometrium. In high producing dairy cows, postpartum uterine infection is a major factor influencing fertility and pregnancy outcome. Lipopolysaccharide (LPS), an endotoxin of Gram-negative bacteria such as Escherichia coli, generates an altered uterine environment by inducing excessive inflammation at the maternal-conceptus interface. Thus, we aimed to investigate whether the endometrial response to IFNT is altered in the presence of LPS. Endometrial explants were isolated from uteri collected at a local abattoir from Holstein Friesian cows (n = 8) during the mid-luteal stage of the estrous cycle, and cultured in RPMI medium for 24 h in 5 % CO 2 in humidified air without (control), or with IFNT (100 ng/mL), a single Day 15 conceptus, LPS (1 μg/mL), both IFNT and LPS, or both a Day 15 conceptus and LPS. Incubation with IFNT and a Day 15 conceptus up-regulated (P < 0.05) well-known classical ISGs (ISG15, OAS1, MX1 and MX2) as well as other candidate ISGs (CMPK2, IFI35, TRIM38 and TNFSF10) and down-regulated expression of IL1B in endometrial explants. Incubation with LPS increased (P < 0.05) abundance of NFKB1 (a key transcription factor involved in inflammatory and immune response), TNFA, IL1B and IL6 (pro-inflammatory cytokines), IL10 (anti-inflammatory cytokine), IL8, CXCL1, CXCL3 and CCL2 (chemokines), and, to a lesser extent, classical ISGs in endometrial explants. However, LPS did not alter endometrial response to IFNT, irrespective of IFNT concentration (1, 10 or 100 ng/mL). Results suggest that the expression of ISGs, up-regulated by conceptus-derived IFNT, is not altered in the endometrium in the presence of LPS; however, the increased expression of inflammation-related genes induced by LPS indicate an altered endometrial immune response that may be associated with compromised pregnancy establishment or pregnancy failure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In vitro embryo production (IVP) is of great importance to the porcine industry, as well as for basic research and biomedical applications. Despite the large efforts made in laboratories worldwide to address suboptimal culture conditions, porcine IVP remains inefficient. Nobiletin (Nob, 5,6,7,8,3',4' hexamethoxyflavone) supplementation to in vitro culture (IVC) medium, enhances in vitro embryo development in various species. However, its impact on the quality and developmental capacity of in vitro-produced pig embryos is yet to be established. This study evaluated the effects of different concentrations (2.5 and 5 μM) of Nob during the early culture of in vitro-produced pig embryos on embryo developmental competence, mitochondrial activity, lipid content, intracellular Reactive Oxygen Species (ROS) and Glutathione (GSH) content, Total Cell Number (TCN) per blastocyst, and expression of genes related to embryo development, quality and oxidative stress. Embryos cultured in medium without Nob supplementation and in medium supplemented with 0.01 % dimethyl sulfoxide (DMSO-vehicle for Nob) constituted the Control and DMSO groups, respectively. Embryo development rates were evaluated on Days 2, 6 and 7 of IVC. Additionally, a representative group of embryos was selected to assess mitochondrial activity, lipid, ROS and GSH content (on Days 2 and 6 of IVC), TCN assessment and gene expression analyses (on Day 6 of IVC). No significant differences were observed in any of the parameters evaluated on Day 2 of IVC. In contrast, embryos cultured under the presence of Nob 2.5 showed higher developmental rates on Days 6 and 7 of IVC. In addition, Day 6 embryos showed increased mitochondrial activity, with decreased levels of ROS and GSH in the Nob 2.5 group compared to the other groups. Both Nob 2.5 and Nob 5 embryos showed higher TCN compared to the Control and DMSO groups. Furthermore, Nob 2.5 and Nob 5 upregulated the expression of Superoxide dismutase type 1 (SOD1) and Glucose-6-phosphate dehydrogenase (G6PDH) genes, which could help to counteract oxidative stress during IVC. In conclusion, the addition of Nob during the first 48 h of IVC increased porcine embryo development rates and enhanced their quality, including the upregulation of relevant genes that potentially improved the overall efficiency of the IVP system., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Seminal plasma (SP) provides essential nutrients, transport, and protection to the spermatozoa during their journey through the male and female reproductive tracts. Extracellular vesicles (EVs) are one of the main components of the SP with several biomolecular cargoes, including miRNAs, that can influence spermatozoa functions and interact with the cells of the female reproductive tract. This study aimed to isolate, characterize, and identify the miRNA expression profiles in the SP-EVs isolated from fertile (F) and subfertile (S) rabbit bucks that could serve as fertility biomarkers. In this study, the methods to isolate and identify EVs including exosomes, from SP of 3 F and S bucks have been developed. Ultracentrifugation and size exclusion chromatography analysis were using to isolate EVs from SP of F and S males that were qualitative and quantitively characterised using transmission electron microscopy, nanoparticle tracking analysis and western blotting. In addition, total RNA, including miRNA, was isolated, sequenced and identified from SP-EVs samples. Different SP-EVs concentrations (8.53 × 10 11  ± 1.04 × 10 11 and 1.84 × 10 12  ± 1.75 × 10 11 particles/mL of SP; P = 0.008), with a similar average size (143.9 ± 11.9 and 115.5 ± 2.4 nm; P = 0.7422) in F and S males, respectively was observed. Particle size was not significantly correlated with any kinetic parameter. The concentration of SP-EVs was positively correlated with the percentage of abnormal forms (r = 0.94; P < 0.05) and with the percentage of immotile spermatozoa (r = 0.88; P < 0.05). Small-RNA-seq analysis identified a total of 267 and 244 expressed miRNAs in the F and S groups, respectively. Two miRNAs (let-7b-5p and let-7a-5p) were the top most abundant miRNAs in both groups. Differential expression analysis revealed that 9 miRNAs including miR-190b-5p, miR-193b-5p, let-7b-3p, and miR-378-3p, and another 9 miRNAs including miR-7a-5p, miR-33a-5p, miR-449a-5p, and miR-146a-5p were significantly up- and downregulated in the F compared to the S group, respectively. The SP from F and S rabbit males contains EVs with different miRNA cargo correlated with spermatogenesis, homeostasis, and infertility, which could be used as biomarkers for male fertility and potential therapies for assisted reproductive technologies., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Mammalian follicles are constituted of a complex structure composed of several layers of granulosa cells surrounding the oocyte and of theca cells that reside beneath its basement membrane. During folliculogenesis, granulosa cells separate into two anatomically and functionally distinct sub-types; the mural cells lining the follicle wall and the oocyte-surrounding cumulus cells, i.e. those in intimate metabolic contact with the oocyte. The cumulus cells connecting with the oocyte have trans-zonal cytoplasmic projections which, penetrating the zona pellucida, form the cumulus-oocyte complex. The connections through gap junctions allow the transfer of small molecules between oocyte and cumulus cells, such as ions, metabolites, and amino acids necessary for oocyte growth, as well as small regulatory molecules that control oocyte development. The bi-directional communication between the oocyte and cumulus cells is crucial for the development and functions of both cell types. Our current knowledge of the relationship between the oocyte and its surrounding cumulus cells continues to change as we gain a greater understanding of factors regulating oocyte development and folliculogenesis. This review will mainly focus on the reciprocal interaction between oocytes and cumulus cells during the latter stages of follicle development i.e. through antral development to periovulatory events including oocyte maturation, expansion, and degradation of the cumulus matrix., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

