bluuebunny/crossref_metadata_2025 huggingface dataset:
198 SUCCESSFUL TRANSPLANTATION OF BUFFALO (Bubalus bubalis) GERM CELLS TO HOMOLOGOUS RECIPIENTS
Authors: Sharma A. , Kumaresan A. , Singla S. , Palta P. , Manik R. S. , Chauhan M. S.
Published: December 2016

Transplantation of isolated germ cells from a fertile donor male into the seminiferous tubules of infertile recipients can result in donor germ cells-derived sperm production. This technique has the potential to be used as an alternative strategy for producing transgenic livestock with higher efficiency and less time and capital requirement than the current methods. The objective of the present study was to investigate whether the homologous transplantation of isolated buffalo germ cells could result in colonization of recipient testes. Germ cells were isolated from prepubertal buffalo testes (4–6 months of age) by using double enzymatic digestion method and filtration through 80- and 60-µm nylon mesh filters. Further enrichment was achieved by differential plating on Datura stramonium agglutinin lectin-coated dishes and after that Percoll density gradient centrifugation as descrived by van Pelt et al. (1996) with minor modifications. A discontinuous density gradient was prepared with 60, 50, 40, 36, 34, 32, 30, 28, and 20% Percoll in a 15-mL centrifuge tube. The enriched germ cells were then labelled with red fluorescent linker dye PKH26 (Sigma, St. Louis, MO, USA) as per the manufacturer’s instructions, and ~10 million cells/testis were transferred into the rete testis of 3 recipients (16–18 months of age) under ultrasonographic guidance. After 45 days, testes were surgically removed and samples were prepared for analysis of labelled cells via wet mount of seminiferous tubules and individual cells isolation. When wet mount specimen were observed under a fluorescence microscope, PKH26-positive cells were identified on the seminiferous tubule basement membrane in all 3 recipients, which indicated that these cells had successfully migrated from the tubule lumen and were likely to be donor germ cells. In freshly isolated cells, clumps of PKH26-positive cells were observed, which indicated either cell division or extensive local colonization of specific areas of the seminiferous tubules. In conclusion, we report successful homologous transplantation of germ cells in prepubertal buffalo testes. Further studies will investigate functionality of transferred testicular cells.
20 EFFECT OF PENICILLAMINE, HYPOTAURINE, AND EPINEPHRINE TREATMENT ON MOTILITY, HYPERACTIVITY AND ACROSOME REACTION OF RAM SPERMATOZOA
Authors: El-Shahat K. , Ismail T. , Badr M. , Zaki K.
Published: December 2016

The aim of this study was to determine the effects of in vitro treatment of freshly ejaculated ram spermatozoa with different concentrations of penicillamine, hypotaurine, and epinephrine (PHE) at different incubation times on motility, hyperactivity (HA) and acrosome reaction (AR). Freshly ejaculated spermatozoa collected from three rams were pooled and then subjected to swim up technique in modified sperm Tyrode’s albumin lactate pyruvate (S-TALP) medium supplemented with different concentrations of PHE (0, 10, 20, 30, 40, 50, 75, and 100 mM). Following incubation (0, 1, 2, 3 and 4 h), sperm motility and hyperactivity were examined under the phase contrast microscope and acrosome reaction was detected by staining of the spermatozoa with silver nitrate. Results showed that treatment of spermatozoa with high concentrations of PHE (30, 40, 50, 75, and 100 mM) significantly increased the motility when compared with the control immediately after dilution (82.0, 82.0, 81.0, 82.0, 82.0 v.76.0%, respectively).This increase existed for the first and second hours of incubation. However, when the incubation time was increased for more than 2 h total motility significantly decreased (P < 0.05, ANOVA) as compared with the control. The same trend was observed in hyperactive motility. Treatment of spermatozoa with 50 and 75 mM of PHE for 1 h significantly increased the percentage of sperm with incomplete AR (20.0 and 27.0% respectively). At 4 h incubation, (49.0%) of spermatozoa treated with 75 mM PHE had undergone complete AR. Furthermore, the maximum value of total acrosome reaction (73.0%) was achieved at 4 h post incubation after addition of 75 mM PHE to ram sperm. In conclusion, under our experimental conditions, 75 mM PHE for 4 h was considered the best concentration of PHE for treatment of ejaculated ram spermatozoa for in vitro induction of acrosome reaction.
205 PRODUCTION OF Cas9-EXPRESSING CATTLE USING DNA TRANSPOSON
Authors: Hahn S.-E. , Yum S.-Y. , Lee S.-J. , Lee C.-I. , Kim H.-S. , Kim H.-J. , Choi W.-J. , Lee J.-H. , Jang G.
Published: December 2016

A genome-editing technology, CRISPR/Cas9 system is proved to be a powerful tool for knockout and knock-in in various species. When 2 components [Cas9 and single guide (sg) RNA] are delivered into cells or embryos, the events of gene editing occur. Because Cas9 is essential for every gene editing based on the CRISPR/Cas9 system, some studies reported that efficiency of gene editing would be increased as Cas9 was integrated into cells or animals. Accordingly, if the Cas9-expressing cattle is born, it would be broadly used for gene editing in cattle. For this study, the Cas9 and RFP genes were cloned into the PiggyBac transposon system. PiggyBac-Cas9-RFP and transposase were microinjected into 1436 IVF embryos and 241 blastocysts were formed. Blastocysts with RFP expression accounted for 14.1% of total formed blastocysts. Five blastocysts were selected and transferred into 5 recipient cow (1 embryo per recipient). After gestation periods, 4 transgenic cattle were delivered without any veterinary assistance. From transgenic cattle, ear skin tissue was collected for primary culture. On those primary cells, sgRNA in DNA form for various genes such as PRNP, RB1, and BLG were transfected with 2 μg of sgRNA per 5 × 105 cells using electroporation. As expected, every group of each sgRNA delivered was confirmed to be mutated by T7E1 assay. The data demonstrated that, for the first time, transgenic cattle with Cas9 expression were born, grown up to date (age = 5 months) and will be a valuable resource for genome editing in cattle.
This work was supported by BK21 PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU 550–20160004).
208 PRODUCTION OF TRANSGENIC CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS CONTAINING HUMAN INSULIN GENE THROUGH HAND-GUIDED CLONING
Authors: Mehta P. , Kaushik R. , Chauhan M. S. , Palta P. , Singla S. , Singh M. K. , Manik R. S.
Published: December 2016

