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Oocyte/embryo selection methodologies are either invasive or noninvasive and can be applied at various stages of development from the oocyte to cleaved embryos and up to the blastocyst stage. Morphology and the proportion of embryos developing to the blastocyst stage are important criteria to assess developmental competence. Evaluation of morphology remains the method of choice for selecting viable oocytes for IVP or embryos prior to transfer. Although non-invasive approaches are improving, invasive ones have been extremely helpful in finding candidate genes to determine oocyte/embryo quality. There is still a strong need for further refinement of existing oocyte and embryo selection methods and quality parameters. The development of novel, robust and non-invasive procedures will ensure that only embryos with the highest developmental potential are chosen for transfer. In the present review, various methods for assessing the quality of oocytes and preimplantation embryos, particularly in cattle, are considered. These methods include assessment of morphology including different staining procedures, transcriptomic and proteomic analyses, metabolic profiling, as well as the use of artificial intelligence technologies.

During artificial insemination in bovine, the deposition of semen into the uterus results in an immune reaction which is based on polymorphonuclear neutrophils activity, including the formation of neutrophil extracellular traps. The formation of neutrophil extracellular traps as a reaction of neutrophils to spermatozoa was recently described. However, it is not completely clear which components of the semen are responsible for this reaction. The objective of this study was to quantify and compare the formation of neutrophil extracellular traps following in vitro incubation of bovine polymorphonuclear neutrophils with semen and extenders of different origins and conditions. We investigated the interactions between bovine polymorphonuclear neutrophils and different semen extenders, various seminal plasma concentrations from young and old bulls as well as sexed and non-sexed semen and their corresponding extenders. Three different semen extenders from two companies in fresh and frozen-thawed conditions were compared. Fresh semen extenders showed higher neutrophil extracellular traps induction than did frozen-thawed ones. The formation of neutrophil extracellular traps were also dependent on the presence of seminal plasma. We could show that seminal plasma alone, without any sperm cells, induced the reaction and that the addition of at least 1% seminal plasma already resulted in the formation of neutrophil extracellular traps. Furthermore, seminal plasma from young bulls led to significant higher neutrophil extracellular traps induction. No difference between non-sex-sorted and sex-sorted sperm and its extenders was observed. Taken together, different semen extenders as well as the amount and origin of seminal plasma influence neutrophil extracellular traps formation, whereas sex-sorted sperm did not affect the reaction.

After insemination of cows, either naturally or artificially, the deposition of semen into the vagina or uterus results in an immune reaction which is based on polymorphonuclear neutrophil activity. Sperm must be resistant to immune system actions of the female for an adequate time to allow fertilization to occur. Neutrophils, however, either directly phagocytize sperm through cell-cell attachment or entrap sperm cells in neutrophil extracellular traps (NETs), structures consisting of neutrophil nuclear DNA and associated proteins. In this review article, the interaction of neutrophils and sperm cells in t cattle will be described, with a special focus on the formation of neutrophil extracellular traps (NETs).

The composition of follicular fluid (FF) has an impact on the developmental capacity of the oocyte and the resulting embryo. FF is composed of blood plasma constituents which cross the blood follicular barrier and the secretory components of granulosa and theca cells. Moreover, it has been shown recently that follicular cells have the ability to synthesize bile acids (BAs). BAs are present in several fluids of mammals especially in bile, blood and urine. FF is an essential impacting factor on the oocyte quality and therefore resulting embryos. To achieve a better understanding of this subject, the presence and concentration of BAs were measured in fluid collected from bovine follicles, categorized according to their size, throughout two entire oestrus cycles and compared to those in blood and urine. The body fluids were collected during the same examination procedure and in total samples from four heifers were obtained. A broad spectrum of 11 BA derivatives was measured applying liquid chromatography–tandem mass spectrometry (LC‐MS/MS). The simultaneous and direct quantification of BAs in different body fluids of cattle are reported. Within the follicular fluid, blood and urine, cholic acid and glycocholic acid are the dominant BA subspecies irrespective of the oestrus cycle stage. Moreover, BA concentrations in blood compared to those in the FF were similar. For the first time these results clearly highlight the presence of different BA subspecies in FF, blood and urine during the oestrus cycle in cattle., Bile acids have been identified recently in follicular fluid of humans and cattle. To achieve a better understanding of bile acids during reproduction, the presence and concentration of bile acids were measured in fluid collected from bovine follicles throughout the oestrus cycle and compared to those in blood and urine.

Steroid hormones are regulators in the fine-tuned process of follicular development. During final maturation in vivo a switch from oestradiol (E2) to progesterone (P4) dominance within the follicle is well-described. This change is accompanied by the resumption of meiosis and results in the maturation of the oocyte. It also suggests the important role of these hormones. However, present in vitro maturation (IVM) systems do not completely mimic the in vivo situation, resulting in oocytes of reduced quality. Aim of the study was to determine the temporal pattern of steroid hormone concentrations in the IVM medium of bovine cumulus-oocyte-complexes (COC) at defined time points. The influence of different gonadotropin supplementations during IVM on oocyte maturation, as well as the molecular quality of the oocytes and their corresponding cumulus cells was investigated. COCs were obtained from abattoir-derived ovaries and matured in medium added with different compounds of gonadotropins (eCG/hCG; FSH/LH, each at 0.05 IU or 0.01 IU; only FSH; without gonadotropins) employing a standard protocol without oil overlay. In experiment 1, medium, oocytes and cumulus cells were collected at different time points (0 h [control], 4 h, 8 h, 12 h, 16 h, 20 h, 24 h) after IVM in just eCG/hCG-supplemented medium. In experiment 2, medium, oocytes and cumulus cells were collected at 0 h (control) and after 24 h of IVM with all above-named supplements. The E2 concentration remained similar during IVM whereas P4 concentration increased during experiment 1. No significant changes could be determined after the addition of different gonadotropins (experiment 2). These results suggest that during IVM the temporal pattern of E2 and P4 did not correspond with the pattern during final maturation in vivo. RT-qPCR was used to assess the relative abundance of developmentally important genes in oocytes (BMP15; GDF9; ZAR1; PGR; PGRMC1/2; G6PD; StAR; ESR1/2; SULT1E1; STS; SOAT) and cumulus cells (ESR1/2; FSHR; LHCGR; CYP19A1; HSD3B1; PGR; PGRMC1/2; SULT1E1; STS; SOAT) at all collection points in both experiments. Most transcripts follow a time-regulated mRNA expression pattern during the entire in vitro maturation period. In addition, the expression of the analyzed transcripts was not influenced by the different gonadotropin supplementations during the IVM period. In all, this underlines that present conditions of IVM do not reflect the in vivo situation and require further optimisation.

Preimplantation epigenetic reprogramming is sensitive to the environment of the gametes and the embryo. In vitro maturation (IVM) of bovine oocytes is a critical step of embryo in vitro production procedures and several factors influence its efficiency, including atmospheric oxygen tension. The possibility that the IVM environment can alter this process is tested by determining whether the global DNA methylation pattern (measured via immunofluorescent labeling of 5-methylcytosine [5meC]) in the parental pronuclei of bovine zygotes produced from cumulus-oocyte complexes matured under low (5%) and atmospheric (~20%) oxygen tension. Normalized 5meC signals differed significantly between maternal and paternal pronuclei of oocytes matured in vitro at 5% oxygen (p ≤ 0.05). There was a significant difference of 5meC between maternal pronuclei of oocytes matured at 5% oxygen and 20% oxygen ( p ≤ 0.05). The relative methylation level (normalized fluorescence intensity of paternal pronucleus divided by the normalized fluorescence intensity of maternal pronucleus) subsequent to maturation in vitro at 5% and 20% oxygen was also significantly altered ( p ≤ 0.05). Our results show that the pattern of global DNA methylation in the maternal pronucleus of bovine zygotes is affected by maturing the oocytes under low oxygen tension which may have an impact on early embryonic development. These data may contribute to the understanding of possible effects of IVM conditions on pronucleus reprogramming.

