bluuebunny/crossref_metadata_2025 huggingface dataset:
A hybrid microfluidic device for on-demand orientation and multidirectional imaging of C. elegans organs and neurons
Authors: Ardeshiri Ramtin , Mulcahy Ben , Zhen Mei , Rezai Pouya
Published: December 2016

C. elegans is a well-known model organism in biology and neuroscience with a simple cellular (959 cells) and nervous (302 neurons) system and a relatively homologous (40%) genome to humans. Lateral and longitudinal manipulation of C. elegans to a favorable orientation is important in many applications such as neural and cellular imaging, laser ablation, microinjection, and electrophysiology. In this paper, we describe a micro-electro-fluidic device for on-demand manipulation of C. elegans and demonstrate its application in imaging of organs and neurons that cannot be visualized efficiently under natural orientation. To achieve this, we have used the electrotaxis technique to longitudinally orient the worm in a microchannel and then insert it into an orientation and imaging channel in which we integrated a rotatable glass capillary for orientation of the worm in any desired direction. The success rates of longitudinal and lateral orientations were 76% and 100%, respectively. We have demonstrated the application of our device in optical and fluorescent imaging of vulva, uterine-vulval cell (uv1), vulB1\2 (adult vulval toroid cells), and ventral nerve cord of wild-type and mutant worms. In comparison to existing methods, the developed technique is capable of orienting the worm at any desired angle and maintaining the orientation while providing access to the worm for potential post-manipulation assays. This versatile tool can be potentially used in various applications such as neurobehavioral imaging, neuronal ablation, microinjection, and electrophysiology.
Physics in 2116: Privacy concerns prompt protests in California
Authors: Hodgson Gabrielle
Published: December 2016

The state’s new law mandating neural-network-based screenings has driven workers statewide to vent their anger.
Physics in 2116: African Arrow sees hints of structure in the fabric of space
Authors: Kornilovich Pavel
Published: December 2016

The cellular nature of space reveals itself in the angular distribution of μ and μ‾ produced at the accelerator.
Elastic-wave propagation and the Coriolis force
Authors: Snieder Roel , Sens-Schönfelder Christoph , Ruigrok Elmer
Published: December 2016

Seismic waves and Foucault pendula share intriguing similarities in the way they respond to Earth’s rotation.
Insights into conceptus elongation and establishment of pregnancy in ruminants
Authors: Spencer T. E. , Forde N. , Lonergan P.
Published: December 2016

This review integrates established and new information on the factors and pathways regulating conceptus–endometrial interactions, conceptus elongation and establishment of pregnancy in sheep and cattle. Establishment of pregnancy in domestic ruminants begins at the conceptus stage (embryo or fetus and associated extra-embryonic membranes) and includes pregnancy recognition signalling, implantation and the onset of placentation. Survival and growth of the preimplantation blastocyst and elongating conceptus require embryotrophic factors (amino acids, carbohydrates, proteins, lipids and other substances) provided by the uterus. The coordinated and interactive actions of ovarian progesterone and conceptus-derived factors (interferon-τ and prostaglandins) regulate expression of elongation- and implantation-related genes in the endometrial epithelia that alter the uterine luminal milieu and affect trophectoderm proliferation, migration, attachment, differentiation and function. A comparison of sheep and cattle finds both conserved and non-conserved embryotrophic factors in the uterus; however, the overall biological pathways governing conceptus elongation and establishment of pregnancy are likely conserved. Given that most pregnancy losses in ruminants occur during the first month of pregnancy, increased knowledge is necessary to understand why and provide a basis for new strategies to improve pregnancy outcome and reproductive efficiency.
Concepts and tools for gene editing
Authors: Josa Santiago , Seruggia Davide , Fernández Almudena , Montoliu Lluis
Published: December 2016

Gene editing is a relatively recent concept in the molecular biology field. Traditional genetic modifications in animals relied on a classical toolbox that, aside from some technical improvements and additions, remained unchanged for many years. Classical methods involved direct delivery of DNA sequences into embryos or the use of embryonic stem cells for those few species (mice and rats) where it was possible to establish them. For livestock, the advent of somatic cell nuclear transfer platforms provided alternative, but technically challenging, approaches for the genetic alteration of loci at will. However, the entire landscape changed with the appearance of different classes of genome editors, from initial zinc finger nucleases, to transcription activator-like effector nucleases and, most recently, with the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas). Gene editing is currently achieved by CRISPR–Cas-mediated methods, and this technological advancement has boosted our capacity to generate almost any genetically altered animal that can be envisaged.
Epidemiological evidence for metabolic programming in dairy cattle
Authors: Opsomer G. , Van Eetvelde M. , Kamal M. , Van Soom A.
Published: December 2016

In humans, there is evidence that metabolic diseases occurring in later life arise in utero as a result of programming of key endocrine systems during suboptimal intrauterine conditions. The process by which prenatal insults lead to permanent changes in tissue structure and function, and finally to low birthweight (BW), is known as developmental programming. Poor nutrition, environmental temperature, oxygen availability and overnutrition all have been shown to significantly affect intrauterine development. Because the placenta is the organ for communication between mother and fetus, placental insufficiency invariably affects embryonic development and health in later life. In order to optimise their income, dairy farmers inseminate their nulliparous heifers at adolescent age, and subsequently strive for calving intervals not longer than 380 days. Hence, heifers are still growing and multiparous animals are still yielding large quantities of milk while pregnant. Dairy cows heavily selected for milk yield have specific endocrinological characteristics, like low peripheral insulin levels and low peripheral insulin sensitivity, both contributing to safeguard glucose for milk production. The reverse of this advanced selection is the high incidence of a wide range of metabolic diseases. Evidence from epidemiological studies is now available demonstrating that milk yield during gestation and environmental factors, such as season of pregnancy and parturition, affect both the size and the intermediary metabolism of the neonatal calf. The latter suggests that further optimisation in terms of production, reproduction, general health and longevity in the dairy sector may be feasible by taking into account environmental factors occurring during pregnancy.
107 RESEARCH PRIORITIES FOR SAFE SANITARY TRADE OF EMBRYO AND SEMEN
Authors: Fieni F. , Grant C. , Gard-Schnuelle J. , Perry G. , Wrenzycki C. , Blondin P.
Published: December 2016

