Automatic recording of individual oestrus vocalisation in group-housed dairy cattle: development of a cattle call monitor.

Oestrus detection remains a problem in the dairy cattle industry. Therefore, automatic detection systems have been developed to detect specific behavioural changes at oestrus. Vocal behaviour has not been considered in such automatic oestrus detection systems in cattle, though the vocalisation rate is known to increase during oestrus. The main challenge in using vocalisation to detect oestrus is correctly identifying the calling individual when animals are moving freely in large groups, as oestrus needs to be detected at an individual level. Therefore, we aimed to automate vocalisation recording and caller identification in group-housed dairy cows. This paper first presents the details of such a system and then presents the results of a pilot study validating its functionality, in which the automatic detection of calls from individual heifers was compared to video-based assessment of these calls by a trained human observer, a technique that has, until now, been considered the 'gold standard'. We developed a collar-based cattle call monitor (CCM) with structure-borne and airborne sound microphones and a recording unit and developed a postprocessing algorithm to identify the caller by matching the information from both microphones. Five group-housed heifers, each in the perioestrus or oestrus period, were equipped with a CCM prototype for 5 days. The recorded audio data were subsequently analysed and compared with audiovisual recordings. Overall, 1404 vocalisations from the focus heifers and 721 vocalisations from group mates were obtained. Vocalisations during collar changes or malfunctions of the CCM were omitted from the evaluation. The results showed that the CCM had a sensitivity of 87% and a specificity of 94%. The negative and positive predictive values were 80% and 96%, respectively. These results show that the detection of individual vocalisations and the correct identification of callers are possible, even in freely moving group-housed cattle. The results are promising for the future use of vocalisation in automatic oestrus detection systems.

Negative effects of oxidative stress in bovine spermatozoa on in vitro development and DNA integrity of embryos.

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 1h at 37°C without or with 100µM 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 60h 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 46h 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.

Birth of healthy calves after intra-follicular transfer (IFOT) of slaughterhouse derived immature bovine oocytes.

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.

Profiling of bile acids in bovine follicular fluid by fused-core-LC-MS/MS.

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.

Effect of suppression of postpartum ovulation on endometrial inflammation in dairy cows.

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 IL1ß (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, IL1ß, IL6, and TNFα than cows without endometritis. In conclusion, suppression of early ovulation by transvaginal FP enhances clearance of uterine inflammation in postpartum cows.

[Importance of reproductive biotechnology in cattle in Europe]. / Bedeutung der Biotechnologie beim Rind in Europa.

Reproductive biotechnology has manifold applications and includes a great innovation potential in livestock. Due to the global changes the new findings and techniques can aid to meet the future challenges. The use of biotechnology in animal production can guarantee enough high quality food for the whole population. Genetic resources of animals can be preserved via sperm and embryo banking. Early diagnosis of hereditary defects, generation of offspring with predetermined sex and the avoidance of animal transports for breeding employing shipment of frozen embryos will improve animal welfare. A special application is the use of animal models for human assisted reproductive technologies. Therefore, not only in Germany research related to the methodologies in reproductive biotechnology and their improvement need to be supported.

Follicular progesterone concentrations and messenger RNA expression of MATER and OCT-4 in immature bovine oocytes as predictors of developmental competence.

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 (P < 0.05) 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 (P < 0.05). 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.

The somatotropic axis during the physiological estrus cycle in dairy heifers--Effect on hepatic expression of GHR and SOCS2.

Pituitary growth hormone (GH) release and hepatic insulin-like growth factor-I (IGF-I) production increase after an injection of 17ß-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.

Diagnostic opportunities for evaluation of the exposure of dairy cows to the mycotoxins deoxynivalenol (DON) and zearalenone (ZEN): reliability of blood plasma, bile and follicular fluid as indicators.

