bluuebunny/crossref_metadata_2025 huggingface dataset:
137 Recovery of quail spermatogenesis by donor spermatogonia transplantation
Authors: Vetokh A. N. , Tomgorova E. K. , Volkova L. A. , Volkova N. A. , Zinovieva N. A.
Published: December 2019

Spermatogonia, the stem cell precursors of male germ cells, are used as convenient biological material for the preservation of genetic resources (cryobanks) and the introduction of recombinant DNA (transgenesis). Donor spermatogonia subsequently differentiate into mature germ cells (spermatozoa), which are used to produce offspring. Our laboratory is investigating methods to improve the efficiency of spermatogonial germ cell transplantation in quail. The objectives of this study were to (1) determine the optimal age for spermatogonia isolation from the testes of donor male quail and (2) identify the most appropriate concentration of busulfan for treatment of recipient quail testes. Statistical analysis was performed using SPSS ver. 15.0 (IBM Corp.; analysis of variance test). In order to determine the optimal age for spermatogonia isolation, testes from male quail at 1-4 weeks of age were isolated and histological studies were performed on a population of spermatogenic cells in the seminiferous tubules. Histological studies of quail testes isolated at different ages showed that the optimal age for obtaining a culture of spermatogonia is a period from 1-2 weeks of age. During this period, spermatogenic cells were represented mainly by spermatogonia (P<0.01). Therefore, testes of 1-week-old quails were used to obtain a culture of spermatogonia. The resulting cell culture consisted mainly of spermatogonia (85%) with a small number of Sertoli cells. Next, a series of experiments introducing busulfan into quail testes was carried out using concentrations from 10-150mg kg−1 of liveweight. Experiments showed that an effective dose to remove the recipient male's own spermatogenic cells was a concentration of 100mg kg−1 of liveweight (P<0.05). Finally, using the optimal parameters described above, spermatogonia cultures were obtained and introduced into the testes of quail recipients (n=6), following administration of busulfan for 2-3 weeks before donor spermatogonia were introduced. The effectiveness of spermatogenesis recovery was assessed based on the analysis of sperm from quail recipients at 3 months after the injection of donor cells. The presence of donor germ cells in the testes of quail recipient drakes was confirmed by microsatellite analysis of DNA isolated from the blood and sperm of recipients as well as the donor cells (spermatogonia). The microsatellite profiles of the blood and sperm DNA in quail recipient males were different, which confirms the restoration of spermatogenesis in the studied recipients due to the development of donor germ cells.
The reported study was funded by RFBR, project number 18-29-07079.
15 Combination of RepSox with histone deacetylation inhibitors on invitro development competence of porcine somatic cell nuclear transfer embryos
Authors: Luo Z.-B. , Xuan M.-F. , Li Z.-Y. , Yin X.-J. , Kang J.-D.
Published: December 2019

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). In this study, we compared histone deacetylase inhibitors combined with the pluripotency inducer RepSox on invitro development of porcine embryos produced via SCNT. Porcine embryos were treated with valproic acid (VPA), mocetinostat, M344 and panobinostat (LBH589) after SCNT, respectively. The porcine embryo invitro-development competence, histone modification level, and pluripotency-related genes expression were analysed. The results showed that LBH589 significantly increased the blastocyst formation rate compared with mocetinostat, M344, and control. In addition, VPA treatment increased the blastocyst formation rate of SCNT porcine embryos; both VPA-treated and the untreated clones developed to term, but offspring from VPA-treated embryos had a lower survival to adulthood than those from control embryos (18.2 vs. 67.0%; P<0.05). Furthermore, cotreatment with 12.5mM RepSox and 50 nM LBH589 (RepSox+LBH589) for 24h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9 vs. 8.5%, respectively; P<0.05). Moreover, RepSox + LBH589 improved epigenetic reprogramming by histone acetylation and methylation. The expression of pluripotency-related genes NANOG and SOX2 was found to be significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming, and improves the invitro development of porcine SCNT embryos.
16 Improvement of porcine cloned embryo developmental competence via KDM4A overexpression and H3K9me3 methyltransferase inhibitor treatment
Authors: Zhang Y.-T. , Liu Y. , Liu Z.
Published: December 2019