Guinea pig in vitro fertilization (IVF) are poorly developed due to the limited accessibility to oocytes and the lack of an efficient method of sperm capacitation. Thus, we aimed to evaluate different capacitation protocols that we validated through sperm analysis and using heterologous (He) IVF with zona-intact bovine oocytes. Spermatozoa of guinea pigs were collected and processed separately by 4 different protocols: A) Spermatozoa were obtained by flushing the lumen of one cauda epididymis and incubated in a minimal culture medium (MCM); B) One epididymis was placed in a prewarmed of M2 medium and gently minced with fine scissors. Spermatozoa were incubated in a modified human tubal fluid medium (HTF). In both protocols, the spermatozoa were capacitated at 37 °C under an atmosphere of 5% CO2 for 2 h. In the protocols C and D, the spermatozoa were collected by flushing the lumen of the cauda epididymis and selected by commercial density gradient Bovipure® (Nidacon Laboratories AB, Göthenborg, Sweden), according to the manufacturer's instructions. Then for Protocol C) spermatozoa were incubated in MCM medium supplemented with 10 mg/mL heparin (MCM-Hep); while for Protocol D) spermatozoa were incubated in FERT medium supplemented 10 mg/mL heparin (FERT-Hep). Incubation of C and D protocols were performed at 38.5 °C under an atmosphere of 5% CO 2 for 2 h. Capacitation protocols C and D showed a higher percentage of viability, total and hyperactive-like motility, and acrosome reaction compared to protocols A and B. For this reason, protocols C and D were used for further He-IVF analysis. Guinea pig sperm and matured zona-intact bovine oocytes were co-incubated at 5% CO 2 and 38.5 °C. Sperm-oocyte interaction was assessed at 2.5 h post-insemination (hpi) and pronuclear formation (PrF) were evaluated at 18, 20, 22, 24 and 26 hpi, while the cleavage rate was evaluated at 48 hpi. In protocol D, PrF was significantly higher than in protocol C (P ≤ 0.05) at every time point evaluated. Also, the cleavage rate at 48 hpi was higher (P ≤ 0.05) in He-IVF protocol D (69.8 ± 1.7%) compared to He-IVF protocol C (49.1 ± 1.1%). In conclusion, we determined the most adequate sperm capacitation conditions for guinea pig that allow zona-intact bovine oocyte penetration and lead to hybrid embryo formation, suggesting that these conditions could be optimal to develop IVF in guinea pigs., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In this study, the transcriptome of oviductal epithelial cells and certain characteristics of their extracellular vesicles of dairy cows were described under thermoneutral and heat stress conditions. Twenty cows were compared in springtime at THI = 65.6 ± 0.90 and in summertime at THI = 78.36 ± 2.73. During each season, the estrous cycles of the cows were synchronized, and on day 3 of the ensuing cycle, a blood sample was collected for progesterone determination, while their oviducts were collected after slaughter. Epithelial cells and oviductal fluid were collected from the oviduct ipsilateral and contralateral to the corpus, respectively. For the gene expression study, a comparative transcriptomic approach, using RNASeq, was performed on cells collected from the ipsilateral and the contralateral oviducts. The size and the concentration of extracellular vesicles (EVs) at both seasons were analyzed using Transmission Electron Microscopy and Nanoparticle tracking analysis and specific proteins were detected by Western blotting. Progesterone concentration was higher during the thermoneutral period. Between seasons, divergent expression of genes related to immune system, contractility, gamete protection and lncRNAs was found. The size and the concentration of the EVs did not differ between seasons, however, the concentration in the ipsilateral oviduct tended to be lower (p = 0.09) from the contralateral one in the summer, but not in the spring. Our results show for the first time that HS could be involved with alterations in the oviductal cells' gene expression and in the changes in concentration of EVs in the oviductal lumen. Our results imply that the altered oviductal environment during HS could be associated with the suppressed summer fertility in dairy cows., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Diacylglycerol acyltransferase-1 (DGAT1) is one of the DGAT enzymes that catalyzes the final step in the synthesis of triacylglycerol, which is a major component of the lipid droplets in embryos. Intracellular lipids accumulated in embryos produced in vitro have been associated with reduced cryotolerance and quality. The objective of the present study was to investigate the influence of DGAT1 inhibition on embryo development, quality, and post-vitrification survival, in addition to expression profiles of selected lipid metabolism-regulating and oxidative stress genes. Bovine cumulus-oocyte complexes were matured and fertilized in vitro and were cultured in synthetic oviduct fluid (SOF) supplemented with 5% fetal calf serum (FCS) alone (Control) or with 1, 5, 10 or 50 μM DGAT1 inhibitor (A922500®; D1, D5, D10, and D50, respectively) or 0.1% dimethyl sulfoxide (CDMSO: vehicle for DGAT1 inhibitor dilution) from 54 h post-insemination until Day 8 post insemination. No differences were found in blastocyst yield on days 7 and 8 in Control, CDMSO, D10, and D50 groups. Embryos cultured with 10 or 50 μM DGAT1 inhibitor had greater mitochondrial activity (P < 0.01), and increased number of cells (P < 0.05), while the cytoplasmic lipid content was reduced (P < 0.01), the latter associated with altered expression profiles of selected genes regulating lipid metabolism or genes related with oxidative stress (transcript abundance increased for SLC2A1 and SLC2A5 and decreased for DGAT1 and GPX1). Importantly, the survival rate of blastocysts produced with 10 μM DGAT1 was higher than that of Control, CDMSO and D50 groups at 72 h after vitrification and warming (73.8 vs 57.1, 55.9 and 56.1%, respectively, P < 0.001). In conclusion, inhibition of DGAT1 synthesis in bovine embryos produced in vitro abrogates the negative effect of FCS by decreasing their lipid content, increasing mitochondria activity and improving embryo cryotolerance, as well as favoring the expression of lipid metabolism regulating and oxidative stress-related transcripts., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Neonatal brain injury (NBI) is a serious adverse outcome in premature neonates. We sought to determine the levels and prognostic value of serum S100B during the first three days of life in premature neonates (<34 weeks) that later developed NBI in the form of either intraventricular hemorrhage (IVH) or periventricular leukomalacia (PVL)., Methods: This is a prospective case - control longitudinal study. Each case (n = 29) was matched according to birthweight and gestational age to one neonate with normal head ultrasound scans., Results: Neonates with NBI, had significantly higher S100B concentration during the first three days of life. In both groups S100B was significantly higher on the first day when compared to the next two days of life showing a downwards trend. Serum S100B on the first day was the best predictor for adverse neonatal outcome such as death or II-IV IVH grade. A cut-off value of 10.51 ng/ml serum S100B performed a sensitivity of 100% and a specificity of 93.9% to predict adverse neonatal outcome., Conclusion: Further research on the predictive value of serum S100B regarding NBI in premature neonates is of great interest and may provide the first clinically useful biomarker for early detection of neonates at high risk., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A comprehensive understanding of the complex embryo-maternal interactions during the preimplantation period requires the analysis of the very early stages of pregnancy encompassing early embryonic development, maternal recognition and the events leading to implantation. Despite the fact that embryo development until blastocyst stage is somewhat autonomous (i.e., does not require contact with the maternal reproductive tract and can be successfully recapitulated in vitro), many studies on ruminant embryo production have focused on the fundamental question of why: (i) only 30%-40% of immature oocytes develop to the blastocyst stage and (ii) the quality of such blastocysts continually lags behind that of blastocysts produced in vivo. Clear evidence indicates that in vitro culture conditions are far from optimal with deficiencies being manifested in short- and long-term effects on the embryo. Thus, enhanced knowledge of mechanisms controlling embryo-maternal interactions would allow the design of novel strategies to improve in vitro embryo conditions and reproductive outcomes in cattle., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The poor fertility of ram semen stored chilled for long periods has encouraged the development of protocols designed to improve the kinetic vigour and cervical barrier-crossing capacity of sperm. The present work evaluated the effect of sperm selection with Sephadex filtration and the supplementation of 2% glycerol (GLY) to extenders based on ultra-heat-treated skimmed milk (UHT) or Tris-Tes-Glucose (TEST) on ram sperm kinetic parameters, plasma membrane integrity, acrosome integrity, mitochondrial function and fertilizing ability, over long chilling times. The results showed that for non-filtered semen, values for progressive sperm motility (%PSM), straight line velocity (VSL, μm/s) and the percentage of sperm with an intact plasma membrane/intact acrosome/a high mitochondrial function index (%IPIAHM) at all times up to 96 h of chilling were higher when the UHT extender (P < 0.01) was used compared to TEST extender irrespective of the presence of GLY. When semen was previously filtered with Sephadex, the addition of GLY to the UHT extender improved total motility (%TM), the %PSM and the VSL at 96 h compared to all other treatments (P < 0.01). The best results of all were obtained with non-filtered semen and UHT either with or without GLY. Heterologous IVF using zona-intact bovine oocytes was used to assess the fertilizing capacity of non-filtered fresh (FS0), chilled-for-24 h (CS24) or chilled-for-48 h (CS48) ram semen diluted in UHT extender (GLY-free). Heterologous IVF showed that ram sperm, either FS0, CS24 or CS48, were equally capable of penetrating zona pellucida intact bovine oocytes, leading to pronuclear formation and hybrid embryo cleavage (46.3 ± 3.2; 48.8 ± 3.2; and 43.3 ± 3.5, respectively). No differences were seen with respect to fresh sperm in terms of sperm binding, penetration, polyspermy, pronucleus formation or cleavage rates (P > 0.05). In conclusion, neither Sephadex filtration nor addition of glycerol provided extra benefits to ram sperm chilled up to 96 h. Chilled, non-filtered sperm extended with UHT without GLY showed better sperm functionality than did similar sperm extended with TEST extenders. Indeed, sperm diluted in UHT extender, maintained fertilizing ability up to 48 h., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