To investigate the usefulness of follicular fluid (FF) in relation to blood plasma and bile as indicators of exposure of dairy cows to ZEN, DON and their metabolites, a dose-response study was performed with 30 dairy cows. The cows, 10 in each group (named CON; FUS-50, FUS-100), received a diet with three different concentrations of Fusarium toxin-contaminated maize. Thereby, the following dietary concentration were reached: CON (0.02 mg ZEN and 0.07 mg DON, per kg dry matter, DM), FUS-50 (0.33 mg ZEN and 2.62 mg DON, per kg DM) and FUS-100 (0.66 mg ZEN and 5.24 mg DON, per kg DM). ZEN, DON and de-epoxy-DON (de-DON) were detected in FF. Based on the linear regression between toxin concentration in plasma and FF, it seems that about 50% (m = 0.5) of ZEN present in plasma is present in FF while an increase of 1 ng/ml DON or de-DON in plasma is paralleled by an increase of 1.5 ng/ml DON or 1.1 ng/ml de-DON in FF. ZEN, DON and their metabolites, except zearalenone (ZAN), were also detected in bile. Contrary to DON and de-DON, ZEN and its metabolites were accumulated in bile so that the concentration of ZEN and metabolites was much higher than for DON and de-DON. The main compound was ß-zearalenol (ß-ZEL). The biliary ZEN, α-zearalenol (α-ZEL) and ß-ZEL concentration correlated linearly with each other with an uncertainty of <15% (r(2) ≥ 0.86), whereas the ratio between ZEN: α-ZEL: ß-ZEL was about 1.5:1:11. With the help of established linear relationship between toxin intake and toxin concentration, bile could be used as diagnostic indicator to assess the exposure of cows.

Maturation environment and impact on subsequent developmental competence of bovine oocytes.

In vitro production (IVP) of bovine embryos has been improved immensely throughout the last decades. Nevertheless, embryos generated in vitro still differ from their in vivo-produced counterparts. It is possible to achieve blastocyst rates of up to 70% if in vivo-matured oocytes are used. In contrast, if oocytes are matured in vitro, blastocyst rates are only half that of those matured in vivo. This rather limited success may be attributed to the heterogeneous population of oocytes which are normally retrieved from follicles of 3-8 mm rather than from preovulatory follicles. In contrast to the in vivo-ovulated oocyte, these oocytes lack development up to the preovulatory stage and are matured in vitro. Therefore, much effort has been devoted to the establishment of non-invasive and non-perturbing means for selecting the most competent oocytes, for example the extensiveness and compactness of the cumulus-corona investment and the granulation of the ooplasm. In vitro culture (IVC) conditions have been enhanced in the last few years, mainly by adjustment of media formulations, whereas the in vitro maturation (IVM) protocols stay invariable. Consequently, maintaining or mimicking the in vivo situation in vitro will aid to improve the quality and developmental competence of the resulting matured oocyte. The scope of this review is to give an overview of the current situation of in vitro maturation of mammalian oocytes with emphasis on the bovine species. Special attention has been paid to the in vivo situation in the follicle and how a better understanding of these intrafollicular factors will aid to improve the in vitro maturation conditions.

Effect of postpartum suppression of ovulation on uterine involution in dairy cows.

The objective of this study was to investigate the effect of time of first postpartum ovulation after calving on uterine involution in dairy cows with and without uterine puerperal disease. Transvaginal follicular puncture (FP) of follicles >6 mm suppressed ovulation and development of a CL until Day 42 after calving. Fifty-three lactating Holstein Friesian cows (3.4 ± 1.2 years old, parity 2.5 ± 1.0 [median ± mean absolute deviation]) were divided into groups on the basis of the presence (UD+) or absence (UD-) of uterine disease and whether FP was carried out (FP+) or not (FP-). Uterine disease was defined as the occurrence of retained fetal membranes and/or metritis. This resulted in the following groups: UD-FP- (n = 15), UD-FP+ (n = 13), UD+FP- (n = 13), and UD+FP+ (n = 12). A general examination, vaginoscopy, transrectal palpation, and transrectal B-mode sonography of the reproductive organs were conducted on Days 8, 11, 18, and 25 and then every 10 days until Day 65 after calving. After hormonal synchronization of ovulation (cloprostenol between Days 55 and 60 postpartum and GnRH 2 days later), cows were inseminated in the next spontaneous estrus. On average, the cows ovulated on Day 21.0 ± 6.0 (UD-FP-), 50.0 ± 4.0 (UD-FP+), 16.0 ± 3.0 (UD+FP-), and 48.0 ± 2.0 (UD+FP+) postpartum. Calving-to-conception interval and first-service conception rates were not affected by FP (P > 0.05). Healthy cows with FP had smaller (P < 0.05) uterine horn and cervical diameters assessed sonographically than cows without FP. FP reduced the prevalence of purulent vaginal discharge and uterine size assessed transrectally in UD+ cows (P < 0.05). The results showed that suppression of an early ovulation by transvaginal FP improved uterine involution in cows with and without uterine disease.