Aberrant epigenetic reprogramming is a major reason for the developmental failure of somatic cell nuclear transfer (SCNT) embryos. Histone H3 lysine 9 trimethylation (H3K9me3), a histone marker for transcriptional repression, is considered a key barrier to the development of cloned embryos. In the present study, we found that H3K9me3 was much higher in SCNT embryos than in IVF embryos at the 4-cell and 2-cell stages; H3K9me3 demethylase KDM4A mRNA was injected into cloned embryos 5h after activation. The intensity of H3K9me3 modification decreased obviously after microinjection of KDM4A mRNA, and the developmental efficiency of porcine cloned embryos was enhanced significantly compared with control (32.2±4.2 vs. 21.0±3.5; P<0.05). Moreover, we evaluated the effect of chaetocin, an inhibitor of histone methyltransferase suv39h1/2, on SCNT embryo development. The results showed that 10 nM chaetocin not only suppressed the H3K9me3 modification in porcine embryonic fibroblast but also downregulated the expression of SUV39H1, SUV39H2, and KDM4D. However, treatment of cloned embryos with 10 nM chaetocin efficiently decreased the H3K9me3 level. Importantly, chaetocin treatment at the 4-cell stage for 6h significantly increased the blastocyst rate (57.8±4.3 vs. 43.5±1.8; P<0.05) compared with the control group. Furthermore, because a recent study showed that a high level of H3K9me3 appears in the reprogramming resistance region of nuclear transfer embryos during zygogene activation (ZGA), we examined the expression levels of ZGA-related genes. The qPCR results showed that the expression of ZGA-related genes increased significantly in SCNT embryos with chaetocin treatment compared with the control. These results suggested that chaetocin treatment can improve the efficiency of SCNT reprogramming during ZGA. In summary, our results suggested that H3K9me3 acts as an epigenetic barrier in porcine SCNT reprogramming and that a suv39h1/2 inhibitor can effectively reduce the H3K9me3 level in the early reprogramming phase and further improve the invitro developmental competence. Due to the existence of a variety of abnormal epigenetic mechanisms during somatic cell reprogramming, the combined use of small-molecule inhibitors is required in future studies. Data analyses were performed using SPSS software. Significance was set at P<0.05 unless otherwise specified. The results are expressed as the mean±standard deviation.
This work was supported by the National Natural Science Foundation of China (grant number 31601942); the Postdoctoral Science Foundation of Heilongjiang Province (grant number LBH-Z17010); and the Fund for the National Key Research and Development program of China-Stem Cell and Translational Research (2016YFA0100200).
165 Comparison of spermatozoa traits and testosterone levels, seminal plasma constituents, and lipid peroxidation of Large White×Landrace and Kolbroek boars
Authors: Netshirovha T. R. , Thomas R. S. , Kanengoni A. T. , Matabane M. B. , Mphaphathi M. L. , Pilane C. M. , Chimonyo M.
Published: December 2019

Reproductive traits in domestic pigs are one of the most important economic characteristics for improved pig production. Seminal plasma (SP) constituents provide a better indicator of male fertility as measured by semen traits. The objective of this study was to compare spermatozoa traits, testosterone concentration, SP constituents, and lipid peroxidation in Large White×Landrace (LW×LR) and Kolbroek boars aged between 8 and 9 months. Kolbroek (n=12) and LW×LR (n=12) boars were used in this study. Semen was collected using the gloved-hand technique. Blood samples were collected from each boar monthly to determine testosterone concentrations. Spermatozoa motility traits was evaluated using computer-aided sperm analysis. Spermatozoa viability was evaluated using the hyperosmotic swelling test, lipid peroxidation was evaluated using the malondialdehyde (MDA) test, and SP protein was evaluated using atomic absorption spectrophotometric analysis. Analysis of variance was used to test the difference between the breeds. The LW×LR boars had higher ejaculation volume and testosterone concentrations (P<0.05) than Kolbroek boars. The average spermatozoa concentration for Kolbroek and LW×LR boars was 1.8±1.0 and 1.9±1.1×109mL−1, respectively (P>0.05). The average genotype spermatozoa total motility was 91.7±4.0 and 90.6±4.1%, respectively (P>0.05). No significant differences were found between genotype in abnormal spermatozoa morphology, rapid spermatozoa, and total spermatozoa motility, spermatozoa velocity on the curve line, and hyperosmotic swelling test. Spermatozoa with distal, head, and midpiece abnormalities were significantly different in Kolbroek boars (17.1±1.4, 5.0±2.0, and 7.0±2.6%, respectively) and LW×LR boars (9.2±0.1, 3.5±1.1, and 2.6±1.2%, respectively). The LW×LR boars had lower levels of MDA than Kolbroek boars (P<0.05). The SP constituents with selenium, sodium, magnesium, and calcium were significantly different in Kolbroek boars (166.6±1.0, 167.2±0.2, 5.0±0.8, and 10.2±0.3%, respectively) and LW×LR boars (155.0±0.0, 140.0±0.5, 12.0±0.2, and 22.3±0.0%, respectively). In conclusion, spermatozoa parameters of Kolbroek boars were similar to those of LW×LR boars except for semen volume, head and distal spermatozoa abnormalities, testosterone concentration, MDA, and seminal plasma. Further studies need to be conducted on the major seminal plasma constituents as well as their physiological roles.
178 Evaluation of polar body extrusion following exposure of cattle oocytes to different concentrations of ethylene glycol cryoprotectant
Authors: Mphaphathi M. L. , Nkadimeng M. , Sithole S. M. , Sebopela M. D. , Ramukhithi F. V. , Tshabalala M. M. , Maqhashu A. , O'Neill H. , Nedambale T. L.
Published: December 2019