Embryo transfer and artificial insemination are utilised nationally and internationally for the introduction, improvement, and preservation of livestock genetics. Embryos present a lower risk of infectious disease transmission than do live animals. In order to maintain the sanitary security and to facilitate the trade of embryos and semen worldwide, the Health and Scientific Advisory Committee of the International Embryo Technology Society designed, developed, and conducted a survey to determine research priority. The survey questionnaire was sent to 32 government representatives and 76 embryo transfer and artificial insemination industry representatives around the world to countries where artificial breeding industries are active and well developed. A total of 16 answers were received, 9/32 (28%) from government representatives and 7/76 (9%) from industry representatives. The global feedback was 15%. The survey indicated that, in terms of research priority, embryos and semen were equally important. With regards to embryo research priorities, the survey results ranked in vitro-produced embryos research as the most important, followed by in vitro-derived embryos, and then oocytes. Apart from scrapie for embryos and Campylobacteriosis for semen, research priorities were similar for the major pathogens of embryos and semen, in particular, bovine viral diarrhoea and paratuberculosis (Johne’s disease; Table 1). Emerging or less common diseases were not forgotten. Other diseases suggested but not listed in Table 1 included bluetongue, foot and mouth disease, lentivirus, arbovirus, bovine tuberculosis, porcine epidemic diarrhoea, porcine reproductive and respiratory syndrome, African swine fever, and ovine pulmonary adenocarcinoma (Jaagsiekte). The survey highlighted the need to focus research largely on ruminant species (Table 1). Other issues identified by the survey included (i) alternative or indirect processes for determining the sanitary quality of in vitro-produced embryo as for in vitro-derived embryos, including standardized testing of discarded oocytes or testing culture media for pathogens with validated PCR techniques, (ii) safe sanitary techniques for the perforation of the zona pellucida for biopsy, for pre-implantation genetics and for diagnostic purposes, (iii) recommendations for international trade in embryos or semen originating from vaccinated donors, and (iv) use of biologicals for disinfecting embryos during washing procedures.

Table 1.

Answers (%) to the questions: Which pathogens (species) represent the most urgent research need? (answers with results <5% are not listed)
129 KNOCKDOWN OF WBP1 DECREASES TROPHECTODERM FORMATION IN THE PRE-IMPLANTATION BOVINE EMBRYO
Authors: Ortega M. S. , Hansen P. J.
Published: December 2016

A single nucleotide polymorphism (SNP) in WBP1 has been previously associated with embryonic development to the blastocyst stage. WBP1 interacts with WW domain containing proteins including YAP1 from the hippo signalling pathway that is involved in trophectoderm (TE) formation. Here we tested whether reduction in mRNA abundance for WBP1 would reduce development to the blastocyst stage and formation of cells in the inner cell mass (ICM) and TE. Knockdown was performed using a GapmeR LNATM antisense oligonucleotide designed to target WBP1. A scrambled version of the same sequence was used as a control. Embryos were produced in vitro from slaughterhouse oocytes and bulls from Bos taurus and Bos indicus breeds. At 20 to 22 h after insemination (hpi), embryos were treated with 5 µM anti-WBP1 GapmeR (KD), 5 µM scrambled GapmeR (SC), or vehicle (CTL). At 72 to 75 hpi (the time of maximal WBP1 expression), groups of 18 to 20 embryos were collected from each treatment to evaluate WBP1 expression. Other cultured embryos (minimum of 50/treatment for each replicate) were cultured until Day 8 after insemination. Cleavage was assessed at Day 3 and blastocyst formation at Day 7 and 8. Embryos were collected at Day 8 to determine ICM and TE cell number by determining nuclear immunoreactive CDX2. All experiments were replicated 5 times. Fold change was calculated relative to the CTL group. Data were analysed by analysis of variance for gene expression and cell number, and through logistic regression for embryonic development. WBP1 expression was reduced (P = 0.04) in KD embryos compared to CTL (least squares means ± SEM: 1 ± 0.19 v. 0.64 ± 0.19 fold change) or SC (1.05 ± 0.19). There was no difference in expression between CTL and SC. Percent of embryos that cleaved was not affected by treatment (P > 0.05); however, percent of inseminated oocytes that became blastocysts tended to be lower in KD compared to CTL and SC at Day 7 (P = 0.09) [10.8 ± 2.8, 20 ± 3.0, and 16.3 ± 3.1% for KD, CTL, and SC, respectively] and 8 after insemination (P = 0.06) [13.7 ± 3.3, 24.2 ± 3.3, and 22.9 ± 3.6%]. Knockdown of WBP1 caused a reduction in number of total (P = 0.0004) and TE (P < 0.0001) cells with no effect on ICM cell number (P = 0.83). Total cell numbers for KD, SC, and CTL were 124.2 ± 6.4, 157.75 ± 7.4, and 124.28 ± 6.4 and numbers of TE cells were 59.7 ± 3.8, 90.0 ± 4.47, and 90.0 ± 4.4. Results show that reduction in mRNA for WBP1 decreases TE formation and tends to reduce competence of embryos to become blastocysts.
This study was supported by USDA AFRI 2013–68004–20365.
131 EFFECT OF PROLONGED PROGESTERONE EXPOSURE OF BEEF COWS ON THE EXPRESSION OF OOCYTE DEVELOPMENTAL COMPETENCE-ASSOCIATED GENES
Authors: Diaz F. A. , Foster B. A. , Hardin P. T. , Gutierrez E. J. , Bondioli K. R.
Published: December 2016