Hepatic mRNA expression of acid labile subunit and deiodinase 1 differs between cows selected for high versus low concentrations of insulin-like growth factor 1 in late pregnancy.

The somatotropic axis is a key metabolic pathway during transition from late pregnancy to early lactation in dairy cows. The first objective of this study was to determine the feasibility of selecting cows with persistent differences in total insulin-like growth factor 1 (IGF-1) concentration by taking only a single antepartum blood sample. The second objective was to elucidate the underlying causes of differences in peripheral IGF-1 concentrations throughout late pregnancy and whether hormonal axes also differed in dairy cows with low versus high IGF-1. Twenty clinically healthy Holstein Friesian cows were chosen based on their plasma IGF-1 concentration at 244 to 254 d after artificial insemination (AI) and other selection criteria (health status, body condition score, number of lactations). These cows were selected from a large-scale farm, transported to the clinic, and monitored daily from 261 to 275 d after AI. The concentrations of IGF-1, growth hormone, IGF binding proteins 2, 3, and 4, insulin, cortisol, thyroid hormones, progesterone, and estradiol were measured. Ultimately, 7 IGF-1-low and 7 IGF-1-high cows were statistically analyzed. Additionally, a liver biopsy was taken on d 270 ± 1 after AI for analysis of gene expression of somatotropic family members, liver deiodinase 1, and suppressor of cytokine signaling-2. It was possible to select cows with different IGF-1 concentrations based upon only 1 blood sample collected in late pregnancy. Concentrations of IGF-1 in IGF-1-low versus IGF-1-high animals (n=7 each) remained significantly different between groups from the day of selection of the animals until d 275 after AI. Second, the differences in total plasma IGF-1 concentration between experimental groups may be attributed to differences in hepatic production of acid labile subunit. The ability of IGFBP-3 to bind IGF-1 declined before calving in all cows. Furthermore, in addition to decreased mRNA expression of growth hormone receptor 1A and IGF-1 relative to calving, serum binding capacities for IGF-1 also decreased. Insulin-like growth factor binding protein 4 mRNA expression was higher in cows with low IGF-1 concentrations; this binding protein inhibits IGF-1 action at the tissue level and therefore may reduce IGF-1 bioavailability. Finally, other endocrine end points (e.g., insulin and thyroid hormones) differed between the 2 groups.

Future consequences and challenges for dairy cow production systems arising from climate change in Central Europe - a review.

It is well documented that global warming is unequivocal. Dairy production systems are considered as important sources of greenhouse gas emissions; however, little is known about the sensitivity and vulnerability of these production systems themselves to climate warming. This review brings different aspects of dairy cow production in Central Europe into focus, with a holistic approach to emphasize potential future consequences and challenges arising from climate change. With the current understanding of the effects of climate change, it is expected that yield of forage per hectare will be influenced positively, whereas quality will mainly depend on water availability and soil characteristics. Thus, the botanical composition of future grassland should include species that are able to withstand the changing conditions (e.g. lucerne and bird's foot trefoil). Changes in nutrient concentration of forage plants, elevated heat loads and altered feeding patterns of animals may influence rumen physiology. Several promising nutritional strategies are available to lower potential negative impacts of climate change on dairy cow nutrition and performance. Adjustment of feeding and drinking regimes, diet composition and additive supplementation can contribute to the maintenance of adequate dairy cow nutrition and performance. Provision of adequate shade and cooling will reduce the direct effects of heat stress. As estimated genetic parameters are promising, heat stress tolerance as a functional trait may be included into breeding programmes. Indirect effects of global warming on the health and welfare of animals seem to be more complicated and thus are less predictable. As the epidemiology of certain gastrointestinal nematodes and liver fluke is favourably influenced by increased temperature and humidity, relations between climate change and disease dynamics should be followed closely. Under current conditions, climate change associated economic impacts are estimated to be neutral if some form of adaptation is integrated. Therefore, it is essential to establish and adopt mitigation strategies covering available tools from management, nutrition, health and plant and animal breeding to cope with the future consequences of climate change on dairy farming.

