bluuebunny/crossref_metadata_2025 huggingface dataset:
24 Survival rates of vitrified biopsied bovine in vitro-produced blastocysts using the VitTrans device
Authors: González N. , Scherzer J. , Reichenbach M. , Otzdorff C. , Zerbe H.
Published: December 2018

In breeding programs, the application of a vitrification method suitable for direct transfer of biopsied embryos can increase the genetic improvement of cattle and help reduce the costs of embryo transfer. The aim of this study was to determine the in vitro survival of biopsied vitrified blastocysts using the new VitTrans device (Morató and Mogas 2014 Cryobiology 68, 288-293), a 1-step in-straw warming system. Immature bovine oocytes were in vitro matured, fertilized, and cultured to the blastocyst stage. A total of 110 grade 1 blastocysts (IETS codes 6 and 7) were randomly allocated to 2 groups: (1) biopsy (n=49) and (2) without biopsy, or control (n=61). Blastocysts were biopsied using a microblade mounted on a micromanipulator. A small portion of the trophoblast, approximately 15%, was cut off and a significant part of the zona pellucida was sliced away. Both groups were then vitrified using the VitTrans device. For vitrification, all blastocysts were exposed to an equilibration medium with 7.5% ethylene glycol+7.5% dimethyl sulfoxide in holding medium (HM) consisting of TCM-199 with 20% FCS, moved into a drop with 16.5% ethylene glycol+16.5% dimethyl sulfoxide+0.5M sucrose in HM, and then placed in a microdroplet on the VitTrans. The VitTrans was plunged into LN and covered with a 0.5-mL straw. For warming, the protective cover was removed from the VitTrans while still submerged in LN. Subsequently, a new 0.5-mL plastic embryo transfer straw was placed on the VitTrans while flushing the warming solution (0.3mL of 0.5M sucrose in HM at 45°C) with a syringe through the lumen of the device. By entering the warming solution into the VitTrans device, the embryo is flushed inside the plastic straw. The straw containing the embryo can then be readily used for transfer after the VitTrans is removed. To recover the embryo in the laboratory, the content of the straw was put into a Petri dish and blastocysts were placed in the culture medium and incubated at 38.5°C in 5% CO2 and 5% O2 in air. Morphology and re-expansion were evaluated 24h post-warming. The embryo survival rate was defined as the ratio of blastocysts that were able to re-expand with regards to the total number of warmed blastocysts. Due to the attachment of embryos inside the straw, a total of 18 embryos were lost during recovery (12 from the biopsied group and 6 from the nonbiopsied group). The ratio of re-expanded blastocysts from the recovered embryos was 40% in the biopsy group and 61% in the control group. In conclusion, vitrification using the VitTrans device showed good results with intact embryos compared with biopsied embryos. In addition, biopsied embryos had a tendency to adhere to the inside of the straw, which is probably due to the damage or loss of the zona pellucida. Additional research is required to minimize the loss of embryos.
28 Effect of deuterium oxide on bovine oocyte cryotolerance
Authors: Salerno F. , Rubessa M. , Gasparrini B. , Wheeler M.
Published: December 2018

It is known that cryopreservation triggers spindle disassembly, increased aneuploidy risk, decreased post-thaw survival, fertilization, and embryo development. We hypothesised that a treatment with D2O before vitrification would slow down oocyte metabolism and reduce ice crystal formation by replacing water inside the cells. The aim of the study was to evaluate the effect of a 4-h treatment with different D2O concentrations (0, 3, 15, and 30%) on cryotolerance of bovine in vitro-matured oocytes. Abattoir-derived bovine oocytes were matured in vitro for 20h in TCM-199 medium with 15% of bovine serum (BS), 0.5µg mL−1 of FSH, 5µg mL−1 of LH, 0.8mM l-glutamine, and 50µg mL−1 of gentamicin at 39°C with 5% of CO2 and randomly divided into 5 experimental groups. A group of non-vitrified oocytes was used as the fresh oocyte control group, whereas the remaining oocytes were incubated for 4h in in vitro maturation medium with 0% (vitrified control; n=205), 3% (n=205), 15% (n=205), and 30% D2O (n=205) before vitrification. The experiment was repeated 4 times. Oocytes were denuded in HEPES-buffered TCM-199 (H199)+5% BS and vitrified using a cryotop freezing straw. The oocytes were incubated in 200μL of H199+20% BS with 7.5% ethylene glycol and 7.5% dimethyl sulfoxide for 3min. After that, oocytes were collected in 50μL of H199+20% fetal bovine serum with 15% ethylene glycol+15% dimethyl sulfoxide and 0.5M sucrose for 20s and plunged into LN2. One month later, oocytes were warmed in thawing media with decreasing concentrations of sucrose (1.35M to 0.31M) and then placed into in vitro maturation medium for 2h before IVF. Matured oocytes were IVF and cultured according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). Cleavage and blastocyst rates were evaluated after 7 days of culture. Data were analysed using the GLM procedure of SPSS (SPSS Inc., Chicago, IL, USA). The least statistical difference post-hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. As expected, both cleavage [60.5±4.6 (fresh control); 36.9±2.6 (0% D2O); 46.3±3.7 (3% D2O); 31.6±2.4 (15% D2O); and 24.4±2.6 (30% D2O)] and blastocyst rates [25.7±0.8 (fresh control); 9.0±0.8 (0% D2O); 9.0±0.7 (3% D2O); 3.6±0.2 (15% D2O); and 4.3±0.8 (30% D2O)] decreased in all vitrified groups compared with the fresh control group. Within vitrified oocytes, cleavage rate increased (P<0.05) with 3% D2O treatment compared with the other groups. However, pretreatment with higher (15-30%) D2O concentrations decreased (P<0.05) blastocyst rates of vitrified-warmed oocytes. In conclusion, a pretreatment with low concentrations (3%) of D2O improved the cleavage rate of bovine vitrified-warmed oocytes, suggesting a potential beneficial effect, whereas deleterious effects were observed using the higher concentrations. Therefore, further studies are required to assess a potential use of D2O to improve oocyte cryotolerance, likely testing different incubation times.
29 Development and survival of bovine vitrified sexed IVF-derived embryos in vitro matured with pituitary or human recombinant follicle-stimulating hormone
Authors: Ferré L. B. , Fresno C. , Kjelland M. E. , Ross P. J.
Published: December 2018