Metabolic syndrome (MetS), which is associated with chronic inflammation, predisposes males to hypogonadism and subfertility. The underlying mechanism of these pathologies remains poorly understood. Homozygous leptin-resistant obese db/db mice are characterized by small testes, low testicular testosterone, and a reduced number of Leydig cells. Here we report that IL-1β, CCL2 (also known as MCP-1), and corticosterone concentrations were increased in the testes of db/db mice relative to those in WT controls. Cultured murine and human Leydig cells responded to cytokine stress with increased CCL2 release and apoptotic signals. Chemical inhibition of CCL2 rescued Leydig cell function in vitro and in db/db mice. Consistently, we found that Ccl2-deficient mice fed with a high-energy diet were protected from testicular dysfunction compared with similarly fed WT mice. Finally, a cohort of infertile men with a history of MetS showed that reduction of CCL2 plasma levels could be achieved by weight loss and was clearly associated with recovery from hypogonadism. Taken together, we conclude that CCL2-mediated chronic inflammation is, to a large extent, responsible for the subfertility in MetS by causing damage to Leydig cells., MCP-1/CCL2 upregulation associates with metabolic syndrome–induced male subfertility in both mice and men.

The reliable detection of estrus is an important scientific and practical challenge in dairy cattle farming. Female vocalization may indicate reproductive status, and preliminary evidence suggests that this information can be used to detect estrus in dairy cattle. The aim of this study was to associate the changes in the vocalization rate of dairy heifers with behavioral estrus indicators as well as test the influence of the type of estrus (natural estrus vs. superovulation-induced estrus). We analyzed 6 predefined estrus-related behavior patterns (standing to be mounted, head-side mounting, active mounting, chin resting, being mounted while not standing, and active sniffing in the anogenital region) and vocalization rates in the peri-estrus period (day of estrus ± 1 d) of 12 German Holstein heifers using audio-visual recordings. Each heifer was observed under natural estrus and a consecutive superovulation induced by FSH and cloprostenol. Estrus was determined by behavioral patterns and confirmed by clinical examination (vaginoscopy and ultrasound imaging of the ovaries) as well as by the concentration of peripheral progesterone. Estrus behavior and vocalization rates were analyzed in 3-h intervals (an average of 19 intervals for each heifer), and an estrus score was calculated based on the 6 behaviors. The interval with the highest estrus score (I0) was considered the estrus climax. We demonstrated similar time courses for the estrus score and vocalization rate independent of estrus type. However, in natural estrus, the maximum vocalization rate (±SE) occurred in the interval before estrus climax (I−1; 42.58 ± 21.89) and was significantly higher than that in any other interval except estrus climax (I0; 27.58 ± 9.76). During natural estrus, the vocalization rate was significantly higher within the interval before estrus climax (I−1; 42.58 ± 21.89 vs. 11.58 ± 5.51) than under superovulation. The results underscore the potential use of vocalization rate as a suitable indicator of estrus climax in automated estrus detection devices. Further studies and technical development are required to record and process individual vocalization rates.

Sulfated steroid hormones, such as dehydroepiandrosterone sulfate or estrone-3-sulfate, have long been regarded as inactive metabolites as they cannot activate classical steroid receptors. Some of them are present in the blood circulation at quite high concentrations, but generally sulfated steroids exhibit low membrane permeation due to their hydrophilic properties. However, sulfated steroid hormones can actively be imported into specific target cells via uptake carriers, such as the sodium-dependent organic anion transporter SOAT, and, after hydrolysis by the steroid sulfatase (so-called sulfatase pathway), contribute to the overall regulation of steroid responsive organs. To investigate the biological significance of sulfated steroid hormones for reproductive processes in humans and animals, the research group "Sulfated Steroids in Reproduction" was established by the German Research Foundation DFG (FOR1369). Projects of this group deal with transport of sulfated steroids, sulfation of free steroids, desulfation by the steroid sulfatase, effects of sulfated steroids on steroid biosynthesis and membrane receptors as well as MS-based profiling of sulfated steroids in biological samples. This review and concept paper presents key findings from all these projects and provides a broad overview over the current research on sulfated steroid hormones in the field of reproduction.

To circumvent the negative impacts of in vitro culture on bovine embryos, we have recently established a new method, the so called intra-follicular oocyte transfer (IFOT), enabling in vivo fertilization and in vivo development of in vitro matured oocytes up to the blastocyst stage as well as to term. In this study, we raised the question whether immature bovine oocytes could also be transferred into a pre-ovulatory follicle to support in vivo maturation prior to subsequent in vivo fertilization, in vivo development as well as to term. To unravel that question, a total of 791 immature oocytes were transferred in groups of ∼50 into pre-ovulatory follicles of 16 recipient heifers. Consequently, we were able to recollect a total of 306 structures 8 days thereafter (38.5%). All in all, 12 heifers (75%) gave embryos developed to the morula or blastocyst stage in addition to the expected native embryos. Among all recollected structures, 40.1% had developed to the morula and/or blastocyst stage, meaning a total efficiency of 17.3% based on all transferred oocytes. Of impact, IFOT-embryos reached significantly higher developmental rates to the Morula and/or blastocyst stage until day 7 compared to in vitro cultured control embryos, despite being derived from the same charge of slaughterhouse ovaries (40.1 vs. 29.3%). This implicates a beneficial effect of the follicular environment for the intrinsic quality of the fertilized embryos during maturation and for subsequent developmental rates up to the blastocyst stage. Finally, the birth of two healthy calves after transfer of frozen-thawed IFOT-derived blastocysts to final recipients established the first proof of principle that IFOT of immature bovine oocytes generates bovine blastocysts bearing developmental capacity to term. Likewise, to the best of our knowledge, these calves are the first calves derived from full in vivo development of immature slaughterhouse derived oocytes. Thus, the results of the present study clearly demonstrate that IFOT of immature slaughterhouse-derived oocytes is now a feasible technique. Since efficiencies following IFOT achieved within the present study were improved compared to previous studies, IFOT now offers an attractive option for designing new scientific experiments.

After in vivo fertilisation, the preimplantation embryo goes through cleavage during migration along the oviduct in mammals or the fallopian tube in a woman and ends up inside the uterus. This study investigates the effect of a protocol aimed at closely reproducing that natural oxygen concentration in the oviduct (7 % O2 from day 1 to day 3 and 2 % from day 3 to day 5), in contrast to the concentrations (5 % or 20 %) widely used in practice in ART using morphokinetic. Female mice (BI6/CBAca) were sacrificed, and zygotes were isolated 20 h after mating and randomly allocated to three parallel groups, which were grown under high atmospheric, low, or sequential oxygen concentrations. Zygotes were cultured in GTL medium (Vitrolife) and observed by the Primovision time-lapse system. Blastocyst rate at 120 h in the sequential group (91.3 %) was significantly increased over the high (76.3 %) and low (74.4 %) groups. Blastocyst size was also enlarged in the sequential group compared to the high and low groups. Moreover, cell division in the sequential group was significantly faster at almost every cleavage stage than it was in the other groups. Notably, the duration of the interims between stages also differed significantly between the groups. This study demonstrated that, in comparison to routinely used high or low oxygen conditions, oxygen concentrations mimicking changes in the oviduct and uterus significantly improve the blastocyst rate and size and accelerate cell division at several stages as well as the interims between cleavage events.