Seminal plasma and seminal plasma proteins added to bulk sorted sperm do not alter the mRNA expression of in vitro produced bovine embryos.

Although sex-sorted sperm have been used for AI and IVF for over a decade there is still need to improve the technology as the results are highly variable. The goal of the present study was to assess the effect of seminal plasma and seminal plasma proteins as a supplement to sorted sperm on subsequent embryonic development, as a beneficial effect of these substances has been reported. In vitro matured oocytes were fertilized in vitro with either unsorted sperm (n = 215; Group 1), bulk sorted sperm (n = 226; Group 2), bulk sorted sperm extended in the presence of 1% seminal plasma (n = 185; Group 3) or bulk sorted sperm supplemented with seminal plasma proteins (4 mg mL(-1); n = 254; Group 4). An additional group of oocytes (n = 307; Group 5) was fertilized with the semen of another bull routinely used for IVF and served as a laboratory standard control. Subsequently, the presumptive zygotes were cultured for 8 days under standard conditions (SOFaa, 39 °C, 5% CO(2), 5% N(2)). Cleavage rates were assessed on day 3 p.i. (post insemination; group 1: 30.5 ± 14.7%; group 2: 28.8 ± 9.8%; group 3: 20.8 ± 14.9%; group 4: 25.7 ± 8.2%; group 5: 54.8 ± 11.5%). Development rates were documented on days 7 p.i. (group 1: 7.3 ± 6.6%; group 2: 5.6 ± 3.1%, group 3: 6.2 ± 7.7%, group 4: 6.7 ± 5.9%, group 5: 20.2 ± 6.9%) and 8 p.i. (group 1: 8.9 ± 7.0%; group 2: 6.0 ± 2.9%; group 3: 8.6 ± 11.3%; group 4: 7.8 ± 6.2%; group 5: 23.3 ± 7.8%), respectively. Significant differences among cleavage and development rates could only be seen for Group 5 compared to all other groups. However, this difference between Groups 1-4 vs. Group 5 regarding the development rates on Day 8 could not be detected when assessing the development rates on base of the number of cleaved embryos instead of the number of oocytes fertilized (group 1: 31.4 ± 17.2%; group 2: 26.0 ± 21.0%; group 3: 33.3 ± 19.05%; group 4: 26.6 ± 17.8%; group 5: 42.6 ± 11.3%). The relative abundance of six different developmentally important gene transcripts (G6PD, HSP1A1, SLC2A3, BAX, BCL2L1, DNMT3A) was determined using single Day 8 expanded blastocysts of all five groups. No significant differences were seen among the embryos of the five groups. Our results show that neither the bulk sorting procedure nor the addition of seminal plasma or seminal plasma proteins, respectively, affected cleavage and development rates when sperm from a specific bull was used. Additionally, sorting and subsequent exposure of sperm to either seminal plasma or seminal plasma proteins did not influence mRNA expression in bovine IVP embryos.

Cryopreservation affects the quality of in vitro produced bovine embryos at the molecular level.