Progesterone is a key hormone in reproduction and plays a crucial regulatory role during the oestrous cycle and pregnancy. The effect of progesterone on the developmental competence of bovine oocytes is yet to be fully understood. Molecular tools might provide further information on the role of progesterone on expression of developmentally important genes. The objective of this study was to evaluate the effect of the prolonged exposure of beef cows (days = 32) to different progesterone levels on the expression of genes associated with oocyte developmental competence. Fifteen cows were used in the experiment and were divided in 3 treatment groups (group n = 5). Treatment 1 consisted of no progesterone supplementation, treatment 2 consisted of supplementation by insertion of one controlled internal drug release device (CIDR) per animal, and treatment 3 consisted of supplementation with the insertion of 2 CIDR per animal. All cows were subjected to follicle ablation 4 days before initiation of the experiment. At Day 0, prostaglandin F2α (PGF2α, 25 mg of dinoprost tromethamine, i.m., Lutalyze®) was administered and CIDR devices were inserted in cows designated to treatments 2 and 3. Cumulus-oocyte complexes were collected through ovum pickup where follicles >2 mm were aspirated. Samples evaluated were obtained at Day 0, 8, 16, 24, and 32. The oocyte developmental competence-associated genes METAP2, FOXM1, HMGA1 were analysed in the study. Polyadenylate polymerase was used as normalization gene. Relative gene expression was calculated utilising a comparative CT method (2−ΔΔCT) where data were calculated as fold increase (>1) or decrease (<1) from a calibrator included in each PCR reaction. Mean values of the fold increase/decrease were compared between treatments and were analysed by repeated-measures ANOVA. No statistical difference was detected between treatments at Day 0, 8, 16, 24, or 32 when analysing independent relative expression of oocyte developmental competence-associated genes METAP2, FOXM1, or HMGA1 (P > 0.05). Similarly, no significant differences between treatments was detected in relative expression of METAP2, FOXM1, or HMGA1 across time (P > 0.05). Prolonged exposure of progesterone at different levels does not affect the expression of METAP2, FOXM1, and HMGA1 genes in bovine cumulus-oocyte complexes as indicated by the results of this experiment.
140 COMPARISON OF 3 DIFFERENT METHODS TO SELECT HIGH SPERM QUALITY FOR IVF
Authors: Rubessa M. , Lotti S. N. , Knox R. V. , Wheeler M.
Published: December 2016

Sperm selection is a fundamental step for IVF, influencing treatment and the health of offspring. Over the years, several techniques have been tested to recover highly motile sperm, including centrifugation with discontinuous gradients and numerous filtration procedures. In our previous paper, we showed that it is possible, in ~20 min, to select a high quality sperm population using an electric charge in a microchannel device. This method produced the same blastocyst rates as a discontinuous gradient (DG), but in less time [25 min less; Rubessa et al. 2016 Andrology (Los Angeles) 5]. This technique takes advantage of one of the physiological characteristics of high quality sperm; that is, the head has a negative electric charge. The aim of the present experiment was to analyse the characteristics of the sperm selected with our device. We compared 2 standard sperm procedures and our device, with semen assessed using a computer-assisted sperm analyzer to evaluate the principal sperm parameters. Standard procedures were used to select sperm by DG and swim-up (SU). Thawed semen was pipetted into the microchannel of our device and then exposed to the electric charge (10 V) for 10 min [Rubessa et al. 2016 Andrology (Los Angeles) 5]. For each replicate, 3 straws of frozen bovine semen were thawed at 37°C for 40 s and pooled. The pooled sample was divided into 3 aliquots: one for each technique, 5 replicates of the experiment were performed. Data were analysed by ANOVA using the general linear model (GLM) procedure (SPSS Inc., Chicago, IL, USA). Bonferroni’s post hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. The results show an effect of the method all motility parameters are not significantly different, although some of the motility parameters were statistically different (P < 0.05). (P < 0.05). The beat cross frequency results were 23 ± 2.4 v. 37.7 ± 2.1 v. 19.5 ± 0.7 (µm/s) for DG, SU, and EC. The same statistical differences were found for the STR (straightness): 75 ± 3.5 v. 84.5 ± 2.0 v. 62.3 ± 2.4 (µm/s), and LIN (linearity): 39.8 ± 4.4 v. 53 ± 1.2 and 31.8 ± 1.4. These results confirmed the potential of this new device, and that it is possible to use this new method to select high motility sperm without harming sperm characteristics. These results showed that our device produced the same quality of sperm obtained with DG and SU but in as little as 20 min.
155 INDUCTION OF FOLLICULAR RECRUITMENT USING DOMINANT FOLLICLE REDUCTION OR 2 DOSES OF GONADOTROPIN-RELEASING HORMONE
Authors: Lindell K. A. , Bowley S. C. , Matsas D. J. , White E. C.
Published: December 2016