Diabetes is a growing disease worldwide and has emerged as a major healthcare problem in India. Insulin is an essential medicine for the treatment of diabetes. Large dairy animals, such as buffaloes and cows, may be used as bioreactors for cost-effective production of human insulin. The present study was aimed to produce transgenic buffalo embryos containing the human insulin gene through hand-guided cloning for production of transgenic animals. Buffalo female fetal fibroblast cells at passage number 3 were transfected using mammary gland- specific expression vector containing the human insulin gene under buffalo β-lactoglobulin promoter by nucleofection method and cultured with G418 drug for 3 weeks to obtain positive transgenic cell clones. Transgene integration into buffalo female fetal fibroblast genome was confirmed by PCR and Southern blotting. Nontransfected and transgene integrated cells were used as nuclear donors to produce embryos by the hand-guided cloning technique. The developmental competence and quality of embryos as judged by total cell number and TUNEL assay were compared among transgenic and nontransgenic (control) embryos. The blastocyst rate was lower (P < 0.05) for transgenic embryos than that of nontransgenic cloned embryos (35.97 ± 2.16 v. 45.80 ± 4.11, respectively). The apoptotic index was found to be lower (P < 0.05) for control blastocysts than that for transgenic blastocysts. However, the total cell number was similar (P < 0.05) among transgenic and control cloned blastocysts. Thus, transgenic cells, and subsequently transgenic embryos containing the human insulin gene, were successfully produced and transferred in recipients. In the future, these may be used for production of transgenic buffalo expressing human insulin in its milk and thus can be further utilised in large-scale production of human insulin.
24 RELATIONSHIP BETWEEN BOAR SPERM TRAITS AND FERTILITY RATE FOLLOWING ARTIFICIAL INSEMINATION UNDER SMALLHOLDER PRODUCTION SYSTEMS
Authors: Matabane M. B. , Nethenzheni P. , Thomas R. , Norris D. , Nephawe K. , Tsatsimpe M. , Nedambale T. L.
Published: December 2016

The prediction of sperm fertility has a great economic importance to the pig breeding industry. The objective of the study was to determine the relationship between boar sperm quality and fertility following artificial insemination (AI) under smallholder production systems. A total of 18 ejaculates were collected from 3 breeding boars using a hand-gloved technique. Aliquots of diluted semen were assessed for sperm motility using a computer aided sperm analysis before AI. Sperm viability was evaluated using Synthetic Binding CD-14 (SYBR-14+)/propidium iodide (PI–), whereas sperm morphology was evaluated using Eosin Nigrosin staining. Fluorescent microscope was used at 100× magnification to count 200 sperm per slide. The semen was extended with Beltsville Thawing Solution and contained 3 × 109 sperm/dose. A total of 73 multiparous sows were inseminated twice. Fertility was measured by conception rate, farrowing rate, litter size and number of piglets born alive following AI. Sperm quality and fertility data were analysed using one-way ANOVA. Spearman’s rank correlation was used to determine the relationship between sperm quality and fertility traits. The mean values for total sperm motility ranged from 93.5 to 96.8%. Progressive and rapid sperm motility differed significantly (P < 0.05) among the boars. However, no significant differences were found for sperm velocity traits. The mean values for morphologically normal sperm ranged from 47.8 to 60.9% and live sperm ranged from 71.8 to 77.2%, but did not differ significantly among the boars (P > 0.05). Conception rate from different boars varied (P < 0.05) from 63.6 to 93.3%. Of all fertility traits studied, conception rate was significantly related to total sperm motility rate (r = 0.34, P < 0.0029), progressive motility (r = 0.29, P < 0.0141) and rapid motility (r = 0.34, P < 0.0032), although relatively low. There was a low positive relationship between morphologically normal sperm and fertility traits (P > 0.05). In conclusion, total, progressive, and rapid sperm motility rate were the only sperm traits significantly related to conception rate. Conversely, litter size and number born alive were not correlated with sperm motility, viability, or morphology traits.
29 POSITRON EMISSION TOMOGRAPHY IMAGING OF BRAIN METABOLISM AND DOPAMINERGIC NEURON DESTRUCTION IN PARKINSON'S DISEASE MODEL PIG
Authors: Oh H. J. , Moon J. , Kim G. A. , Lee S. , Paek S. H. , Kim S. , Kim H. , Kim J. H. , Lee B. C.
Published: December 2016