There are still immense differences in the quality of in vitro produced embryos compared to their in vivo generated counterparts. These differences include a higher sensitivity of in vitro produced embryos towards cryopreservation. The quality of such embryos has been evaluated by morphological examination as well as the assessment of total cell numbers, pregnancy rates and in few cases through the analysis of their gene expression. The aim of the present study was to determine whether different cryopreservation methods have an influence on the quality of in vitro produced embryos after thawing. Bovine blastocysts were produced in a standard culture system (SOFaa). Having reached the stage of an expanding blastocyst on day 7, embryos were randomly either vitrified (n = 106) or cryopreserved conventionally (n = 131). Reexpansion (24h post thawing; 86/106 [81.1 ± 20.3%] of the vitrified embryos, 104/131 [79.4 ± 16.5%] of the conventionally cryopreserved embryos respectively) and hatching rates (48 h post thawing; 67/106 [63.2 ± 24.5%] of the vitrified embryos, 80/131 [61.1 ± 29.3%] of the conventionally cryopreserved embryos respectively) were similar. No significant differences in total cell numbers as well as live-dead cell ratio could be seen in hatched blastocysts of either cryopreservation group as well as in a control group (embryos cultured up to the stage of a hatched blastocyst and not cryopreserved by either method). Additionally, RT-qPCR was used to assess the relative abundance of eight developmentally important genes (HSPA1A, SLC2A1, SLC2A3, DSC2, CDH1, TJP1, DNMT3A, IFNT2) in hatched embryos of all groups. The results revealed significant differences in 4 of 8 (HSPA1A, SLC2A1, TJP1, DSC2) gene transcripts when comparing vitrified embryos to embryos of the control group and in 6 of 8 gene transcripts (HSPA1A, SLC2A1, TJP1, SLC2A3, DNMT3A, IFNT2) when comparing slow frozen embryos to embryos of the control group. Furthermore, the comparison of vitrified embryos to those frozen conventionally solely showed significant differences in the relative abundance of IFNT2. These results indicate that although not visible in gross morphology, but in the relative amount of gene transcripts, vitrification may be the more suitable method to cryopreserve in vitro produced bovine embryos cultured in SOF media.

Quantitative mRNA expression in ovine blastocysts produced from X- and Y-chromosome bearing sperm, both in vitro and in vivo.

Artificial insemination (AI) of sex-sorted sperm results in decreased fertility, compared with non-sorted sperm, in most species. However, this has not been the case in sheep, where the low-dose AI of sex-sorted ram sperm produced similar, if not superior, fertility to non-sorted controls. The aim of the present study was to determine the impact of sex-sorting technology on ovine embryo gene expression following embryo production in vivo and in vitro. After semen collection, ejaculates were split and either sex-sorted by flow cytometry and frozen, or diluted and frozen. Embryos were produced in vivo by inseminating superovulated ewes with either X- or Y-chromosome enriched sperm, or non-sorted control sperm, and collected by uterine flushing on Day 6 after AI. Embryos were produced in vitro using the same sperm treatments and cultured in vitro for 6 d. The relative abundance of selected gene transcripts was measured in high-grade blastocysts, defined by morphological assessment, using RT-qPCR. The mRNA expression of DNMT3A and SUV39H1 was upregulated in embryos cultured in vitro, compared to those cultured in vivo (DNMT3A: 3.61 ± 1.08 vs 1.99 ± 0.15; SUV39H1: 1.88 ± 0.11 vs 0.88 ± 0.07; mean ± SEM; P < 0.05). Both G6PD and SLC2A3 transcripts were reduced in embryos produced from sex-sorted sperm, in vivo (SLC2A3: 0.23 ± 0.03 vs 0.64 ± 0.10; G6PD: 0.32 ± 0.04 vs 1.01 ± 0.16; P < 0.05). The expression of DNMT3A was up-regulated in male (3.85 ± 0.31), compared to female embryos (2.34 ± 0.15; P < 0.05). This study contributes to the growing body of evidence citing aberrant patterns of gene expression resulting from in vitro culture. Whereas the process of sex-sorting altered the expression of several of the genes examined, no effect on embryo development was detected.

Epigenetic profile of developmentally important genes in bovine oocytes.