Improving in vitro production efficiency involves the development of effective oocyte in vitro maturation conditions. Although >80% of cumulus-oocyte complexes (COC) undergo nuclear maturation, only approximately 30 to 35% of immature bovine COC develop to the blastocyst stage. Also, animal-sourced FSH is typically used in IVF, so an effective alternative using recombinant DNA technology is desirable. The aim of this study was to compare the effect of porcine (p) and recombinant human (rh)FSH concentrations on in vitro performance and post-thaw survival. Ovaries were collected from an abattoir and oocytes were aspirated from 2- to 6-mm follicles. The COC with compact and complete cumulus cell layers were selected and matured in groups of 25 COC in 200µL of M199 medium supplemented with alanyl-glutamine (0.1mM), Na pyruvate (0.2mM), gentamicin (5µg mL−1), epidermal growth factor (50ng mL−1), pLH (5µg mL−1), cysteamine (0.1mM), and 10% FBS for 22 to 24h in humidified air and 5% CO2. Oocytes were divided into the following groups: 1× pFSH (2µg mL−1), 1× rhFSH (0.01 UI mL−1), 1× pFSH+1× rhFSH, 2× pFSH, and 2× rhFSH. After 22 to 24h, fertilization (Day 0) was carried out using female sexed-sorted semen selected with a mini single-continuous 80% layer (PureSperm, Nidacon International AB, Mölndal, Sweden) and diluted to 1×106 sperm mL−1. The SOF-FERT medium was supplemented with fructose (90µg mL−1), penicillamine (3µg mL−1), hypotaurine (11µg mL−1), and heparin (20µg mL−1). After 18h, presumptive zygotes were denuded and cultured under low oxygen tension in groups of 15 to 20 in 50-µL drops of SOF-BSA for 7 days. Also, 2% FBS was added post-fertilization on Day 3.5. Expanded blastocysts were selected based on IETS standards at Day 6.5 to 7 of culture. Only grade 1 expanded blastocysts were vitrified (Cryotop, Kitazato, Tokyo, Japan). Vitrification medium was 15% (vol/vol) ethylene glycol+propylene glycol. Vitrified embryos were thawed in a solution of H199+20% FBS and 0.25M sucrose at 39°C. Thawed embryos were cultured in SOF-BSA+10% FBS under cumulus/granulosa cell monolayer co-culture. Embryo assessment involved post-thaw survival (0h), re-expansion and development progress (24-48h), and hatching of the zona pellucida (72h). A minimum of 4 replicates were performed. Data were analysed using a generalized linear mixed model with logit-link binomial distribution. Media treatment differences were determined using Fisher’s least significant difference test with the Bonferroni correction (α-level=0.05). The FSH origin affected cleavage and embryo development rate but not cryotolerance (Table 1). The results support previous research on low dose versus high dose rhFSH effectiveness and interspecies interaction of FSH on follicular receptors.

Table 1.Cleavage, embryo development rate, and cryotolerance of FSH of various origins
31 Effect of fetal calf serum on production and cryotolerance of in vitro bovine embryos from Ecuadorian Creole heifers
Authors: Méndez M. S. , Soria M. E. , Galarza L. R. , Perea F. P. , Argudo D. E.
Published: December 2018

In the Ecuadorian Andes there is a Creole bovine biotype whose population is disappearing. In vitro embryo production and cryopreservation is an important biotechnology that allows the conservation of animals threatened with extinction. The objective of this study was to determine the in vitro production and cryopreservation of embryos from creole heifers raised in the highlands of Ecuador. Immature cumulus-oocyte complexes were retrieved by ovum pickup from 10 Creole heifers (OPU) and from abattoir ovaries (control). The experiment was completed within 8 replicates. Cumulus-oocyte complexes were cultured in a maturation medium (TCM-199 supplemented with 10% fetal bovine serum, 100µg mL−1 of sodium pyruvate, 0.75mg mL−1 of l-glutamine, 4µg mL−1 of FSH-p, 100µM cysteamine, and 250µg mL−1 of gentamicin) following IVF (SOF medium supplemented with 10µg mL−1 heparin) and in vitro culture (citrate SOF medium). After denudation (Day 1 after IVF), presumptive embryos from each oocyte source (OPU and control) were split into 2 groups: with (FCS+) and without (FCS−) FCS (2.5%), which was added on Day 5 after IVF. On Day 7, embryos were evaluated, and those with quality 1 were vitrified. After warming, embryo re-expansion at 2h and embryo re-expansion and hatching at 24 and 48h were evaluated. Data were analysed by logistic regression in SAS software (SAS Institute Inc., Cary, NC, USA). Results of embryo rate at Day 7 and rates of vitrified, re-expanded, and hatched embryos are shown in Table 1. Regardless of the oocyte source, the addition of 2.5% FCS decreased embryo re-expansion at 2h and reduced embryo hatching at 48h in the OPU group. In conclusion, FCS did not improve embryo production and adversely affected the cryotolerance of embryos produced in vitro from Ecuadorian creole heifers.