Due to similarities between human and porcine, pigs have been proposed as an excellent experimental animal for human medical research. Especially in paediatric brain research, piglets share similarities with human infants in the extent of peak brain growth at the time of birth and the growth pattern of brain. Thus, these findings have supported the wider use of pigs rather than rodents in neuroscience research. Previously, we reported the production of porcine model of Parkinson's disease (PD) by nuclear transfer using donor cell that had been stably infected with lentivirus containing the human α-synuclein gene. The purpose of this study was to determine the alternation of brain metabolism and dopaminergic neuron destruction using noninvasive method in a 2-yr-old PD model and a control pig. The positron emission tomography (PET) scan was done using Biograph TruePoint40 with a TrueV (Siemens, Munich, Germany). The [18F]N-(3-fluoropropyl)-2β-carbomethoxy-3β-(4-iodophenyl) nortropane (FP-CIT) was administrated via the ear vein. Static images of the brain for 15 min were acquired from 2 h after injection. The 18F-fluorodeoxy-D-glucose PET (18F-FDG PET) images of the brain were obtained for 15 min at 45 min post-injection. Computed tomography (CT) scan and magnetic resonance imaging (MRI) were performed at the same location of the brain. In both MRI and CT images, there was no difference in brain regions between PD model and control pigs. However, administration of [18F]FP-CIT was markedly decreased in the bilateral putamen of the PD model pig compared with the control pigs. Moreover, [18F]FP-CIT administration was asymmetrical in the PD model pig but it was symmetrical in control pigs. Regional brain metabolism was also assessed and there was no significant difference in cortical metabolism of PD model and control pigs. We demonstrated that PET imaging could provide a foundation for translational Parkinson neuroimaging in transgenic pigs. In the present study, a 2-yr-old PD model pig showed dopaminergic neuron destruction in brain regions. Therefore, PD model pig expressing human α-synuclein gene would be an efficient model for human PD patients.
This study was supported by Korea IPET (#311011–05–5-SB010), Research Institute for Veterinary Science, TS Corporation and the BK21 plus program.
44 SUCCESSFUL KIDDING AFTER ULTRARAPID VITRIFICATION OF GOAT EMBRYOS
Authors: Toishibekov Y. , Yermekova M.
Published: December 2016

This work evaluated methods for goat morulae cryopreservation by using cryoloop: vitrification (V) and super-cooling ultra-rapid vitrification (SCURV). The vitrification method was applied according to the method described by Vajta et al. (1998 Mol. Reprod. Dev. 51, 53–58). Both treatments used a vitrification solution [VS: 20% (3.6 mol L−1), ethylene glycol (EG), 20% (2.4 mol L−1) dimethylsulfoxide (Me2SO)] 0.5 mol L−1 sucrose in DPBS with 10% BSA in both methods. In our experiment, we used the Vit-Master™ (MTG, Bruckberg, Germany). The super-cooled LN facilitates heat transmission between LN and the cryosolution interface and this is efficient for bovine semen and blastocyst cryoconservation (Arav et al. 2002 Mol. Cell. Endocrinol. 187, 77–81). By surgical flushing 25 super stimulated goats, 127 transferable morulae were harvested; 39 morulae were transferred fresh to synchronized recipients (control) and the others were cryopreserved by V (n = 46) or SCURV (n = 42), respectively thawed or warmed, and transferred to recipients. Embryos were vitrified using the cryoloop. They were first incubated in 50% VS for 2 min and then transferred for 30 s into 100% VS. Each embryo was loaded by cryoloop, which was immediately submerged into and stored in liquid nitrogen. Warming was done by placing the narrow end of the cryoloop into DPBS + 0.25 M sucrose for 5 min. Embryos were then transferred into DPBS + 0.125 M sucrose for 3 min and finally to DPBS until transfer. The SCURV morulae were then exposed to 50% and 100% VS at 37°C for 2 min and 30 s, respectively. Embryos after saturation in VS were transferred by cryoloop and using negative pressure of liquid nitrogen in the chamber for freezing with the VIT-Master. Thawing vitrify embryos was accomplished by placing the vitrified embryos in solutions of sucrose 0.25 M and 0.125 M with 2- and 3-min exposures accordingly. After thawing, embryos were transferred. Statistical analysis was done using Student’s test. The kidding rate following transfer of fresh, frozen-thawed vitrification, and SCURV methods were 22, 16, and 16 kids, respectively. No statistical difference was found for the percentage of does kidding following transfer thawed after vitrification (34.7 ± 4.5%a), and SCURV methods (38.1 ± 5.9%b). The survival rate following transfer of fresh embryos (56.4 ± 4.9c) was higher and in line with previous findings using VS. Differences were statistically significant (ac, bc P < 0.05). Importantly, our data suggested that the SCURV method can be used for cryopreservation of goat morulae and has similar success to the vitrification method. While further work on the developmental competence of embryos cryopreserved with the SCURV method is needed, we hypothesise that SCURV, with a faster freeze rate and potentially a lower level of cryoprotectants, may be able to minimize ice crystal formation; SCURV should be further evaluated as a routine mechanism for cryopreserving goat embryos.
46 VITRIFICATION OF IMMATURE AND MATURE BOVINE OOCYTES
Authors: Hardin P. T. , Diaz F. A. , Foster B. A. , Gutierrez E. J. , Bondioli K. R.
Published: December 2016