Deposition of sperm during artificial insemination in the bovine female reproductive tract results in early host innate immune reactions of polymorphonuclear neutrophils (PMNs). Furthermore, sperm-mediated neutrophil extracellular trap (NET) formation (NETosis) was recently reported to occur in different mammalian species, including humans. We, here, investigated the interactions of bovine PMN with different semen-derived samples and analyzed in more depth molecular aspects of this effector mechanism. Overall, confrontation of PMN with sperm/cell preparation (SCP) resulted in a rapid and dose-dependent NET formation leading to effective spermatozoa entrapment. Thereby, spermatozoa induced different phenotypes of NETs. Immunostaining analyses revealed the presence of histones (H3), neutrophil elastase (NE), and pentraxin (PTX) in sperm-triggered NET structures. Fresh SCP strongly induced NETosis than frozen-thawed ones. The level of NETosis was not related to spermatozoa viability. SCP as well as purified sperm cells (SCs) and supernatant (SN) induce NETosis, although the reaction in SC was lower. Enhanced levels of oxygen consumption and proton leak in PMN revealed sperm SNs but not purified SCs as PMN activators. Functional inhibition experiments revealed sperm-triggered NETosis as an NADPH oxidase- and peptidylarginine deiminase 4-dependent process and proved to be dependent on intra- and extracellular Ca++ influxes while myeloperoxidase activity and as ERK1/2- and PI3K-related signaling pathways did not seem to play a pivotal role in this effector mechanism. From these findings, we speculate that sperm-derived NETosis might also occur in vivo during artificial insemination and might therefore play a role related to reduced fertility.

Supplementation of rumen-protected amino acids (RPAA) has proven to be an effective tool to supply limiting AA in dairy diets. Methionine and lysine are the two most limiting AA for lactating dairy cows. Recently, it has been shown that methionine supplementation seems to affect pre-implantation embryos collected from superovulated cows enhancing their developmental competence because there is strong evidence that endogenous lipid reserves serve as an energy substrate (Acosta et al. 2016 Theriogenology 85, 1669–1679). Moreover, higher concentrations of methionine were determined in the follicular fluid of the first dominant follicle postpartum in cows supplemented with rumen-protected methionine and rumen-protected choline from 21 days before calving to 30 days postpartum and it was assumed that higher methionine concentrations in the follicular fluid could affect oocyte quality (Acosta et al. 2017 Theriogenology 96, 1–9). There is no information available so far regarding the effect of a combined methionine and lysine supplementation (each rumen-protected) on oocyte quality. Therefore, the objective of this study was to evaluate the effect of a combined methionine and lysine supplementation during early to mid-lactation on the developmental competence of oocytes collected from lactating dairy cows (days 0 to 100 p.p.). Thirty pregnant multiparous German Holstein dairy cows were grouped 3 weeks before their expected calving date, receiving identical diets. After calving, they were randomly allocated to 2 groups fed a total mixed ration supplemented with (N = 14 cows; RPAA) or without (N = 16 cows; CON) LysiGEMTM (encapsulated lysine; Kemin Industries) and Metasmart DryTM (isopropyl ester of the hydroxylated analogue of methionine adsorbed onto a silicon dioxide carrier; Adisseo). Starting from 45 days p.p., animals from both groups were submitted to an ovum pickup (OPU) session once a week for at least 8 weeks. Collected cumulus–oocyte complexes (COC) were subjected to a standard invitro production (IVP) protocol (Stinshoff et al. 2014 Reprod. Fertil. Dev. 26, 502–10) including IVM, IVF, and invitro culture (IVC). Cleavage and developmental rates up to the morula/blastocyst stage were recorded on Days 3, 7, and 8. In total, 1211 follicles have been aspirated from RPAA animals compared with 1413 from CON animals, from which 742 and 885 COC were collected, respectively. The calculated recovery rate based on the number of aspirated follicles and collected COC was similar for both groups (61.3 ± 29.4% vs. 62.6 ± 33.5%). Cleavage and developmental rates based on 240 (RPAA group) and 299 (CON group) COC also showed similar results [RPAA: 84.1 ± 5.9% (202/240), 18.3 ± 4.4% (44/240), 18.8 ± 4.7% (45/240); CON: 81.9 ± 8.6% (245/299), 15.4 ± 8.9% (46/299), 16.7 ± 8.4% (50/299)]. In conclusion, supplementation of RPAA (methionine and lysine) had no beneficial effect on the developmental competence of COC obtained from these animals compared with those collected from cows fed the diet without RPAA supplementation.

Over the past decades in vitro production (IVP) of bovine embryos has been significantly improved. Nevertheless, embryos generated in vitro still differ from their in vivo produced counterparts. Embryos must adjust to multiple microenvironments at preimplantation stages. Consequently, maintaining or mimicking the in vivo situation in vitro will aid to improve the quality and developmental competence of the resulting embryo.

Under natural conditions, mammalian oocytes and zygotes are never exposed to daylight or artificial light. During assisted reproductive procedures such as invitro production (IVP), oocytes and pre-implantation embryos are exposed to light from time to time before being transferred to recipients. The detrimental effect of visible light is not directly related to the intensity and exposure time, but is also a function of the spectral composition of the light. Recently, a green pass light filter was used during all steps of bovine IVP. Results indicated the protective effect of the filter against harmful blue and infrared regions of the light (Korhonen et al. 2009 Hum Reprod. 24, 308-314; https://doi.org/10.1093/humrep/den432). Data regarding either blue or infrared light are missing. Therefore, the effect of environmental light will be examined on the developmental capacity of bovine oocytes and pre-implantation embryos produced invitro. Three different experimental conditions were set during all IVP steps: (i) light from the microscope filtered with a red pass filter, no artificial light, and no daylight, (ii) artificial light without day light, light from the microscope without a filter, and (iii) artificial and daylight, light from the microscope without a filter, as normally used in the laboratory (control). Bovine cumulus-oocyte complexes were recovered from abattoir-derived ovaries by the slicing method. After 24h of maturation, fertilisation was realised and after 19h of co-culture of cumulus-oocyte complexes and sperm, presumptive zygotes were cultured in SOFaa for 8 days. Cleavage and developmental rates were recorded at Day 3 and Day 7/8 (Day 0=IVF, 10 IVP runs). Blastocysts from all groups were individually stored at −80°C until analyses. Then RT-qPCR (at least 12 replicates) was performed as described previously (Stinshoff et al. 2014 Reprod. Fertil. Dev. 26, 502-510; https://doi.org/10.1071/RD12372) for gene transcripts related to cellular stress (BAX, BCL2L1, HSPA1A, SOD1) and embryo developmental capacity (SLC2A3). The morphological quality of expanded blastocyst was assessed via a differential staining procedure (with propidium iodide and Hoechst, 4 replicates) to determine the number of inner cell masses (ICM), trophectoderms (TE), and total cells and to calculate the ICM/TE ratio. Data were analysed by analysis of variance followed by a Tukey test. Cleavage and developmental rates were similar in all groups of embryos (P>0.05). The relative abundance of all analysed gene transcripts was significantly increased (P ≤ 0.05) in blastocysts stemming from settings (i) and (ii) compared with those from the control group. Blastocysts generated using only artificial light or artificial and daylight did show a significantly increased number of ICM and total cells (86.3±3.2, 56.3±1.4; 183.6±4.0, 161.8±2.8) compared with embryos produced under filtered microscope light without any other light (35.3±0.8; 133.8±1.4; P ≤ 0.05). The number of TE cells was similar (P>0.05) in blastocysts of all groups (i: 98.5±1.3; ii: 97.3±2.6; iii (control): 105.5±2.4). These data suggest that day light exposure might influence bovine embryos at the molecular level, whereas the morphological quality seems to be unaffected when compared with embryos exposed to only artificial light.

The objective of this study was to investigate the effect of long-term exposure to a Fusarium toxin deoxynivalenol (DON, 5 mg/kg DM) on the energy metabolism in lactating cows fed diets with different amounts of concentrate. In Period 1 27 German Holstein cows were assigned to two groups and fed a control or mycotoxin-contaminated diet with 50% concentrate for 11 weeks. In Period 2 each group was further divided and fed either a diet containing 30% or 60% concentrate for 16 weeks. Blood samples were collected in week 0, 4, 8, 15, 21, and 27 for calculation of the Revised Quantitative Insulin Sensitivity Check Index and biopsy samples of skeletal muscle and the liver in w 0, 15, and 27 for analysis by real-time RT-qPCR. The DON-fed groups presented lower insulin sensitivities than controls at week 27. Concomitantly, muscular mRNA expression of insulin receptors and hepatic mRNA expression of glucose transporter 2 and key enzymes for gluconeogenesis and fatty acid metabolism were lower in DON-fed cows compared to the control. The study revealed no consistent evidence that DON effects were modified by dietary concentrate levels. In conclusion, long-term dietary DON intake appears to have mild effects on energy metabolism in lactating dairy cows.