This study examines the effects of the histone deacetylation inhibitor scriptaid (SCR) on preimplantation embryo development in vitro and on imprinting gene expression. We hypothesized that SCR would increase histone acetylation levels, enhance embryonic genome activation, and regulate imprinting and X-chromosome inactivation (XCI) in in vitro produced bovine embryos. Zygotes were cultured in vitro in presence or absence of SCR added at different time points. We assessed cleavage and blastocyst rates as well as the quality of blastocysts through: (i) differential cell counts; (ii) survival after vitrification/thawing and (iii) gene expression analysis -including imprinted genes. Blastocyst yields were not different in the control and experimental groups. While no significant differences were observed between groups in total cell or trophectoderm cell numbers, SCR treatment reduced the number of inner cell mass cells and improved the survival of vitrified embryos. Further, genes involved in the mechanism of paternal imprinting (GRB10, GNAS, XIST) were downregulated in presence of SCR compared with controls. These observations suggest SCR prevents deacetylation of paternally imprinting control regions and/or their up-regulation, as these events took place in controls. Whether or not such reductions in XIST and imprinting gene expression are beneficial for post implantation development remains to be clarified., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In this study, we successfully described for the first time a vitrification of epididymal Iberian ibex spermatozoa. Spermatozoa from epididymis were obtained from 15 Iberian ibex. The right epididymis' semen sample was vitrified and the left one was frozen. After thawing/warming, samples were selected by density gradient. Sperm characteristics from each treatment were evaluated. To test the spermatozoa fertilization ability, heterologous IVF was carried out using bovine oocytes. Despite of the observation of a decrease of about 40% for motility sperm between pre-freezing and post-thawing (75.0 ± 5.2 and 45.0 ± 6.0) and pre-vitrification and post-warming (78.2 ± 5.2 and 33.9 ± 6.2) (P < .05), after the washing, an improvement of sperm motility was found when using the vitrification treatment compared to frozen-thawed. Heterologous IVF showed that Iberian Ibex spermatozoa, either frozen-thawed or vitrified-warmed, were equally capable of penetrating ZP intact bovine oocytes, leading to pronuclear formation (%) and hybrid embryo cleavage (%), (31.3 ± 27.2 and 45.1 ± 24.4, respectively). As expected, in the homologous IVF group, higher percentages of penetration, pronuclei formation and cleavage were found compared to heterologous groups using Iberian ibex frozen and vitrified sperm (P < 0,5). The highest pronuclei formation was found after 20 h post insemination in both heterologous IVF groups (30.2 ± 6.7 and 31.7 ± 21.5 thawed and vitrified group). Consequently, the cleavage rate (48 h) followed the same results to homologous and thawed and vitrified groups (76.1 ± 15.9; 31.3 ± 27.2 and 45.1 ± 24.4, respectively) (P < .05). In conclusion, Iberian ibex sperm vitrification is a promising and useful alternative to conventional methods resulting in good quality spermatozoa post-thaw, and an adequate in vitro fertilizing ability., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Pre-eclampsia and eclampsia are parts of the broader spectrum of hypertensive disorders complicating pregnancy. This study aims to examine the association between serum inhibin and leptin levels and pre-eclampsia., Methods: This study included 98 consecutive cases of pregnant women with pre-eclampsia, together with their 98 pregnant controls, matched for age, gestational week and time period of delivery. Maternal venous blood samples were obtained within 24h before delivery. In addition to serum inhibin and leptin, birth order, multiple pregnancy, maternal age, maternal overweight/obesity, maternal education, maternal smoking and family history of diabetes/hypertension, were examined as risk factors. Multivariate logistic regression analysis was performed., Results: At the univariate analysis, serum inhibin and leptin levels were significantly higher in cases vs., Controls: Pre-eclampsia occurred more frequently in primiparous women, whereas overweight and obesity were also associated with pre-eclampsia. At the multivariate analysis, higher serum inhibin levels were associated with pre-eclampsia (multivariate OR=1.09, 95%CI: 1.03-1.17, p=0.004, increase per 0.1ng/mL). On the other hand, leptin was not independently associated with the occurrence of pre-eclampsia (multivariate OR=1.02, 95%CI: 0.95-1.09, p=0.631, increase per 10ng/mL)., Conclusions: Elevated serum inhibin levels seem to be associated with pre-eclampsia, reflecting placental dysfunction. Increased serum leptin levels may merely reflect an elevated maternal body mass index, which is a well-known risk factor for pre-eclampsia., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Assisted reproductive technologies have provided a very useful tool for studying early embryonic development. The exchange of signals between the embryo and maternal environment during this period is critical to successful development, but most mechanisms involved remain to be elucidated. Understanding how the mother communicates with gametes and embryos is a major scientific challenge but in vivo studies are difficult to perform, especially in cattle, since they are expensive, the amount of material is limited, and it is not possible to differentiate between the outcome of fertilization and early embryonic death. In addition, the local interactions of the embryo with the maternal epithelium may not be detectable because of the small size of the embryo and the difficulty of identifying its exact position in the oviduct. On the basis of current knowledge gained from in vivo studies, the challenge now is to identify appropriate in vitro models to facilitate the study of early embryo-maternal communication., (Copyright © 2016 Elsevier Inc. All rights reserved.)