While vitrification has become a valuable system used in oocyte and embryo preservation, there is still much to be learned in optimizing this protocol. Both mature and immature oocytes can be vitrified but each presents challenging aspects. Mature oocytes have microfilaments that are not yet developed in immature oocytes, which are fragile and may be disrupted by ice crystal formation during freezing. Further, currently many different cryoprotectants are used in different concentrations, most being combinations of dimethyl sulfoxide (DMSO), glycerol, and ethylene glycol. This study aimed to determine if vitrification solutions composed of ethylene glycol and either dimethyl sulfoxide or glycerol resulted in more-competent post-thaw oocytes, and to determine if maturation stage affected optimal vitrification solution. As validation of the IVF protocol, fresh mature oocytes from a commercial source were fertilized and proportion, with pronuclei formation 48 h post-IVF was recorded. Two experiments evaluated 2 cryoprotectant solutions by analysing post-vitrification and thaw competence of in vitro-fertilized oocytes to form pronuclei. Oocytes in both studies were exposed to 2 sequential vitrification solutions containing 10% DMSO or glycerol, 10% ethylene glycol and 0.5 M sucrose, and then 20% DMSO/glycerol and ethylene glycol and 0.5 M sucrose, before vitrification on cryolocks. In the first study, immature bovine oocytes (n = 200) were vitrified. Following thawing and IVM, they were analysed for pronuclei formation, with 8.49% and 0% fertilization following vitrification in DMSO and glycerol, respectively (P < 0.01). In the second study, mature oocytes were vitrified (n = 200), thawed, and fertilized using the same methods as in study 1. In total, 12.62% and 3.4% of the mature oocytes were successfully fertilized following vitrification in DMSO and glycerol, respectively (P < 0.05). Fisher’s exact test was used for all statistics in both studies. These results suggest that DMSO in combination with ethylene glycol may be superior to glycerol for vitrification of both immature and mature bovine oocytes.
48 EFFECT OF DIMETHYL SULFOXIDE- OR GLYCEROL-BASED VITRIFICATION PROTOCOLS ON THE DNA METHYLATION OF BOVINE CUMULUS-OOCYTE COMPLEXES
Authors: Gutierrez E. J. , Diaz F. A. , Foster B. A. , Hardin P. T. , Bondioli K. R.
Published: December 2016

Although vitrification is becoming increasingly common for oocyte preservation, there has been recent evidence that some cryoprotectants may alter DNA methylation and so result in decreased oocyte developmental competence and abnormal embryonic development post-warming. The objective of this study was to determine the effect of dimethyl sulfoxide (DMSO)- or glycerol-based vitrification protocols on DNA methylation in bovine cumulus-oocyte complexes (COC). The vitrification protocols evaluated included a combination of ethylene glycol (EG) with either DMSO or glycerol (GLY). Cumulus-oocyte complexes were first exposed to equilibration solution (ES) consisting of 7.5% DMSO or GLY and 7.5% EG for 9 min at room temperature (RT) before being transferred to vitrification solution (VS) containing 15% DMSO or GLY, 15% EG, and 0.5 M sucrose. While in VS, 3 to 4 COC were loaded into an open system vitrification device (Cryolock®, BioTech Inc.) and plunged into LN within 1 min. For warming, COC were exposed to dilution solution 1 consisting of 0.5 M sucrose (37°C) for 2.5 min and to dilution solution 2 consisting of 0.25 M sucrose for 2.5 min (RT). Base media for all solution was PBS supplemented with 20% FBS. Cumulus-oocyte complexes (n = 137) were collected from crossbred cows by ovum pick-up on 3 separate days (repetitions), with half of the oocytes each day being matured to metaphase II (MII) stage before vitrification and the other half vitrified as germinal vesicle (GV) COC. The resulting treatments were DMSO GV v. Glycerol GV and DMSO MII v. Glycerol MII. Fresh COC (GV and MII stage, respectively) were utilised as controls. DNA methylation analysis of oocytes was performed through fluorescent staining using a primary antibody anti-5 mC (1:1000) and secondary antibody Alexa Fluor 488 (1:500). Fluorescence intensity of samples was measured using ImageJ software (NIH, Bethesda, MD, USA). Data were analysed through ANOVA with post hoc Tukey’s test. No differences between the vitrification groups or controls were found when analysing DNA methylation of GV stage COC (P = 0.1825). Metaphase II stage COC showed statistical difference between groups (P < 0.001) where glycerol-treated MII oocytes exhibited higher DNA methylation compared with DMSO MII and fresh MII COC. Results of the experiment showed that the glycerol-based vitrification protocol causes alteration of DNA methylation of bovine metaphase II cumulus-oocyte complexes, and as such may not be an optimal choice in mature oocyte vitrification protocols.
58 RADIO-FREQUENCY IDENTIFICATION INTELI-STRAWS: GAMETES AND EMBRYO PACKAGING, STORAGE, AND INFORMATION RECOVERY
Authors: Kjelland M. E. , Loper T. , Woodley C. , Swannack T. M. , Stroud T. K. , Romo S.
Published: December 2016