Cryoprotectant (CPA) toxicity has been recognised as a critical barrier to further advancement of cattle oocytes. Furthermore, CPA at high concentration might be toxic to oocytes, leading to osmotic shock and cell death (Arav et al. 1993). Selection of quality oocytes among a heterogeneous pool is mostly done subjectively. The use of Brilliant Cresyl Blue (BCB) stain for oocyte selection before ethylene glycol (EG) CPA toxicity test on cattle oocytes might be a useful tool. The objectives of this study were to elucidate the toxicity of EG penetrating CPA to cattle oocytes and the effectiveness of BCB on oocyte selection. Ovaries from cows of unknown reproductive status were collected from the local slaughterhouse and transported in warmed (37°C) saline water to the laboratory within 2h of slaughter. The oocytes (n=374) were exposed to 26mM BCB for 90min at 38.5°C under an atmosphere of 5% CO2. The other oocytes (n=450) were not exposed to BCB solution or CPA (positive control: no BCB and no CPA exposure). Oocytes were classified as BCB positive (+) with a varying degree of blue cytoplasm or BCB negative (−) with no blue cytoplasm. Oocytes were exposed in EG at different CPA concentrations as follows: Toxicity test 1 (TT1) was 0, 5, 10, and 15%, followed by exposure to TT2 as follows: 10, 20, and 30% (stepwise increased CPA). The oocytes were then invitro matured (IVM) as per treatment groups for 22h. After maturation, oocytes were removed from the maturation medium and denuded of granulosa cells by vortexing. The polar body extrusion was evaluated with the aid of Oosight Imaging System (Hamilton Thorne) connected to an inverted research microscope. Treatment means were compared using Fisher protected t-test least significant difference. There was a drastic decline of oocytes with polar body extrusion as CPA concentration increased (P<0.05): 40.5% (positive control, no BCB and no CPA exposure), 33.3% [(control, CPA exposure (EG 5 + 10%)], 23.1% [BCB− with CPA toxicity test (EG 5 + 10%)], 12.5% [(BCB− with CPA toxicity test (EG 10 + 20%)] and 4.9% [(BCB− with CPA toxicity test (EG 15 + 30%)]. The BCB+ groups (EG 5 + 10% and EG 10 + 20%) had significantly more oocytes with polar body extrusion (68.9% and 51.9%) compared with the positive control (40.5%), respectively (P<0.05). In conclusion, higher EG cryoprotectant concentrations compromise oocyte polar body extrusion following IVM. We recommend that BCB be used for selection of suitable oocytes before the CPA toxicity test because of its ability to stain larger and more competent oocytes from cattle.
193 Effect of clinical metritis on oocyte recovery, oocyte quality, and early invitro developmental competence of embryos in Bos indicus dairy cattle
Authors: Saleem M. , Sarwar Z. , Saad M. , Zahoor I. , Ahmad N. , Riaz A.
Published: December 2019

Unhygienic practices at the time of parturition or AI lead to uterine infections. The uterine infections ultimately result in genetic drain by culling the elite animals. The invivo developmental competence of embryos is compromised in clinically metritic animals. The genetic potential of problematic females could be harvested by invitro embryo production (IVEP). Therefore, the objective of the present study was to evaluate the effect of clinical metritis on oocyte recovery, oocyte quality, and early invitro developmental competence of embryos in Bos indicus dairy cattle. This experiment was carried out from December 2017 to April 2018. Ovaries were collected from a local abattoir (Bos indicus; 5- to 8-year-old dairy cattle, body condition score 2.75±0.25, mixed parity). These ovaries (n=982) were divided into two groups: (1) clinically metritic (n=184), and (2) healthy (n=798), based upon the presence or absence of pus in the uterine lumen. Oocytes were aspirated from follicles using an 18G needle attached to a 10-mL syringe. Cumulus-oocyte complexes (COCs) were categorized into A, B, C, and D grades based on the number of layers of cumulus cells and integrity of ooplasm. The oocytes of grades A and B were subsequently transferred in groups (10/group) in four-well plates containing 100-μL droplets. The droplets with oocytes were covered with prewarmed mineral oil and incubated for 24h at 38.5°C, 5% CO2, and 95% relative humidity. The oocytes were evaluated for IVM on the basis of cumulus expansion. Frozen semen was thawed and prepared using the sperm swim-up procedure for each group. Spermatozoa and oocytes were incubated together for a period of 18h. The presumptive zygotes were invitro cultured for 4 days in a CO2 incubator under similar culture conditions. The cleavage rate, 4-cell, and 8-cell stages were recorded on Days 2, 3, and 4 after the day of insemination, respectively. Data on oocyte recovery, oocyte quality, IVM, cleavage rate, and 4-cell and 8-cell stages were analysed by Chi-squared test using SPSS software (version 20; IBM Corp.) for Windows. Results demonstrated that recovery rate was lower (63.8% vs. 71.7%; P<0.05) in clinically metritic compared with healthy cattle. Similarly, oocytes of grade A and B quality were lower (41.0% vs. 51.1%; P<0.05), whereas those of C and D quality were higher (59.0% vs. 48.9%; P<0.05) in clinically metritic compared with the healthy group. Moreover, 4-cell (38.2% vs. 54.8%) and 8-cell stage embryos (11.3% vs. 29.1%), were lower (P<0.05) in the clinically metritic compared with the healthy group, respectively. However, maturation rate and cleavage rate did not differ (P>0.05) between groups. In conclusion, metritis in slaughterhouse ovaries negatively affects oocyte recovery rate, oocyte quality, and early invitro developmental competence of embryos in Bos indicus dairy cattle.
203 High environmental temperatures during early fetal life may impair the ovarian reserve in cattle
Authors: Succu S. , Sale S. , Ghirello G. , Ireland J. , Evans A. , Atzori A. , Mossa F.
Published: December 2019