To evaluate the effects of incubator door openings by the sensitive measurement of morphokinetics using time-lapse imaging.To mimic lab practice and to evaluate minimal changes in culture conditions a randomized parallel group study with mouse embryos was designed. 249 murine zygotes derived from 31 mice were randomly distributed into two groups. Embryos of the control group were non-invasively monitored with a Primovision time-lapse system (Vitrolife) in the incubator that was completely closed for 5 days, while the conditions for the embryos in the treatment group were interrupted by two door openings per day of 10 s each.Door opening twice per day did not lead to significant differences in blastocyst formation rate (p = 0.820), however significantly more embryos hatched in a shorter duration (p = 0.011), and the embryos transitioned more quickly from 2C to 3C, from 3C to 4C, from 2C to 5C and from 5C to blastocyst stage (p 0.001 in cc2, p = 0.037 in cc3, p = 0.002 in t2 to t5, and p = 0.015 in t5 to blastocyst). Size of the blastocysts from the treatment group was 1.562 μm smaller compared to that of control embryos (p = 0.016).Even disruptions in culture conditions as simple as incubator door openings lead to measurable, significant changes in morphokinetics that would have been missed with standard parameters such as blastocyst rate. This underlines the relevance of undisturbed and stable culture conditions and emphasizes the need for further research in this field.

Over the past decades in vitro production (IVP) of bovine embryos has been significantly improved and in particular bovine IVP is now widely applied under field conditions. This in vitro technique provides new opportunities for cattle producers, particularly in the dairy industry, to overcome infertility and to increase dissemination of animals with high genetic merit. Improvements in OPU/IVP resulted in large-scale international commercialization. More than half a million IVP embryos are generated on the yearly basis demonstrating the enormous potential of this technology. These advances and the fact that bovine and human early development is remarkably similar have also prompted the use of bovine embryos as a model system to study early mammalian embryogenesis including humans. Despite all the improvements, embryos generated in vitro still differ from their in vivo derived counterparts. Embryos must adjust to multiple microenvironments at preimplantation stages. Consequently, maintaining or mimicking the in vivo situation in vitro will aid to improve the quality and developmental competence of the resulting embryo. The successful clinical application of the techniques in reproductive biotechnology requires both species-specific clinical skills and extensive laboratory experience. The recent advances in transcriptome profiling have also provided deeper insight into RNA expression and regulation at an unprecedented resolution. The development of these high-throughput DNA sequencing methods has resulted in new approaches for both mapping and quantifying transcriptomes. The aim of this review is therefore to summarize the available data related to gene expression analyses as well as in vitro embryo production procedures and to provide ideas about future concepts.

Oxidative stress in spermatozoa has effects on subsequent embryo development. The aim of the present study was to elucidate whether sperm oxidative stress results in increased DNA damage in the embryo. To this end, bovine spermatozoa were incubated for 1 h at 37°C without or with 100 µM H2O2, resulting in non-oxidised (NOX-S) and oxidised (OX-S) spermatozoa respectively. Non-incubated spermatozoa served as the control group (CON-S). After IVF, developmental rates 30, 46 and 60 h and 7 days after IVF were assessed. DNA damage was analysed in embryos using the comet assay and a DNA damage marker (γH2AX immunostaining); the apoptotic index was determined in blastocysts. Exposure of spermatozoa to H2O2 induced a significant amount of sperm chromatin damage. The use of OX-S in IVF resulted in significantly reduced cleavage and blastocyst rates compared with the use of CON-S and NOX-S. Furthermore, in embryos resulting from the use of OX-S, a developmental delay was evident 30 and 46 h after IVF. γH2AX immunostaining was lower in blastocysts than in early embryos. In blastocysts, the comet and apoptotic indices were significantly higher in embryos resulting from the use of OX-S than CON-S and NOX-S. In conclusion, oxidative stress in spermatozoa induces developmental abnormalities and is a source of DNA damage in the resulting embryos.

Over the past decades, in vitro production (IVP) of bovine embryos has been significantly improved, and in particular bovine IVP is now widely applied under field conditions. This in vitro technique provides new opportunities for cattle producers, particularly in the dairy industry, to overcome infertility and to increase dissemination of animals with high genetic merit. Improvements in OPU/IVP resulted in large-scale international commercialization. More than half a million IVP embryos are generated on the yearly basis demonstrating the enormous potential of this technology. These advances and the fact that bovine and human early development is remarkably similar have prompted the use of bovine embryos as a model system to study early mammalian embryogenesis including humans. In horses, OPU/IVP is also an established procedure for breeding infertile and sports mares throughout the year. It requires ICSI because conventional IVF does not work in this species. In small ruminants, application of IVP on the commercial and research basis is low compared to other livestock species.

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

Pituitary growth hormone (GH) release and hepatic insulin-like growth factor-I (IGF-I) production increase after an injection of 17β-estradiol (E2) in ovariectomized dairy cattle. However, whether endogenous sexual steroid hormones also influence the hepatic GH receptor (GHR) signaling pathway during a physiological estrus cycle remains unclear. The aim of this study was to analyze the hepatic GHR signaling pathway during the luteal phase and after a period of increased E2 concentrations (after ovulation) as well as in 7 heifers before ovulation. Ovarian ultrasounds were performed daily during repeated physiological cycles (n = 56) of 30 Holstein Friesian heifers to determine ovulation [before ovulation (n = 7, bOv) and after ovulation 24-60 h after the appearance of estrus signs (n = 49, aOv)] and luteal phase (CLP; d 12 ± 1 after ovulation). Blood samples and liver biopsies were obtained, and blood concentrations of E2, P4, insulin-like growth factor (IGF)-I, IGF-II, and GH were measured. In the liver biopsies, we determined mRNA expression of the estrogen receptor α (ERα), GHR, Janus kinase 2 (JAK2), signal transducer and activator of transcription 5B (STAT5B), suppressor of cytokine signaling (SOCS)2 and 3, IGF-I, and IGF-II by quantitative reverse transcription-PCR. The concentration of E2 was higher bOv than aOv and CLP, as expected. The concentrations of IGF-I and GH were higher bOv and aOv compared with CLP. In contrast, concentrations of IGF-II were lower aOv compared with bOv and CLP. The mRNA expression of GHR was higher in liver biopsies obtained bOv compared with aOv and CLP. Notably, the expression of SOCS2 was higher bOv than aOv and in the CLP. Increased hepatic expression of SOCS2 during estrus was detectable when IGF-I concentrations were high; this result might indicate that SOCS2 expression attenuates the GHR signal transduction pathway during the phase of increased pituitary GH release. In conclusion, hepatic GHR and SOCS2 mRNA expression appeared to be promptly and sensitively regulated by increased E2 levels before ovulation of dairy heifers.