The assisted reproduction industry involving sales and services for gametes and embryos for domestic animals of commercial value is a large market totaling millions of dollars annually. The objective of this study was to develop and test gamete and embryo packaging—Inteli-Straws (I-S) equipped with radio-frequency identification (RFID) technology. Specifically, French straws (0.25 and 0.5 mL) were modified to include extreme cold-tolerant RFID microchips. Two groups of I-S were formed: Group (G)1: RFID chips that were autoclaved (n = 49), and G2: RFID chips that were not autoclaved (n = 47). Both groups had a control that was not exposed to liquid nitrogen (LN). Each group was exposed to LN up to 4 times: 2 slow freezes first and then 2 fast (i.e. vitrification) freezes, and I-S RFID chip survival was determined. I-S detection and readability (non-autoclaved) was also measured, placing I-S just above LN (in vapors, n = 43) or just below LN (n = 38). Statistical differences (α = 0.05) were determined using Fisher’s exact test. The results between G1 and G2 were not significantly different (P = 0.108) after 4 rounds of cryopreservation (and thawing). For G1, 98% (48/49) of the I-S RFID chips remained operational, and control and treatment were not significantly different (P = 1.000). For G2, 89.4% (42/47) of the autoclaved RFID chips remained operational, and control and treatment not significantly different (P = 0.099). RFID chip readability results; that is, the ability to detect the I-S versus not able to detect the I-S, comparing placement just above liquid nitrogen (LN) versus the placement just below LN were not significantly different (P = 0.105). Notably, detection differences varied within each group, with I-S in G1 (mean = 9.5; SD = 3.5 cm) readable at a larger distance, 5.2 cm farther than the mean of G2 (mean = 4.3; SD = 1.9 cm). During AI or embryo transfer (ET), a technician may not clearly identify the label or colour of straw, may incorrectly record the information, or may take more time than desirable to record it. Increased exposure times may lead to decreased viability of gametes and embryos. The results show that by using the I-S, one may quickly scan the straw within LN or LN vapors, thereby automatically detecting information and even uploading it to a database (e.g. scanner sophistication). We are not aware of comparable device to I-S for locating and retrieving associated information without removing the gamete/embryo packaging from LN or LN vapors; unlike traditionally labelled straws (e.g. laser etched or ink labels). Also, for AI and ET, the I-S can be quickly scanned and the straw information automatically detected and uploaded to a database.
64 EFFECTS OF NANOPURIFIED BOAR SEMEN FOR ARTIFICIAL INSEMINATION ON PROTEIN DETECTION IN SWINE OFFSPRING MUSCLE AND FAT TISSUE
Authors: Moorhead W. A. , Durfey C. L. , Liao S. , Devost-Burnett D. , Gastal G. D. A. , Ryan P. L. , Willard S. T. , Feugang J. M.
Published: December 2016

Standard extended semen contains both viable and non-viable spermatozoa. Magnetic nanoparticles have proven to be effective in the purification of boar semen by targeting nonviable spermatozoal cells (Feugang et al. 2015 IVF Reprod. Med. Genet. 3, 2), allowing potentially greater efficiency within the pork production industry. Previous research lacks data regarding the biochemical effects on offspring produced from such nanopurified semen. Here, we aim to determine whether there is a difference in protein expression between offspring produced with standard and nanopurified semen. Myoglobin (MYO) and fatty acid synthase (FAS) were chosen as the protein markers for this study because they are often studied in reference to meat quality. Myoglobin provides meat its red colour, and FAS assembles fatty acids contributing to tenderness and palatability. The results produced here will provide a baseline for further research in the meat quality and the safety of consuming meat produced with this nanopurification method. Six sows maintained on our experimental farm were inseminated with standard (CTRL) and nanopurified (NANO) boar semen, leading to the birth of viable full-term piglets. At weaning, 10 pigs (5 male and 5 female) were randomly selected from each group and allowed to grow to market weight. Samples of longissimus muscle and SC back fat were then collected from each pig and prepared for protein analyses. Western immunoblotting and immunofluorescence of tissue samples were performed using anti-MYO and anti-FAS antibodies. Images were appropriately captured and quantified (ImageJ). Data (mean ± SEM) were analysed (ANOVA/Wilcoxon) with P < 0.05 set as threshold of significance. Western immunoblotting confirmed the specificity of each antibody. Myoglobin and FAS proteins were highly detected in muscle and fat tissues, respectively, and there were no differences between groups for each protein. Compared with the CTRL group, immunofluorescence signals of MYO in fat tissues and FAS in muscle tissues were significantly reduced in NANO group pigs, irrespective of the sex. However, MYO immunofluorescence levels in fat tissues and FAS levels in all tissues were significantly reduced in females of the NANO group compared with their counterparts in the CTRL group. In conclusion, MYO and FAS proteins were confirmed as good markers for muscle and fat tissues, respectively, with similar immunofluorescence levels between CTRL and NANO groups. Current preliminary data also show that sperm nanopurification (1) may decrease FAS protein expression in muscle tissue with the possibility to affect meat quality and (2) mainly affected females in this study. Further studies are needed for confirmation of these early findings.
70 XBP1 DYSREGULATION BY CRISPR/Cas9-MEDIATED GENE EDITING DURING PORCINE EMBRYO EARLY DEVELOPMENT
Authors: Gutierrez K. , Glanzner W. G. , Dicks N. , Bohrer R. C. , Currin L. G. , Michalovic L. , Agellon L. B. , Bordignon V.
Published: December 2016

Early developing embryos are very sensitive to their developmental milieu. For instance, variations in temperature, pH, or culture media composition can trigger endoplasmic reticulum (ER) stress. Endoplasmic reticulum stress has been shown to reduce early embryo development and embryo quality. In response to ER stress, embryos activate coping mechanisms, such as the unfolded protein response, to re-establish ER homeostasis. The X box binding protein (XBP1) is one of the main transducers of the unfolded protein response. Under ER stress, XBP1 mRNA is unconventionally spliced by IRE1α to yield its activated isoform (XBP1s), which allows expression of genes involved in protein folding, transport, and degradation. XBP1s has been detected in oocytes and early stage embryos of different species, including Drosophila, Xenopus, zebrafish, mice, and pigs, suggesting an important role during early embryo development. In this study, we used the CRISPR/Cas9 gene editing technology to investigate the effect of XBP1 dysregulation during development of porcine embryos in vitro. Pig zygotes were produced by intracytoplasmic sperm injection using in vitro-matured oocytes. Treatments consisted of (a) Cas9 mRNA (Cas9) + 1 single guide RNAs targeting XBP1 gene region 1 (sgRNA-1); (b) Cas9 + 1 single guide RNAs targeting XBP1 gene region 2 (sgRNA-2); (c) Cas9 + sgRNA-1 + sgRNA-2; (d) Cas9 alone; and (e) sgRNA-1 + sgRNA-2. After injection, embryos were cultured in vitro for 5 to 7 days to assess development and cell numbers. Experiments were repeated 5 or more times, and data were analysed by ANOVA and means compared using Student’s t-test or Tukey–Kramer Honestly Significant Difference test. Embryo cleavage was similar between the groups (a = 59.8 ± 4.9%, b = 58.8 ± 5.3%, c = 68.86 ± 2.2%, d = 66.4 ± 5.9%, and e = 70.10 ± 1.9%), but development to the blastocyst stage was substantially reduced (P < 0.05) in the groups injected with Cas9 + sgRNAs (a = 18 ± 4.5%, b = 16 ± 1.5%, and c = 5.3 ± 2.8%) compared with controls (d = 33.7 ± 6.2% and e = 31.4 ± 1.2%). Moreover, we observed that only 22.7% of the embryos treated with Cas9 + sgRNA-1 + sgRNA-2 were able to develop beyond 8-cell stage compared with 62.5% in the control group injected with Cas9 alone. These findings suggest that XBP1 activity is required for maintenance of ER homeostasis and development of porcine embryos beyond the main period of embryo genome activation.
75 PLACENTA PROTEIN PROFILE CHARACTERIZATION BY PLACENTOME SIZE AND GESTATIONAL AGE IN CATTLE
Authors: Ramirez M. M. , Martins J. F. , Villamil P. R. , Bertolini M. , Bertolini L. R. , Moura A. A.
Published: December 2016