The causes of the inherently high variation in number of follicles and oocytes in ovaries of mammals are unknown. Evidence suggests that the ovarian reserve (total number of healthy follicles and oocytes in ovaries) can be programmed by events occurring during fetal life. For instance, maternal nutritional restriction during the first trimester of pregnancy negatively affects the size of the ovarian reserve in calves. The aim of the present study was to establish whether exposure of pregnant dairy cows to high environmental temperatures from conception to the end of the first trimester of pregnancy impairs establishment of the ovarian reserve in their offspring. This work was conducted in four commercial dairy farms with similar nutrition and farming systems located in Sardinia, Italy, on a total of 310 Holstein-Friesian dairy heifers (16 months old) that were conceived and born at different times of year coincident with different environmental temperatures. We tested whether exposure of the heifer's dams to a mean temperature-humidity index (THI) >68 from conception to the end of the first trimester of pregnancy resulted in a diminished ovarian reserve in their offspring. To estimate the size of the ovarian reserve, a single blood sample was collected to measure serum anti-Müllerian hormone (AMH; n=310), and the number of follicles >3mm (antral follicle count, AFC) was assessed using transrectal ovarian ultrasonography (n=258) on a random day of the oestrous cycle (16.09±0.07 months of age). Relations among variables were analysed with Pearson correlation with SAS (SAS Institute Inc.). Anti-Müllerian hormone and AFC were analysed with a mixed model (PROC MIXED of SAS) considering the main effects of season during the first trimester of fetal life and age at sampling; the effect of farm was included as a random effect. Tukey's test was used for comparisons. Circulating AMH concentrations and AFC were highly positively correlated (P<0.0001), as previously reported. The results also showed that both AMH concentrations and AFC were lower (419.27±22.81 pgmL−1, 9.32±0.42 follicles; P<0.0001) in young adult heifers of the dams exposed to a THI >68 compared with dams exposed to an average THI of 55 (634.91±47.60 pgmL−1, 11.84±0.46 follicles). Neither AMH nor AFC were influenced by farm and age at sampling of the daughters. In conclusion, maternal exposure to THI >68 (typical high temperatures during summers in Sardinia) during the first trimester of pregnancy has a negative effect on the development of the ovarian reserve in female fetuses, which may subsequently impair their reproductive performance as adults.
Research was funded by Regione Autonoma della Sardegna, Legge Regionale 7, Bando 2015.
208 Effect of growth factors and reprogramming molecules on induction to multipotency of dermal fibroblasts from colocolo (Leopardus colocolo)
Authors: Echeverry D. , Rojas D. , Aguilera C. , Rodriguez-Alvarez L. , Castro F.
Published: December 2019

Reprogramming of terminally differentiated cells to higher plasticity levels can be achieved with small molecules. This can be of value for somatic cell nucleus transfer, deriving multi and pluripotent cells and conservation purposes. Recently, induced mesenchymal stem cells were derived from differentiated human and mouse cells by using small molecules and growth factors. The pampas cat or colocolo (Leopardus colocolo) is a South American felid categorrized as near threatened by the International Union for Conservation of Nature (IUCN) Red List of Threatened Species. Major historical threats to the pampas cat include illegal hunting, habitat loss or transformation, and conflict retaliation for poultry predation. Here, we tested 5-azacytidine (an epigenetic modifier) and A8301 (a potent inhibitor of transforming growth factor-β type I receptor superfamily), linked to platelet-rich plasma (PRP) and platelet-derived growth factor (PDGF-B) to induce changes in the expression of pluripotency genes and differentiation capacity of colocolo fibroblasts towards other mesodermal lineages. For this, dermal fibroblasts were treated with (I) 5-azacytidine + PRP + A8301 + VitC, or (II) 5- azacytidine + VitC + A8301 + PDFG for 12 days. On Days 0, 5, and 12 of reprogramming, expression of OCT4, NANOG, E-cadherin and SNAIL was evaluated by reverse transcription-PCR, and tri-lineage differentiation was induced. For treatment I, no statistical difference was found in the expression of OCT4 and NANOG. Chondrogenic and osteogenic differentiation was observed. In treatment II, significant expression of OCT4 and NANOG (P<0.05) was induced, and reprogrammed fibroblasts were differentiated into chondrogenic and osteogenic lineages. Immunohistochemistry positivity for OCT4 was detected in treatment II. In summary, we showed that dermal fibroblasts of pampas cat can be reprogrammed into cells with multipotent characteristics, particularly when a cocktail of 5-azacytidine + VitC + A8301 + PDFG was used. Treatment I probably failed because of other growth factors and proteins present in PRP, which might inhibit successful reprogramming or activate other pathways leading to a nonmultipotent phenotype. Further refinements of these protocols are required to improve the reprogramming protocol. This in turn should help us obtain cells that can be used in nucleus transfer or cellular therapies in endangered felid species.
216 Oxygen levels and pluripotency maintenance supplements affect cellular reprogramming of bovine fibroblasts
Authors: Bessi B. , Botigelli R. , Recchia K. , Pieri N. , Barbosa G. , de Castro R. , de Souza A. , Meirelles F. , Bressan F.
Published: December 2019