Induction of follicular recruitment is a common method of treatment in bovine donors before initiation of superstimulatory protocol for transvaginal ultrasound-guided oocyte recovery (TVOR; Bo and Mapletoft 2014 Theriogenology 81, 38–48). Two types of induction are frequently applied: dominant follicle reduction (DFR) performed on Day −6 before TVOR, or 2 injections of gonadotrophin releasing hormone (GnRH), 100 μg each, on Day −6 and −13 before TVOR. The objective of this study is to compare the efficacy of DFR v. 2 GnRH injections for inducing follicular recruitment before TVOR in cattle. Dominant follicle reduction is the mechanical aspiration of follicles >5 mm and is a reliable method of initiating follicular recruitment (Blondin 2012 IETS Innovation Workshop 1, St Augustine, FL, USA). This procedure also allows the practitioner to evaluate donors before beginning any treatments. For several reasons (expense, time, and logistics), DFR may be impractical, making a double GnRH regimen appealing. Administering 2 doses of GnRH is simpler but is not considered as reliable; reported ovulation in response to GnRH are ~40% and 60% in heifers and cows, respectively. The donor populations in this study consist of cows and heifers privately owned by farms in the northeastern United States. Donors received an average of 14 to 15 mL (280–300 mg) Folltropin total, split between 6 injections given every 12 h. Cows averaged a 43-h coasting period (time of the last FSH injection to TVOR), and heifers averaged a 31-h coasting period. Oocytes were collected March 2015 to June 2016 by the same practitioner using the same equipment under the same procedure at 2 ovum pickup locations, and submitted to our partnering IVF laboratory, Boviteq USA (Madison, WI, USA). Comparing DFR to 2 doses of GnRH in Table 1, there was no statistically significant difference in total follicles aspirated, total transferable embryos produced or blast rate by either t-test or non-parametric Wilcoxon rank-sum test (P > 0.05). The use of 2 doses of GnRH is an acceptable method for the induction of follicular recruitment before TVOR. Additional statistical models are currently being applied to evaluate which, if any, differences are present in the 2 donor populations.

Table 1.

Raw data and results1
158 FIRST LLAMA BORN BY IN VITRO FERTILIZATION
Authors: Landeo L. , Mendoza J. , Manrique L. , Taipe E. , Molina R. , Contreras J. , Ruiz J.
Published: December 2016

The aim was to transfer alpaca and llama embryos obtained by IVF into recipient llamas and evaluate pregnancy and birth rates. Gametes were collected from samples of ovaries and testes collected from a local abattoir. Cumulus-oocyte complexes (COC) were recovered by aspiration of ovarian follicles using a 5-mL syringe, where oocytes with at least 3 layers of cumulus cells and a homogenous cytoplasm were matured in TCM-199 supplemented with 10% FCS, FSH (0.1 IU), and oestradiol 17β for 30 and 36 h with 5% CO2 in air, for alpaca and llama, respectively. After the maturation time, COC were placed in FERT-TALP solution 30 min before in vitro insemination with epididymal sperm of alpaca and llama as appropriate, the sperm were selected by swim-up method with centrifuging twice in 2 mL of SPERM-TALP supplemented with 6 mg mL−1 of fatty-free BSA, in an incubator with 5% CO2 in air set at 39°C for 45 min, oocytes were co-incubated with sperm concentration of 3 × 106 mL−1 for 18 to 20 h at 39°C with 5% CO2 in air. The IVF was carried out the day of ovulation induction of recipients. A group of 15 morphologically intact Day-8 blastocysts derived from in vitro culture with SOF serum were transferred nonsurgically into the uterine horn ipsilateral to the corpus luteum of 15 synchronized recipient llamas with an intravaginal device (controlled internal drug release) for 8 days. Then, 6 days after controlled internal drug release removal, ovulation was induced in recipients with the application of 1 mL of GnRH with previous ultrasound confirmation of the presence of a dominant follicle greater than 7 mm in diameter. Nine alpaca blastocysts and 6 llama blastocysts were transferred. The pregnancy rate was assessed by ultrasound at 45 days after transfer. The results obtained were as follows: for pregnancy rate, 33.3% (3/9) and 50% (3/6) for alpaca and llama embryos, respectively; for birth rate, 0.0% (0/9) and 16.7% (1/6) for alpaca and llama embryos, respectively. An alpaca fetus and 2 llama fetuses were aborted between 7 and 10 months of pregnancy, and only a llama gestation ended successfully, producing the first birth of the world of a llama bred by IVF in South American camelids, demonstrating that it is possible to obtain viable offspring in these species using this biotechnology.
164 CARNITINE IMPROVES POST-THAWING SPERM MOTILITY BY INCREASING ADENOSINE TRIPHOSPHATE CONTENT IN BUFFALO (Bubalus bubalis)
Authors: Longobardi V. , Zullo G. , Albero G. , De Canditiis C. , Salzano A. , D'Onofrio N. , Gasparrini B.
Published: December 2016

Semen cryopreservation plays a critical role for a wide application of both AI and in vitro embryo production in buffalo. In this species, spermatozoa are more susceptible to hazards during freezing and thawing than cattle spermatozoa, thus resulting in lower fertilizing potential (Andrabi et al. 2008 Anim. Reprod. Sci. 104, 427–433). Carnitine is a quaternary ammonium compound with antioxidant capacities, able to reduce the availability of lipids for peroxidation by transporting fatty acids into the mitochondria for β-oxidation to generate adenosine triphosphate (ATP) energy (Tanphaichitr and Leelahagul 1993 Nutrition 9, 246–54). It is known that cryopreservation processes decreases the intracellular concentration of carnitine in spermatozoa (Reyes-Moreno et al. 2000 J. Androl. 21, 876–86). In cattle, supplementation of semen extender with carnitine improves sperm motility and DNA integrity (Bucak et al. 2010 Cryobiology 61, 248–53). The aim of this study was to evaluate whether supplementation of semen extender with carnitine would increase ATP content in buffalo sperm and affect post-thawing motility. Eight ejaculates from 4 bulls were used for the trial. Each ejaculate was split into 3 equal aliquots and diluted at 37°C with BioXcell extender containing 0 (control), 2.5, and 7.5 mM carnitine to a final concentration of 30 × 106 spermatozoa/mL. After 4 h at 4°C, the straws were frozen in an automated system. At thawing, sperm motility was evaluated by phase contrast microscopy at 40× magnification (Gillan et al. 2008 Anim. Reprod. Sci. 103, 201–204). Adenosine triphosphate content was measured using a Colourimetric ATP Assay Kit (Biovision, Milpitas, CA, USA). Briefly, Percoll-separated spermatozoa were homogenised and then deproteinized using 10-kDa spin columns. Samples were incubated at RT for 30 min and absorbance was measured at 570 nM in a microplate reader. Differences in sperm motility and ATP content among groups were analysed by ANOVA. Both concentrations of carnitine increased post-thawing sperm motility compared with the control (44.4 ± 3.5, 53.1 ± 3.9, and 52.5 ± 3.6, respectively, with 0, 2.5, and 7.5 mM carnitine; P < 0.05). Interestingly, carnitine increased ATP content of buffalo frozen–thawed sperm in a dose-dependent manner (4.1 ± 0.1, 5.3 ± 0.1, and 8.2 ± 0.4 nM × 108 sperm, respectively, with 0, 2.5, and 7.5 mM carnitine; P < 0.01). In conclusion, the enrichment of semen extender with carnitine improved post-thawing motility of buffalo sperm by boosting mitochondrial ATP production, hence providing energy for use by spermatozoa.
167 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES DERIVED FROM SMALL- OR MEDIUM-SIZED FOLLICLES AND DENUDED OF CUMULUS CELLS BEFORE AND DURING IN VITRO MATURATION
Authors: Ferré P. , Nguyen K. X. , Wakai T. , Funahashi H.
Published: December 2016