During the past 50 years, the fertility of high-producing lactating dairy cows has decreased, associated with intensive selection for increased milk production. The physiological and metabolic changes associated with high milk production, including decreased (glucose, insulin, IGF-I) or increased (nonesterified fatty acids, ketone bodies) concentrations of circulating metabolites during nutrient partitioning associated with negative energy balance as well as uterine and nonuterine diseases have been linked with poor reproductive efficiency. Fertilization is typically above 80% and does not seem to be the principal factor responsible for the low fertility in dairy cows. However, early embryonic development is compromised in high-producing dairy cows, as observed by most embryonic losses occurring during the first 2 weeks after fertilization and may be linked to compromised oocyte quality due to a poor follicular microenvironment, suboptimal reproductive tract environment for the embryo, and/or inadequate maternal-embryonic communication. These and other factors related to embryo development will be discussed., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background and Aims: We aimed to assess the prognostic value of free androgen index (FAI) and its change over time in arterial stiffness progression, endothelial function and hypertension in postmenopausal women., Methods: Postmenopausal women (n = 180) without clinically overt cardiovascular disease or diabetes were consecutively recruited and followed for a median of 29 months. The main outcome measures were changes over time in endothelial function (FMD), reflected waves, localized and systemic (PWV) arterial stiffness and hypertension., Results: Increased baseline FAI was significantly associated with new onset hypertension (OR for each SD, 2.71, 95% CI 1.14-6.41, p = 0.024), deterioration of pulse wave velocity (PWV) (0.414 m/s per SD), flow-mediated dilation (FMD) (-0.42% per SD), systolic (2.5 mmHg per SD) and pulse pressure progression (2.3 mmHg per SD, p < 0.05 for all). Baseline FAI remained an independent predictor of changes in PWV (p = 0.006), FMD (p = 0.02), peripheral pulse pressure (p = 0.028), transition to new onset hypertension (p = 0.001) and higher BP category (p = 0.012), after adjustment for age, changes in systolic blood pressure, traditional risk factors, vasoactive medication or total testosterone. Baseline FAI improved reclassification for the risk of transition into higher BP category (NRI = 47.5 ± 20.3%, p = 0.02) and abnormal PWV (NRI = 53.4 ± 23.2%, p = 0.021). Similarly, in a subgroup of patients with measured FAI at follow-up, its changes over time predicted changes in PWV, peripheral pulse pressure and hypertension status (p < 0.05 for all)., Conclusions: In apparently healthy postmenopausal women, FAI could be a novel biomarker superior to total testosterone for accelerated vascular aging and hypertension status., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Assisted reproductive technologies are of great importance for increasing the genetic diversity in captive animals. The use of bovine or murine oocytes in heterologous IVF provides advantages compared to homologous IVF in nondomestic animals, such as the accessibility to oocytes and the availability of well-developed in vitro maturation systems. The aim of this study was to determine the heterologous IVF parameters using cryopreserved dolphin spermatozoa and zona-intact bovine or murine oocytes and to examine the nuclear chromatin status of the dolphin spermatozoa. All the processes involved in the fertilization including embryo cleavage were observed by confocal microscopy and hybrid embryo formation was confirmed by polymerase chain reaction. Heterologous bovine IVF showed no polyspermy, lower percentages of pronuclear formation, and a lower cleavage rate compared to homologous IVF group (34.8% vs. 89.3%). Heterologous murine IVF showed a lower cleavage rate than homologous IVF (9.6% vs. 77.1%). With respect to dolphin sperm chromatin, it was more stable, i.e. more resistant to EDTA-SDS decondensation than the bovine sperm chromatin. This study revealed the stability of the dolphin sperm chromatin and the ability of the dolphin spermatozoa to penetrate zona-intact bovine and murine oocytes, leading to hybrid embryo formation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Reactive oxygen species (ROS) are physiologically generated during mitochondrial respiration and are involved in several signaling mechanisms. However, under pathological conditions, the concentration of ROS may exceed the antioxidant scavenging systems and subsequently lead to cell damage. High ROS levels have been proven to be detrimental to spermatozoa and furthermore compromise sperm function through lipid peroxidation, protein damage, and DNA strand breakage. Although the oral administration of antioxidants has been demonstrated to improve the semen quality in subfertile men, it is still a matter of debate if it can positively influence fertilization outcome and embryo developmental competence. Studies carried out in suitable animal models could resolve these fundamental questions. Hence, the main aims of the present study were to evaluate: (1) the effects of zinc, d-aspartate, and coenzyme Q10, included in the dietary supplement Genadis (Merck Serono), on bull sperm motility and DNA fragmentation; and (2) whether treated spermatozoa have a superior competence in fertilization and in supporting the development of healthy embryos. Our data indicate that this treatment prevents the loss of sperm motility and the rise in sperm DNA fragmentation over time. Moreover, blastocyst rate was found to be significantly higher in oocytes fertilized by treated spermatozoa, and these blastocysts harbored a significantly lower percentage of apoptotic cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Studies concerning oocyte quality markers, oocyte/embryo metabolism or commercial OPU settings treating donors with low oocyte yields, indicate a need for optimization of IVP protocols to culture single oocytes to the blastocyst stage. However, culture conditions for single oocyte usually impair development, although previous research showed that single oocyte culture on a monolayer of cumulus cells can lead to similar developmental competence than group oocyte culture. Aiming to develop a fully single IVP procedure, Experiment 1 and 2 revealed that individual maturation, fertilization and culture in 20 μL droplets, using a monolayer of heterologous (SSSm, Exp 1) or autologous cumulus cells in coculture (SSSa, Exp 2), resulted in 23.9% and 15.1% of blastocysts 8 days p.i., respectively, which is significantly less compared to regular group IVP (GGGc, 33.5% (Exp 1) and 26.2% (Exp 2), respectively). In a third Experiment, day 7 p.i. blastocyst quality was analyzed in four treatment groups: regular group IVP (GGGc), group IVP with coculture (GGGm), in group produced zygotes, singly cultured on a heterologous cumulus cell monolayer (GGSm) and individually matured and fertilized zygotes, singly cultured on a monolayer (SSSm). Mean cell number and apoptotic cell index, were similar for all treatment groups. Moreover, mRNA abundance relative to H2AFZ was equal for 9 qualitatively linked genes (TP53, BAX, SHC1 SHC, IGF2R, PTGS2, AKR1B1, PLAC8, SLC2A1, and MNSOD). Only GPX1, involved in detoxification and mtDNA protection to oxidative stress, was significantly downregulated (ANOVA, P < 0.05) in singly produced blastocysts (SSSm), compared to the other treatments. In conclusion, a valuable individual IVP system was established and autologous cumulus cells in coculture showed to partly neutralize hampered individual culture conditions. Additionally, to our knowledge this is the first report in which blastocyst quality, in terms of cell number, apoptosis and gene expression, of singly produced embryos was investigated and shown to be similar to in group produced embryos, implicating that the single IVP system can be applied as a tool in oocyte and embryo quality studies., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Leptin is a metabolic hormone related to body condition and nutritional status that influences fertility in assisted reproductive technologies modulating oocyte and embryo quality. The aim of the present study was to establish the effect of various leptin concentrations (0, 10, 100 ng/mL) during in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on bovine embryo development and quality in terms of gene expression. The relative mRNA abundance of the genes encoding solute carrier family 2 (facilitated glucose transporter) member 1 (SLC2A1), Bcl-2-associated X protein (BAX), placenta-specific 8 (PLAC8), aldo-keto reductase family 1 member B1 (AKR1B1) and leptin receptor (LEPR) were determined on Day 7 blastocysts by qRT-PCR. Cleavage rate (P < 0.005) and blastocyst yield (P = 0.05) was significantly lower when cumulus-oocyte complexes (COCs) were matured with 100 ng/mL leptin compared to 0 or 10 ng/mL leptin. No significant effect of different concentrations of leptin added during IVC on blastocyst yield was observed. The presence of 100 ng/mL leptin in both IVM and IVC further decreased cleavage rate (P < 0.005) and blastocyst yield compared to the control group without leptin (P = 0.05) and those supplemented with 10 ng/mL leptin or FCS (P < 0.005). There was no evidence of any leptin-induced difference in the relative transcript abundance of SLC2A1, BAX and PLAC8 genes in Day 7 blastocysts. Expression of AKR1B1 was significantly lower in blastocysts from COCs matured with 100 ng/mL leptin compared to those matured with 0 or 10 ng/mL leptin (P < 0.005). LEPR expression was up regulated when leptin concentration was increased from 0 ng/mL during IVM to 10 ng/mL during IVC, but it was down-regulated in the opposite situation (P < 0.005). In conclusion, high leptin concentrations possibly related to obesity seem to be more detrimental rather than the absence of this hormone for preimplantation embryo survival; this effect is independent of LEPR gene expression and it does not influence expression of SLC2A1, BAX and PLAC8 genes in Day 7 blastocysts., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The oviduct provides the optimal environment for the transport of sperm and oocyte at the earliest stages of mammalian embryo development. During the early postfertilization period, several major developmental events occur in the embryo including (i) the first cleavage division, (ii) activation of the embryonic genome, (iii) compaction of the morula, and (iv) formation of the blastocyst. Most of these events are initiated in the oviduct. The absence of assistance from the oviduct may compromise the developmental ability of the cattle embryo under in vitro culture conditions. The oviducts of several mammalian species, including rabbits, cow, sheep (in situ), and mice (organ culture), can sustain early bovine embryos and yield blastocysts of better quality compared with those of culture conditions in vitro, leading to normal pregnancy rates in recipient animals. This review focuses on the use of oviducts in vitro or in vivo as intermediate hosts for postfertilization culture environment of bovine in vitro-produced zygotes with emphasis on the mouse model., (2010 Elsevier Inc. All rights reserved.)