Different supplements are used during invitro cellular reprogramming, usually acting on pluripotency maintenance and/or differentiation inhibition, such as basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), and 2i (MEK inhibitor: PD0325901 + GSK3 inhibitor: CHIR99021). Another important factor affecting the reprogramming process is the oxygen (O2) tension because O2 levels can modify cellular metabolism and epigenetic markers, which are known to modulate pluripotency. Our objective was to evaluate the efficiency of reprogramming bovine fibroblasts in combination with different oxygen tensions (high O2, hO2×low O2, lO2) in different cell differentiation inhibitors: bFGF and bFGF + LIF + 2i (FL2i). Bovine fibroblasts were transduced with lentivirus harbouring mouse OSKM transcription factors (OCT4, SOX2, KLF4, and cMYC). Three clonal lineages were analysed for each experimental group. Pluripotency was characterised by morphology, detection of alkaline phosphatase, formation of embryoid bodies, and analysis of gene expression. As an initial pluripotency test, all colonies were positive for alkaline phosphatase (AP) activity in passages 5 to 6. Colonies were cultured for at least 15 passages (±140 days) with the exception of bFL2i colonies cultured in lO2, which did not grow beyond 7 to 8 passages. For gene expression analysis, samples of each colony in passages 5, 10, and 15 were used. When gene expression was analysed, both endogenous NANOG and OCT4 were increased in the bFGF group when cultured in hO2, and bFGF cultured in lO2 was higher than in the FL2i group (P<0.05). Also, NANOG was increased in early passages compared with late passages (P<0.05); SOX2 and FGF5 were increased in lO2 groups (P<0.05). The bFGF treatment increased STELLA expression compared with bFL2i (P<0.05) at both oxygen tensions. Interestingly, exogenous vector expression increased in the bFGF group compared with bFL2i (P<0.05) but was not affected by oxygen tension (P>0.05). All colonies tested were able to form embryoid bodies. In conclusion, it was not possible to maintain bovine induced pluripotent stem cells (biPS) in bFL2i treatment cultured in lO2 because these colonies were not able to remain viable after 8 passages. Moreover, small molecule supplementation strongly affected pluripotency gene expression. Further analysis on epigenetic changes, metabolism, and self-renewal is necessary to understand the pluripotent state in biPS under our experimental conditions.
We acknowledge FAPESP for funding (grant 2015/26816-5 and fellowship 2018/24520-7).
227 Is the antral follicle count on a random day of the oestrous cycle correlated with superovulatory responses in Santa Inês ewes?
Authors: Pupin M. , Vergani G. , Lima M. , Silva K. , Monteiro A. , Ramos A. , Batista R. , Vicente W. , Oliveira M. , Fonseca J.
Published: December 2019

Antral follicle count (AFC) performed after an oestrus synchronization protocol has been studied as a tool to select ewes with high potential for invivo embryo production (Pinto et al. 2018 Theriogenology 113, 146-152). However, it would be interesting to know whether AFC assessed on a random day of the oestrous cycle correlates with the superovulatory response. The present study was conducted to evaluate the correlation between AFC at the beginning of progesterone (P4)-based oestrus synchronization protocol used as basis of superovulatory treatment and the number of corpora lutea (CL) 12h before recovery of embryos in Santa Inês ewes. The study was conducted during September and October in northeast Brazil (03°40′26″S and 40°14′20″W) using 8 adult Santa Inês ewes. On a random day of oestrous cycle (Day 0) all ewes received an intravaginal device (CIDR) of progesterone (0.3g, Eazi-breed, Zoetis), which remained for 9 days. On Day 7, the pFSH (133mg, Folltropin V, Vetoquinol) treatment began, with 6 decreasing doses (25, 25, 15, 15, 10, and 10%) injected IM at 12-h intervals. On Day 9, 2 equal doses of D-cloprostenol were injected at a 12-h interval (37.5µg, Prolise, Agener União). All ewes showed oestrus and were mated by fertile rams. Flunixin meglumine (24.9 mg; Banamine, MSD Animal Health) was administered IM on Days 12, 13, and 15. On Day 16, non-surgical embryo recovery (NSER) was performed after cervical dilation using D-cloprostenol and oestradiol benzoate at 16h and oxytocin 20min before. Transrectal B-mode ultrasound evaluations (Z5 Vet, Mindray), frequency 7.5MHz, were performed on Day 0 and 7 and 12h before NSER to evaluate the ovarian population present. Pearson correlation analysis (P<0.05) was performed using Bioestat 5.3 software. The number of AFC per ewe at the beginning of the protocol and on Day 7 were 9.9±2.7 and 11±3.2, respectively. The numbers of CL, recovered embryos, and viable embryos were 14.0±3.5, 8.2±10.9, and 6.0±11.0, respectively. There was no correlation of AFC on a random day of oestrous cycle with the number of AFC on Day 7 (P=0.42), number of corpora lutea (P=0.44), number of recovered embryos (P=0.18), or number of viable embryos (P=0.11) in superovulated ewes. In conclusion, we did not find significant correlations between AFC on a random day of oestrous cycle and the superovulatory/embryos response in Santa Inês ewes.
Financial support for this study was provided by Embrapa (02.13.06.026.00.02 and 02.13.06.026.00.04) and FAPEMIG (PPM 00201-17).
230 Cryopreservation for Canis lupus conservation: Evaluating protocols to freeze grey wolf testicular tissue
Authors: Andrae C. , Ferraz M. , Songsasen N. , Nagashima J.
Published: December 2019