This experiment was undertaken to assess the meiotic and developmental competences of oocytes derived from different sized follicles and denuded of cumulus cells 0, 20, and 44 h after the start of culture for in vitro maturation (IVM). Groups of 60 oocyte-cumulus complexes from small- (SF; <3 mm) and medium-sized follicles (MF; 3–6 mm) were cultured for IVM in porcine oocyte medium with 50 μM β-mercaptoethanol supplemented with 1 mM dibutyryl-cyclic adenosine monophosphate, 10 IU mL−1 of eCG, and 10 IU mL−1 of hCG for 20 h at 39°C and 5% CO2 in air. Then, after washing, they continued culture in fresh β-mercaptoethanol without dibutyryl-cyclic adenosine monophosphate and gonadotropins under the same conditions for another 24 h. At 0, 20, and 44 h of IVM, cumulus cells were removed with 0.1% (wt/vol) hyaluronidase and the denuded oocytes continued IVM culture following the protocol. Mature oocytes with the first polar body were selected, parthenogenetically activated with a single electrical pulse (DC: 1.2 kV/cm, 30 µs), incubated with 4% (wt/vol) BSA and 5 μM cytochalasin B for 4 h, and cultured in porcine zygote medium for 5 days. Cleavage and blastocyst formation rates were observed on Day 2 and 5, respectively. Blastocysts were stained with 4’,6-diamidino-2-phenylindole for cell count assessment. The experiment was replicated 5 times and analysed with a 1- or 2-way ANOVA. If P < 0.05 in ANOVA, a Tukey multiple comparisons test was performed. Regardless of the time of cumulus cell removal, oocytes from MF had significantly higher in rates of maturation, cleavage, and blastocyst rates, as compared with those from SF, whereas there were no significant differences in the cell number of blastocysts between SF and MF (32 v. 34 cells, respectively). When oocytes were denuded before IVM culture, rates of oocyte maturation (37.6% in SF and 50.8% in MF), and blastocyst formation (2.7% in SF and 27.3% in MF) were significantly lower than controls (51.2% in SF and 76% in MF; 25.8% in SF and 48.5% in MF, respectively). When oocytes were denuded 20 h after the start of IVM, oocyte maturation rates were significantly increased (64.1% in SF and 82.5% in MF) as compared with controls, whereas no significant differences were observed in cleavage and blastocyst formation rates in comparison with controls. These results conclude that removing cumulus cells from oocyte-cumulus complexes 20 h after the start of IVM improves the meiotic competence of oocytes derived from both SF and MF, without any reduction of developmental competence of the oocytes following parthenogenetical activation.
17 SPERM FERTILITY RATE ASSESSED BY EMBRYO PRODUCTION IN VIVO AND IN VITRO IN SOUTH AFRICAN BULLS
Authors: Mapeka M. H. , Ramukhithi F. V. , Pilane C. M. , Norris D. , Banga C. , Lehloenya K. C.
Published: December 2016