The ability of bovine embryos to develop to the blastocyst stage and to implant and generate healthy offspring depends greatly on the competence of the oocyte. Oocyte competence is attributed to its close communication with the follicular environment and to its capacity to synthesize and store substantial amounts of messenger RNA. Higher developmental competence of bovine oocytes has been associated with both the expression of a cohort of developmental genes and the concentration of sex steroids in the follicular fluid. The aim of this study was to identify differences in the expression of FST in cumulus cells and OCT-4 and MATER in oocytes and the influence of the follicular progesterone and follicular estrogen concentration on the competence of bovine oocytes retrieved 30 minutes or 4 hours after slaughter. Cumulus-oocyte complexes (COCs) were left in postmortem ovaries for 30 minutes (group I) or 4 hours (group II) at 30 °C. Aspirated oocytes were then subjected to IVM, IVF, and IVC or were evaluated for MATER and OCT-4 messenger RNA abundance by quantitative real-time polymerase chain reaction. Total RNA was isolated from pools of 100 oocytes for each experimental replicate. Progesterone and estradiol concentration in follicular fluid was evaluated by immunoassay using an IMMULITE 2000 analyzer. Three repeats of in vitro embryo production were performed with a total of 455 (group I) and 470 (group II) COCs. There were no significant differences between the cleavage rates (72 hours postinsemination [hpi]) between both groups (63.5% vs. 69.1%). However, blastocyst (168 hpi) and hatching (216 hpi) rates were higher (P0.05) in group II compared with those of group I (21.3% vs. 30.7% and 27.6% vs. 51.5%, respectively). Group II oocytes exhibited the highest MATER and OCT-4 abundance (P0.05). Follicular estradiol concentration was not different between both the groups, whereas the progesterone concentration was lower (P ≤ 0.05) in group II follicles. These results indicate that retrieving COCs 4 hours after slaughter could increase bovine in vitro developmental competence, which is linked to higher levels of oocyte MATER and OCT-4 transcripts and lower follicular progesterone concentration. Moreover, the results of the present study contribute to the identification of factors involved in the developmental competence of immature oocytes.

Historically sulfonated steroids were primarily considered as inactive metabolites destined for elimination. However, more recently they have been increasingly recognized as precursors for the production of bioactive steroids in target tissues and as functional molecules without preceding hydrolysis. In order to comprehensively characterize their occurrence in cyclic cows and their formation and hydrolysis in bovine ovarian steroidogenesis, ovaries from cyclic cows were screened for the expression of oestrogen sulfotransferase (SULTE1) and steroid sulfatase (STS) by Western blot and immunohistochemistry. Moreover, a broad spectrum of 13 sulfonated steroids was measured applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) in blood samples collected from three cycling heifers during defined stages of the ovarian cycle and in fluid obtained from ovarian follicles of different size. SULT1E1 was undetectable in ovarian tissues. For STS only a weak immunostaining was found predominantly in granulosa cells of larger follicles. However, no specific band occurred in Western blot. In blood, concentrations of all sulfonated steroids investigated were below the limit of quantification (LOQ). In follicular fluid, only cholesterol sulfate was measured in considerable concentrations (328.3 ± 63.8 ng/ml). However, the role of cholesterol sulfate in bovine follicular steroidogenesis remains unclear as concentrations were obviously unrelated to follicular size. The remaining sulfonated steroids investigated were undetectable or only slightly exceeded LOQ in a minor proportion of samples. The results are clearly contrary to a role of sulfonated steroids as important precursors, intermediates or products of bovine ovarian steroidogenesis.

The sex ratio of newborn calves and embryos produced in vivo is ~1:1. However, numerous studies on bovine in vitro-produced embryos suggest that the sex ratio may differ from 1:1 and that the rate of development may be influenced by the sex of the embryo under certain culture conditions. The duration of sperm-oocyte interaction and sperm pre-incubation also affect the sex ratio of bovine embryos produced in vitro. It is well documented that in vitro male embryos reach the more advanced stages earlier than do their female counterparts. Selection of developmentally more advanced embryos in anticipation that they have a greater developmental capacity may be one of the underlying causes of the disproportionate number of males among offspring born after transfer of in vitro-produced embryos. The aim of the present study is to test whether a pre-incubation of sperm before IVF might improve the developmental rates and also influence the sex ratio of the resulting embryos. Bovine cumulus-oocyte complexes were recovered from abattoir-derived ovaries by the slicing method. After 24h of maturation, fertilization was realised using a standard protocol. Prior to IVF, sperm cells from 2 different bulls were treated as follows: sperm within group A were pre-incubated in IVF medium for one hour. This step was omitted for sperm in group B (control). After 19h of co-culture of COC and sperm, presumptive zygotes were cultured in SOFaa for a period of 7 days. Cleavage and developmental rates were recorded at Day 3 and 7 (Day 0=IVF). Day 7 blastocysts from all groups were sexed using bovine and Y chromosome-specific primers. Data were analysed by ANOVA. As shown in Table 1, sperm pre-incubation did not affect the cleavage and developmental rates for the individual bull (P>0.05). On average, at Day 7 of development a higher number of blastocysts was determined when embryos had been produced from pre-incubated sperm (P ≤ 0.05). This held true for both bulls. The shift in favour of male embryos was detectable in all groups of embryos, with a drastic one for bull 1 after sperm pre-incubation. In conclusion, sperm pre-incubation accelerated embryo development and possibly enhanced the proportion of male embryos, which was already shifted toward males. Table 1.Developmental rates, developmental kinetics and sex ratio of embryos after sperm pre-incubation before IVF (mean±standard deviation)

Zielsetzung: Ziel des Projektes war die Entwicklung eines Mikroskops zur kontinuierlichen digitalen Fotografie der Eizell- und Embryonalentwicklung in vorhandenen Inkubatorsystemen mit innovativen Analysemoglichkeiten im Vergleich zu bereits erhaltlichen kommerziellen Systemen. Durch Einsatz eines Polarisationsfilters und einer neu entwickelten Fokussierungstechnik mit einer quasi 3D-Abbildungstechnik, ergeben sich vollig neue Dimensionen der Beurteilung eines Embryos und seiner Praimplantationsentwicklung. Es werden, unter anderem, spezifische Beurteilungen der Polkorper, des Spindelapparates und der Zona pellucida ermoglicht die neue Einblicke in Zellteilung- und Entwicklungsprozesse bieten. Material und Methoden: Zur Beobachtung unter Inkubatorbedingungen wurde ein konfokales Mikroskopiesystem von Grund auf neu entworfen und gebaut. Darin wurde neben einer elektrisch fokussierbaren Polymerlinse, die dazu verwendet wird verschiedene Ebenen der Oozyten aufzunehmen, eine Polarisierungstechnik implementiert. Diese Technik wurde zuerst von Shribak und Oldenbourg in 20031 vorgestellt und ermoglicht es durch nachtragliche computerbasierte Bildverarbeitung besonders geringe Doppelbrechungen – wie es bei Oozyten oder biologischen Praparaten im Allgemeinen haufig der Fall ist – sichtbar zu machen. Ergebnisse: Der derzeit genutzte Prototyp des Mikroskops und der Software liefert stabile Bilderserien und es lassen sich die verschiedenen doppelbrechenden Strukturen im zeitlichen Verlauf dokumentieren. Das Mikroskop verbleibt dabei im Inkubator und die Zellentwicklungsraten (Blastozystenraten) sind nicht unterschiedlich zu denen der Vergleichsgruppe. Zusammenfassung: Anhand der ersten Ergebnisse konnen nun weitere Experimente zur Verifikation der dargestellten Strukturen und Korrelationen zu derzeit verwendeten „time-lapse score's“ erfolgen.