Because of the severe decline in grey wolf (Canis lupus) abundance, preservation of genetic diversity within the species is essential to prevent inbreeding depression. Cryopreservation is a valuable tool in endangered species conservation, because it allows genetic material to be rescued from rare individuals for future use. The ability to preserve gonadal tissue is particularly useful in seasonal breeders, such as grey wolves, when mature gametes are not available during the non-breeding season. Optimization of species-specific cryopreservation protocols is necessary due to the toxicity of cryoprotectants. Here, we aimed to investigate three protocols to cryopreserve grey wolf testicular tissue, as a model for endangered canid species. Testicular tissue samples from four adult grey wolves were obtained and cryopreserved using slow-freezing (SF) and needle vitrification (nVT) techniques. Slow-frozen samples were exposed to 15% dimethylsulfoxide (DMSO) or 7.5% DMSO + 7.5% ethylene glycol (EG) in minimum essential medium (MEM) at room temperature, placed in a Mr. Frosty Freezing Container (Thermo Fisher Scientific) and cooled to −80°C overnight, before storage in liquid nitrogen. Needle-vitrified samples were equilibrated in a 7.5% DMSO + 7.5% EG solution in MEM for 10min at 4°C, then treated with 15% DMSO + 15% EG + 0.5M sucrose for 10min at 4°C before plunging into liquid nitrogen. Each sample was thawed using serial dilutions of sucrose (1, 0.5, and 0.25) into MEM at 37°C for 5min each, fixed in 4% paraformaldehyde, and embedded in paraffin wax. Five-micron-thick cross-sections of tissue samples were stained using haematoxylin and eosin. Tubule areas and quantity of each cell type (Sertoli cells, spermatogonia, spermatocytes, spermatids, and abnormal cells) were recorded and compared with a fresh control group for each wolf. Cell populations and tubule areas were analysed using a nonparametric Wilcoxon test through JMP statistical software (SAS Institute Inc.), and are presented as means±standard deviations. There was no difference in tubule areas between the treatment groups (fresh=5200±2870, nVT=5655±2370, SF DMSO=3607±814, SF DMSO+EG=3905±1450µm2; P>0.05). Although there was an increase in abnormal cells/tubule area (µm2) for nVT, SF DMSO, and SF DMSO+EG compared with the fresh samples (0.0015±0.0014, 0.0014±0.0013, 0.0016±0.0011, and 0.0007±0.0004, respectively; P>0.05), differences were not statistically significant. Moreover, the number of Sertoli cells, spermatogonia, and spermatocytes were reduced in the cryopreserved groups compared with fresh tissues, but the differences were not statistically different (P>0.05). The data suggest that the three tested protocols preserved testicular structure and cell populations of cryopreserved testicular tissue and could be useful in the genetic preservation of grey wolves.
30 Supplementation of invitro culture medium with linoleic acid albumin improves bovine embryo survivability in low-temperature storage at 4°C
Authors: Jung S. K. , Nishisouzu T. , Dochi O. , Imai K.
Published: December 2019

The objective of this study was to investigate the addition of linoleic acid albumin (LAA) to invitro culture medium to improve the development of blastocysts with low permeability and fluidity in serum-supplemented medium. Currently, vitrification and slow freezing are performed as cryopreservation methods of bovine embryos. Recently, it was reported that invivo embryos stored at 4°C for 168h retained their pregnancy competence. For IVF, embryo development and cryotolerance are highly influenced by the presence of serum in the culture medium. Thus, this study aimed to confirm the survivability of invitro fertilized embryos cultured in medium supplemented with LAA after low-temperature storage. The cumulus-oocyte complex collected from slaughterhouse-derived ovaries was matured for 20h in tissue culture medium-199 supplemented with 0.02 AUmL−1 FSH and 5% calf serum (CS). After cumulus-oocyte complexes were incubated with 5×106 spermatozoa per mL for 6h and the presumptive zygotes were removed, their cumulus cells were cultured with CR1aa supplemented with 5% CS (control) or CR1aa supplemented with 0.3mgmL−1 LAA and 5% CS (LAA group) for 7 or 8 days. The code 1 or 2 blastocysts that developed on Days 7 and 8 were used in this study. The developed blastocysts were equilibrated to the low temperature storage medium (25mM HEPES buffered tissue culture medium-199 supplemented with 50% fetal calf serum (FCS)) for 20min and stored in a refrigerator at 4±1°C for 48 or 72h. After low-temperature storage, the blastocysts were diluted in a buffer (Dulbecco's phosphate-buffered saline) supplemented with 10% FCS for 10min, washed three times with tissue culture medium-199 supplemented with 20% FCS and 0.1mM β-mercaptoethanol, and cultured in the same medium. Thereafter, blastocyst survival (re-expansion) and hatching were observed for up to 72h. The data were analysed using the chi-squared test with Yates's correction. The results of 48-h low-temperature storage of blastocysts for the control (n=55) and LAA (n=28) groups showed significant differences (P<0.01) in survival (41.8 and 89.3%, respectively) and hatched rates (20.0 and 89.3%, respectively). The results of 72-h low-temperature storage of blastocysts for the control (n=13) and LAA (n=38) groups showed significant differences (P<0.01) in survival (7.7 and 52.6%, respectively) and hatched rates (7.7 and 52.6%, respectively). These results suggested that supplementation of culture media with LAA improved the survivability of expanded blastocysts after low-temperature storage.
44 Analysis of abnormal chromatin configuration induced by inhibiting MEK at the 1-cell stage
Authors: Magara K. , Naruto S. , Watanabe R. , Wakayama T. , Kishigami S.
Published: December 2019

After fertilisation, mammalian oocytes resume meiosis, leading to pronuclear (PN) formation. It has been known that the mitogen-activated protein kinase pathway negatively regulates PN formation. However, it remains largely unknown how the mitogen-activated protein kinase pathway is involved in the dynamic organisation of chromatin structure in PNs. Here, to address how MEK1/2 activity is involved in PN formation and transcriptional regulation, we examined whether treatment of fertilised oocytes with MEK1/2 inhibitor (PD0325901; PD) during the 1-cell stage affects their global epigenetic modifications and heterochromatin formation in mice. We performed IVF using superovulated oocytes from and ICR strain female mouse and treated IVF embryos with 1 µM PD for 10h from insemination to observe global epigenetic modifications such as H3K9me3 or H3K27me3, localization of heterochromatin protein. First, we found that PD treatment enhanced PN formation with abnormal pronuclear sizes, that is, larger female and smaller male PNs. Further, PD treatment for 10h after fertilisation caused significantly lower global H3K9me3 or H3K27me3 modifications in female PN [H3K9me3: 1.00±0.01 vs. 0.75±0.01 (N>10); P=1.0×10−2, H3K27me3: 1.00±0.01 vs. 0.55±0.01 (N>10); P=6.0×10−7], suggesting failure of establishment of normal histone modifications for heterochromatin. Consistently, we found loss of HP1b around the nucleolus precursor body, which is normally associated with pericentric heterochromatin, suggesting that MEK1/2 activity is required for normal PN and heterochromatin formation. Regardless, embryos treated with PD at the 1-cell stage recovered such global histone modifications through the pre-implantation embryos to reach the blastocyst stage. Together, our data show that MEK1/2 activity plays a pivotal role not only in regulation of PN formation but also heterochromatin formation during the 1-cell stage in mice.
5 Transport and cholinergic innervation in the bovine oviduct are dysregulated in cystic ovary disease
Authors: Scully D. , Campion D. , McCartney F. , Reese S. , Kölle S.
Published: December 2019