This study evaluated the effect of two oxygen concentrations (20 and 5%) on bovine embryo development (kinetics of first cleavage and blastocyst development) during maturation (M) and fertilization (F) and analysed differences in gene expression between cumulus-oocyte complexes (COC) matured at 5 or 20% oxygen and the resulting blastocysts. A total of 1179 COC were divided into four groups according to the oxygen tension used (M5F5, M5F20, M20F5 and M20F20). Relative poly(A) mRNA abundance of GLUT1, GAPDH, LDHA, G6PD, MNSOD, GPX1, IGFR2, BAX, CCNB1, PTGS2 and GREM1 was analysed in COC, whereas 10 quality-related genes were analysed in blastocysts. M20F5 group developmental rates were significantly lower than all other groups (one-way ANOVA, P < or = 0.05). Two-way ANOVA showed a beneficial effect of low oxygen tension during in-vitro maturation on developmental rates, whereas the opposite situation was obtained in fertilization (P < or = 0.05). GAPDH, IGFR2, CCNB1, and GREM1 were up-regulated in the oocytes matured in low oxygen, whereas GLUT1, GAPDH, LDHA and GREM1 were up-regulated and PTGS2 down-regulated in the cumulus cells from the M5 group (P < or = 0.05). No differences were observed in blastocysts. Low oxygen tension during maturation alters the expression of genes related to oocyte competence and glucose metabolism and significantly (P < or = 0.05) improves embryo development, but not blastocyst quality., (Copyright 2009 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Background: Cytokines are significant mediators of the immune response to surgery and also play a role in parturition. The aim of the study was to investigate the impact of the anesthetic technique for cesarean section on plasma levels of cytokines IL-6 and TNF-alpha., Methods: Thirty-five parturients scheduled for elective cesarean section were randomly assigned to general (n=18) or neuraxial (n=17) anesthesia. The general anesthesia group received thiopental 4 mg/kg, succinylcholine 1-1.5 mg/kg and 1% end-tidal concentration of sevoflurane in nitrous oxide and 50% oxygen. The neuraxial anesthesia group received intrathecal 0.5% levobupivacaine 1.8-2.2 mL and epidural fentanyl 1 microg/kg. Blood samples were taken for IL-6 and TNF-alpha immediately after positioning the parturient on the operating table, after uterine incision and before the umbilical cord clamping and 24h after surgery (T(1), T(2) and T(3) respectively)., Results: The two groups did not differ in IL-6 (P=0.15) or TNF-alpha (P=0.73) serum concentrations at any time point. In the general and neuraxial anesthesia groups, IL-6 serum concentrations were significantly higher in the third blood sample, T(3) (12.2+/-5.0 and 15.2+/-4.3 pg/mL), than in T(1) (0.41+/-0.38 and 0.29+/-0.10 pg/mL) and T(2) (0.37+/-0.47 and 0.24+/-0.05) respectively (P<0.001). Within each group, serum TNF-alpha concentrations did not differ significantly over time (P=0.44)., Conclusions: Under the present study design anesthetic technique did not affect IL-6 or TNF-alpha concentrations in parturients undergoing elective cesarean section. Serum IL-6 levels increased 24 h postoperatively independently of anesthetic technique.

Postpartum dairy cows (n=35) were used to determine the effects of feeding propylene glycol (PG) on metabolic variables related to ovarian function and on oocyte developmental competence. Starting on Day 7 postpartum, each animal received an oral dose (500 ml) of either PG or water once daily. Blood samples were collected on Days 5, 15, 25 and 35 pp to measure insulin, non-esterified fatty acids (NEFAs), beta-hydroxybutyrate (BHB) and glucose concentrations. Oocytes were recovered by ultrasound guided follicular aspiration starting on approximately Day 30 postpartum and submitted to in vitro fertilization. Ovarian follicular activity was examined daily by ultrasonography from Day 7 until ovulation or Days 35-40 postpartum. Animals receiving PG had elevated insulin concentrations over the subsequent 90 min following dosing (P<0.05) compared to control animals. Glucose concentrations followed a similar pattern. Irrespective of treatment, concentrations of NEFA declined significantly from Days 15 to 35 postpartum. Administration of PG resulted in a decrease in NEFA (P<0.001) and BHB (P<0.001) over the subsequent 90 min compared to control animals. Treatment with PG had no effect on follicular dynamics, mean days to emergence of the first cohort of follicles postpartum, or days to dominance and duration of dominance for any follicular wave recorded postpartum. There was also no difference in mean days to first ovulation or in size of the preovulatory follicle between treatments. Oocyte quality as measured by blastocyst development after IVF was not affected by treatment. These results suggest that administration of PG has the ability to positively alter the systemic concentrations of a number of metabolic variables which have been related to fertility. However, we did not observe an effect of PG treatment on follicular dynamics or the length of the postpartum interval. An effect on oocyte developmental competence remains to be proven.

This study was conducted to examine pregnancy progression and fetal characteristics following transfer of vitrified bovine nuclear transfer versus in vivo-derived embryos. Nuclear transfer (NT) was conducted using cumulus cells collected from an elite Holstein-Friesian dairy cow. Expanding and hatching blastocysts on Day 7 were vitrified using liquid nitrogen surface vitrification. Day 7 in vivo embryos, produced using standard superovulation procedures applied to Holstein-Friesian heifers (n=6), were vitrified in the same way. Following warming, embryos were transferred to synchronized recipients (NT: n=65 recipients; Vivo: n=20 recipients). Pregnancies were monitored by ultrasound scanning on Days 25, 45 and 75 and a sample of animals were slaughtered at each time point to recover the fetus/placenta for further analyses. Significantly more animals remained pregnant after transfer of in vivo-derived embryos than NT embryos at all time points: Day 25 (95.0 versus 67.7%, P<0.05), Day 45 (92.8 versus 49.1%, P<0.01) and Day 75 (70.0 versus 20.8%, P<0.0). There was no significant difference (P=0.10) in the weight of the conceptus on Day 25 from NT transfers (1.14+/-0.23 g, n=8) versus in vivo transfers (0.75+/-0.19 g, n=8). On Day 45, there was no significant difference in the weight of either fetus (P=0.393) or membranes (P=0.167) between NT embryos (fetus: 2.76+/-0.40, n=12; membranes: 59.0+/-10.0, n=11) or in vivo-derived embryos (fetus: 2.60+/-0.15, n=6; membranes: 41.8+/-5.2, n=4). However, on Day 75 the weight of the fetus and several of the major organs were heavier from NT embryos. These data suggest that morphological abnormalities involving the fetus and the placenta of cloned pregnancies are manifested after Day 45.

Objective: We investigated the dose-related effect of dopexamine and dopamine on free radical production and lipid peroxidation estimated by MDA measurements in an ischaemia-reperfusion model of supraceliac aortic repair., Design: Prospective, randomized, blinded experimental study., Materials: Twenty-five healthy pigs., Methods: All experiments were performed under general endotracheal anaesthesia. Supraceliac aortic cross clamping was performed in all pigs. The pigs were randomly assigned into five groups (n=5 in each group) and received a continuous intravenous infusion of normal saline (CTL), dopamine 2 microg kg(-1)min(-1) (dopa 2), dopamine 8 microg kg(-1)min(-1) (dopa 8), dopexamine 2 microg kg(-1)min(-1) (dopex 2), dopexamine 8 microg kg(-1)min(-1) (dopex 8). Cardiac output, mean arterial pressure, arterial blood gas analysis and blood sampling for plasma MDA measurements (to reveal lipid peroxidation) were recorded after induction of anaesthesia (baseline), 60 and 120 min after cross-clamping of aorta (ischaemia phase), and 60 and 120 min after restoration of flow (reperfusion phase)., Results: Dopexamine and dopamine at 8 microgkg(-1)min(-1) reduced MDA at 60 and 120 min after reperfusion., Conclusion: Dopexamine seems superior to dopamine in reducing oxygen free radicals and subsequent lipid peroxidation during reperfusion after supraceliac aortic cross clamping in pigs.