Assisted reproductive technologies are associated with an increased incidence of epigenetic aberrations, specifically in imprinted genes. Here, we used the bovine oocyte as a model to determine putative epigenetic mutations at three imprinted gene loci caused by the type of maturation, either in vitro maturation (IVM) in Tissue Culture Medium 199 (TCM) or modified synthetic oviduct fluid (mSOF) medium, or in vivo maturation. We applied a limiting dilution approach and direct bisulfite sequencing to analyze the methylation profiles of individual alleles (DNA molecules) for H19/IGF2, PEG3, and SNRPN, which are each associated with imprinting defects in humans and/or the mouse model, and are known to be differentially methylated in bovine embryos. Altogether, we obtained the methylation patterns of 203 alleles containing 4,512 CpG sites from immature oocytes, 213 alleles with 4,779 CpG sites from TCM-matured oocytes, 215 alleles/4,725 CpGs in mSOF-matured oocytes, and 78 alleles/1,672 CpGs from in vivo-matured oocytes. The total rate of individual CpGs and entire allele methylation errors did not differ significantly between the two IVM and the in vivo group, indicating that current IVM protocols have no or only marginal effects on these critical epigenetic marks. Furthermore, the mRNA expression profiles of the three imprinted genes and a panel of eight other genes indicative of oocyte competence were determined by quantitative real-time PCR. We found different mRNA expression profiles between in vivo-matured oocytes versus their in vitro-matured counterparts, suggesting an influence on regulatory mechanisms other than DNA methylation.

Characterization of differentially methylated regions in 3 bovine imprinted genes: a model for studying human germ-cell and embryo development.

Correct imprinting is crucial for normal fetal and placental development in mammals. Experimental evidence in animal models and epidemiological studies in humans suggest that assisted reproductive technologies (ARTs) can interfere with imprinted gene regulation in gametogenesis and early embryogenesis. Bos taurus is an agriculturally important species in which ARTs are commonly employed. Because this species exhibits a similar preimplantation development and gestation length as humans, it is increasingly being used as a model for human germ-cell and embryo development. However, in contrast to humans and mice, there is relatively little information on bovine imprinted genes. Here, we characterized the bovine intergenic IGF2-H19 imprinting control region (ICR) spanning approximately 3 kb. We identified a 300-bp differentially methylated region (DMR) approximately 6 kb upstream of the H19 promoter, containing a CpG island with CTCF-binding site and high sequence similarity with the human intergenic ICR. Additional differentially methylated CpG islands lie -6 kb to -3 kb upstream of the promoter, however these are less conserved. Both classical bisulfite sequencing and bisulfite pyrosequencing demonstrated complete methylation of the IGF2-H19 ICR in sperm, complete demethylation in parthenogenetic embryos having only the female genome, and differential methylation in placental and somatic tissues. In addition, we established pyrosequencing assays for the previously reported bovine SNRPN and PEG3 DMRs. The observed methylation patterns were consistent with genomic imprinting in all analyzed tissues/cell types. The identified IGF2-H19 ICR and the developed quantitative methylation assays may prove useful for further studies on the relationship between ARTs and imprinting defects in the bovine model.

Epigenetic modifications and related mRNA expression during bovine oocyte in vitro maturation.

The present study investigated the global pattern of two histone modifications and methylation of DNA during in vitro maturation of bovine oocytes retrieved from follicles of two different sizes (<2 mm and 2-8 mm). The methylation status of histone H3 at position lysine K9 (H3K9 me2), the acetylation status of histone H4 at position lysine K12 (H4K12ac) and the methylation of DNA were assessed by immunocytochemistry. In parallel, the relative abundance of mRNAs coding for proteins specifically involved in reprogramming, including HLA-B associated transcript 8 (G9A), suppressor of variegation 3-9 homolog 1 (SUV39H1), the somatic isoform of DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3b (DNMT3b) and zygote arrest 1 (ZAR1) was determined by RT-PCR. The alpha-H3K9 me2 signal was present in the GV stage and remained detectable until the end of the maturation period. alpha-H4K12ac antibody gave a stronger signal in GV and GVBD oocytes and markedly decreased after GVBD. The signal showing the methylation of DNA was present during the entire maturation period. The five transcripts showed a gene-specific expression profile. Results revealed the global patterns of H3K9 me2, H4K12ac, DNA methylation and the mRNA pool profiles of genes critically involved in epigenetic modifications during bovine oocyte maturation and their possible relationship with the acquisition of oocyte developmental competence and follicular development.