Cystic ovary disease (COD) is one of the most common causes of bovine infertility and affects 7-14% of cows in European herds. The morphological and molecular causes of this disease are largely unknown. Because the oviduct is pivotal to gamete transport, fertilization, and early embryonic development, the aim of this study was to analyse the effects of COD on the function of the oviduct. Oviducts were excised from cows affected by COD (n=29) as well as from healthy cows in mid diestrus (n=20) immediately after slaughter. A unique digital live cell imaging system established in our laboratory was used to capture real-time videos of ciliary beat and tubal fluid flow under near invivo conditions. For ciliary beat frequency, the differences in grayscale of beating cilia were transformed into frequencies using ImageJ (National Institutes for Health) and AutoSignal (SyStat Software Inc.). For particle transport speed (PTS), polystyrene beads were added to the buffer media and were automatically tracked using ImagePro (Media Cybernetics). Additionally, smooth muscle contraction and epithelial ion transport were investigated using organ baths and Ussing chambers. Our results showed that PTS was significantly decreased in oviducts from cows affected by COD as compared with controls (P=0.01, unpaired t-test). Further, in healthy control cows, PTS was consistently increased in the oviduct ipsilateral to ovulation as compared with the contralateral oviduct (P=0.03, paired t-test). This was not the case in cows affected by COD (P=0.47, paired t-test). Reduced PTS in oviducts from cows with COD was not due to changes in ciliary beat frequency. Although smooth muscle contraction was similar in oviducts from healthy and COD cows, the contractile response (mN) to the cholinomimetic drug carbachol (10−7-10−4 M) was significantly reduced in COD as compared with the controls (P<0.0001, nonlinear regression best fit analysis). Active epithelial ion transport, as measured by the change in short circuit current (μA cm−2), was stimulated by carbachol. Carbachol-induced active ion transport in the oviducal epithelium of COD cows was significantly decreased as compared with controls (P=0.03, unpaired t-test of area under the curve). These results suggest, for the first time, that the transport of the gametes and the early embryo might be impaired in COD. Further, inadequate or insufficient cholinergic innervation may result in dysregulated fluid flow, which might be associated with alterations in oviducal metabolism and gamete or embryo nutrition. This knowledge is pivotal to establish novel therapeutic concepts for successful treatment of infertility in individuals affected by COD.
55 Identification of microRNAs associated with sex determination in bovine amniotic fluid and maternal blood plasma
Authors: Sánchez J. M. , Gómez-Redondo I. , Browne J. A. , Planells B. , Gutiérrez-Adán A. , Lonergan P.
Published: December 2019

In most eutherian mammals, sex determination is the process through which a bipotential gonad (also known as genital ridges) develops into a testis or ovary depending on the sex chromosome content of the embryo, specifically by the presence of the SRY/Sry gene (sex-determining region of the Y chromosome). MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression and are involved in diverse functional roles including development, differentiation, apoptosis, and immunity. We hypothesised that the expression of miRNAs in amniotic fluid (AF) and maternal blood plasma (MP) would be affected by the sex of the embryo around the time of sex determination. Amniotic fluid and MP were collected from 6 crossbred beef pregnant heifers (3 carrying a single male and 3 carrying a single female embryo) following slaughter on Day 39 (when the peak of SRY expression occurs in cattle). All heifers had been synchronized and inseminated with semen from the same beef bull. A total of 12 samples (6 AF and 6 MP) were profiled using the miRCURY LNA miRNA Serum/Plasma Focus PCR Panel (Qiagen; 179 assays targeting relevant miRNAs). Data were analysed by GeneGlobe Data Analysis Center (Qiagen). A threshold cycle cut-off of 35 was applied and data were analysed using an unpaired t-test. Gene ontology enrichment analysis was performed using the WebGestaltR package to explore the possible functions of differentially expressed (DE) miRNAs. In this study, DE miRNAs were identified in male vs. female AF (n=5; 3 upregulated and 2 downregulated; P<0.05) and MP (n=57; 54 upregulated and 3 downregulated; P<0.05). Although no enrichment was detected for DE miRNAs in AF (in either sex) or in MP in heifers carrying a female embryo, 37 biological processes were enriched by DE miRNAs in MP of heifers carrying a male embryo (false discovery rate<0.05). Interestingly, the top five most enriched biological processes were male gonad development, development of primary male sexual characteristics, signal transduction in absence of ligand, actomyosin structure organisation, and male sex differentiation, suggesting a potential role of these miRNAs in reproductive traits. Results from this study highlight unique aspects of sex determination in cattle such as the role of miRNAs in gonad development. Moreover, although it is well known that AF provides a protective space around the developing embryo/fetus that allows its movement and growth; here we provide evidence suggesting that its components may play important roles in fetal development. Finally, miRNAs in MP may offer new opportunities to investigate biomarkers for early prediction of embryo/fetal sex in commercial practice.
This research was supported by the Science Foundation Ireland (13/IA/1983) and the European Union H2020 Marie Sklodowska-Curie Innovative Training Network project Biology and Technology of Reproductive Health - REP-BIOTECH - 675526.
Dynamic calibration method for reactive gases
Authors: Sari Saxholm , Timo Rajamäki , Jussi Hämäläinen , Panu Hildén
Published: December 2019