No laboratory test exists that can reliably predict differences among rams in field fertility after artificial insemination (AI) with frozen-thawed semen. In vitro fertilisation (IVF) has been proposed as a method of predicting these differences. The objectives of this study were to evaluate whether IVF system could discriminate among rams of different fertility in vivo after AI using frozen-thawed semen. Also, to examine effects of lowering sperm concentration on discrimination power between rams used for IVF. The aim of Experiment 1 was to evaluate the effect of altering the sperm concentration from 2 x 10(6) to 0.03125 x 10(6) spermatozoa/mL on subsequent cleavage rate and blastocyst rate in vitro. In Experiment 2, six rams (three High and three Low in vivo fertility; average pregnancy rates of 37.6% and 21.8%, respectively) were compared for their fertilising ability in IVF. Spermatozoa from each of the six rams were added to ewe oocytes using a concentration of either 2 x 10(6) or 0.0625 x 10(6)/mL. There were six replicates with 25 oocytes per well and two wells per ram per replicate. Cleavage rate was monitored at 48 h post-insemination (p.i.) and blastocyst rate determined on Days 6-8 p.i. In Experiment 1, cleavage rate increased with increasing sperm concentration and blastocyst rate was not affected by sperm concentration on any day. When the six rams were tested using 2 x 10(6) spermatozoa/mL, no significant differences were found between High and Low fertility groups for cleavage rate or blastocyst rate on Days 6, 7, or 8 p.i. (P>0.05). When the experiment was repeated using 0.0625 x 10(6) spermatozoa/mL, no differences were found between High and Low group rams for blastocyst rate on any of Days 6, 7 or 8 p.i. (P>0.05). However, there was a significant difference between High and Low fertility rams for percentage of oocytes cleaved (16.4, S.E. 2.02%; P<0.01) and the correlation between fertility in vivo and cleavage rate in vitro was significant (P=0.013). Replicate of IVF was a source of significant variation for both cleavage rate and blastocyst rate and conditions need to be further controlled. However, we suggest that using a low concentration of spermatozoa (0.0625 x 10(6)/mL) for IVF may be a useful method for predicting field fertility of frozen-thawed ram semen.

Ewe breed has been shown to have a major effect on pregnancy rates following cervical AI using frozen-thawed semen. The main objective of this study was to examine the differences between purebred Belclare and Suffolk ewes (multiparous) in fertilization rate, number of accessory sperm and stage of embryo development on day 6 after cervical or laparoscopic AI with frozen-thawed semen. In experiment 1, Belclare and Suffolk ewes were synchronized for 12 days and were either cervically inseminated (year 1: n=28 and 31; year 2: n=16 and 15, respectively) or laparoscopically inseminated (year 2: n=13 and 14). In experiment 2, superovulated Belclare (n=4) and Suffolk (n=13) ewes were laparoscopically inseminated. All ewes were slaughtered 6 days after AI; oocytes/embryos were recovered, morphologically graded and stained to assess the number of cells and accessory spermatozoa. Data from both experiments were combined for statistical analysis. The proportion of ewes with fertilized oocytes was significantly higher following laparoscopic AI compared with cervical AI (54% versus 19%). More Belclare than Suffolk ewes yielded fertilized oocyte(s) after cervical AI (34% versus 10%, P<0.02) but there was no difference after laparoscopic AI (62% versus 60%). From the ewes that yielded at least one fertilized oocyte the proportion of Belclare ewes with embryos at the morula/blastocyst stage was significantly greater than for Suffolk ewes (94% versus 59%, P<0.02). A higher proportion of Belclare than Suffolk ewes had evidence of sperm reaching the site of fertilization following cervical AI (39% versus 15%, P<0.02) but there was no difference after laparoscopic AI (62% versus 64%, P>0.8). Amongst the ewes with evidence of sperm at the site of fertilization, laparoscopic AI resulted in a higher number of sperm per oocyte/embryo or per ewe than cervical AI (P<0.01). These results suggested that the difference in pregnancy rate between Suffolk and Belclare ewes following cervical AI was due to: (i) sperm traversing the cervix and uterus in a higher proportion of Belclare than Suffolk ewes, leading to a higher incidence of fertilization and (ii) the lower developmental competence of fertilized oocytes from Suffolk ewes.

The final steps of oocyte capacitation and maturation are critical for embryonic development but detailed information is scarce on how the oocyte is affected during this period. In this study, 2033 oocytes were collected from 106 superovulated cattle at four different time points before ovulation. Follicular characteristics were measured and oocyte quality was assessed by morphology, mRNA expression of eight marker genes or developmental ability after in vitro/in vivo maturation and subsequent in vitro fertilization and culture. Approaching ovulation, expected increases in follicular size and cumulus expansion suggested progression of oocyte maturation. No differences were found in the expression patterns of analyzed genes, except for heat-shock-protein (Hsp) that was lower in in vivo matured oocytes collected shortly before ovulation. Oocytes collected at this time also had higher developmental ability measured as blastocyst rates (57.6%) after in vitro production while no differences were found between oocytes recovered earlier at the first three time points (39.3-41.5%). We conclude that oocytes recovered late in the preovulatory period are more developmentally competent than oocytes recovered at the pre-capacitation and the capacitation period, probably due to the former having matured in vivo. However, a precisely defined time for aspirating immature oocytes for subsequent in vitro development seems not to be crucial.

The aims of the present study were to compare (1) Holstein-Friesian heifers versus early postpartum lactating cows, and (2) different age categories of crossbred beef heifers versus cows, in terms of oocyte yield, morphological quality and developmental competence. Four experiments were designed to test the associated hypotheses. In Experiment 1, ovum pick up was carried out twice weekly for a period of 5 weeks on Holstein-Friesian heifers (n = 8) and early postpartum cows (n = 8). Oocytes were submitted to in vitro maturation (IVM), fertilization and culture. Significantly more follicles were punctured on the ovaries of heifers than cows (10.4 versus 7.8, P < 0.001). This was reflected in a significantly higher number of total oocytes (4.7 versus 2.8, P < 0.001) and grade 1-2 oocytes recovered/animal from heifers than from cows (3.0 versus 1.8, P < 0.05). There was no significant difference in the percentage of oocytes cleaving after fertilization, or in the percentage reaching the blastocyst stage between heifers and cows. In Experiment 2, oocytes were obtained by manual aspiration from the ovaries of slaughtered crossbred beef heifers (under 30 months, n = 1241) and cows (over 4 years old, n = 1125), and processed in vitro as above. No significant difference was observed between the two groups in terms of the number of aspirated follicles or oocytes recovered. A significantly higher proportion (P < 0.01) of cow oocytes than heifer oocytes reached the blastocyst stage (Day 8: 46.5% versus 33.4%). In Experiment 3, ovaries were separated according to age of heifer into three groups: (1) 12-18 months, (2) 19-24 months and (3) 25-30 months, and compared with cow oocytes. There was no significant difference in the blastocyst yield between the different age groups of heifers. Irrespective of heifer age, the blastocyst yield on Day 8 was significantly lower than that from cow oocytes (35.0, 35.2, 36.5 and 48.3%, respectively, P < 0.05). In Experiment 4, a significantly higher proportion (P < 0.001) of presumptive zygotes derived from abattoir-derived cow oocytes reached the blastocyst stage following culture in vivo in the ewe oviduct than those derived from heifer oocytes (Day 8: 53.1% versus 25.2% for cow and heifer oocytes, respectively). In conclusion, the origin of the oocyte has a significant impact on its subsequent developmental potential. These results would suggest that in an in vitro production system, cow oocytes should be preferentially used over those from heifers in order to maximize blastocyst development.