Neutrophils are recruited into the female reproductive tract following insemination to eliminate excess spermatozoa and bacteria introduced by the breeding process. In natural breeding, bovine semen is deposited in the vagina and sperm migrate into the uterus, leaving the bulk of seminal plasma (SP) behind. Current artificial insemination protocols introduce variable amounts of SP and semen extender into the uterus. Beside phagozytosis and secretion of immune modulators, polymorphonuclear neutrophils (PMN) are able to form neutrophil extracellular traps (NETs) extruding their DNA into the extracellular environment and ensnare pathogens including sperm cells. Recently, a time-dependent increase in bovine NETs formation has been reported. In the absence of SP and extender only a low degree of NETs formation by the sperm cells alone was detected (Fichtner et al. 2017 Reprod. Domest. Anim. 52(S1), 16). The aim of the present study was to investigate the effect of different semen extenders with various supplements or varying SP concentrations on NETs formation. Bovine PMN were isolated via Ficoll gradient centrifugation from peripheral blood. Semen extenders, purchased from 2 companies, were supplemented either with no animal protein or fresh egg yolk or “egg yolk-like substances” (gamma-irradiated sterile egg yolk or phospholipids as liposomes). The SP collected from ejaculates of eight 5-year-old bulls was added to the incubation medium in concentrations of 1, 3, 5, 10, 15, and 20%. For NETs induction, PMN and semen extenders or SP concentrations were co-cultured for 60 min; PMN and incubation medium alone served as negative controls. After incubation, the samples were treated with micrococcal nuclease and stained with PicoGreen. Quantification was performed by spectrofluorometric analyses using an automated plate monochrome reader (Varioscan Flash; Thermo Scientific, Waltham, MA, USA). Relative fluorescence intensities (FI in arbitrary units, AU) were calculated by subtracting the values of the negative controls from the ones obtained from the different samples. For statistical analyses, a one-way ANOVA followed by Tukey’s HSD test was used. Experiments were repeated at least 9 times. No significant differences in the relative FI were detected between the 2 animal protein-free extenders. A significantly higher FI was observed in one of the extenders supplemented with egg yolk compared with the other. The same held true for one extender completed with an egg yolk-like substance. Relative FI significantly increased from 1 to 5% SP, stayed constant up to 10%, followed by a slight decrease up to a concentration of 20%. These data indicate that formation of NETs in vitro depends on the composition of the extender itself and the protein source used by different companies. Furthermore, formation of NETs is also dependent on the dose of SP. From these results, it could be speculated that semen extenders plus additives and SP may affect fertility. The financial support of the Förderverein Bioökonomieforschung e.V. (FBF) is gratefully acknowledged.

Bile acids (BAs) are present in follicular fluid (FF) from humans and cattle. This fact has triggered an interest on the role BAs might play in folliculogenesis and their possible association with fertility. To achieve a better understanding about this subject, new methods are needed to provide reliable information about concentrations of the most important BAs in FF. In this context, liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers high specificity with a relatively simple sample workup. We developed and validated a new assay for the quick profiling of the 9 most abundant BAs in follicular fluid from cattle. The method uses 200μl of FF and can quantify cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and their glycine (G) and taurine (T) conjugates. Lithocholic acid (LCA), its conjugates GLCA and TLCA, and sulfated forms, were present in some samples, but their concentration was low compared to other BAs (in average, below 60ng/ml for LCA, GLCA or TLCA and below 20ng/ml for their corresponding sulfates). Method performance was studied at three quality controls for each compound in consonance with their physiological concentration. Excellent linearity and recovery were found for all compounds at every control level. Intra-day and between-day precisions (%CV) and accuracies (relative errors) were below 15% for all the compounds. Matrix effects were negligible for most of the analytes. Samples undergoing freeze-thaw showed no degradation of their BAs. The method makes use of a fused-core phenyl column coupled to a triple quadrupole tandem mass spectrometer to achieve chromatographic separation within 5min. We quantified BAs grouped in four different follicle sizes (3-5mm, 6-8mm, 9-14mm, >15mm), obtaining a similar relative BA profile for all the sizes, with CA always in higher concentration, ranging between 1600 and 18000ng/ml, approximately, followed by its conjugate glycocholic acid, GCA, which ranged between 800 and 9000ng/ml. The highest concentration in CA, DCA or CDCA was always detected in FF stemming from follicles of 6-8mm. To our knowledge, this is the first report in which BAs subspecies have been detected and quantified in bovine follicular fluid.

The objective of this study was to investigate the effect of time of first postpartum ovulation on endometrial inflammation in dairy cows with and without uterine disease during the early puerperal period. Transvaginal follicular puncture (FP) was carried out to suppress postpartum ovulation and formation of a CL until Day 42. Fifty-three lactating Holstein Friesian cows were divided into four groups on the basis of presence (UD+) or absence (UD-) of uterine disease, which was defined as retained fetal membranes and/or metritis, and whether FP had (FP+) or had not been (FP-) carried out. This resulted in the following groups: UD-FP- (n = 15), UD-FP+ (n = 13), UD+FP- (n = 13), and UD+FP+ (n = 12). Cloprostenol was given on Days 55 to 60 postpartum, and GnRH was administered 2 days later for synchronization of ovulation. In the FP- groups, endometrial swab and biopsy samples were collected during the second estrus (approximately Day 40) and during the estrus after synchronization. In the FP+ groups, the same samples were collected during the first estrus (approximately Day 49) and during the estrus after synchronization. The prevalence of positive bacteriologic cultures of the endometrium was not affected by FP (P > 0.05). Histologic signs of endometritis were more severe in UD+FP- cows at second sampling than in UD+FP+ cows (P ≤ 0.05). Endometrial expression of IL1α (in UD- after first sampling and in UD+ after second sampling) and IL1β (in UD- and UD+ after first sampling) was higher (P ≤ 0.05) in FP- cows than in FP+ cows. Regardless of group, cows with histopathologic evidence of endometritis had higher expression (P ≤ 0.05) of IL1α, IL1β, IL6, and TNFα than cows without endometritis. In conclusion, suppression of early ovulation by transvaginal FP enhances clearance of uterine inflammation in postpartum cows.

During final maturation (between LH surge and ovulation) in vivo, a switch from oestradiol to progesterone dominance within the follicle is well described. The aim was to mimic the in vivo situation during in vitro maturation via the supplementation of different gonadotropins. Groups of 30 cumulus-oocyte complex (abattoir-derived ovaries) were matured in TCM 199 plus different gonadotropins (eCG/hCG; FSH/LH, each in 0.05 or 0.01 IU; only FSH 0.05 IU; without gonadotropins) using a standard protocol without oil overlay. In Experiment 1, denuded oocytes were collected at 0 h (immature) and after 24 h of in vitro maturation (IVM; exhibit first polar body). In Experiment 2, oocytes were collected at different time points [0 (immature), 4, 8, 12, 16, 20, and 24 h] after IVM in eCG/hCG-supplemented medium. They were individually stored at −80°C until analyses. Transcripts of developmental competence (BMP15, GDF9, ZAR1), glucose or steroid metabolism (G6PD, STAR), and progesterone receptors (PGR, PGRMC1/2) were examined in individual oocytes via quantitative RT-PCR (n = 5). For statistical analyses, 1-way ANOVA followed by a Tukey test was used. Relative abundance of BMP15 transcripts was significantly lower (P ≤ 0.05) in oocytes of the group matured for 24 h with FSH/LH 0.01 IU, FSH 0.05 IU, and without gonadotropins than in immature oocytes. Relative amount of G6PD and PGRMC2 mRNA was significantly lower (P ≤ 0.05) in mature oocytes of the group with FSH/LH 0.01 IU, FSH 0.05 IU, and without gonadotropins than in immature ones. Relative abundance of GDF9, STAR, and ZAR1 transcripts was significantly lower (P ≤ 0.05) in oocytes of the group with eCG/hCG, FSH/LH 0.01 IU, FSH 0.05 IU, and without gonadotropins compared with immature oocytes. Relative abundance of PGR mRNA was significantly higher (P ≤ 0.05) in mature oocytes of the group with eCG/hCG than in immature oocytes, FSH/LH 0.01IU, FSH 0.05 IU, and without gonadotropins (Experiment 1). Relative amount of GDF9 transcripts was significantly lower (P ≤ 0.05) in mature oocytes collected after 24 h than in immature ones. Relative abundance of PGR mRNA was significantly higher (P ≤ 0.05) in oocytes collected after 20 and 24 h of IVM than in immature ones. Relative amount of ZAR1 transcripts was significantly lower (P ≤ 0.05) in oocytes collected after 16, 20, and 24 h of IVM than in immature oocytes; likewise, they were significantly lower (P ≤ 0.05) in oocytes collected after 12, 16, 20, and 24 h than in oocytes collected after 4 h of IVM. Relative amount of STAR mRNA was significantly lower (P ≤ 0.05) in oocytes collected after 24 h than in immature ones, and significantly lower (P ≤ 0.05) in oocytes collected after 16, 20, and 24 h than in oocytes collected after 4 h of IVM (Experiment 2). The results suggest a down-regulation of most transcripts during the period of IVM with different gonadotropin supplements with exception of PGR. Furthermore, most transcripts follow a timely regulated mRNA expression pattern during the entire IVM period. We gratefully acknowledge the financial support of the German Research Foundation (DFG; FOR 1369, WR 154/3–1).