Table 1.Production and cryotolerance of in vitro bovine embryos
35 The effect of fatty acid-free BSA supplementation on the ability of low-temperature-preserved chicken semen
Authors: Kim S. W. , Kim C. , Jeon I. S. , Ko Y. G.
Published: December 2018

The dilution of chicken semen is one of the important processes for low-temperature storage and cryopreservation and is considered to be a highly applicable method for semen exchanges between small farms in developing countries. However, studies on chicken semen preservation have been limited for several reasons. First, dilution shocks chicken semen, with more than 4 to 6 times deterioration of sperm qualities. For example, approximately 8 to 10 times dilution reduces the activity and vitality of chicken semen. The dilution factor of chicken semen could not compete with that of mammals such as cow, ram, and goat. Second, the use of glycerol as a cryoprotectant causes serious problems such as infertility of spermatozoa; therefore, farmers are reluctant to use cryopreserved semen. To increase dilution factors of chicken semen without loss of fertility, the use of fatty acid-free BSA (FAF-BSA) in Beltsville poultry semen extender (BPSE) diluent was studied with Leghorn semen. In this study, fresh semen of 9 leghorn cocks was obtained using the side collection method. The semen was diluted 10 times using BPSE medium supplemented with 0.1% FAF-BSA as a final concentration and preserved at 17°C to test fertility and hatchability of diluted chicken semen. The semen was stored at 17°C for 6h to test transportation time and used for AI to 18 layers with 3 repeats. After AI with 100µL of preserved semen (total: 60~120×106 spermatozoa), the fertilized eggs were harvested for 10 days and incubated at 37.8°C for 21 days. The fertility of the eggs was confirmed at 5 days and the hatchability was confirmed at 21 days of incubation. The supplementation of FAF-BSA in dilution medium induced higher fertility rates (53.2% v. 38.3%; P<0.05), and hatching rates also increased (90.9% v. 78.33%; P<0.05) compared with the control group. All data were analysed by Student’s t-test. These results showed that FAF-BSA supplementation could improve semen utilisation by low-temperature preservation. Small farmers who want to increase the ability of their herd could utilise FAF-BAS containing diluents to exchange the semen.
39 General motility and mitochondrial cytochemical activity of post-thawed semen of pasture-fed Nelore bulls supplemented with palm and soybean oils
Authors: Tsuneda P. P. , Hatamoto-Zervoudakis L. K. , Motheo T. F. , Zervoudakis J. T. , Nichi M.
Published: December 2018

This study aimed to evaluate general motility and mitochondrial cytochemical activity of post-thawed semen of pasture-fed Nelore bulls supplemented with palm and soybean oils (protected by calcium salts). Twenty-four young male Nellore bulls were randomly assigned to 2 groups: control (CO; corn supplement and distillery grains without the addition of protected fat) and palm and soybean oils association (OP+OS; control supplement+145g of protected soybean oil+145g of protected palm). The experiment lasted 84 days, and semen was collected for cryopreservation. Post-thawed general sperm motility was assessed by computer-assisted sperm analysis using a computerized analyser (HTR Ivos II; Hamilton Thorne, Beverly, MA, USA). Motility (%), progressive motility (%), velocity average path (μm s−1), linearity (%), and lateral displacement of sperm head (μm) were analysed. Additionally, mitochondrial cytochemical activity was evaluated by co-incubation with 3,3′-diaminobenzidine (DAB; 1mg mL−1 of PBS) at 37°C (water bath) for 1h under reduced lighting conditions. Sperm cells were classified based on the mitochondrial activity of their midpiece in a 4-class scale: class 1 (DAB 1; all mitochondria are active; sperm cells with midpiece completely stained), class 2 (DAB 2; sperm cells with active and inactive segments but with prevalence of active stained segments, indicating average to high mitochondrial activity), class 3 (DAB 3; spermatozoa with less than half of the mitochondrial sheath active), and class 4 (DAB 4; spermatozoa totally inactive, with midpiece completely discolored). Data were analysed using SAS statistical program (SAS 9.3, SAS Institute Inc., Cary, NC, USA), and Tukey’s test was used to identify treatments. Study design was completely randomized, and effects were declared significant at P<0.05. Increased percentage of sperm motility (CO: 30.27±5.44v. OP+OS: 38.90±4.68) and progressive motility (CO: 17.27±3.84v. OP+OS: 22.90±3.88) were noticed in post-thawed samples after supplementation. Nevertheless, the addition of palm and soybean oils to the feed promoted a decrease in the percentage of DAB 3 sperm cells (CO: 4.77±1.03v. OP+OS: 2.06±0.51; P=0.0267). Therefore, supplementation with palm and soybean oils improves sperm kinetics in post-thawed semen of grazing Nellore bulls.
4 Induction of ovulation by kisspeptin in llamas
Authors: Carrasco R. A. , Leonardi C. E. , Hutt K. D. , Singh J. , Adams G. P.
Published: December 2018