Recent studies comparing bovine oocyte maturation, fertilization and embryo culture in vivo and in vitro have demonstrated that the origin of the oocyte is the main factor affecting blastocyst yield, while the post-fertilization culture environment is critical in determining blastocyst quality, measured in terms of cryotolerance and relative transcript abundance, irrespective of the origin of the oocyte. Production of embryos in vitro, particularly when using an extended period of in-vitro culture, may predispose the embryo to phenomena such as the large offspring syndrome, which is likely to alter gene expression, particularly of imprinted genes. It is clear now that the post-fertilization culture environment has a profound effect on the relative abundance of gene transcripts within the embryo, and culture under suboptimal conditions for as little as 1 day can lead to perturbations in the pattern of expression.

Background: After birth, apoptosis rates might slow down, compared to those in utero. Thus, factors, attenuating the apoptotic process, like the soluble forms of Fas/FasL system, may increase. AIM-STUDY DESIGN: Soluble Fas (sFas) and soluble Fas ligand (sFasL) concentrations were measured in maternal serum (MS), umbilical cord (UC) and neonatal serum in the first (1N) and fifth (5N) days after birth in order to evaluate the alterations of these molecules during the early neonatal period., Subjects and Methods: Soluble molecules were estimated in 35 healthy, appropriate for gestational age, full-term neonates, their mothers and in 25 healthy, nonpregnant women, age-matched to the mothers (controls), using enzyme immunoassays., Results: sFas concentrations in MS (p < 0.01), UC (p < 0.0001), 1N (p < 0.0003) and 5N (p < 0.02) were lower than those in controls. Neonatal sFas concentrations showed a significant increase from UC to 5N (p < 0.001). In contrast, sFasL concentrations were significantly elevated in all neonatal samples (UC, 1N and 5N) compared to those in MS and controls (p < 0.0001), showing also a significant elevation from UC to 5N (p < 0.0001)., Conclusion: Our results demonstrate increasing serum concentrations of the soluble molecules sFas and sFasL during the first days after birth, indicating possibly a gradual decrease of apoptosis in early neonatal life.

The objectives of this study were to evaluate the effect of sperm dose and sire on the fertilization rate, cleavage rate and blastocyst yield following insemination in vitro, to examine the relationship between these parameters and field fertility in cattle, and to examine the relationship between blastocyst quality and sire used in IVF. Frozen semen from four bulls with 150-day nonreturn rates ranging from 57 to 78% was used. In Experiment 1, oocytes were inseminated with sperm from one of the four bulls at concentrations ranging from 0.016 to 0.5 x 10(6)sperm/ml. A proportion of presumptive zygotes were fixed at 17 h post-insemination (hpi), while the remainder was transferred to in vitro culture (IVC) in droplets of synthetic oviduct fluid (SOF). Cleavage at 48 hpi and the percentage of oocytes reaching the blastocyst stage by Day 8 were recorded. In Experiment 2, to assess blastocyst quality, after insemination with semen from one of the four bulls, presumptive zygotes were cultured in SOF until Day 7. Blastocysts for each bull were removed and vitrified/warmed and survival was recorded at 24, 48 and 72 h after warming. Regardless of bull used, a concentration of 0.125 x 10(6)sperm/ml or above resulted in higher blastocyst yields than any lower concentration used. Fertilization and cleavage rates were also higher at higher sperm concentrations. The best predictor of field fertility was fertilization rate at a concentration of 0.5 x 10(6)sperm/ml (r=0.94, P<0.0001). There was also a significant correlation between cleavage rate at a concentration of 0.5 x 10(6)sperm/ml and nonreturn rate (r=0.90, P<0.0001). In Experiment 2, blastocysts derived from one bull, HTA, were of superior quality as measured by survival 24h after thawing, although these differences were less significant at the subsequent time points measured. In conclusion, these data show that differences between the field fertility of bulls can be determined at sperm concentrations routinely used in IVF. Lowering the sperm concentration does not increase the likelihood of optimizing the differences in fertility or cleavage rate between bulls of different field fertility. We have also demonstrated that the bull can have a significant effect on the quality of blastocysts produced using IVF techniques.

The aim of these experiments was to investigate the effect of duration of IVM, duration of gamete co-incubation, and of sperm dose on the development of bovine embryos in vitro. In addition, the speed of sperm penetration of six bulls of known differing in vivo and in vitro fertility was examined. In Experiment 1, following IVM for 16, 20, 24, 28 or 32 h, cumulus oocyte complexes (COCs) were inseminated with 1 x 10(6) spermatozoa/ml. After 24 h co-incubation, presumptive zygotes were denuded and placed in droplets of synthetic oviduct fluid (SOF). In Experiment 2, following IVM and IVF, presumptive zygotes were removed from fertilization wells at 1, 5, 10, 15 or 20 h post insemination and placed in culture as described above. In Experiment 3, following IVM, COCs were inseminated with sperm doses ranging from 0.01 x 10(6) to 1 x 10(6) spermatozoa/ml. Following co-incubation for 24 h, presumptive zygotes were placed in culture as described above. In Experiment 4, following IVM, oocytes were inseminated with sperm from six bulls of known differing field fertility. To assess the rate of sperm penetration, oocytes were subsequently fixed every 3 h (up to 18 h) following IVF. Based on the results of Experiment 4, in Experiment 5, following IVM for 12, 18 or 24 h, COCs were inseminated with sperm from two sires with markedly different penetration speeds. After 24 h co-incubation, presumptive zygotes were denuded and placed in culture. The main findings from this study are that (1) the optimal duration of maturation of bovine oocytes in vitro to maximize blastocyst yield is 24 h, (2) sperm-oocyte co-incubation for 10 h is sufficient to ensure maximal blastocyst yields, (3) sperm concentrations of 0.25 x 10(6) and 0.5 x 10(6) spermatozoa/ml yielded significantly more blastocysts than any other concentration within the range of 0.01 x 10(6) 1 x 10(6) spermatozoa/ml, (4) there are marked differences in the kinetics of sperm penetration between sires and this may be a useful predictor of field fertility, and (5) the inferior development associated with slower penetration rates may in part be overcome by carrying out IVF at a time when the actual penetration is most likely to coincide with the completion of maturation.

Immunomediators seem to have a central role in the immune system of both human milk and newborn infants. CD31/PECAM-1 is an adhesion molecule, member of Ig gene superfamily, mediating cell-cell adhesion in both homophilic and heterophilic ways. Levels of the soluble form of PECAM-1 (sPECAM-1) were evaluated on the 2nd and 5th day postpartum in breast milk and serum paired samples from 20 lactating women as well as in time-matched serum from their single, term, healthy neonates. Concentrations of sPECAM-1 in breast milk (median, range) on both the 2nd (2.05 ng/ml, 0.0-7.2) and 5th day postpartum (0.89 ng/ml, 0.0-3.6) were about 10 and 20 times lower than those (mean +/- SD) in controls (healthy adults) (19.83 +/- 5.17, p<7 x 10(-8)), showing a significant fall from the 2nd to the 5th day postpartum (p<0.0005). Maternal serum sPECAM-1 values (mean +/- SD) were significantly lower on the 2nd day postpartum (14.21 +/- 5.15 ng/ml) than those in controls (p<0.002), but reached control values on the 5th day postpartum after a significant rise (p<0.0075). Neonatal serum sPECAM-1 values with no significant difference between the 2nd (14.4 +/- 4.11 ng/ml) and 5th day of life (14.54 +/- 4.99 ng/ml) were significantly lower than those in controls (p<0.002). Values of sPECAM-1 in milk and sera of lactating mothers and their neonates on the 2nd day postpartum depended on the mode of delivery, being significantly lower after caesarean section (p<0.034, p<0.0075 and p<0.035, respectively). In conclusion, our findings in the early postpartum period demonstrate that: (a) sPECAM-1 is present in human milk in low and decreasing concentrations; (b) the shedding of sPECAM-1 is an established component of the neonatal immune system from birth, though in lower concentrations than in adults, possibly reflecting its immaturity; and (c) the mode of delivery has a significant effect on sPECAM-1 values in milk and sera of lactating mothers and their neonates; the lower values after caesarean section may reveal a deranged endothelial homeostasis.