The aim of this study was to determine the sperm fertility rate by embryo production in vivo and in vitro in South African bulls and further compare the embryo quality developed from different oocyte recovery methods. A total of 15 frozen semen straws (5 Bonsmara; 5 Nguni; 5 Boran) were thawed and evaluated for sperm motility characteristics using sperm class analyzer. The fertilizing ability of frozen–thawed semen was assessed by performing AI and in vitro fertilization. For AI, 6 cows were superovulated and inseminated with frozen–thawed semen followed by flushing on Day 7 post-insemination and then evaluated for embryo developmental stages. For IVF, oocytes were retrieved using two recovery methods namely ovum pick-up (OPU) and ovary aspiration. A total of 383 (106, OPU; 277, ovary aspiration) oocytes were matured in M199 + 10% fetal bovine serum (FBS) maturation medium at 38.5°C for 24h. Oocytes were washed in Bracket and Oliphant’s fertilization medium, co-incubated with frozen–thawed (Boran) semen at 38.5°C for 6 h, and then cultured in SOF-BSA medium, incubated at 38.5°C, 5% CO2 for 7 days, and further evaluated for embryo development. Data were analysed by ANOVA. Total sperm motility was >70% in all breeds. Boran had a significantly (P < 0.05) higher total post-thaw sperm motility (93.2 ± 3.6) compared with Nguni (75.1 ± 4.2) and Bonsmara (80.7 ± 6.9). Furthermore, Boran had higher (P < 0.05) progressive motility (39.7 ± 3.4) and rapid motility (36.1 ± 5.9) compared with other breeds. Interestingly, Boran produced significantly (P < 0.05) higher blastocyst rate (56.34%) compared with Bonsmara (38.03%) Nguni (31.08%). Superovulation and OPU resulted in a significantly higher (P < 0.05) number of blastocysts (10.5 ± 3.3 and 10.5 ± 3.3) respectively, compared with aspiration (1.3 ± 3.3). Moreover, the OPU method yielded a significantly higher (P < 0.05) number of grade 2 blastocyst (3.0 ± 0.1) compared with aspiration (0.50 ± 0.1). However, there was no significant (P > 0.05) difference in the number of grade 1 and grade 3 blastocysts obtained when the 3 recovery methods were used. In conclusion, the Boran breed showed better a sperm fertility rate following in vivo and in vitro embryo production. The superovulation and OPU methods resulted in higher numbers and better quality blastocysts compared with aspiration.
173 MELATONIN ALLEVIATES THE ENDOPLASMIC RETICULUM STRESS THROUGH THE REGULATING OF UNFOLDING PROTEIN RESPONSE SIGNALING DURING PORCINE OOCYTE MATURATION IN VITRO
Authors: Park J.-Y. , Park H.-J. , Kim J.-W. , Park S.-Y. , Yang S.-G. , Jung J.-M. , Kim M.-J. , Koo D.-B.
Published: December 2016

Unfolding protein response (UPR) is a defence mechanism during endoplasmic reticulum (ER) stress in mammalian cells. Especially, UPR genes and regulation of reactive oxygen species is involved in ER stress response on porcine oocyte maturation in vitro. Some studies have shown that melatonin treatment results in reducing oxidative stress, a protective function of free radical damage in oocyte maturation and embryo development. Also, melatonin has an important role in reducing reactive oxygen species and ER stress. However, it is unknown how the changes of UPR genes expression levels are affected the porcine oocyte maturation. In addition, there are no reports about ER stress recovery mechanism by melatonin during porcine oocyte maturation. Here, we investigated the UPR signal genes (Bip/Grp78, Atf4, p90/p50Atf6, and Xbp1) and ER-stress mediated apoptosis factors (Chop and Cleaved caspase 3) in porcine oocyte maturation in vitro. Expression of Chop and Cleaved caspase 3 mRNA levels were significantly increased (P < 0.01) in matured oocytes (metaphase II; 44 h) in vitro. Porcine oocytes were cultured in maturation medium with ER stress inducer, tunicamycin (Tm), and supplemented with various concentrations (1, 5, and 10 μg mL−1) of Tm for 0 to 44 h. Our results indicated that the proportion of matured oocytes was significantly decreased in Tm-treated groups in a dose-dependent manner (60.1 ± 1.3, 46.5 ± 2.1, and 38.9 ± 5.1% at 1, 5, and 10 μg mL−1 of Tm) compared with the control group (76.6 ± 1.9%). Likewise, mRNA expression of UPR regulator genes (Grp78/Bip, Aft4, Xbp1, Chop, and Cleaved caspase 3) was decreased by melatonin treatment (0.1 μM, 22–44 h) after pretreatment of Tm (5 μg mL−1, 0–22 h) during oocyte maturation. Our results demonstrated that the roles of melatonin as UPR signaling regulator for reducing ER stress are essential for promotion of porcine oocyte maturation and cumulus cell expansion of cumulus-oocyte complex. Moreover, the current study was initiated to confirm a functional link between effect of melatonin and regulating of UPR signaling in porcine oocytes maturation. These results suggest that melatonin improve the oocyte maturation and cumulus cells expansion by regulating of UPR signal genes against the ER stress during the porcine in vitro maturation process.
This work was supported by grants from the Next-Generation BioGreen 21 Program (PJ01117604) and the Bio-industry Technology Development Program (316037–04–1-HD020) through the Rural Development Administration, the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.
192 BEHAVIOR OF PORCINE MESENCHYMAL STEM CELLS ON A COLLAGEN-GLYCOSAMINOGLYCAN HYDROGEL SCAFFOLD FOR BONE AND CARTILAGE TISSUE ENGINEERING
Authors: Womack S. A. , Milner D. J. , Weisgerber D. W. , Harley B. A. C. , Wheeler M. B.
Published: December 2016