After mating, female camelids ovulate in response to nerve growth factor (NGF) present in semen (formerly referred to as ovulation-inducing factor). The ovulatory effect appears to be induced by stimulation of hypothalamic gonadotropin-releasing hormone (GnRH) neuronal activity and LH secretion. Recent studies have identified kisspeptin as an important mediator of GnRH secretion in several species. In the present study, we tested the hypothesis that kisspeptin is involved in the ovulatory pathway in llamas and investigated the mechanism of this effect. In Experiment 1, ovarian function in non-pregnant, non-lactating adult female llamas was synchronized by intramuscular administration of a GnRH analogue (50µg of gonadorelin acetate; Fertiline, Vetoquinol, Quebec, QC, Canada). When a growing dominant follicle ≥8mm in diameter was detected, llamas were assigned randomly to 3 treatment groups and given an intravenous dose of purified seminal NGF (1mg, single dose; n=5), kisspeptin (0.1mg kg−1 of body weight, 2 doses 1h apart; n=5), or PBS (n=4). The bioactive 10 amino acid fragment of murine kisspeptin was used. Ovulation and corpus luteum development were assessed by transrectal ultrasonography every other day from the day of treatment (Day 0) to Day 8. In Experiment 2, ovarian function among female llamas was synchronized, as in Experiment 1. When a growing dominant follicle ≥8mm in diameter was detected, llamas were given kisspeptin (0.1mg kg−1 of body weight IV, 2 doses 1h apart) beginning 2h after pretreatment with either a GnRH receptor blocker (cetrorelix acetate, 1.5mg per llama IV; Sigma, Oakville, ON, Canada; n=6) or saline (n=6). Llamas were examined 48h later by transrectal ultrasonography to detect ovulation and 8 days later to determine the presence of a corpus luteum. Chi-square tests were used to compare ovulation rates, and ANOVA for repeated measures was used to compare diameter profiles of the corpus luteum. In Experiment 1, ovulation rate did not differ between the NGF and kisspeptin groups (5/5 in each; 100%) and was greater than in the control group (0/4; 0% P<0.05). Corpus luteum diameter did not differ between llamas that ovulated in response to treatment with NGF or kisspeptin (13.2±0.8 and 14.0±1.2mm on Day 8, respectively). In Experiment 2, none of the llamas pretreated with cetrorelix ovulated in response to kisspeptin treatment (0/6; 0%), whereas all of the llamas pretreated with saline ovulated in response to kisspeptin treatment and had a corpus luteum at Day 8 (6/6; 100%; P<0.05). Results supported the hypothesis that kisspeptin induces ovulation in llamas. Because a GnRH receptor antagonist blocked ovulation, our interpretation is that the site of action of kisspeptin is upstream of the pituitary gland and involves control of GnRH release from the hypothalamus. These findings raise the possibility that kisspeptin mediates the ovulation-inducing effect of NGF.
Research was supported by the Natural Sciences and Engineering Research Council of Canada.
40 Toward a standardised annotation of morphokinetical parameters for an automatic early prediction of the in vitro development potential of bovine embryos
Authors: Reis A. P. , Brocart G. , Belghiti M. , Le Brusq N. , Messoudi S. , Le Guienne B. M. , Laffont L. , Ruffini S. , Canon E. , Adenot P. , Duranthon V. , Trubuil A.
Published: December 2018

In a previous work, we proposed an algorithm to select a subset of discriminant morphokinetical parameters within a larger predefined set (116 parameters) and to predict 6 major bovine embryo profiles of in vitro development. The algorithm relies on flexible combinations of the discriminant parameters selected within the four first cleavage cycles. The retained profiles were arrested embryos (embryos without mitotic activity, showing signs of life); dead embryos (embryos with all cells dead); anarchic embryos (embryos with abnormal morphological and/or kinetical development: some of these embryos can result in a blastocyst); not hatched blastocysts (blastocysts not hatching by 8 dpi); hatching blastocysts (blastocysts hatching in vitro from 7.3 to 8 dpi); and early hatching blastocysts (blastocysts hatching from 6 to 7.2 dpi) (Reis et al. 2018 Anim. Reprod.). The aim of the present work was to develop a ready-to-use software (EasyPickAndPredict) allowing the extension of this methodology to other embryologists. The software of high portability was built with the JAVA language and embedded with the classifier (R language, R Foundation for Statistical Computing, Vienna, Austria) and a user’s help for annotation (Adobe Premiere Pro CS5, San Jose, CA, USA). The predictive software is easy to handle, fast to load, and has high portability. The “manual annotation” function is based on click actions to annotate the discriminant parameters. The “prediction” function calls the embedded classifier. The “report” function creates customised reports including the embryo classification, the summary of the measures, and the accuracy of the prediction (vote system). The “help” function calls an audiovisual guideline with annotation specifications for all the morphokinetic parameters currently described (including the discriminant subset of parameters). This document includes help to produce a good time-lapse video (16min 51s); annotation specifications for the cleavage cycles 1 to 4 (36 min 26s); and a summary with examples of the 6 major embryonic morphokinetical profiles (16min 47s). The predictive graphical interface is easy to manipulate and automatically extracts the value of each discriminant parameter on the time-lapse picture as validated by the user click. The functions “manual annotation”, “automatic prediction”, “help”, and “report” supply embryologists with a standard approach to predict and analyse morphokinetical profiles of their embryos. This standardised approach is necessary to improve the capacity of comparison of morphokinetical works in different laboratories and enhance knowledge about in vitro-produced bovine embryos.
41 Delineating the molecular connections between mitotic aneuploidy, micronucleation, and cellular fragmentation in pre-implantation bovine embryos
Authors: Brooks K. E. , Daughtry B. L. , Fei S. S. , Yan M. Y. , Davis B. , Carbone L. , Chavez S. L.
Published: December 2018