In humans, intracytoplasmic sperm injection (ICSI) has become widely applied to overcome infertility. In addition to its clinical usefulness, ICSI is also a valuable research tool for studying fundamental aspects of gamete interaction during fertilization. Oocyte activation is a prerequisite for decondensation of the sperm head and formation of the male pronucleus regardless of whether the spermatozoon enters the oocyte by injection or by fertilization. In several species, the injection procedure itself is sufficient to activate the oocyte. However, the bovine oocyte is rarely activated by the mechanical stimulation of injection alone. Therefore, additional stimuli are required during ICSI, which may also initiate parthenogenetic development. Because blastocyst origin, either via IVF or after parthenogenetic activation, could not be distinguished at the morphological level via microscopic evaluation, the paternal genome needs to be determined, employing a molecular approach. Therefore, the aim of this study was to validate the expression of neuronatin (NNAT) mRNA, a paternally expressed gene, via RT-PCR, to verify whether bovine blastocysts were generated by fertilization or parthenogenesis. Bovine cumulus-oocyte-complexes were recovered from abattoir-derived ovaries by the slicing method. After 24 h of maturation, fertilization was achieved using a standard protocol. Presumptive zygotes were cultured in SOFaa for a period of 8 days. To generate parthenogenetic blastocysts, oocytes were activated, employing the following protocol. After 24 h of maturation, the oocytes were exposed to 5 µM ionomycin for 5 min and were then incubated in 1.9 mM 6-dimethylaminopurine for 4 h. Afterwards, treated oocytes were cultured in SOFaa for 8 days. Blastocysts from both experimental groups (IVF-derived and parthenogenetic) were stored at −80° until analysis. RT-PCR was performed as described previously (Stinshoff et al. 2014 Reprod. Fertil. Dev. 26, 502–510) for 6 embryos stemming from the IVF-group and 3 embryos out of the chemical-activated group. The NNAT mRNA was expressed in 5 blastocysts stemming from the IVF group; one blastocyst did not show NNAT mRNA expression, suggesting that it was derived from parthenogenesis. Conversely, NNAT mRNA could not be detected in all blastocysts derived from chemical activation, indicating that only the maternal genome was present. These results clearly demonstrate that NNAT mRNA expression is a reliable method to prove the paternal participation in early bovine embryo development.

Steroid hormones are regulators in the fine-tuned mechanism of follicular development in cattle. Their concentration and property can be modulated via different processes. Sulfoconjugation via sulfotransferases (SULT) changes them from being hydrophobic to hydrophilic molecules, thereby preventing them from diffusing freely across the lipid bilayer and necessitating a transport system like the sodium-dependent organic anion transporter (SOAT; SLC10A6). In addition, sulfated steroids can no longer bind to their nuclear receptors, rendering them biologically inactive. Steroid sulfatase (STS) removes the sulfate moiety from conjugated steroids, transforming them to the free active forms. Data about the concentration of sulfated steroids in follicular fluid and the expression of the related enzymes are limited in horses and cattle. Recently, transcripts of the corresponding enzymes have been detected in cumulus cells of immature bovine cumulus-oocyte complex. Furthermore, it has been shown that small amounts of sulphated steroids are present in bovine follicular fluid. The objective of the study was to investigate the steroid metabolizing enzymes at the protein level via immunohistochemistry (IHC). Bovine ovaries collected at a slaughterhouse were categorized according to their oestrus cycle stage. Three pairs of ovaries of each cycle stage (proestrus, oestrus, postestrus, interestrus) were collected. Morphological criteria (ovaries: presence/absence of follicles and/or CL, size and number of follicles; closure of the cervix, amount of mucus) were employed to categorize them. Samples for IHC were fixed in formalin. After washing and dehydration, the samples were finally embedded in paraffin and mounted onto blocks. Indirect immunoperoxidase staining methods were applied using the streptavidin-biotin technique for signal enhancement following standard procedures. Tissue sections of 3 µm were mounted onto glass slides, and a polyclonal rabbit anti-human STS and a polyclonal rabbit anti-bovine SULT1E1 primary antibody were used to detect STS or SULT1E1, respectively. Negative controls were included using nonspecific rabbit IgG. The evaluation of the staining was descriptive in relation to a positive control (bovine placenta). Immunostaining for STS was detected in granulosa cells of antral and secondary follicles and in the endothelium of blood vessels, irrespective of the stage of the oestrus cycle. Moreover, staining for SULT1E1 was restricted to granulosa cells in antral follicles, again irrespective of the stage of the oestrus cycle. However, staining intensity for STS and SULT1E1 was only weak. These data indicate for the first time the presence of the steroid metabolising enzymes STS and SULT1E1 in bovine ovaries at the protein level. However, due to the low expression level, further studies are needed to clarify the function. We gratefully acknowledge the financial support of the German Research Foundation (DFG; FOR 1369, WR 154/3–1).

Many techniques for in vitro production of embryos frequently make use of dimethyl sulfoxide (DMSO) as a solvent for compounds that have little or no solubility in water. DMSO is also used as a cryoprotectant. Based on its high glass-forming characteristics, it is also essential for vitrification. Although it is known that high concentrations of DMSO could be embryo toxic, less attention has been focused on whether an effect could be detected from the small concentrations present in culture media when used as a vehicle. Earlier studies deemed concentrations of up to 0.4% in in vitro maturation and 0.1% in in vitro culture (IVC) as safe with regards to morphological criteria. In the present study, bovine in vitro-produced embryos employing standard protocols were exposed to the following DMSO concentrations during IVC: 0% (control), 0.05%, 0.10%, 0.15%, 0.20%, and 0.25%. At Day 8 cleavage and developmental rates were recorded. The morphological quality of expanded Day 8 blastocyst was assessed with determination of the total cell number, live-dead (live-dead ratio), and TUNEL staining (apoptotic index). Fat accumulation was analysed by red-oil staining. Cleavage and developmental rates did not differ (P > 0.05) between embryos of the various groups. Mean cleavage and development rates (%) averaged 58.3 ± 10.6 and 28.4 ± 9.2 (0%), 59.5 ± 11.5 and 26.1 ± 7.4 (0.05%), 57.6 ± 6.6 and 21.7 ± 7.1 (0.10%), 58.1 ± 7.8 and 27.8 ± 5.6 (0.15%), 56.6 ± 7.3 and 24.5 ± 7.0 (0.20%), 56.3 ± 10.9 and 23.5 ± 9.9 (0.25%). Total cell numbers were similar [123.9 ± 26.9 (0%), 115.3 ± 21.5 (0.05%), 114.8 ± 22.3 (0.10%), 125.4 ± 22.4 (0.15%), 122.9 ± 24.7 (0.20%), 115.9 ± 19.7 (0.25%)]; P > 0.05. The live/dead cell ratio was significantly higher (P ≤ 0.05) in those embryos derived from the 0.1% group (40.1 ± 23.1) than that from embryos of the other groups [22.6 ± 13.5 (0%), 23.4 ± 10.4 (0.05%), 24.2 ± 14.6 (0.15%), 22.7 ± 14.0 (0.20%), 20.3 ± 9.9 (0.25%)]. Apoptotic cells were significantly lower in embryos exposed to 0.10% and 0.20% DMSO than in those of other groups, and the number of apoptotic cells in embryos of 0.05% was also slightly lower compared with those of the control group (P = 0.08). Apoptotic index tended to be lower in embryos out of the groups supplemented with 0.1% and 0.2% DMSO compared with those of the control group (0%: 3.8 ± 1.6; 0.05%: 2.6 ± 1.6; 0.10%: 2.3 ± 1.8; 0.15%: 3.2 ± 1.5; 0.20%: 2.2 ± 1.5; 0.25%: 3.1 ± 1.7; P = 0.09; P = 0.06). Fat accumulation was significant higher (P ≤ 0.05) in embryos stemming from the group supplemented with 0.15% DMSO (0%: 6617 ± 2703 µm2; 0.05%: 7346 ± 1981 µm2; 0.10%: 6976 ± 1848 µm2; 0.15%: 9301 ± 1703 µm2; 0.20%: 8675 ± 2271 µm2; 0.25%: 8301 ± 2711 µm2). These findings show that DMSO concentrations of 0.10% and 0.20% used during in vitro culture influence the quality of embryos at the morphological level. However, further analyses to verify these results at the molecular level via RT-qPCR are still needed. The financial support of the Förderverein Bioökonomieforschung e.V. (FBF) is gratefully acknowledged.