The placenta plays a key role in normal conceptus development and in mediating effects of the maternal system on the fetus. Changes in the environment or in placental function may affect fetal developmental and lead to what is known as “developmental origins of health and disease.” Thus, the aim of this study was to analyse the protein profile of bovine placentomes of different sizes at distinct gestation ages to determine spatial and temporal differences or similarities in the protein pool of biological significance to the conceptus and the newborn. Samples of placentomes (n = 36) representing small, medium, and large sizes among placentomes at 60, 90, 120, and 160 days of gestation were collected at a local slaughterhouse and freeze-dried for protein analysis. For that, 5 mg of each tissue sample was used for protein extraction with 1% Triton X and sonication for 10 min at 4°C, with protein concentration per sample determined by the Bradford method. A total of 300 μg of protein was subjected to 2D-SDS PAGE electrophoresis (GE Healthcare®), and gels were stained with Coomassie Blue G-250. Spots were analysed using the PDQuest, with data compared by ANOVA or the Kruskall-Wallis test (SAS). In silico protein identification was performed using ExPASy. On average, 74 proteins were detected in the gels, regardless of placentome sizes and gestational ages. No interactions were detected between placentome sizes and gestational periods. However, differences were observed in the mean number of proteins between small (n = 69), medium (n = 90), and large (n = 64) placentomes (P = 0.002), and between Days 60 (n = 113), 90 (n = 65), 120 (n = 44), and 160 (n = 74) of gestation (P = 0.001). One specific spot (32.45 kDa, 4.6 pI) represented 1.0, 10.8, 12.1, and 2.1% of the intensity of all valid spots for Day 60, 90, 120, and 160 placentomes, respectively, indicating a bell-like expression. For small placentomes, all the spots had similar intensities, whereas for medium placentomes, the 32.45 kDa (4.6 pI) spot represented 3.0% of the intensity of all valid spots. For large placentomes, the 32.45 kDa (4.6 pI) spot and a 46.19 kDa (5.4 pI) spot represented 5.8 and 5.0% of the total intensity for all spots, respectively. Six and 10 spots were differentially identified between placentome sizes and between gestational periods, respectively, from which male-enhanced antigen-1 proteins and angio-associated migratory cell protein were identified by in silico analysis, with the former only present on Day 90 placentomes, and the latter for 120 days of gestation. Further analyzes on the identities of such placental proteins and their profiles is underway, which will be crucial to the comprehension of placentome function and growth during pregnancy, and for the understanding of physiological mechanisms and processes associated with normal conceptus development and life ex utero.
77 TRANSCRIPTOME PROFILING IN OOCYTES-EMBRYO AND GRANULOSA CELLS FROM BOVINE
Authors: Sirard M. A. , Fournier É. , Dufort I. , Gilbert I. , Robert C.
Published: December 2016

The EmbryoGENE Network (2008–2013) has assembled data from more than 764 DNA arrays covering the entire transcriptome of bovine oocytes, embryos, and somatic cells, such as granulosa and cumulus cells. The data were obtained using the same extraction-amplification-hybridization-analysis protocols enabling normalization and statistical analysis across time, tissues, and experiments. To provide the scientific community with this information in an organised and functional matter, we have created a web application (http:// http://emb-bioinfo.fsaa.ulaval.ca/granulosaIMAGE/) where the information on 40,000 genes products from 4 tissues are currently available: oocytes (mainly at the germinal vesicle stage), blastocysts in different culture conditions (namely in vitro, in vivo, different concentrations of lipids, free radicals and glucose, as well as blastocysts from cloning, vitrification, and different breeds). Also represented is the complete panel of late folliculogenesis from 3 mm upward including growing, plateau, and atretic phase oocytes, as well as their response to LH and several types of ovarian stimulation. In addition, a postpartum panel of dominant follicles (PGF2α synchronized at Day 30, 60, 90, 12) with high or low BHB (beta-hydroxybutyrate) or vitamin treated (B-6 and B-9) is represented. In each of the panels, a red background indicates differential expression for any given gene or isoform (40,000 on the EmbryoGENE array). To complete the information, a RNAseq experiment was performed on each stage from the germinal vesicle oocyte to the blastocyst (including early and late 8 cell) and presented as an absolute expression level, which is a measure of the total number of RNA molecules for the selected family of transcripts. The web application also provides relative expression values, which measure the proportion of the total transcripts represented by the selected family of transcripts. This information is provided as a graphical representation from the germinal vesicle to the blastocyst stage. This source of information may be cited either by referring to the website or by citing each individual analysis (>20 published papers from >20 principal investigators from Canada and abroad, all publicly available) used to assemble the profiles. We anticipate that this site will be useful to all scientists in bovine reproduction.
The EmbryoGENE network was supported by NSERC Canada and Semex Canada for the bovine side.
84 PTEROSTILBENE CAN REDUCE THE PERCENTAGE OF LIPIDS AND REACTIVE OXYGEN SPECIES IN IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Sosa F. , Fernando de la Torre J. , Álvarez H. , Pérez S. , Kjelland M. E. , Romo S.
Published: December 2016