Whole chromosomal abnormalities (aneuploidy) that arise during early embryo development are a major contributor to in vitro fertilization failure. It is estimated that ~50 to 80% of human embryos contain aneuploid cells, which contribute to high levels of chromosomal mosaicism detected by pre-implantation genetic screening. Previous studies estimate that 32 to 88% of bovine embryos are aneuploid at the 2-cell stage, advocating cattle as a physiologically relevant model to study the mechanisms mediating meiotic and/or mitotic errors. In cleavage-stage human embryos, a process called cellular fragmentation is associated with aneuploidy, and when used in conjunction with assessment of early mitotic timing, can largely distinguish chromosomally normal and abnormal embryos. We recently demonstrated that some cellular fragments contain chromosomal material that likely began as mis-segregated chromosomes that were encapsulated into micronuclei. Given that bovine embryos exhibit cellular fragmentation, albeit to a lesser extent than human embryos, we hypothesise that cellular fragmentation is a response to micronucleation and represents a conserved mechanism to eliminate mis-segregated chromosomes from the pre-implantation embryo. Using a combination of live-cell imaging, single-cell DNA-sequencing, whole-embryo RNA-sequencing, quantitative RT-PCR, and multicolour confocal microscopy, we aim to further investigate the correlation between these phenomena using in vitro-produced bovine embryos. Similar to humans, the first three mitotic divisions are able to successfully predict progression to the blastocyst stage (N=84). Bovine embryos frequently contained multi-/micro-nuclei, and DNA-sequencing of individual bovine blastomeres up to 12 cells confirmed that ~58 to 87% of cleavage-stage bovine embryos are aneuploidy (N=38) and often detectable by abnormal cell divisions. Transcriptional profiling of fragmented versus non-fragmented bovine embryos via RNA-sequencing identified a small subset of differentially abundant genes at the 4-cell stage. Pathway analysis showed reduced abundance of genes associated with the cytoskeleton, microtubules, and spindle in 4-cell embryos with cellular fragmentation as well as enrichment of membrane targeting and vesicle fusion pathways. The potential role of these cellular components in micronucleation and cellular fragmentation is being assessed by microinjecting bovine zygotes with fluorescently labelled mRNA mCherry-H2B (chromatin marker) and mCitrine-LaminB1 (nuclear envelope marker), followed by overnight live-cell multicolour confocal imaging (Zeiss LSM 880 with AiryScan; Zeiss, Thornwood, NY, USA). Results from these studies contribute to our knowledge of early embryogenesis with translational application to help ameliorate embryonic loss in women and cattle.
42 Nrf2 and nuclear factor kappa B cross-talk in bovine granulosa cells under lead challenge
Authors: Aglan H. S. , Gebremedhn S. , Salilew-Wondim D. , Neuhoff C. , Tholen E. , Held E. , Hoelker M. , Schellander K. , Tesfaye D.
Published: December 2018

Lead (Pb2+), one of the pervasive and protracted environmental heavy metals, is known to affect the female reproductive system by inducing oxidative stress. The Nrf2 and NF-κB are two key transcriptional factors (TF) known to regulate cellular redox status and response against stress and inflammation, respectively. Evidence showed that these TF undergo functional cross-talk. Although the properties of Pb2+ have been extensively studied, little is known about its effect on bovine granulosa cells with respect to Nrf2/NF-κB interaction. The aim of this study was to investigate the impact of Pb2+ on the response of bovine granulosa cells with regard to the cross-talk between Nrf2 and NF-κB. For this, bovine granulosa cells aspirated from small growing follicles (3-5mm of diameter) from ovaries obtained from a local slaughterhouse were in vitro cultured at 37°C and 5% CO2. Sub-confluent cells were exposed to different doses of lead acetate [Pb (C2H3O2)2] (1, 2, or 3µg mL−1) for 2h. Following this, the accumulation of intracellular reactive oxygen species was measured using the H2DCFDA fluorescent probe (Life Technologies, Darmstadt, Germany). The cell viability was assessed using Cell Counting Kit-8 (CCK-8, Dojindo, Munich, Germany) 24h after treatments. Moreover, cells were harvested and subjected to total RNA isolation using miRNeasy Mini Kit (Qiagen, Hilden, Germany) followed by cDNA synthesis using First Strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) and the gene expression was determined using quantitative PCR. Data were analysed using comparative threshold cycle (ΔΔCT) method and normalized against the expression of GAPDH and β-actin genes. Furthermore, the protein lysate was isolated from cells to assess carbonylated protein using OxyBlot protein oxidation detection kit (abcam, Cambridge, MA, USA). Results showed that exposure of granulosa cells to lead acetate provoked intracellular reactive oxygen species accumulation and protein carbonylation. This was accompanied by cell cycle arrest at G0/G1 phase, reduction in cell viability, and decrease in the expression of cell proliferation marker genes (CCND2 and PCNA). Moreover, lead acetate elicited down-regulation of both Nrf2 and NF-κB and their downstream antioxidant genes (SOD and CAT), whereas no significant difference was shown in the level of other genes involved in both TF pathways (Keap-1, HO-1, Trx, IKK, and TNF-α). In addition, lead acetate challenge increased the expression of endoplasmic reticulum stress marker genes (GRP78 and CHOP) and the pro-apoptotic gene (Caspase-3), whereas it reduced the anti-apoptotic gene (BCL-2). Our findings suggest that Pb+2 driven-oxidative stress deregulates granulosa cell viability and proliferation, enhances endoplasmic reticulum stress, induces cell cycle arrest, and mediates apoptosis probably via disruption of Nrf2/NF-κB cross-talk.
44 Misregulation of ten-eleven translocation 3 CXXC domain leads to abnormal formation of 5-hydroxymethylcytosine and expression of pluripotency genes in pig embryos
Authors: Uh K. , Ryu J. , Miko H. , Carey K. , Lee K.
Published: December 2018