In this study an evaporation method is applied to dynamically generate reference gas mixtures with an accurately known concentration of reactive compounds. As a representative application, the method is used to generate several reference gas mixtures for oxidized mercury. The reliability of online monitoring of gases is dependent on the availability of accurate and metrologically traceable calibration standards. For reactive gases this is a challenge since these sticky gases easily adsorb to surfaces, which complicates and can even prevent the use of traditional static tools. In the evaporation method, a solution with well-known concentration is dosed using, e.g. an automatic syringe pump or a peristaltic pump, and mixed with the carrier gas flow. Mixing and evaporation are key parts of this process, along with temperatures and materials in different parts of the system and gas flow rate, which play an important role in the response time optimization. To estimate the applicability of this method we test its operation in the case of oxidized mercury by investigating the key operational parameters: calibrator response time and stability, possible non-linear behaviour of selected molecules, and also sensitivity to interference caused by other reactive gas compounds. We also compare its outcome to a traceable source for Hg0. Based on the achieved results the evaporation method technique is both a potential and feasible tool for the dynamic preparation of calibration gas mixtures for reactive components, in both laboratory and field conditions.
Composite stacks for reliable > 17 T trapped fields in bulk superconductor magnets
Authors: Huang Kai Yuan , Shi Yunhua , Srpčič Jan , Ainslie Mark D , Namburi Devendra K , Dennis Anthony R , Zhou Difan , Boll Martin , Filipenko Mykhaylo , Jaroszynski Jan , Hellstrom Eric E , Cardwell David A , Durrell John H
Published: December 2019

Trapped fields of over 20 T are, in principle, achievable in bulk, single-grain high temperature cuprate superconductors. The principle barriers to realizing such performance are, firstly, the large tensile stresses that develop during the magnetization of such trapped-field magnets as a result of the Lorentz force, which lead to brittle fracture of these ceramic-like materials at high fields and, secondly, catastrophic thermal instabilities as a result of flux movement during magnetization. Moreover, for a batch of samples nominally fabricated identically, the statistical nature of the failure mechanism means the best performance (i.e. trapped fields of over 17 T) cannot be attained reliably. The magnetization process, particularly to higher fields, also often damages the samples such that they cannot repeatedly trap high fields following subsequent magnetization. In this study, we report the sequential trapping of magnetic fields of ∼ 17 T, achieving 16.8 T at 26 K initially and 17.6 T at 22.5 K subsequently, in a stack of two Ag-doped GdBa2Cu3O7-δ bulk superconductor composites of diameter 24 mm reinforced with (1) stainless-steel laminations, and (2) shrink-fit stainless steel rings. A trapped field of 17.6 T is, in fact, comparable with the highest trapped fields reported to date for bulk superconducting magnets of any mechanical and chemical composition, and this was achieved using the first composite stack to be fabricated by this technique. These post-melt-processing treatments, which are relatively straightforward to implement, were used to improve both the mechanical properties and the thermal stability of the resultant composite structure, providing what we believe is a promising route to achieving reliably fields of over 20 T.
Fading channel estimation for free-space continuous-variable secure quantum communication
Authors: Ruppert László , Peuntinger Christian , Heim Bettina , Günthner Kevin , Usenko Vladyslav C , Elser Dominique , Leuchs Gerd , Filip Radim , Marquardt Christoph
Published: December 2019

We investigate estimation of fluctuating channels and its effect on security of continuous-variable quantum key distribution. We propose a novel estimation scheme which is based on the clusterization of the estimated transmittance data. We show that uncertainty about whether the transmittance is fixed or not results in a lower key rate. However, if the total number of measurements is large, one can obtain using our method a key rate similar to the non-fluctuating channel even for highly fluctuating channels. We also verify our theoretical assumptions using experimental data from an atmospheric quantum channel. Our method is therefore promising for secure quantum communication over strongly fluctuating turbulent atmospheric channels.
Corrosion behavior of hybrid coconut shell powder and silica carbide reinforced aluminium composite towards aggressive biofilm attack
Authors: Suriani M J , Zulkifli F , Khairul M A , Azaman M D , Jarkoni M N K
Published: December 2019

This study enveloped innovative uses of a hybrid agro waste and silica carbide incorporated with aluminium as a new hybrid composite materials for marine application. In this study, the purpose of adding coconut shell powder (CSP) from the agro waste and silicon carbide (SiC) as reinforcement aluminium composites materials for combating corrosion problem. The CSP was incorporated into the molten aluminium alloys varies from 0%, 5%, 10%, 15% and 20% of weight percentages. The corrosion behaviour was determined by weight gain measurement (WGM), electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization (PP). As a results, 5% CSP reinforcement in hybrid aluminum composites contribute to excellent corrosion resistance performance by shifting the polarization curve towards the positive region, increases the value of polarization resistance, R

p
and reduces the I

corr
value. It can be concluded that, by adding CSP into aluminium composite has significantly increase the corrosion resistance towards a severe biofilm attack.