Insulin-like growth factor 1 (IGF1) is a key regulator in early embryonic development, influencing physiological processes and stimulating growth and development (Fowden et al. 2003). Supplementing IGF1 during in vitro culture of bovine embryos improved cleavage and developmental rates while it reduced apoptosis (Byrne et al. 2002). The signal transduction of IGF1 is performed by its binding to the insulin-like growth factor 1 receptor (IGF1R). At the mRNA level, IGF1R is expressed throughout pre-implantation embryonic development and was identified as a potential marker of good quality embryos (Yaseen et al. 2001). However, information on protein level is rare. Therefore, protein expression of the IGF1R during early embryonic development in vitro was analysed in the present study by immunofluorescence staining. Furthermore, the mRNA expression of the IGF1R was investigated by RT-qPCR. In vitro derived embryos of different stages (2-cell, 4-cell, 8-cell, 16-cell stage, morula, blastocyst, and expanded blastocyst) were either directly subjected to immunofluorescence staining or frozen at –80°C for use in RT-qPCR. Staining was performed with a peptide antibody against two peptide sequences of the bovine IGF1R α unit, which was specifically produced. Pixel intensity of immunofluorescence was measured and a mean grey value was calculated using the cellsens® software (Olympus, Hamburg, Germany). Data were analysed by one-way ANOVA followed by a Tukey's test using SigmaStat 3.5 Software (Systat Software GmbH, Erkrath, Germany). The detection of the IGF1R mRNA and protein was possible in all stages of embryonic development beginning at the 2-cell stage up to the expanded blastocyst. The maximal mRNA expression could be observed in 2- and 4-cell embryos. It significantly decreased to the 8-cell stage, followed by an increase up to the expanded blastocyst. The IGF1R protein was mainly localised in the plasma membrane of single blastomeres and also weakly in the cytoplasm. Mean grey values are highest in the 2-cell stage, showing a significant decline up to the 16-cell stage and an increase again until the expanded blastocyst. The mRNA and protein expression showed similar patterns during early embryonic development. IGF1R expression started to increase at the 8-cell stage (mRNA) and 16-cell stage (protein) indicating a link to the maternal-embryonic transition. For the first time, these results show that in bovine embryos, the IGF1R expression is related to the activation of the embryonic genome. We gratefully acknowledge the financial support of the H. Wilhelm Schaumann Foundation (Hamburg, Germany).

Steroid hormone concentration and property can be modulated via different processes. Sulfoconjugation via sulfotransferases (SULT) changes steroids from hydrophobic to hydrophilic, necessitating a transport system such as the sodium-dependent organic anion transporter (SOAT; SLC10A6). Steroid sulfatase (STS) removes the sulfate moiety from conjugated steroids, transforming them to the free active ones. Moreover, present in vitro maturation systems do not completely mimic the in vivo situation resulting in oocytes of reduced quality. The present study investigates the local effects of sulfated steroids during follicular and oocyte development in vivo and in vitro. Follicles of bovine abattoir-derived ovaries were categorized according to their size (3 to 5, 6 to 8, 9 to 14, and >15 mm) after dissection and measurement via a caliper. Only nonatretic follicles were used (Kruip and Dieleman, 1982). Follicular fluid was collected via aspiration and analysed for the presence of steroids and their sulfated counterparts via LC-MS/MS. Moreover, oocytes were in vitro maturated with a standard protocol. The medium was measured via radioimmunoassay after 0, 4, 8, 12, 16, 20, and 24 h to detect 17β-oestradiol (E2) and progesterone (P4). Data were tested using analysis of variance (ANOVA) followed by multiple pairwise comparisons using Tukey's test. A P-value of ≤0.05 was considered significant. It was possible to detect 17β-oestradiol, progesterone, testosterone, 17β-oestradiol sulfate, estrone sulfate, pregnenolone sulfate, cholesterol sulfate (Table 1), and furthermore androstendione, estrone, androsterone, and 17OH-pregnenolone. During IVM, P4 significantly increased in the medium (4 h: 3.3 ± 1.0 ng mL–1; 24 h: 9.8 ± 1.7 ng mL–1), whereas the E2 concentration did not change (4 h: 52.8 ± 12.1 pg mL–1; 12 h: 68.4 ± 3.7 pg mL–1; 24 h: 66.9 ± 19.7 pg mL–1). In addition, preliminary data suggest that transcripts of the steroid metabolizing and transporting enzymes (SULT1E1, STS, SLC10A6) were present in cumulus cells from immature bovine COC. These results indicate for the first time that only small amounts of sulfated steroids are present in bovine follicular fluid. However, the related enzymes are present at the mRNA level. Further studies are underway to analyse the protein level. Table 1.Steroid hormone concentration in follicular fluid We thank Prof. Dr Wudy and A. Sánchez Guijo for the LC-MS/MS analysis. Furthermore, we gratefully acknowledge the financial support of the German Research Foundation (DFG; FOR 1369).

The in vitro production (IVP) of bovine embryos is a well-established technique that has been available for nearly 20 years. However, there remain major differences between IVP-derived blastocysts and their in vivo-derived counterparts. Many studies have pointed out that most of these differences are due to the in vitro developmental environment. To circumvent these negative effects due to in vitro culture conditions, a new method – intrafollicular oocyte transfer (IFOT) – was established in the present study. Using modified ovum pick-up (OPU) equipment, in vitro-matured oocytes derived from slaughterhouse ovaries were injected into the dominant preovulatory follicle of synchronised heifers (follicular recipients) enabling subsequent ovulation, in vivo fertilization, and in vivo development. A total of 810 in vitro-matured oocytes were transferred into 14 heifers. Subsequently, 222 embryos (27.3%) were recovered after uterine flushing at Day 7. Based on the number of cleaved embryonic stages, 64.2% developed to the blastocyst stage, which did not differ from the IVP-derived embryos (58.2%). Interestingly, lipid content of IFOT-derived blastocysts did not differ from the fully in vivo-produced embryos, whereas IVP-derived blastocysts showed significantly higher lipid droplet accumulation compared with fully in vivo-derived and IFOT-derived blastocysts (P

International audience; Epigenetic mechanisms play a crucial role in many biological processes, such as regulation of gene expression especially after fertilization and during early embryonic development. Indeed, the parental genomes that carry special epigenetic signatures, undergo important chromatin remodelling through epigenetic modifications during the first embryonic cleavages, some of which are crucial for the production of healthy embryos. It is therefore very important for breeders and embryologists to understand how parentally inherited genomes may be epigenetically altered by animal biotechnologies as it could affect embryo quality and further development. This chapter introduces some of the basic epigenetic parameters underpinning early embryonic development and how they could be affected during the processes of embryo in vitro production, somatic cell nuclear transfer or stem cells derivation