Ten-eleven translocation (TET) methylcytosine dioxygenases are considered to play an important role in regulation of DNA methylation patterns by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). TET3 protein, a member of TET family, is enriched in mature oocytes and early stage embryos and contributes to DNA demethylation of the paternal genome in zygotes. N-terminal CXXC domain of TET3 is thought to be important in catalysing 5mC oxidation through its DNA binding potential. However, it is not clear whether specific DNA binding of CXXC domain is required for 5hmC conversion in mammalian embryos. Here, we investigated the role of TET3 CXXC domain in controlling 5hmC formation in fertilized pig embryos by injecting TET3 CXXC domain into mature pig oocytes as a dominant negative to inhibit the direct binding of TET3 to the genome through the CXXC domain. The CXXC domain of pig TET3 was identified through bioinformatics comparison of TET3 sequences among different species and cloned from mature pig oocyte-derived cDNA. To construct the green fluorescent protein (GFP)-CXXC fusion protein, CXXC sequence was subcloned into N-terminal GFP fusion vector, and then mRNA was synthesised by in vitro transcription. The GFP-CXXC mRNA (100 ng/µL) was injected into oocytes matured in vitro for 36 to 37h. Then, the oocytes were fertilized at 42h post-maturation. Water-injected oocytes served as a control. At 17h post-fertilization, zygotes were collected to analyse 5hmC level by immunocytochemistry. The level of 5hmC was analysed using ImageJ (https://imagej.nih.gov/ij/). Expression of pluripotency-related genes at Day 7 blastocysts was examined through quantitative RT-PCR; ΔΔCt method was used to analyse the quantitative RT-PCR data and Student’s t-test was used for statistical analysis. All experiments were conducted at least three times and P-values of less than 0.05 were considered significant. The GFP-CXXC was exclusively localised in pronuclei, indicating that the CXXC domain may lead to nuclear localization of TET3. The level of 5hmC in zygotes was not altered by the overexpression of GFP-CXXC. Interestingly, in 2-cell stage embryos, the 5hmC level was reduced in GFP-CXXC injected embryos compared with the control group, suggesting that CXXC domain is important for 5hmC formation post-DNA replication. There was an increase in transcript abundance of NANOG and ESRRB in blastocysts developed from GFP-CXXC injected oocytes compared with control blastocysts (P<0.05). There was no difference in the expression of POU5F1 and SOX2. In this study, we found that CXXC domain of TET3 is critical in maintaining the level of 5hmC formation at 2-cell stage and proper level of pluripotency-related genes (NANOG and ESRRB) in blastocysts. Future studies will focus on elucidating mechanisms behind the changes after overexpression of GFP-CXXC in pig embryos.
46 Morphologic and functional characterization of the early fetal equine gonads
Authors: Scarlet D. , Walter I. , Handschuh S. , Ellerbrock R. , Canisso I. , Aurich C.
Published: December 2018

In the equine embryo, putative primordial germ cells appear between 20 and 30 days and the gonadal primordium can first be identified at Day 30 after ovulation, respectively. Subsequently, sexual differentiation of the gonad occurs and completes by Day 45 of pregnancy. The objectives of this work were to describe the morphology and function of the fetal equine ovary and testis at the beginning of the fetal stage of pregnancy. For this purpose, 12 equine fetuses (6 males and 6 females) were collected at 45 days (n=1, female), 50 days (n=1, male), and 60 days (n=10, 5 males and 5 females) after ovulation, respectively. A high attention was given to Day 60 because it is the representative time for fetal sex determination in horses by transrectal ultrasonography. Conceptuses were collected transcervically by uterine lavage and fixed in 4% formaldehyde before being prepared for morphology analysis and immunohistochemistry assay. Gonads were identified and immunostained for anti-Müllerian hormone (AMH), Ki67, CD117, LIN28, vimentin, cytokeratin, and laminin. In all fetuses, gonads were situated in a sublumbar localisation and connected with the mesonephros. In females, primordial germ cells were localised close to the surface germinal epithelium, whereas in males the primordial germ cells were organised in cord-like clusters-the future seminiferous tubules. At this stage, interstitial cells predominate in the testes. The AMH staining was strongly expressed in the fetal testis, but was completely absent from the fetal ovary. Protein expression of mitosis marker Ki67 was localised in primordial germ cells of both sexes. Moreover, stem cell markers LIN28 and CD117 were also present in the gonads. In females, these proteins were not only localised in some of the primordial germ cells, but also in the surface germinal epithelium, whereas in males LIN28 and CD117 were immunolocalized in the seminiferous tubules, distant from the surface epithelium. Vimentin was strongly expressed in the interstitial cells of the gonads of both sexes. Using laminin staining, basal membrane of the seminiferous tubules in males and of primordial germ cells in females could be visualised. In females, the basal membrane of primordial germ cells also stained positive for cytokeratin, whereas in males no cytokeratin staining was seen around seminiferous tubules. Moreover, the surface germinal epithelium of both sexes stained positive for cytokeratin. This study widely extends existing knowledge about morphology, development, and function of the early fetal equine gonad. Presence of stem cells could be clearly demonstrated in the gonads of both sexes, whereas AMH staining clearly distinguished between males and females, confirming the important role of this hormone for gonadal and reproductive tract differentiation.
51 Effects of modulating early luteal phase progestin concentration on endometrial function in early pregnant mares
Authors: Aurich C. , Beyer T. , Scarlet D.
Published: December 2018