The pig is an ideal species for use in tissue engineering studies of bone and cartilage defect repair. Novel collagen-glycosaminoglycan hydrogel (CG) scaffolds have shown promise for supporting bone and cartilage growth from mesenchymal stem cells. In order to determine the suitability of these scaffolds for use in porcine models for bone and cartilage tissue engineering, we have begun to investigate the behaviour of porcine mesenchymal stem cells on this material. The purpose of this study was to determine if mesenchymal stem cells from fat (ASC) or bone marrow (BMSC) displayed better adherence and penetration into the CG scaffold material. The BMSC and ASC isolated from young adult Yorkshire pigs were cultured in DMEM with 10% fetal bovine serum. The ASC and BMSC were then trypsinized and used to seed ~3 mm diameter CG scaffolds with 140,000 cells/scaffold. Scaffolds were then cultured for 10 days by 3 different methods: roller culture, free-floating non-adherent dishes (floating), or attached to tissue culture-treated dishes (static). At the conclusion of the incubation period, the scaffold pieces were then fixed with 4% paraformaldehyde, embedded for cryosectioning, and sliced into 10 µm cryosections. Sections were stained for vimentin and 4’,6-diamidino-2-phenylindole (DAPI) to label cells. Stained sections were observed on a Leica DMB4200 microscope (Leica Microsystems, Wetzlar, Germany) and images acquired using ImagePro Plus software (Media Cybernetics Inc., Rockville, MD, USA). The DAPI-stained cells were counted to determine cell density and expressed as average number of nuclei per millimeter squared for each cell and culture type. Data were analysed by ANOVA utilising a post hoc Holm multiple comparison analysis. Samples from roller cultures did not display adhered cells for either BMSC or ASC. In contrast, floating and static culture allowed both ASC and BMSC to adhere to the scaffold and migrate to the centre of the scaffold equally well. However, significant differences in cell densities were noted between ASC and BMSC on CG scaffolds, with BMSC growing to higher densities than ASC in both floating and static culture. For floating cultures, BMSC-loaded scaffolds exhibited a cell density of 105.7 compared with 53.3 cells/mm2 for ASC (n = 4; P < 0.05). For static cultures, BMSC-loaded scaffolds exhibited a cell density of 128.3 compared with 36.8 cells/mm2 for ASC-loaded samples (n = 3; P < 0.01). Thus, BMSC grow to greater densities more rapidly than ASC and may be more efficient for use in forming bone and cartilage on these scaffolds. Current experiments underway will compare osteogenic and chondrogenic differentiation potential of ASC and BMSC on CG scaffolds, and will attempt to engineer osteochondral interface tissue on CG scaffolds from co-cultures of chondrocytes and stem cells.
196 USE OF A MICRO-BIOREACTOR TO PROMOTE 3-DIMENSIONAL CELL REARRANGEMENT AND INDUCE, MAINTAIN, AND STABILIZE HIGH PLASTICITY IN EPIGENETICALLY ERASED FIBROBLASTS
Authors: Manzoni E. F.M. , Pennarossa G. , Zenobi A. , Ledda S. , Gandolfi F. , Brevini T. A. L.
Published: December 2016

Development and cell differentiation are driven by complex epigenetic mechanisms that regulate chromatin structure and specific gene transcription programs. We recently demonstrated that it is possible to modify the epigenetic signature of terminally differentiated cells, switching their phenotype into one of higher plasticity, through the use of molecules that remove epigenetic marks from DNA and histones (Pennarossa et al. 2013 Proc. Natl. Acad. Sci. 110, 8948–8953; Brevini et al. 2014 Stem Cell Rev. 10, 633–642). Here we drive mammalian fibroblasts into a high plasticity state using the epigenetic eraser, 5-aza-cytidine (5-aza-CR), and investigate whether the simultaneous use of a micro-bioreactor culture system is able to promote three-dimensional (3D) cell rearrangement, boost the induction of high plasticity, and stably maintain it. To this purpose, fibroblasts were either plated on plastic dishes (Group A) or encapsulated in a liquid marble micro-bioreactor (polytetrafluoroethylene powder; Sigma 430935, St. Louis, MO; Group B). Both groups were erased with 5-aza-CR and cultured in embryonic stem cell medium for 28 days. Morphological analysis was carried out for the entire length of the experiment. The OCT4, NANOG, and REX1 expression levels were assessed by real-time PCR at different time points. Exposure to 5-aza-CR induced a dramatic change in morphology in Group A fibroblasts. Cells became rounded, with larger and granulated nuclei and retained a monolayer distribution for the entire length of the experiment. The same changes in cell and nuclear morphology were observed also in cells encapsulated in liquid marble (Group B). In addition, these cells formed 3D spherical structures that were stably maintained until Day 28. These morphological rearrangements were accompanied by the active expression of the pluripotency markers, OCT4, NANOG, and REX1, in both groups. However, while Group A cells progressively down-regulated their expression by Day 6, Group B cells steadily transcribed these genes until Day 28, when cultures were arrested. Altogether, the data confirm that epigenetic erasing induces a high plasticity state in terminally differentiated fibroblasts with the expression of pluripotency related genes. Striking morphological changes accompanied the removal of epigenetic marks. These were influenced by the use of an adequate 3D in vitro culture system, with the induction of distinctive cell rearrangements and the formation of spherical structures that boosted and maintained cell plasticity. These results suggest a correlation between the mechanotransduction pathways induced by the micro-bioreactor culture system and the epigenetic regulation of cell phenotype.
Study was supported by Carraresi Foundation. Authors are members of the COST Actions FA1201, BM1308 and CM1406.
204 POTENTIAL OF GREEN FLUORESCENT PROTEIN LOCUS FOR GENE EDITING IN DNA TRANSPOSON-PRODUCED TRANSGENIC CATTLE
Authors: Yum S.-Y. , Lee S.-J. , Kim H.-M. , Lee C.-I. , Kim H.-S. , Kim H.-J. , Choi W.-J. , Hahn S.-E. , Lee J.-H. , Kim S.-J. , Jang G.
Published: December 2016

Recently, we published on the efficient production of transgenic cattle using the DNA transposon system (Yum et al. 2016 Sci. Rep. 6, 27185). In that study, 8 transgenic cattle were born following transposon-mediated gene delivery system (Sleeping Beauty and Piggybac transposon) via microinjection of zygotes. In the analysis of their genomic stability using next-generation sequencing, there was no significant difference in the number of genomic variants between transgenic and nontransgenic cattle. In this study, we have described current status of those transgenic cattle in term of health, germ-line transmission, and application. All the transgenic cattle have grown up to date (the oldest being 30 months old, the youngest being 12 months old) without any health issue. In general blood analysis, there were not any significant changes between transgenic cattle and wild type. Because the transgene (green fluorescent protein; GFP) expression is constitutively active and has strong expression, it could be visualised without fluorescence equipment. One of transgenic male cattle reached puberty and semen was collected. Over 200 frozen semen straws were produced and some were used for IVF. In every IVF replication, around 80% blastocysts expressed the GFP. Over 36 GFP blastocysts were frozen for embryo transfer in the future, and we are planning to crossbreed for generating homozygotic transgenic cattle. Another application is to use the GFP locus to gene-edit the transgenic cattle, as long-term expression of transgene did not affect their health. In 1 cell stage, embryos produced using GFP frozen-thawed semen, single guide RNA for GFP, Cas9, together with donor DNA that included RFP and homology arms to link the double-strand break of single guide RNA target site, were co-injected and RFP was observed. Knockout/-in for editing GFP locus using CRISPR-Cas9 might be a valuable approach for the next generation of transgenic models by microinjection. In conclusion, we demonstrated that transgenic cattle via transposon are healthy to date and germ-line competence was confirmed. The GFP locus will be used as the target region for future gene engineering via genome-editing technology. Finally, all those animals could be a valuable agricultural and veterinary science resource for studying the effects of gene manipulation on disease resistance and food production.
This work was supported by BK21 PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU 550–20160004).
212 A SIMPLE PROTOCOL TO EXTRACT DNA FROM SMALL NUMBERS OF CELLS
Authors: Lopez N. A. , Schreiber S. , Lotti S. N. , Rubessa M. , Wheeler M. B.
Published: December 2016