The actual challenge for the majority of research centers involves the embryo culture medium, since it is known that the culture medium plays a large role in determining embryo quality. Pterostilbene (PT) is a natural analogue of Resveratrol, an antioxidant that can reduce lipids in embryos, but no reports exist of PT being used with IVF-produced embryos or gametes. The objective of the present research was to evaluate the effect of PT in culture media CDM1 and CDM2 on embryo production, cell count, lipid accumulation, and reactive oxygen species (ROS). A total of 4 concentrations of PT and a control were evaluated, i.e. 3, 1, 0.33, 0.11, and 0 μM, in 2 separate experiments. The first experiment was performed using 6 replicates (n = 204) to evaluate blastocyst production (n = 201) and determine the percentage of lipids using the stain Sudan-Black, (n = 100). Hoechst 33258 and propidium iodide were used for determining cell counts. The second experiment was performed using 7 replicates, the effect of using PT (0.33 μM) was compared with a control with 2 O2 concentrations (5 and 20%) for evaluating ROS production (n = 124). Blastocysts without zona pellucida were incubated 48 h at 38.5°C in PBS (without polyvinyl alcohol) with 60 μL of ro-green fluorescent protein. After incubation, 25 μL of 4′,6-diamidino-2-phenylindole (1 mg mL−1) was added and incubated for 5 min. A fluorescence microscope was used and positive ROS particles digitized using Photoshop CS6 and quantified using the program ImageJ®. The data were transformed to arcsin values for subsequent analysis. In the first experiment, ANOVA and least significant difference tests were used to determine statistical significance (Statgraphics). No significant differences were found among treatments (P > 0.05) for the internal cell mass: 3, 1, 0.33, 0.11 μM PT (29.2 ± 5.2; 28.9 ± 3.8; 22.2 ± 3.2; 29.0 ± 1.7, respectively) and the control (27.0 ± 1.7). The cells of the trophectoderm did not differ (P > 0.05) between treatments (31.7 ± 4.8; 31.3 ± 3.9; 38.6 ± 3.5; 30.8 ± 1.8) and control (33.4 ± 2.1). Total cells did not differ (P > 0.05) between treatments (72.0 ± 9.8; 82.6 ± 4.3; 94.8 ± 12.8; 73.2 ± 9.2) and control (83.8 ± 7.7). Embryo production (Day 8) was greater for control (33.5 ± 3.0) versus treatments (14.1 ± 1.7; 19.4 ± 1.9; 21.1 ± 2.6; 20.8 ± 2.1) (P < 0.05); however, PT reduced the percentage of lipids (11.0 ± 0.8; 10.7 ± 0.9; 11.6 ± 1.3; 11.3 ± 1.1) within the cytoplasm of the embryos (P < 0.05) versus control (17.01 ± 1.20). In the second experiment, a factorial 2 × 2 matrix demonstrated that the O2 concentration did not have an effect on ROS (P > 0.05); however, the PT had a significant effect on the reduction of ROS (P < 0.05), i.e. a negative correlation, r = −0.835. In summary, we determined that PT did not improve the production of blastocysts but resulted in a significant reduction of ROS and lipids.
91 DISRUPTION OF TET1 DURING PORCINE EMBRYOGENESIS USING CRISPR/Cas9 SYSTEM
Authors: Uh K. , Ryu J. , Ray C. , Lee K.
Published: December 2016

Ten-eleven translocation (TET) enzymes catalyse oxidation of 5-methylcytosine to 5-hydroxymethyl cytosine. This TET-mediated conversion of 5-methylcytosine to 5-hydroxymethyl cytosine is implicated in initiating the DNA demethylation process, observed post-fertilization. Three members (TET1–3) of the TET family are differentially expressed during embryo development and appear to have different roles. Previous studies in mice suggest that TET1 is a key regulator in maintaining pluripotency in embryonic stem cells by managing epigenetic marks such as DNA methylation. This would imply that TET1 should be a regulator of epigenetic marks during embryo development, although this has not been demonstrated. Previously, we have cloned porcine TET1 from blastocysts (GenBank accession number KC137683) and demonstrated that the level of TET1 (mRNA and protein) was high in blastocysts. The protein level was greater in the inner cell mass compared with the trophectoderm. In this study, we generated TET1 knockout porcine embryos using CRISPR/Cas9 system to study the role of TET1 in controlling epigenetic marks during porcine embryo development. First, 2 sgRNA, immediately downstream of the presumable translation initiation site, were designed and synthesised; location of the sgRNA were nucleotide position at 2 to 21 bp and 23 to 42 bp, respectively (KC137683). Then, sgRNA (10 ng μL−1 each) and Cas9 mRNA (20 ng μL−1) were injected into the cytoplasm of IVF zygotes, and Day 7 blastocysts were genotyped. All embryos carried mutations on both alleles of TET1 (10/10), one homozygous and 9 biallelic mutations. However, immunocytochemistry analysis of other CRISPR/Cas9 injected embryos revealed that TET1 was not removed (10/10), indicating that the sgRNA may have not introduced a premature stop codon 3′ to the presumable translation initiation site. Therefore, 2 new sgRNA were designed to generate a premature stop codon at the 5′ side of a key functional domain, the 2-oxoglutarate-Fe(II)-dependent oxygenase domain (4690 to 5160 bp); the locations of the 2 sgRNA were 4450 to 4469 bp and 4501 to 4520 bp, respectively. Similarly, all of the embryos carried mutations in TET1 (7/7), 2 homozygous and 5 biallelic mutations. In addition, TET1 proteins were not detected in 11 of 16 blastocysts, confirmed by immunocytochemistry. In this study, we successfully generated embryos lacking TET1 by introducing designed CRISPR/Cas9 system during embryogenesis. Presence of TET1 from the first injection experiment suggests that the presumable translation initiation site is not accurate. Discrepancy between genotyping and immunocytochemistry results from the second injection experiment indicates that embryos possessing TET1 protein probably have mutations in triplets, thus no premature stop codon was synthesised. Further studies will focus on identifying the role of TET1 in maintaining pluripotency and epigenetic modification during pre-implantation stage using these embryos.
92 DOES BLASTOCENTESIS AFFECT CRYOPRESERVATION SURVIVAL OF IN VITRO-PRODUCED BOVINE EMBRYOS?
Authors: Tutt D. A. , Lyons R. E. , Holland M. K.
Published: December 2016