Progesterone prepares the endometrium for pregnancy. This requires down-regulation of progesterone receptors in the endometrial epithelium as a prerequisite for the expression of pregnancy-associated proteins. We investigated effects of modulated peripheral progestin concentration in early luteal phase mares on endometrial function on Day 14 of pregnancy. Genitally healthy oestrous mares (n=8; age 4 to 14 years) were inseminated until ovulation and treated with either altrenogest (0.044mg kg−1 once daily orally) on Days 5 to 10 after ovulation (ALT), cloprostenol (125mg once daily intramuscularly) on Days 0 to 3 after ovulation (CLO) or left untreated (CON). The ALT and CLO treatments were chosen to increase and decrease total peripheral progestin concentration, respectively. Each treatment was given to every mare in consecutive cycles at random order. On Day 14 after ovulation, endometrial fluid was collected with a cotton roll (Salivette, Sarstedt, Germany) inserted into the uterus and an endometrial biopsy for immunohistological examination was collected. In endometrial fluid, free amino acid concentrations were analysed by ion exchange liquid chromatography with an amino acid analyser (Institut Kuhlmann, Analytik-Zentrum Ludwigshafen, Germany). Cell nuclei staining positive for the progesterone receptor were determined in the luminar and glandular epithelium as well as in the stroma. Statistical analysis was performed by non-parametric Friedman test with subsequent Wilcoxon test. Values are given as mean±standard error of mean. Pregnancy rate was 0.6±0.1 (13 cycles/8 pregnancies), 1.0±0 (8 cycles/8 pregnancies), and 0.7±0.1 (11 cycles/8 pregnancies) in CON, ALT, and CLO cycles, respectively (P=0.062). Conceptus size between Days 10 and 14 did not differ among treatments. The percentage of luminal epithelial cells staining positive for progesterone receptor differed among treatments (CON 72.8±4.1, ALT 70.7±4.7, and CLO 84.1±1.9%; P<0.05) and was higher in CLO than in ALT and CON cycles (P<0.05). Free amino acids glutamic acid and glycine were most abundant in endometrial fluid, but their concentrations did not differ among treatments. The concentrations of the amino acids isoleucine (CON 0.17±0.03, CLO 0.14±0.02, and ALT 0.23±0.04 µmol) and lysine (CON 0.27±0.08, CLO 0.18±0.05, and ALT 0.44±0.13 µmol) were influenced by treatment (P<0.05) and lower in CLO than in ALT and CON cycles. In conclusion, impaired luteal function due to CLO treatment during the early luteal phase of pregnant mares delayed down-regulation of progesterone receptors in the endometrial epithelium on Day 14. This influenced endometrial function as reflected in lower concentrations of the amino acids lysine and isoleucine in endometrial secretions.
52 Decellularization of goat uterus as a promising 3-dimensional homing matrix of biological scaffold: A pilot study
Authors: Ghiringhelli M. , Verdile N. , Brevini T. A. L. , Gandolfi F.
Published: December 2018

Female infertility or absence of a functional uterus has a prevalence of 3 to 5% in the general human population. Nowadays most patients diagnosed with absolute uterine factor infertility remain untreatable. Moreover, the world population is aging rapidly, and as people live longer, uterus frailty increases the risk of developing age-related diseases. Whole-organ decellularization and recellularization, a novel technique within the field of regenerative medicine, could eventually provide another solution compared with the reality of gestational surrogacy. Extracellular matrix to enable recellularization has been described using different animal models, where decellularization has been performed of whole uteri. The aim of the present study was to offer a new perspective with a goat uterus as a large whole reproductive organ model. For this purpose, uteri were collected from 6 goats after slaughter and attached to the perfusion circuit. Decellularization was achieved by 2 methods of whole-uterus perfusion, namely with Triton-X100 and dimethyl sulfoxide (group 1; n=3), or with sodium deoxycholate (group 2; n=3), both compared with a control group (n=1). Morphological, immunofluorescence, real-time quantitative PCR analysis, coupled with vascular corrosion cast preparation, were used to assess protocol efficiency. Statistical analysis was carried out to identify significant differences between the 2 groups, using ANOVA and, when required, a post-hoc test (Bonferroni method). Morphological analysis showed different opacity between protocols. In both group 1 and 2, the removal of cells and cellular debris was confirmed with DNA quantification. Immunofluorescence confirmed the presence of the major extracellular matrix components, with collagen type I being the most abundant. Vascular tree network cast of the decellularized organ was successfully perfused in all the uteri. In conclusion, this preliminary study gives a new perspective in whole uterus bioengineering, utilising goats as a biological model to increase the knowledge in veterinary medicine as well as toward the in vitro and in vivo research of regenerative medicine in the field of reproduction.
Research was funded by the Carraresi Foundation.
53 Pre-implantation exposure to bisphenol A and 4-tert-octylphenol result in disruption of calcium channels
Authors: Tran D. N. , Lee J.-H. , Yoo Y.-M. , Jung E.-M. , Ahn C. , Park S. Y. , Lee B. , Jeon B.-H. , Nguyen T. H. T. , Jeung E.-B.
Published: December 2018

Miscarriage due to blastocyst implantation failure occurs in up to two-thirds of all miscarriage cases in humans. Calcium (Ca2+) has been shown to involve many cellular signal transduction pathways as well as regulation of cell adhesion, which is necessary for the physiology process of endometrial epithelial cell transformation and stromal cell decidualization during embryo implantation. Exposure to endocrine-disrupting chemicals (ED) can regulate the expression of genes associated with calcium transport in during pregnancy such as TRPV5, TRPV6, PMCA, and NCX1. Additionally, exposure to ED during early gestation results in disrupted intrauterine implantation, uterine receptive, leading to implantation failure. In this study, oestrogen (E2), bisphenol A (BPA), octylphenol (OP), and/or ICI 182,780 (oestrogen receptor antagonist, ICI) were injected subcutaneously from gestation Day 1 to gestation Day 3 post coitus. The number of implantation sites was significantly lower in the OP group, and no implantation site was observed in the E2 and ED+ICI groups. There were differences in the expression of calcium transient transport channel between maternal uterine and implantation. The level of TRPV6 and TRPV5 mRNA and protein was significantly increased by ED and/or ICI treatment in the uterus. The levels of TRPV5 and TRPV6 gene expression were significantly increased by ED with/without ICI treatment in the uterus. However, TRPV5 and TRPV6 gene expression was significantly lower in implantation site samples. The NCX1 and PMCA1 mRNA levels were significantly decreased by OP and BPA in the implantation site samples. Both mRNA and protein levels of MUC1 were markedly higher in all groups, except the BPA group when compared with the vehicle group in the uterus. The LIF and HOXA-10 mRNA were significantly low in E2; BPA+ICI; OP; and/or ICI in both the uterus and implantation site. Expression of the oestrogen receptor (ERa) and progesterone receptor (PR) was significantly lower in all groups except the BPA group when compared with the vehicle group. Taken together, E2, BPA, and OP disrupt the success of implantation through altered expression of calcium transport genes.
57 Non-targeted metabolomic profiles within the uterine milieu of porcine pregnancies containing populations of uniform or diverse spherical, ovoid, or tubular conceptuses during initiation of embryo elongation
Authors: Miles J. , Wright-Johnson E. , Walsh S. , Corey C. , Yao L. , Rempel L. , Pannier A.
Published: December 2018