The aim of this study was to assess the effect of a bovine in vitro culture system on blastocyst yield and quality after vitrification. In Experiment 1, IVM/IVF zygotes were cultured in either synthetic oviduct fluid (SOF) in 5% CO2, 5% O2, 90% N2; or TCM199-granulosa cells (TCM199-GCM) in 5% CO2 in air. In vivo blastocysts were used as a control. Culture in SOF resulted in a significantly higher blastocyst yield on both Day 7 (31.3 vs 13.2%, P < 0.001) and 8 (36.8 vs 23.7%, P < 0.001) than did culture in TCM199-GCM. After vitrification, survival at 72 h of in vivo blastocysts was significantly higher than both in vitro groups, while significantly more blastocysts produced in TCM199-GCM survived compared to those produced in SOF (0, 43.5, 78.3% for SOF, TCM199-GCM and in vivo, respectively P < 0.01). In Experiment 2, SOF-GCM proved to be the best post-warming culture system of those tested and was adopted as the post-warming medium for all subsequent experiments. In Experiment 3, zygotes were cultured in SOF or SOF-GCM, in either 5% CO2 in air, or 5% CO2, 5% O2, 90% N2. In agreement with Experiment 1, culture in SOF in 5% O2 resulted in significantly more blastocysts at Day 7 (26.4 vs 17.3%, P < 0.01) and Day 8 (31.5 vs 23.2%, P < 0.01) than did culture in SOF-GCM. However, survival at 72 h post vitrification was significantly higher for SOF-GCM (44 vs 8.3%, P < 0.001). Increasing the O2 concentration to 20% significantly reduced the blastocyst eld from SOF (31.5 vs 17.3%, P < 0.001). In addition, the quality of blastocyst produced was reduced in terms of survival post vitrification (8.3 vs 0%, P < 0.05). In contrast, there was no difference in blastocyst yield (23.2 vs 25.2%) or survival (44.0 vs 36.9%) in SOF-GCM, irrespective of O2 concentration. Experiment 4 examined the duration of exposure to GCM necessary to acquire improved blastocyst quality. Zygotes were cultured in SOF; SOF until Day 3, followed by SOF-GCM for the remainder of the culture; SOF until Day 5, followed by SOF-GCM for the remainder of the culture; or SOF-GCM for the entire culture. Survival at 72 h post vitrification was significantly higher (P < 0.05) in Groups 2 (50.0%, 13/26) and 4 (55.3%, 26/47) than in Groups 1 (21.7%, 10/46) and 3 (10.8%, 4/37). In conclusion, culture system can affect blastocyst yield and quality and crytolerance is a useful indicator of blastocyst quality.

Bovine blastocysts were produced using 6 different systems: 5 commonly used in vitro culture systems (synthetic oviduct fluid medium - SOF- without fetal calf serum, SOF supplemented with 10% serum for the entire culture period, SOF supplemented with 10% serum from Day 4 of culture, M199 coculture with bovine oviduct epithelial cells, M199 coculture with granulosa cell monolayer) and 1 in vivo culture system involving collection of blastocysts from superovulated bovine donors at Day 7. Zygotes obtained from IVM/IVF were assigned randomly to 1 of the 5 systems tested and were cultured for 9 d (Day 0= day of insemination). Cleavage, development to the blastocyst stage and blastocyst sex ratio were assessed in all treatments. In addition, the effect of the IVC system on the kinetics of blastocyst development and sex ratio was assessed on Days 6, 7, 8, and 9. The presence of fetal calf serum in SOF not only resulted in faster development (19.1% of blastocysts in SOF supplemented with serum vs 7.1% in absence of serum at Day 6; P < 0.05) and increased blastocyst production (47.5% of blastocysts in SOF supplemented with serum vs 34.4% in absence of serum; P < 0.05) but it also enhanced overall male survival. The coculture systems produced fewer blastocysts than culture in SOF (27.6 to 28.3% in coculture vs 47.5% in SOF supplemented with serum; P < 0.05), but similar to SOF without fetal calf serum, they had no effect on blastocyst sex ratio.

Both E- and L-selectin are cell adhesion molecules. E-selectin is expressed by activated endothelial cells, whereas L-selectin by quiescent leukocytes and is rapidly cleaved off after activation. Both selectins take part in the first step of the 'adhesion cascade', the 'rolling of leukocytes', leading to the extravasation of the white cells to the sites of inflammation, infection or damage. For this reason their soluble forms (sE- and sL-selectin, respectively), are considered early and reliable markers of the immune activation and response. Moreover, sE-selectin has been reported to be a potent angiogenic factor and a reliable marker of infection and sepsis in neonates, as well as endothelial activation, while sL-selectin of the leukocyte function and maturity. Following informed maternal consent, we evaluated prospectively by ELISA, sE- and sL-selectin in the serum of 40 (19 females, 21 males), healthy, term, infection-free neonates, on the second and fifth day of life, and compared them with the respective values in 20 healthy adults (10 females, 10 males), with the purpose of examining the pattern of their values in the early postpartum days, and to establish reference values for both selectins. Values (mean+/-S.D.) of sE-selectin both on the second (139+/-48 ng/ml) and fifth day of life (111+/-35 ng/ml) were found to be highly increased, as compared with those in controls (48+/-13 ng/ml; P<4 x 10(-11) and P<4 x 10(-10), respectively), while sL-selectin values on both the second (674+/-223 ng/ml) and the fifth day of life (684+/-221 ng/ml), were significantly lower than those in controls (938+/-181 ng/ml); P<0.0001 and P<0.0003, respectively). A significant decrease was noted in sE-selectin values, from the second to the fifth day of life (P<10(-7)), while sL-selectin values showed no significant change in the same time interval. A strong correlation was found between values on the second and the fifth day of life of both sE- and sL-selectin (r(P)=0.885 and r(P)=0.813, respectively; P<0.00001). Neonatal values of both sE- and sL-selectin on the second or on the fifth day of life, did not depend on the perinatal factors, neonatal sex, or birth weight, mode of delivery, and maternal age or parity. In conclusion, in the very early neonatal period, our findings of highly increased sE-selectin, while low sL-selectin, suggest an immune and more specifically endothelial activation and an immature and decreased leukocyte function.

Inflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were measured in the serum of healthy, term neonates on the first (N1), fifth (N5) and 40th (N40) day after birth, compared with those in maternal serum (MS), umbilical cord (UC) and in adult controls. All three cytokines were significantly elevated in N1 and N5, compared with those in UC and adults (P < 0.0001). IL-1beta and IL-6 declined significantly from N1 to N40 (P < 0.0001), while TNF-alpha increased significantly from N1 to N5 and declined thereafter. TNF-alpha values in UC were significantly higher than in adults, but lower than in N40 (P < 0.0001), while IL-1beta and IL-6 values in UC did not differ from those in N40 and in adults. IL-1beta and IL-6, but not TNF-alpha values in MS were significantly higher than those in controls (P < 0.0001). IL-1beta values in MS were significantly higher than those in N1 (P < 0.0001), while those of IL-6 and TNF-alpha were significantly lower (P < 0.0001). Moreover, IL- 1beta values were dependent on the mode of delivery in N1 (P < 0.001), in MS (P < 0.02) and in UC (0.03), while IL-1beta and TNF-alpha values in N1 were strongly interrelated (r = 0.7; P < 0.01). In conclusion, the increased values of IL-1beta, IL-6 and TNF-alpha during the perinatal period might reflect a newborn immune response to the stress of delivery and to environmental changes after birth.