The cattle industry primarily employs embryo bisection in order to obtain genetic samples for pre-implantation screening and selection of embryos. Although practical and rapid, bisection is invasive and adversely affects embryo viability and cryopreservation. An alternative biopsy approach is to aspirate the blastocoele fluid (referred to as blastocentesis), which not only provides a genetic sample, but also has the potential to improve cryopreservation (Palini et al. 2013 Repro. Biomed. 26, 603–610). This study investigates blastocentesis as a low impact biopsy procedure to rapidly sample bovine blastocysts with limited effect on embryo cryopreservation survival. In vitro-produced embryos were selected at expanded blastocyst stage and placed in a 50-μL drop of holding media on an inverted microscope. The embryo was held using a glass holding pipette attached to a micromanipulator, oriented so that the inner cell mass was toward the bottom of the view. A 7-μm spiked intracytoplasmic sperm injection pipette attached to the other micro-manipulator was used to pierce the blastocoele cavity and aspirate the blastocoele fluid. Once removed, the aspirate was transferred into 4-μL TE buffer for later genetic analysis. Collapsed blastocysts were then vitrified in ~7 μL 16.5% ethylene glycol, 16.5% dimethyl sulfoxide in TCM-199 (Hanks salts) with 20% FCS and 0.5 M sucrose. Embryos were held for a minimum of 1 week and then thawed and assessed for survival. Post-cryopreservation embryo survival was measured as the proportion of embryos that re-expanded after 48 h in culture. One-way ANOVA was used for statistical testing. A total of 181 control (intact) and 182 blastocentesis embryos were vitrified over 6 replicates. In all but one replicate, non-biopsied control embryos had higher re-expansion rates. Overall, the re-expansion rate was significantly (P = 0.05) higher for control embryos (73.5%) than blastocentesis embryos (61.5%) (Table 1). Initial experiments would suggest embryo survival is affected by the biopsy procedure; however, because this was not the case with every replicate, this may be batch or technician/human error dependent. Further study is required to assess full effect of blastocoele fluid aspiration on embryo cryopreservation, particularly investigating effectiveness for in vivo-produced embryos and subsequent effect on pregnancy rates. Likewise, further investigation is required to assess whether the sample collected is sufficient to allow accuracy over a variety of genetic tests. More than 20 embryos can easily be sampled in an hour using this technique, making it a rapid and efficient process. Given the speed and compatibility with cryopreservation, this sampling procedure may offer an alternative to current techniques used for cattle embryo genetic assessment.

Table 1.

Post-thaw survival rates of in vitro-produced embryos vitrified after blastocentesis1
Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase
Authors: McAndrew Ryan P. , Sathitsuksanoh Noppadon , Mbughuni Michael M. , Heins Richard A. , Pereira Jose H. , George Anthe , Sale Kenneth L. , Fox Brian G. , Simmons Blake A. , Adams Paul D.
Published: December 2016

Significance

NOV1 is a stilbene cleavage oxygenase (SCO). SCOs cleave the central double bond of stilbenes, forming two phenolic aldehydes. Many stilbenes, such as resveratrol, are produced by plants as secondary metabolites. They are also formed from lignin during kraft pulping. SCOs are related to carotenoid cleavage oxygenases (CCOs), which cleave β-carotene or apocarotenoids. Carotenoids play important roles in photosynthesis and light perception in the eye. We present the structure of an SCO and the structure of a CCO-related enzyme in ternary complex with oxygen and substrate. This structure allows us to propose a mechanism relevant to both SCOs and CCOs, where the substrate is activated for reaction with a ferric-superoxo electrophile by active site base-catalyzed deprotonation of a phenol group.
Unexpected relations which characterize operator means
Authors: Osaka Hiroyuki , Wada Shuhei
Published: December 2016

We give some characterizations of self-adjointness and symmetricity of operator monotone functions by using the Barbour transform

f
↦

t
+
f

1
+
f

f \mapsto \frac {t+f}{1+f}

and show that there are many non-symmetric operator means between the harmonic mean

!
!

and the arithmetic mean

∇
\nabla

. Indeed, we show that there exists a non-symmetric operator mean between any two symmetric operator means.