Reproductive biotechnology frequently requires improvements in our ability to isolate DNA from fewer and fewer cells. It is now possible to find several commercially available kits to meet this demand, but they frequently come at a relatively high cost. The aim of this project was to evaluate the efficiency of a simple handmade protocol to extract DNA from a small number of cells. To evaluate this method we chose 3 different bovine cell lines, Mac T-cells, sperm, and fibroblasts, for the analysis. Five different quantities (30, 100, 150, 300, and 500 cells) were evaluated for each cell line. The endpoints examined were the purity of the extracted DNA by NanoDrop analysis and visualisation of bands on agarose gels after PCR. The samples were initially diluted in 20 µL of PBS. The cell lysis buffer contained 15 mM Tris-HCl (pH 8.9), 50 mM KCl, 2.5 mM MgCl2, 0.1% Triton X-100, and 150 µg mL−1 of proteinase K. Twenty microlitres of lysis buffer were added to each sample. The tubes were then incubated at 55°C for 1 h. Last, proteinase K was inactivated by incubation at 90°C for 10 min. After extraction, 3 samples from each cell quantity for each replicate were evaluated by NanoDrop. After NanoDrop analysis, a standard PCR procedure was carried out using the homologue gene, tRep-137, as the amplification target. As a control, the Gene Wizard DNA extraction kit was used for all cell treatments. The DNA fragments resulting from PCR were separated for visualisation on agarose gels. Five replicates of the experiment were performed with 3 individual samples for each cell quantity evaluated. There were 80 total samples evaluated, including 5 control samples (1 per replicate). The PCR results were analysed using chi-square analysis with Fisher correction. An α-level of 0.05 was selected a priori for significant differences. The agarose gel analysis showed that each cell line gave different results. The data presented in Table 1 shows that this protocol is less effective for Mac T-cells, but satisfactory for both sperm and fibroblast cells. When the quality of the DNA extracted was evaluated with the NanoDrop method, DNA was found in all samples, but the purity of the samples was very low. None of the samples had a 260/280 ratio over 1, the range being 0.6 to 0.8. This high impurity could explain why not all samples showed PCR product bands on the gel. The next step will be to purify the DNA by removing all of the protein from the samples after isolation from the cells. In conclusion, these data show that it is possible to extract DNA at a low cost from a small number of cells.

Table 1.
The mean (SD in parentheses) percentage of samples with PCR bands for each cell line and each cell quantity
38 PRODUCTION OF TRANSGENIC DOGS THAT OVEREXPRESS PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-ALPHA IN A MUSCLE-SPECIFIC MANNER
Authors: Kim M. J. , Oh H. J. , Setyawan E. M. N. , Choi Y. B. , Lee S. H. , Kwon M. S. , Koo B. C. , Kim T. , Lee B. C.
Published: December 2016

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. Among 3 major types of PPAR (PPARα, PPARβ, and PPARγ), PPARα is expressed in tissues having a high rate of β-oxidation, such as brown adipose tissue, liver, kidney, heart, and skeletal muscle. The purpose of this study was to produce transgenic dogs that overexpress PPARα in a muscle-specific manner. Male beagle fetal fibroblasts were infected with viruses containing PPARα under a human myoglobin promoter and green fluorescent protein (GFP) under an EF1 promoter. Stable infectants were isolated with puromycin, and stored in liquid nitrogen until somatic cell nuclear transfer. Natural oestrus and ovulation of 22 dogs (13 oocyte donors and 9 recipients) was monitored by serum progesterone concentration, and in vivo-matured oocytes were recovered by surgical oviduct flushing. After cumulus cells were removed by repeated pipetting, nuclear materials of the denuded oocytes were removed. A GFP-expressing cell was selected under ultraviolet light, and injected into the enucleated oocyte. Then, the oocyte-cell couplets were fused electrically and activated chemically. Immediately after activation, the cloned embryos were transferred into the oviducts of recipient dogs. Pregnancy diagnosis was performed at least 27 days after embryo transfer by ultrasonography, and serum progesterone concentration, rectal temperature, and fetal heartbeat were monitored for safe delivery. As results, 152 in vivo oocytes were recovered from oocyte donor dogs, and 116 cloned embryos were produced with the infected fetal fibroblast cells. Among them, 113 cloned embryos were transferred into 9 recipient dogs, and 3 were diagnosed as pregnant. Three healthy transgenic cloned puppies were produced, and they could serve as a canine model having enhanced physical performance and obesity resistance.
This study was supported by NRF (#2014R1A1A2059928), RDA (#PJ010928032016), IPET (#311062-04-3SB010), Nature Cell, Research Institute for Veterinary Science, Natural Balance Korea and the BK21 plus program.