Alterations in the signalling of critical molecular factors within the uterine milieu result in deficiencies in embryo elongation, leading directly to embryonic loss as well as delayed elongation. The objective of this study was to identify metabolites within the uterine environment from populations of uniform and diverse porcine conceptuses as they transition between spherical, ovoid, and tubular conceptuses during the initiation of embryo elongation. White crossbred gilts (n=38) were bred at standing oestrus (designated Day 0) and again 24h later and randomly assigned to collection group. At Day 9, 10, or 11 of gestation, reproductive tracts were collected immediately following harvest and flushed with 40mL of RPMI-1640 media. Conceptus morphologies were assessed from each pregnancy to assign to 1 of 5 treatment groups based on these morphologies: (1) uniform spherical (n=8); (2) diverse spherical and ovoid (n=8); (3) uniform ovoid (n=8); (4) diverse ovoid and tubular (n=8); and (5) uniform tubular (n=6). Subsequently uterine flushings from these pregnancies were submitted for non-targeted profiling by gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) techniques. Raw spectral data were processed using the XCMS package in R (R Foundation for Statistical Computing, Vienna, Austria) and features were clustered using RAMclustR. Unsupervised multivariate principal component analysis was performed in R using pcamethods package, and univariate ANOVA was performed in R with a Benjamini-Hochberg false discovery rate adjustment. Principal component analysis of the GC-MS and UPLC-MS data identified 153 and 104 metabolites, respectively. Of the identified metabolites, 51 and 71 metabolites from the GC-MS and UPLC-MS analysis, respectively, corresponded to known compounds. After false discovery rate adjustment of the GC-MS and UPLC-MS data, 38 and 59 metabolites from the GC-MS and UPLC-MS analysis, respectively, differed (P<0.05) in uterine flushings from pregnancies for the 5 conceptus stages. Some metabolites were greater (P<0.05) in abundance for uterine flushings containing earlier stage conceptuses (i.e. spherical) such as uric acid, tryptophan, 5-hydroxy-L-tryptophan, and L-tryosine. In contrast, some metabolites were greater (P<0.05) in abundance for uterine flushings containing later stage conceptuses (i.e. tubular) such as creatinine, serine, isovaleryl-I-carnitine, and lauric diethaolamide. These data illustrate several putative metabolites that change within the uterine milieu as porcine embryos transition between spherical, ovoid, and tubular conceptuses.
Funding was provided by USDA-NIFA-AFRI Grant no. 2017-67015-26456.
58 Transcervical transfer of blastocysts reveals detrimental effect on implantation rate in di (2-ethylhexyl) phthalate-exposed mice
Authors: Parra-Forero L. Y. , Mojica-Villegas A. , Alfaro-Pedraza E. , Hernández-Ochoa I.
Published: December 2018

Transcervical transfer is widely used in assisted reproduction techniques, such as cloning and the production of transgenic animals. Using this technique, the number of transferred blastocysts and implanted embryos may be quantified, representing a useful method for assessing the effect of xenobiotics on quality and functionality of the blastocyst. Di (2-ethylhexyl) phthalate (DEHP) is one of the most widely used plasticizers worldwide that causes several detrimental effects, such as decreased embryo quality and a low implantation rate when laboratory animals are treated with very high doses, ranging from 3000 to 3500 µg·kg−1d−1. The aim of this research was to evaluate the effect of environmentally relevant doses of DEHP on blastocyst functionality and ability to implant in an ex vivo study. The CD-1 mice were exposed subchronically (1 month) to 0, 20, 200, and 2000 µg·kg−1d−1 of DEHP via deposition in the mouth, and then they were subjected to a transcervical transfer procedure. The donor female mice were superovulated with pregnant mare serum gonadotropin (5IU) IP, followed by hCG (5 UI) IP 48h later. They were then mated with males of proven fertility. The recipient females (unexposed mice) had the same superovulation protocol as donors, but were mated with vasectomized males. Blastocysts were obtained 96h post-hCG by flushing the oviduct with Gardner-blastocyst medium and were classified according to morphological characteristics. Only expanded blastocysts were transferred transcervically under general anaesthesia protocol using isoflurane (induction: 3-5% and maintenance 2-2.5%). Four days after transcervical transfer, recipient females were injected with 200µL of Chicago Blue 1% IV, killed by cervical dislocation 2h later, following which implantation sites were counted and the implantation rate was calculated with the following formula (implanted blastocysts×100/transferred blastocysts). A decreased implantation rate was evident in 2000 µg·kg−1d−1 DEHP-treated females compared with controls (DEHP 2000: 11.67%±9.64v. control: 71.67%±6.0; P<0.05 according to 1-way ANOVA analysis followed by the Bonferroni post-hoc test). Taken together, these data suggest that the dose of 2000 µg·kg−1d−1 DEPH impairs embryo implantation. We confirm that transcervical transfer is a useful technique in toxicological studies to assess detrimental effects on implantation.